Journal of Chemical Neuroanatomy 31 (2006) 2–36

www.elsevier.com/locate/jchemneu

The organization of the brainstem and spinal cord of the mouse:

Relationships between monoaminergic, cholinergic,
and spinal projection systems
Veronique G.J.M. VanderHorst a,b,c,*, Brun Ulfhake a
a
Department of Neuroscience, Karolinska Institutet, Stockholm, S-17177, Sweden
b
Department of Pathology, University of Groningen Medical Center, University of Groningen, Groningen NL-9700 RB, The Netherlands
c
Department of Medicine, University of Massachusetts Medical Center, Worcester, MA 01655, USA
Received 12 December 2004; received in revised form 31 July 2005; accepted 1 August 2005
Available online 23 September 2005

Abstract

Information regarding the organization of the CNS in terms of neurotransmitter systems and spinal connections in the mouse is sparse,
especially at the level of the brainstem. An overview is presented of monoaminergic and cholinergic systems in the brainstem and spinal cord
that were visualized immunohistochemically in inbred C57BL/6 and outbred CD-1 mice. This information is complemented with data on
spinal cord-projecting systems that were characterized using retrograde tracing, spinal hemisections, and double labeling techniques.
Attention is given to differences in these systems related to spinal levels. The data are discussed with reference to studies in the rat, and to
standardized information as provided in the atlas of the mouse brain.
Although the overall organization of these systems in the mouse is largely similar to those in the rat, species differences are present in
relative location, size and/or connectivity of cell groups. For example, catecholaminergic neurons in the (ventro)lateral pons (A5 and A7 cell
groups) in the mouse project to the spinal cord mainly via contralateral, and not ipsilateral, pathways. The data further supplement information
as provided in standardized brainstem sections of the C57BL/6 mouse [Paxinos, G., Franklin, K.B.J., 2001. The mouse brain in stereotaxic
coordinates. Academic Press, San Diego], especially with respect to the size and/or location of the catecholaminergic retrorubral field (A8
group), A5, A1, and C1 cell groups, and the serotonergic B4 group, reticulotegmental nucleus (B9 group), lateral paragigantocellular nucleus
and raphe magnus nucleus (B3 group). Altogether this study provides a comprehensive overview of the spatial relationships of
neurochemically and anatomically defined neuronal systems in the mouse brainstem and spinal cord.
# 2005 Elsevier B.V. All rights reserved.

Keywords: Noradrenergic; Adrenergic; Dopaminergic; Serotonergic; Immunohistochemistry; C57BL/6; CD-1; Retrograde tracing

Abbreviations: 3N, oculomotor nucleus; 3V, third ventricle; 4N, trochlear nucleus; 4V, fourth ventricle; 5HT, 5-hydroxy tryptamine; 6N, abducens nucleus;
7N, facial nucleus; 10N, dorsal motor nucleus of the vagus; 11N, accessory nucleus; 12N, hypoglossal nucleus; A1, A1 noradrenergic cells; A10-dc,
dopaminergic cells in the dorsocaudal part of the A10 cell group; A5, A5 noradrenergic cells; A7, A7 noradrenergic cells; Acs5, accessory trigeminal nucleus;
Acs7, accessory facial nucleus; Amb, nucleus ambiguus; AP, area postrema; APT, anterior pretectal nucleus; APTD, anterior pretectal nucleus dorsal part;
APTV, anterior pretectal nucleus ventral part; aq, aqueduct; Arc, arcuate nucleus; B9, B9 serotonergic cell group; Bar, Barrington’s nucleus; BIC, brachium of
the inferior colliculus; C1, C1 adrenergic cells; C2, C2 adrenergic cells; C7, seventh cervical segment; CER, cerebellum; CerN, deep cerebellar nuclei; CG,
central gray; ChAT, choline acetyltransferase; cic, commissure of the inferior colliculus; Cli, caudal linear nucleus; CN, cochlear nucleus; CnF, cuneiform
nucleus; cp, cerebral peduncle; CTB, cholera toxin subunit B; Cu, cuneate nucleus; DCN, cochlear nucleus; dorsal part; dh, dorsal horn; dl-PAG, dorsolateral
periaqueductal gray; DMSp5I, dorsomedial part of the interpolar spinal trigeminal nucleus; DMTg, dorsomedial tegmental area; d-PAG, dorsal periaqueductal
gray; DPGi, dorsal pontine gigantocellular nucleus; DpMe, deep mesencephalic nucleus; DR, dorsal raphe nucleus; DR-C, dorsal raphe nucleus; caudal part
* Corresponding author. Present Address: Department of Neurology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, KS406, Boston, MA
02215, USA. Tel.: +1 617 877 0458; fax: +1 617 667 3499.
E-mail address: vvanderh@bidmc.harvard.edu (Veronique G.J.M. VanderHorst).

0891-0618/$ – see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchemneu.2005.08.003
 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36 3

1. Introduction stem, and of monoaminergic and cholinergic fibers at

With the introduction of genetically modified mice, this
species is becoming a standard model to study the function
of the nervous system. However, with respect to the
organization of the murine brainstem and spinal cord, we
heavily rely on information gathered in the rat. Precise and
comprehensive information on monoaminergic and choli-
nergic systems in the brainstem and spinal cord, and on
brainstem–spinal pathways in mice is far from complete.
Available data have mainly been gathered in the context of
functional studies (Ure and Rodriguez, 2002; Camand et al.,
2004). This scarcity in information might be due to relative
difficulties in performing tracing experiments in mice, as
a result of their small size, and the enormous body of
information gathered over decades in the rat. Although the
overall organization of neuronal populations in the CNS is
similar among mammalian species, the exact boundaries of
distinct cell groups, their spatial relation to each other, and
their connectivity vary significantly among species. The
analysis of anatomical results obtained in mice, based on rat
information, may therefore lead to interpretational difficul-
ties or even mistakes.
In the present study we focus on the organization of
spinal cord-projecting systems in the murine brainstem. In
the first immunohistochemical part of our study, we will
provide an overview of the distribution of serotoninergic,
catecholaminergic and cholinergic neurons in the brain-
different levels of the spinal cord. In the second part of the
study, this information is supplemented with a description
of the location of spinal cord-projecting neurons in the
brainstem. In this part, spinal hemisections are used to
distinguish between populations that project by way of
ipsilaterally or contralaterally descending pathways. This
data might be relevant for studies on functional recovery
following spinal cord injury or multiple sclerosis (Ure and
Rodriguez, 2002; Camand et al., 2004). Finally, we will
determine which subpopulations of monoaminergic or
cholinergic neurons in the brainstem project to the spinal
cord.
These issues were addressed by combining tracing
experiments to identify spinal cord-projecting neurons with
conventional and fluorescent immunohistochemical tech-
niques to visualize tyrosine hydroxylase (TH; as a marker
for adrenergic, noradrenergic and dopaminergic neurons),
choline acetyltransferase (ChAT), vesicular acetylcholine
transporter (VAChT), and serotonin (5HT). The experi-
ments were performed in female and male mice of a
frequently used inbred strain (C57Bl/6) and of an outbred
strain (CD-1).
The results are presented and discussed in such a way that
thorough knowledge of the anatomy is not required. The
results are put in the context of previous studies in the rat and
information provided in the atlas of the C57Bl6 mouse brain
(Paxinos and Franklin, 2001).

Abbreviations (Continued): DR-D, dorsal raphe nucleus; dorsal part; DR-I, dorsal raphe nucleus; inferior part; DR-V, dorsal raphe nucleus; ventral part;
DR-VL, dorsal raphe nucleus; ventrolateral part; DTg, dorsal tegmental nucleus; ECIC, external cortex of the inferior colliculus; ECu, external cuneate nucleus;
ER, estrogen receptor; EW, Edinger–Westphal nucleus; f, fornix; fr, fasciculus retroflexus; Gi, gigantocellular reticular nucleus; GiA, gigantocellular reticular
nucleus; pars alpha; GiV, gigantocellular reticular nucleus; pars ventralis; Gr, gracile nucleus; IC, inferior colliculus; icp, inferior cerebellar peduncle; IF,
interfascicular nucleus; IML, intermediolateral cell column; In, intercalated nucleus of the medulla; InCo, intercollicular nucleus; InG, intermediate gray layer
of the superior colliculus; InWh, intermediate white layer of the superior colliculus; IO, inferior olive; IP, interpeduncular nucleus; IRt, intermediate reticular
nucleus; KF, Kölliker–Fuse nucleus; L4, fourth lumbar segment; L5, fifth lumbar segment; L6, sixth lumbar segment; LC, locus coeruleus; LCP, lateral
collateral pathway; LDTg, laterodorsal tegmental nucleus; LDTgV, laterodorsal tegmental nucleus; ventral part; lfp, longitudinal fasciculus of the pons; LG,
lateral geniculate nucleus; LH, lateral hypothalamus; ll, lateral lemniscus; LL, nucleus of the lateral lemniscus; l-PAG, lateral periaqueductal gray; LPGi, lateral
paragigantocellular nucleus; LRt, lateral reticular nucleus; LVe, lateral vestibular nucleus; M, mammillary nucleus; mcp, middle cerebellar peduncle; MdD,
medullary reticular nucleus; dorsal part; MdV, medullary reticular nucleus; ventral part; Me5, mesencephalic trigeminal nucleus; MG, medial geniculute
nucleus; MGD, medial geniculute nucleus; dorsal part; MGV, medial geniculute nucleus; ventral part; ml, medial lemniscus; mlf, medial longitudinal
fasciculus; MnR, median raphe nucleus; Mo5, motor trigeminal nucleus; mt, mammillothalamic tract; MVeMC, medial vestibular nucleus; magnocellular;
MVePC, medial vestibular nucleus; parvicellular; NRA, nucleus retroambiguus; NTS, nucleus of the solitary tract; Op, optic nerve layer of the superior
colliculus; Pa5, paratrigeminal nucleus; PAG, periaqueductal gray; PBG, parabigeminal nucleus; PBN, parabrachial nucleus; PBP, parabrachial pigmented
nucleus; pc, posterior commissure; PCRt, lateral parvicellular field; PDTg, posterodorsal tegmental nucleus; PH, posterior hypothalamic area; PMV,
premammillary nucleus; ventral part; Pn, pontine nuclei; PnC, caudal pontine reticular field; PnO, oral pontine reticular field; PnR, pontine raphe nucleus;
PP, peripeduncular nucleus; PPTg, pedunculopontine tegmental nucleus; Pr, nucleus prepositus hypoglossi; Pr5, principal sensory trigeminal nucleus; Pr5DM,
principal sensory trigeminal nucleus; dorsomedial part; Pr5VL, principal sensory trigeminal nucleus; ventrolateral part; py, pyramidal tract; RAmb, nucleus
retroambiguus; Rli, rostral linear nucleus of the raphe; RMC, red nucleus; magnocellular part; RMg, nucleus raphe magnus; Ro, nucleus of Roller; ROb, raphe
obscurus nucleus; RPa, raphe pallidus nucleus; RPC, red nucleus; parvicellular part; RPO, rostral periolivary region; RRF, retrorubral field (A8 dopaminergic
cells); RtTg, reticulotegmental nucleus of the pons; RVL, rostral ventrolateral medulla; S1, first sacral segment; S2, second sacral segment; s5, sensory root of
the trigeminal nerve; SC, superior colliculus; scp, superior cerebellar peduncle; SNC, substantia nigra; compact part; SNL, substantia nigra; lateral part; SNR,
substantia nigra; reticular part; SO, superior paraolivary nucleus; Sol, nucleus of the solitary tract; Sp5, spinal trigeminal nucleus; sp5, spinal trigeminal tract;
Sp5C, spinal trigeminal nucleus; caudal part; Sp5I, spinal trigeminal nucleus; interpolar part; Sp5O, spinal trigeminal nucleus; oral part; Sp5ODM, spinal
trigeminal nucleus; oral part dorsomedial subdivision; SPN, sacral parasympathetic nucleus; SpVe, spinal vestibular nucleus; SubB, subbrachial nucleus; SubC,
subcoeruleus region; SubCA, subcoeruleus region; pars alpha; SuG, superficial gray layer of the superior colliculus; SuM, supramammillary nucleus; SuVe,
superior vestibular nucleus; T7, seventh thoracic segment; TH, tyrosine hydroxylase; Tz, nucleus of the trapezoid body; VAChT, vesicular acetylcholine
transporter; VCN, ventral cochlear nucleus; vl-PAG, ventrolateral periaqueductal gray; VLTg, ventrolateral tegmentum; VTA, ventral tegmental area; VTg,
ventral tegmental nucleus; WGA–HRP, wheat germ agglutinin–horseradish peroxidase; xCST, decussation of the corticospinal tract; xscp, decussation of the
superior cerebellar peduncle; ZI, zona incerta
4 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36

2. Material and methods
2.1. Animals
A total of 39 mice of both sexes were used (males: n = 5;
females: n = 34; Table 1). The handling and housing of the
animals, all surgical procedures, and postoperative monitor-
ing of the animals were performed according to the European
Guidelines for Animal Research. The experimental protocol
was reviewed and approved by the Stockholm Animal Ethical
Committee (permits 106/02, 349/02, and 399/03).
The experiments were performed on inbred (C57Bl/6;
B&K Universal, Stockholm; n = 35) and outbred (CD-1;
Charles River; n = 4) strains. The C57Bl/6 mouse was chosen
because it is one of the most widely used inbred mouse strains.
The outbred CD-1 mice were included as controls to study
whether major strain-related differences in the CNS would
exist, as these are frequently found between inbred strains.
Similarities in the results between these two unrelated strains
would suggest that the organization of monoaminergic and
cholinergic systems in the brainstem is conserved.
Nine mice were used for immunohistochemistry only. In
each of these cases, 4 complete series of (1:8, 40 mm)
consecutive brainstem and spinal cord sections were used for
visulization of VAChT, ChAT, 5HT and TH, respectively.
Seventeen mice were exclusively used for tracing experi-
ments (wheat germ agglutinin–horseradish peroxidase;
WGA–HRP-injected cases). In each of these cases, two
series of 40 mm (1:4) consecutive brainstem sections were
used for visualization of the tracer. Finally, in 13 mice, tracer
experiments were combined with immunohistochemistry
(12 cholera toxin subunit B or CTB-injected animals and one
WGA–HRP injected mouse). In the WGA–HRP injected
case, a 1:4 series was used for visualization of the tracer and

Table 1
Overview of animals
Level of spinal cord injection Case Strain Sex Injection Hemisection
Tracer Side Level Side
None 6 C57Bl6 F – – – –
None 33 C57Bl6 F – – – –
None 34 C57Bl6 F – – – –
None 42 C57Bl6 F – – – –
None 43 C57Bl6 F – – – –
None 44 C57Bl6 M – – – –
None 63 C57Bl6 F – – – –
None 64 C57Bl6 F – – – –
None 65 C57Bl6 F – – – –
C1–medulla 25 C57Bl6 F WGA–HRP L – –
C6–7 69 C57Bl6 F WGA–HRP R – –
C7, medial 35 C57Bl6 F WGA–HRP R – –
T7–8 52 C57Bl6 F WGA–HRP R – –
T7–8 66 C57Bl6 F WGA–HRP R – –
T9–11 9 C57Bl6 F WGA–HRP R – –
T9–11 37 C57Bl6 F WGA–HRP R T6–7 L
T10–12 15 C57Bl6 F WGA–HRP R T7–8 L
T10–L1 17 C57Bl6 F WGA–HRP R T7–8 L
L1–2 67 C57Bl6 F WGA–HRP R T9–10 L
L1–2 68 C57Bl6 F WGA–HRP R T8–9 R
L3–4 10 C57Bl6 F WGA–HRP R – –
L3–4, dorsal 7 C57Bl6 F WGA–HRP R – –
L3–4 14 C57Bl6 F WGA–HRP R T9–10 L
L4–5 38 C57Bl6 F WGA–HRP R – –
L4–5 51 C57Bl6 F WGA–HRP R – –
L5–6 16 C57Bl6 F WGA–HRP bi – –
S1–2 13 C57Bl6 F WGA–HRP bi – –
T7–8 47 C57Bl6 F CTb R – –
T7–8 98 C57Bl6 F CTb R – –
T7–8 106 C57Bl6 M CTb R – –
T7–8 84 CD-1 M CTb R – –
T7–8 89 CD-1 F CTb R – –
T13–L1 46 C57Bl6 F CTb R T7–8 L
T13–L1 61 C57Bl6 F CTb R – –
T13–L1 62 C57Bl6 F CTb R – –
L1–2 99 C57Bl6 F CTb R T7–8 L
L1–2 107 C57Bl6 M CTb R T7–8 L
L1–2 83 CD-1 M CTb R T7–8 L
L1–2 88 CD-1 F CTb R T7–8 L
 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36 5

TH. In the CTB-injected mice, separate 1:8 series of 30 mm

sections were used to visualize TH only (n = 10), 5HT only
(n = 4), VAChT only (n = 3), ChAT only (n = 3), CTB only
(n = 13), CTB-TH (n = 10), CTB 5HT (n = 6), CTB-VAChT
(n = 2), CTB-ChAT (n = 8), TH-5HT (n = 2), VAChT-TH
(n = 2).
In female mice, variation in endocrine status might
induce changes in gene expression profiles, resulting in
changes in intensity of labeling (Kritzer and Kohama, 1998,
1999). To control for this variability, all eight CTB-injected
females as well as the eight females that were used
exclusively for immunohistochemistry were ovariectomized
2–6 weeks prior to the perfusion. Although the purpose of
this study is not to study estrogen related quantitative
differences in monoaminergic and cholinergic systems, two
of the ovariectomized females were treated with estradiol
benzoate (dissolved in sesame oil; 5 mg in 0.05 ml; daily
subcutaneous injections for 4–5 days prior to perfusion;
VanderHorst et al., 2005) to exclude any major differences in
the distribution of monoaminergic and cholinergic neurons
in the brainstem and spinal cord. Similarly, males were
included to control for any major differences related to
gender.

2.2. Surgical procedures
2.2.1. Retrograde tracing experiments
In 30 of the 39 mice (Table 1), tracer injections were
placed into the spinal cord. Before all surgical procedures,
the animals were anaesthetized with isoflurane (induction:
4%; maintenance: 1.6–2.3%; Baxter, Kista, Sweden).
After the surgical procedures, the animals received
analgesics (Buprenorphine 0.025 mg/kg, s.c.; Temgesic,
Schering-Plough, Brussels, Belgium) and were returned to
their home cages where they remained until they were
sacrificed.
To inject tracer into the spinal cord, a midline incision
was made in the skin of the back. Muscle tissue was gently
cleaned away from the vertebrae and with a forceps the
dorsal part of one vertebra was removed, thus giving access
to three spinal segments (i.e. the one underlying the vertebra
and the caudally and rostrally adjacent segments). Using a
glass pipette (tip inner diameter approximately 25 mm), a
total amount 200–1400 nl of WGA-HRP (Sigma, St. Louis,
MO; 1–2%, diluted in distilled water) or CTB (List
Biological Lab., Campbell, CA; 0.2–2%; diluted in distilled
water) was injected into one side of the spinal cord using a
pressure pump (Microinjector 5242, Eppendorf, Hamburg,
Germany). The total volume was divided over multiple
smaller injections that were placed to cover 2–3 rostro-
caudally adjacent spinal segments by keeping the pipette
under an angle. The cervical and lumbar enlargement were
injected with larger volumes than the sacral and thoracic
cord (see Table 1 for individual cases). After the injections,
the area was rinsed with saline, and the overlying muscle
layer and skin were closed.
Fig. 1. Line drawings showing the experimental design used to distinguish
between contralaterally and ipsilaterally descending pathways. In cases in
which the hemisection is placed contralateral to the tracer injection (A),
labeled neurons are found ipsilateral and contralateral to the injection. The
ipsilaterally located neurons project to the spinal cord via an ipsilaterally
descending pathway, whereas the contralaterally located neurons have
axons that cross the midline before they descend. A unilateral injection
combined with a hemisection ipsilateral to the injection (B) identifies
neurons that give rise to ipsilaterally or contralaterally descending pathways
with axons crossing the midline at the level of termination.
2.2.2. Spinal hemisections
In 11 experiments (Table 1), tracer injections into the
lower thoracic or upper lumbar cord were preceded by a
hemisection of the mid-thoracic spinal cord (Fig. 1). The
hemisections were made using microsurgical scissors and
forceps, and were placed contralateral (n = 10) or ipsilateral
(n = 1) to the tracer injection site. This approach, in
combination with the data provided in the non-hemisected
cases, allowed the identification of populations of brainstem
neurons that project to the spinal (i.e. upper lumbar) cord by
way of an ipsilateral or contralateral pathway (VanderHorst
and Holstege, 1995; VanderHorst et al., 2000)
2.2.3. Perfusion
After an appropriate survival time (1–21 days) the
animals were anaesthetized with chloralhydrate (0.3 ml; i.p.;
8% in saline) and transcardially perfused with saline (at
room temperature; 20 ml) followed by fixative (at room
temperature; 20 ml). In experiments in which WGA–HRP
was used, the fixative contained 2% glutaraldehyde, 0.5%
paraformaldehyde, and 4% sucrose in 0.1 M phosphate
6 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36
buffered saline (PBS; pH 7.4). In the CTB-injected cases, the
fixative contained 4% paraformaldehyde and 4% sucrose in
PBS. The brain, brainstem and spinal cord were removed as
one piece, post fixed for 1 h, and cryo protected in 20%
sucrose in PBS (at 4 8C; 1–4 days). The brainstem and C1-2
spinal segments were cut out as one block and together with
the spinal segments (cut into blocks of segments: C3–6, C7–
T2, T3–6, T7–10, T11–L3, L4–S2, S3–Co3) frozen and
stored at –80 8C.
2.3. Histological procedures
2.3.1. Immunohistochemistry
The lower brainstem and C1–2 segments, the spinal
segments in which the injections and hemisections were
placed (n = 30), and all spinal segments of the cases without
injections (n = 9) were cut into transverse sections in a
cryostat. Four to eight series of consecutive, free-floating
40 mm or 30 mm sections were collected for each case
(Table 1). Care was taken to cut the brainstem perpendicular
to the long axis, comparable to brainstem sections in other
species. At the level of the rostral midbrain, this plane starts
to deviate due the slight angle that is present between
midbrain and hypothalamus.
WGA–HRP injection sites (n = 18) were incubated in
diaminobenzidine (DAB, Sigma; St. Louis, MO; 10 mg per
25 ml of TBS) and reacted with 0.3% H2O2 (20 ml per 2 ml
of DAB-solution). Antero- and retrogradely WGA–HRP
labeled axons and neurons in the brainstem were visualized
using a standard protocol (Olucha et al., 1985; VanderHorst
et al., 2000) in the dark, at 4 8C). The WGA–HRP reaction
product was stabilized with CoCl2 (Rye et al., 1984) which
resulted in a black, temperature and light-stable precipitate.
For each WGA–HRP injected case (see Table 1), two series
of brainstem sections were processed, one of which was
counterstained with cresyl fast violet.
For immunohistochemical processing, tissue was pre-
incubated for two hours in normal donkey serum (NDS; 5%;
Jackson ImmunoResearch Laboratories, West Grove, PA) in
0.05 M tris buffered saline containing 0.5% Triton X-100
(pH 7.6; TBS+; at room temperature). Using single or double
labeling immunohistochemical protocols, series of tissue
were then incubated with a primary antibody directed
against: tyrosine hydroxylase (TH; 2332; raised in rabbit;
1:3000; Markey et al., 1980), choline acetyl transferase
(ChAT; 2221; raised in rabbit; 1:2000; German et al., 1985),
vesicular acetylcholine transporter (VAChT; raised in goat;
1:3000; Chemicon, Temecula, CA), serotonin (5HT; 1927;
raised in rabbit; 1:3000; Verhofstad and Jonsson, 1983), or
CTB (List Biological Lab., Campbell, CA; raised in goat;
1:6000 to 1:7000). The primary antibodies were diluted in
TBS+ containing 2% NDS, and the incubations were done
over one to two nights, at 4 8C. To obtain a stable, brown
reaction product, tissue was subsequently incubated with
biotinylated secondary antibodies raised in donkey (1:500 in
TBS+; 1 h; at room temperature), avidin–biotin–complex
peroxidase (ABC) solution (1:500 in TBS; Vector Elite Kit,
Vector Laboratories, Burlingame, CA; 1–2 h; at room
temperature), and finally in 0.025% DAB (in TBS with
0.002% H2O2 for 2–5 min at room temperature). After all
incubation steps, tissue was rinsed three times in TBS.
To obtain unamplified fluorescent signals, fluorescein,
Cyanine-5, and/or rhodamine Red X-labeled secondary
antibodies were used that were directed to the hosts in which
the respective primary antibodies were raised (Jackson
Immuno Research Laboratories, West Grove, PA; 1:80; in
TBS+; 2 h; at room temperature). This latter method was
used for double labeling studies in which primary antibodies
were used that had been raised in different species.
To simultaneously visualize markers for which the
primary antibodies were raised in the same species (CTB
and VAChT, or TH and 5HT), the concentration of one of
these antibodies was decreased 10–20-folds, after which the
signal of this antibody was amplified first as follows. After
incubations with the appropriate biotinylated secondary
antibodies and ABC solution (see above), the tissue was
reacted using a tyramide signal amplification kit (Perkin
Elmer Life Sciences, Boston, MA; fluorescein or tetra-
methyl rhodamine; 1:100 for 4–10 min). After the comple-
tion of the amplification procedure, the second marker
was then visualized using a different fluorophore, using
conventional immunofluorescent techniques as described
above. For this particular technique, appropriate controls
were included. These controls included omission of either
the primary antisera or secondary antibodies, and compar-
ison to parallel series of tissue in which the markers were
visualized separately with conventional fluorescent techni-
ques.
After the incubations, the tissue was mounted on glass
slides, dehydrated in ethanol, cleared in xylene, and
coverslipped using DePeX (EMS, Fort Washington, PA)
mounting medium. A selection of tissue was counterstained
with cresyl fast violet. Tissue processed using immuno-
fluorescent techniques was mounted, coverslipped with
glycerol containing 2.5% of anti-fading agent DABCO (1,4-
di-aza-bi-cyclo-2,2,2-octane, Sigma) and stored at 20 8C.
2.4. Microscopical analysis and reconstruction
The tissue was analyzed and digitally photographed using
a Zeiss or Nikon microscope (under brightfield or
fluorescent illumination) or a Bio-Rad Confocal micro-
scope. Brightness and contrast were adjusted using Adobe
Photoshop 7.0 (general microscopy) or NIH-image 1.4
(confocal microscopy) software. The illumination condi-
tions during the capturing of pictures as well as the
subsequent adjustment were standardized.
2.4.1. Reconstruction of the brainstem
Line drawings that show the distribution of labeled
neurons were made in one animal using a camera lucida
attached to a Nikon microscope. The rostrocaudal interval
 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36
between individual drawings through the brainstem was
320 mm (of a 1:8 series of 40 mm). For the plottings of
cholinergic and monoaminergic neurons, adjacent series of
consecutive sections of the same animal (ovariectomized
female) were used. The information of these sections,
which were located no further than 80 mm from the level
represented by the drawing, was plotted into these drawings.
The drawings were digitzed using Adobe Illustrator 10.0
software and an Intuos digitizing tablet (WACOM, Krefeld,
Germany). The nomenclature of the anatomical structures is
largely according to Paxinos and Franklin (2001).
In addition to this original data, an effort is made to place
this data into standardized drawings from the C57BL/6
brainstem as provided with the atlas of Paxinos and Franklin
(2001). To place the line drawings on the atlas sections, the
most appropriate rostrocaudal atlas level was chosen based
upon the shape of the sections, without paying attention to
the interval size of 320 mm. The size of the line drawings
was then adjusted mediolaterally and dorsoventrally to
match the left side of the underlying atlas sections. The atlas
sections were not adjusted. The adjustments ranged from a
relative increase of size for medullary sections to a decrease
in size for midbrain sections. The plotted data was then
shown superimposed on standard atlas sections on one side
and the adjusted line drawings on the other side. These
adjustments were necessary because the sections of the
medulla oblongata in the atlas were obtained from two
different animals, were displayed at irregular intervals, and
represented a total length (from rostral midbrain to rostral
cervical cord) of 5.44 mm in the atlas versus 6.72 mm in the
present study.
2.4.2. Reconstruction of spinal segments
In order to loose only a minimum amount of tissue during
the cutting procedure, the spinal cord was cut into blocks
of segments rather than into individual segments. The
segmental level was determined based upon the morphology
of the spinal gray matter of two standard series. For these
standard series, the spinal segments of two mice were cut
separately. For each of the segments, a line drawing was
made of one representative section that had been counter
stained with cresyl fast violet.
3. Results
3.1. Location of monoaminergic and cholinergic
neurons in the brainstem and spinal cord and fiber
distributions in the spinal cord
3.1.1. ChAT and VAChT immunoreactivity
3.1.1.1. Neurons. From rostral to caudal (Fig. 2; with level
0 defined as the level at which the aqueduct of Sylvius opens
into the 4th ventricle; Fig. 3), large neurons immunoreactive
for ChAT or VAChT were found in regions containing
somatic motoneurons, i.e. the oculomotor nucleus (levels 3
7
and 4), trochlear nucleus (level 2), main and accessory motor
nuclei of the trigeminal complex (levels 0 to 2), abducens
nucleus (level 3), accessory facial nucleus (levels 3 and
4), facial nucleus (levels 4 to 6), nucleus ambiguus
(levels 7 to 9), hypoglossal nucleus (levels 8 to 11),
and neck muscle motor nuclei of the upper cervical cord and
caudal medulla (levels 12 to 14). A few ChAT-IR or
VAChT-IR neurons were found in the ventrolateral
medullary tegmentum caudal to the compact formation of
the Amb, extending as far caudally as the caudal nucleus
retroambiguus (NRA) at the level of the decussation of the
corticospinal tract (xCST; levels 10 to 12). In the spinal
cord, large sized ChAT-IR or VAChT neurons were found
throughout the ventral horn (lamina IX).
In addition to these large sized, presumably somatic
motoneurons, populations of small to medium-sized ChAT-
and VAChT-IR neurons were found in the brainstem and
spinal cord. Three such groups were found in the
mesopontine tegmentum, the most rostral of which was
present in the pedunculopontine tegmental nucleus (PPTg;
Fig. 2, levels 1 and 2). This group was located immediately
caudal to the group of catecholaminergic neurons in the
retrorubral field (RRF), and dorsal to the decussation of the
superior cerebellar peduncle at the level of the trochlear
motor nucleus. The caudal part of this group was positioned
in between the nucleus of the lateral lemniscus and another
group of cholinergic neurons, the ventral part of the
laterodorsal tegmental nucleus (LDTgV; Fig. 2, levels 1 to
1). This latter nucleus was located ventral to the ventral
periaqueductal gray (v-PAG) or pontine central gray.
Dorsomedially to this group, in the ventral PAG and pontine
central gray, the remaining part of the laterodorsal tegmental
nucleus was located (LDTg; Fig. 3B and C). The caudal pole
of the LDTg was found medial to Barrington’s nucleus and
the rostral part of the locus coeruleus (LC), and extended
further caudally than the more ventrally located group of
cholinergic neurons in the LDTgV. Medium-sized to small
ChAT-IR or VAChT-IR neurons were also found in the
ventral part of the cochlear nucleus (Fig. 3L), in the motor
nucleus of the dorsal vagal complex (Fig. 2, levels 8 to
11; Fig. 3F and G), spinal laminae X and medial VII
(Fig. 3I and J), and the thoracic intermediolateral cell
column (IML; Fig. 3I). The latter group of neurons was
lightly labeled. No ChAT–IR or VAChT-IR neurons were
present in Edinger–Westphal nucleus in the midbrain, in the
sacral parasympathetic nucleus (SPN; Fig. 3K), or in the
spinal dorsal horn (Fig. 4). Compared to ChAT-IR staining,
VAChT-IR neurons were less intensely labeled in the PPTg
and LDTg, but more strongly labeled in regions containing
somatic motoneurons (Fig. 3H and J).
3.1.1.2. Fibers. With respect to the fiber labeling we will
focus on the spinal cord and caudal nucleus of the spinal
trigeminal complex only, as explained in the introduction.
VAChT-IR fiber labeling was present as a coarse or a fine
punctate reaction product. In the spinal cord, coarse punctate
8 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36

Fig. 2. Line drawings of transverse sections through the midbrain, pons and medulla oblongata (case 34) that show the distribution of ChAT-IR neurons. Each
drawing is of one 40-mm thick section, and each dot represents one labeled profile. Level 0 is defined as the level at which the aqueduct of Sylvius opens into the
4th ventricle. The distance between levels represents 320 mm. For abbreviations, see list of abbreviations. Bar = 1mm.
V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36 9

Fig. 2. (Continued ).
10 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36

Fig. 3. Photomicrograph of ChAT-IR neurons in the PPTg, LDTg, and LDTgV in the mesopontine tegmentum (A–C) and in the ventral part of the cochlear
nucleus (L). In D–G: VAChT-IR neurons in the oculomotor (D), facial (E), hypoglossal (F and G), accessory (G) nuclei, and motor nucleus of the dorsal vagal
complex. Arrow head in G points to an occasional VAChT-IR neuron in the medullary lateral tegmentum; H–K: examples of VAChT and ChAT-IR neurons in the
spinal cord. Note that VAChT-IR staining of motoneurons is more prominent than the ChAT-IR staining (H and J), whereas small sized ChAT-IR neurons are
clearly visible in lamina X (J), the thoracic IML (I), but not in the SPN (K). All pictures (A–L) are taken in case 33. Bar = 100 mm.

VAChT-IR labeling was present in all groups of somatic the spinal cord and the caudal nucleus of the spinal
motoneurons (Fig. 3H), but not around the remaining trigeminal complex (Sp5C; Fig. 4A, D, G, J, and M). The
VAChT-IR neurons (i.e. in lamina X and the IML; Fig. 4G). intensity of the reaction product in lamina II was dependent
Fine punctate VAChT-IR was most prominent in lamina II of on spinal level and mediolateral position (Fig. 4). In addition
V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36 11
12 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36

to this diffuse pattern, a distinct concentration of fine
punctate VAChT-IR was present in the lateral collateral
pathway (LCP; levels L5–S1), extending from the dorsal
horn into the SPN and intermediate gray (Fig. 4M). Some
immunoreactivity was also found in lamina X, and the
intermediate zone (laminae V and VII). No labeling was
present in the fiber tracts of the spinal white matter.
3.1.2. 5HT immunoreactivity
3.1.2.1. Neurons. 5HT-IR neurons (Figs. 5 and 6) were
most numerous in the dorsal raphe nucleus (DR; Fig. 5, level
3 to 2). Within the DR they were most extensively
distributed at the level of the trochlear nucleus (level 2),
where in addition to the dorsal and ventral cluster in the
center (dorsal and ventral subdivisions of the central nucleus
of the DR), a lateral region was also labeled (Fig. 6A). More
rostrally and caudally, the majority of 5HT-IR neurons in
the DR were confined to the central region (Fig. 6B).
A significant group of 5HT neurons was also found
immediately outside the PAG, (ventro)lateral to the medial
longitudinal fasciculus.
At the level of the trigeminal motor nucleus (Fig. 5, levels
0 to 2), 5HT-IR neurons were found in the inferior DR in
the midline of the pontine gray, and in the pontine raphe
nucleus immediately ventral to the pontine gray (Fig. 6C).
More ventrally in the mesopontine tegmentum, 5HT-IR
neurons formed two groups: one located in the midline
extending from the rostral pole of the motor nucleus of the
trigeminal complex to the caudal pole of the oculomotor
nucleus in the median raphe nucleus (MnR) and adjacent
tegmentum (Fig. 5, levels 3 to 0; Fig. 6D) and the other group
located in the more laterally positioned reticulotegmental
nucleus (RtTg; Fig. 6D). This latter group was most
prominent at the caudal pole of the interpeduncular nucleus
(IP; Fig. 5, level 3).
At the levels of the superior olivary complex and the
facial nucleus (Fig. 5, levels 2 to 6; Fig. 6E and F), some
5HT-IR neurons were found in the midline raphe magnus
nucleus (RMg) and adjacent tegmentum. In addition, a few
5HT-IR neurons were located further laterally, dorsal to the
trapezoid body. From the level of the caudal pole of the
facial nucleus at levels 7 to 11, distinct groups of 5HT-IR
neurons were located in the midline raphe pallidus and
obscurus (RPa and ROb; Fig. 6G and H) as well as in the
more ventrolaterally located lateral paragigantocellular
nucleus (LPGi; Fig. 6F and G). These cell groups extended
caudally to the level of the xCST (Fig. 5, levels 11–13). At
this level, significant numbers of 5HT-IR neurons were
diffusely spread throughout the ventral and ventrolateral
medulla (Fig. 6H). No 5HT-IR neurons were found in the
rostral midbrain or in the spinal cord caudal to C1.
3.1.2.2. Fibers. In the spinal cord, 5HT-IR fibers were most
pronounced in the intermediolateral and intermediomedial
cell columns of the thoracic cord (Fig. 4H). Fiber labeling
was also present in lamina IX (Fig. 6I) and in the superficial
layers of the spinal dorsal horn and Sp5C (mainly lamina I
and the outer subdivision of lamina II; Fig. 4, middle
column). The intensity of 5HT-IR in the dorsal horn varied to
some extent along the different spinal segments, though not
as pronounced as for VAChT- and TH-IR fibers labeling
(Fig. 4). Some 5HT-IR fiber labeling was present in the
lumbosacral LCP or in the SPN (Fig. 4N). However, in
contrast to VAChT- and TH-IR fibers in the LCP, staining
was not robust.
In the spinal white matter, the majority of 5HT-IR fiber
tracts were found in the periphery of the dorsolateral and
lateral funiculi.
3.1.3. Tyrosine hydroxylase immunoreactivity
3.1.3.1. Neurons. In the diencephalon and rostral midbrain
(Fig. 7, levels 4 to 7) large numbers of TH-IR neurons were
found in the pars compacta of the substantia nigra (SN;
Fig. 7A) and in the more medially located ventral tegmental
area (VTA; Fig. 8A). In the VTA, TH-IR neurons were most
numerous dorsal to the rostral half of the interpeduncular
recessus, whereas rostrally some were present lateral to the
fasciculus retroflexus. A few neurons were located in the
premammillary and supramammillary nuclei (Fig. 7, levels 5
and 6), pars reticularis of the SN, and lateral SN (Fig. 7,
levels 4 to 6). Just caudal to the red nucleus (Fig. 7, level 3),
many TH-IR neurons were present in the retrorubral field
(RRF). This region extended rostrally to a position lateral to
the caudal part of the pars compacta of the SN.
Another distinct TH-IR cell group was located in the
ventral PAG in the dorsocaudal division of the A10 region
(A10-dc; Figs 7 and 8B and C). These neurons were present
from levels 7 to 2, but were most numerous at the level of the
oculomotor nucleus (i.e. at levels 3 and 4). At levels 2 and 3,
this group overlapped somewhat with 5HT-neurons in the

Fig. 4. Photomicrographs of VAChT-IR (A, D, G, J, M; case 33), 5HT-IR (B, E, H, K, N; case 34), TH-IR (C, F, I, L, O; case 33) fibers and neurons in the caudal
nucleus of the spinal trigeminal complex, C7, T7, L4, and L6 segments. TH-IR neurons were present in the LCP (magnification in O). VAChT-IR: Note that
VAChT-IR labeling was more distinct at thoracic levels (G), and least distinct in the cervical dorsal horn and Sp5C (A and D), with an intermediate signal at
lumbosacral levels (J and M). In lamina II, it was heavier laterally than medially in the enlargements (D and J), whereas it was more evenly distributed in the
thoracic and lower lumbosacral cord. Also, VAChT-IR labeling in lamina II appeared more prominent in the outer subdivision than in the inner subdivision.
5HT-IR: Note that the 5HT-IR signal in lamina I was slightly more prominent medially than laterally, and clearly more pronounced at lower lumbar and thoracic
levels than in the enlargements. TH-IR: Note that TH-IR labeling the dorsal horn was more evident at lumbosacral and thoracic levels (I and O) than at mid-
lumbar or cervical levels (F and L). In the Sp5C, even fewer TH-IR fibers were found in lamina II (C). The TH-IR labeling pattern in the dorsal horn was slightly
more prominent medially than laterally, especially in the thoracic and lumbar segments. In the thoracic dorsal horn, TH-IR fibers formed a plexus extending
from laminae II to IV (3I). Bar = 100 mm for all panels except insert in O for which the bar represents 50 mm. See figure on previous page.
 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36 13

Fig. 5. Line drawings of transverse sections through the midbrain, pons and medulla oblongata (case 34) that show the distribution of 5HT-IR neurons. Each
drawing is of one 40 mm thick section, and each dot represents one labeled profile. Level 0 is defined as the level at which the aqueduct of Sylvius opens into the
4th ventricle. The distance between levels represents 320 mm. For abbreviations, see list of abbreviations. Bar = 1 mm.
14 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36

Fig. 5. (Continued ).
 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36 15

Fig. 6. Photomicrographs of 5HT-IR neurons: A–C: in the dorsal raphe nucleus (DR) and PnR from rostral (A) to caudal (C); D: in the MnR and RtTg; E–H:
RMg, LPGi, ROb, and RPa; I: spinal ventral horn. Arrow heads in H point to 5HT-IR neurons are located close to the xCST and in the lateral tegmentum.
Bar = 100 mm.

DR (see also Fig. 13A), but the majority of TH-IR neurons
were located dorsal to the DR. At level 3, TH-IR neurons
were also present more ventrally in the midline in the caudal
linear nucleus (CLi). These neurons were positioned mainly
between the serotoninergic DR and MnR, whereas a few
dispersed neurons were found between the RRF and the CLi.
A scattered group of TH-IR neurons in the lateral pontine
tegmentum extended rostrocaudally from levels 1 to 3.
Rostrally these cells formed a rather dense cluster just rostral
to the motor nucleus of the trigeminal complex in the
Kölliker–Fuse/A7 region (see discussion; Fig. 7, level 1;
Fig. 8D). Caudally, neurons in the lateral pontine tegmentum
were distributed in a more dispersed way and extended as far
caudal as the level of the facial nerve (also called A5 region,
see discussion; Fig. 7, level 3; Fig. 8F).
In the dorsolateral pons medial to the PBN and
immediately lateral to Barrington’s nucleus, TH-IR neurons
formed a dense cluster in the locus coeruleus (LC; Fig. 7,
levels 0 to 2; Fig. 8E). TH-IR neurons were also found
more ventrally in the lateral pontine tegmentum (at this level
also called subcoeruleus region).
At the pontomedullary junction, a small number of TH-
IR neurons were found ventral to the facial nucleus (levels
4 and 5), and in the gray matter dorsolateral to the 4th
ventricle. Somewhat more caudally a few TH-IR neurons
were present in the midline, ventral to the 4th ventricle at
the level of the facial nucleus (Fig. 7, level 6; Fig. 8J,
arrow).
The majority of TH-IR neurons in the caudal medulla
oblongata were found in two regions: in the ventrolateral and
in the dorsomedial medulla. In the ventrolateral medulla
oblongata, TH-IR neurons formed a continuous column
extending from the level of the caudal pole of the facial
nucleus into the upper cervical cord (Figs. 7 and 8G–I).
These levels correspond to the levels where numerous 5HT-
IR neurons were found in the RPa/ROb and in the LPGi. At
16 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36

Fig. 7. Line drawings of transverse sections through the midbrain, pons and medulla oblongata (case 34) that show the distribution of TH-IR neurons. Each
drawing is of one 40-mm thick section, and each dot represents one labeled profile. Level 0 is defined as the level at which the aqueduct of Sylvius opens into the
4th ventricle. The distance between levels represents 320 mm. For abbreviations, see list of abbreviations. Bar = 1 mm.
V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36 17

Fig. 7. (Continued ).
18 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36

Fig. 8. Photomicrographs of TH-IR neurons (case 33) in: A: the VTA and SNC; B: ventral rostral PAG, CLi and VTA; C: A10-dc cell group dorsal to the DR; D:
the lateral pontine tegmentum; E: LC in the dorsolateral pons; F: medial to the facial nerve (n7; A5 cell group); G–I: ventrolateral medulla oblongata from rostral
(G) to more caudal levels (I). Note that TH-IR neurons form a more compact group caudally. Arrows in G and I point to TH-IR neurons that were found outside
these clusters; J–L: dorsomedial medulla oblongata from rostral (J; including the C3 cell group indicated by arrow head) to caudal (L) levels. They are present in
the area postrema (AP), motor nucleus of the dorsal vagal complex (10N) and commissural nucleus of the solitary complex (Sol). Bar = 100 mm.

the level of the caudal pole of the facial nucleus (level 6) (level 9) to the compact formation of the Amb (Fig. 8H).
TH-IR neurons were found medial to the facial nucleus Caudal to the compact formation of the Amb (Fig. 8I), TH-
(Fig. 8G). Further caudally, this group was positioned lateral IR neurons were located in the so-called A1 region, close to
to the LPGi and ventromedial (levels 7 and 8) or ventral the lateral reticular nucleus (LRt; Fig. 7, dorsomedial at level
 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36 19

10, and shifting to a dorsolateral position further caudally

at levels 11 and 12). This column of TH-IR neurons
continued to levels caudal to the xCST, i.e. as far caudal as
the third cervical segment. At this latter level, they were
located in the white matter, ventral to the lateral tip of the
dorsal horn.
In the dorsal part of the caudal medulla oblongata
(Fig. 8J–L), TH-IR neurons were present in the motor
nucleus of the dorsal vagal complex (Fig. 7, levels 6 and
7), the area postrema (AP; levels 8 and 9), in the
medial part of the nucleus of the solitary tract (Sol; levels 7
to 11) and in the commissural nucleus of the Sol (Fig. 8L,
levels 10 to 14). TH-IR neurons in the Sol were most
numerous in the medial part just caudal to the obex. Caudal
to the xCST, a few TH-IR neurons were found in lamina X.
This group extended as far caudal as the third cervical
segment. In the caudal medulla, a few TH-IR neurons were
in addition found in the lateral tegmentum (intermediate
reticular nucleus; IRt), distributed between the two groups of
TH-IR neurons in the Sol and the ventrolateral medulla (see
also Fig. 12F).
Between spinal levels C3 and L4, no TH-IR neurons were
observed. However, a distinct population of small, oval
shaped TH-IR neurons was present in the L5–S2 segments
(Figs. 4O and 9). These neurons were mainly located in the
LCP that extends from the lumbosacral lateral superficial
dorsal horn into the SPN. A few neurons were also present
medially in the superficial dorsal horn or in the intermediate
zone just dorsolateral to the central canal.

3.1.3.2. Fibers. Throughout the spinal cord, TH-IR fibers

were found in all laminae, but were somewhat more
prominent in the dorsal horn, especially in the inner
subdivision of laminae II and III (at all levels; Fig. 4, right
column). Similar to VAChT-IR labeling, the abundance
of TH-IR fibers in the dorsal horn varied significantly
depending on spinal level and mediolateral position. In
addition to pronounced labeling of laminae II and III,
prominent TH-IR fiber labeling was also present throughout
the thoracic IML (Fig. 4I). However, the most distinct TH-IR
fiber labeling in the spinal cord was present in the
lumbosacral LCP (Fig. 4O).
In the spinal white matter, the majority of TH-IR fiber
tracts were found in the dorsolateral funiculus (Fig. 4, right
column), but labeled fibers were also present in the lateral,
ventrolateral, and ventromedial funiculi.
3.2. Distribution of retrogradely labeled neurons after
spinal cord injections
3.2.1. Injections and hemisections
In 30 mice (Table 1), the tracers WGA–HRP (n = 18) or
CTB (n = 12) were injected at different spinal levels to
retrogradely label neurons in the brainstem. The injections
were combined with a hemisection rostral to the level of
injection in 11 cases (Table 1). This approach served to
Fig. 9. Line drawings of transverse sections through the L5–S2 spinal
segments showing the distribution of TH-IR neurons (case 44). The majority
of neurons in this population is located in the LCP. In the C3–L4 segments,
TH-IR neurons were absent. Each drawing is of one 40-mm thick section,
and each dot represents one labeled profile. Bar = 1 mm.
distinguish ipsilaterally from contralaterally descending
pathways (Fig. 1). Although the majority of the hemisections
was complete, some were incomplete in the dorsomedial (case
14) and/or ventromedial funiculus (cases 14 and 15).
First, we will give an overview of the results in these
hemisection experiments (with injections into the upper
lumbar and thoracic cord) in order to divide populations of
retrogradely labeled neurons into those with an ipsilateral or
contralateral pathway. Next, we will present the results
obtained from tracer injections at different spinal levels
(with or without hemisection).
20 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36

Fig. 10. Line drawings of transverse sections through the midbrain that show the distribution of neurons that project to the thoracic cord (green; case 37; a
thoracic injection with a contralateral hemisection), and upper lumbar cord (red; case 67; a unilateral upper lumbar injection with a contralateral hemisection;
blue, case 68; a unilateral upper lumbar injection with an ipsilateral hemisection). Dots on the left represent neurons that make use of contralaterally descending
pathways, whereas the dots on the right represent neurons with ipsilaterally descending pathways. Note that in many regions the distribution of neurons with
 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36 21

ipsilaterally descending pathways is different from those with contralaterally descending pathways. Each drawing is of one 40-mm thick section, and each dot
represents one labeled profile. Level 0 is defined as the level at which the aqueduct of Sylvius opens into the 4th ventricle. The distance between levels represents
320 mm. For abbreviations, see list of abbreviations. Bar = 1 mm.
22 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36
 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36 23

3.2.2. Retrogradely labeled neurons with
contralaterally descending pathways
The most rostrally located group of neurons giving rise
to a contralaterally descending pathway was present in the
magnocellular part of the red nucleus (Fig. 10, levels 4 and
5; Fig. 11B). In the lateral pontine tegmentum, labeled
neurons were found in the Kölliker–Fuse region (Fig. 11D)
and more ventrally a few small sized neurons were found
(Fig. 10, levels 2 to 3). In the dorsal pons, a few medium
sized neurons were found in the LC, LC pars alpha
(Fig. 11G). More ventrally some were found in the
subcoeruleus region and A5 region (levels 1 and 2;
Fig. 11J). At the level of the rostral medulla oblongata,
large neurons were found in the gigantocellular reticular
formation (Gi; mainly levels 4 to 6; Fig. 11M), and in
the spinovestibular nucleus (levels 6 and 7; Fig. 11I).
In the caudal medulla, a few medium-sized neurons were
present in the ventrolateral and commissural Sol (levels 9
to 13; Fig. 11L), whereas some small neurons were
present immediately caudal to the compact formation of
the Amb (only after injections in or rostral to the thoracic
cord; see also VanderHorst, 2005). A very distinct cluster
of mainly medium sized neurons was present in the caudal
NRA at the level of the xCST (Fig. 10, levels 11 to 13;
Fig. 12E; see also VanderHorst, 2005).
Neurons that gave rise to contralaterally descending
axons that recrossed the midline in the spinal cord (caudal to
the hemisection; Fig. 1, right panel) were found in all cell
groups described above as giving rise to contralaterally
descending pathways, but they formed only a small portion
of the respective cell groups.
3.2.3. Retrogradely labeled neurons with ipsilaterally
descending pathways
In the midbrain, labeled neurons giving rise to an
ipsilaterally descending pathway were located in the PAG
(Fig. 10, levels 5 to 3; Fig. 11C) and in the Edinger–
Westphal region (EW; Fig. 11C). In the lateral pontine
tegmentum (levels 1 and 2), a rather diffuse group of
retrogradely labeled neurons was present. Compared to the
group of contralaterally projecting neurons at the same level,
this group was more extensive, located more medially and
their neurons had larger somata (Fig. 11D and E). At level 0,
the group of ipsilaterally projecting neurons in the pontine
tegmentum was not only located more medially, but also
more dorsally (Fig. 10).
In the dorsolateral pons (Fig. 11H), numerous retro-
gradely labeled neurons were found in Barrington’s
nucleus, LC, LC pars alpha, and subcoeruleus region
(levels 1 and 2). Labeled neurons in Barrington’s
nucleus were smaller than those in surrounding regions.
Slightly more caudally (levels 1 to 3), large retro-
gradely labeled neurons were present in the pontine medial
tegmentum (Fig. 11K), whereas a few smaller neurons
were present more laterally.
In the medulla oblongata, large numbers of retrogradely
labeled neurons were found in different regions of the
ventral medullary tegmentum (Figs. 10 and 11M–O).
Medium sized neurons were found in the ventral medullary
medial tegmentum (levels 3 to 13; GiA rostrally, and
GiV at the level of the inferior olivary complex), whereas
some larger sized neurons were present more dorsally (Gi;
levels 3 to 7). Small sized neurons were found in the
midline RMg (levels 3 to 6) and ROb/RPa (levels 7 to
11), and in the LPGi (levels 6 to 11).
Other populations of retrogradely labeled neurons at the
medullary level were found in the lateral vestibular nucleus
(large neurons; Fig. 10, levels 3 to 5; Fig. 11F), the
ventrolateral and commissural Sol (medium-sized neurons;
Fig. 10, levels 7 to 12; Fig. 11L), the dorsal part of the
medullary lateral tegmentum (levels 10 to 12), and the
intermediate reticular formation (IRt) in between the
compact formation of the Amb and the caudal NRA
(Fig. 10, level 10; see also VanderHorst, 2005). Finally, a
few but very large neurons were found at the border between
the medullary tegmentum and the Sp5C.
A unilateral injection was combined with a hemisection
ipsilateral to injection to identify neurons whose axons
descend ipsilaterally, but with axons crossing the midline
at the level of termination (i.e. neurons located contral-
ateral to the injection and hemisection; Fig. 1). Retro-
gradely labeled neurons that give rise to ipsilaterally
descending axons that recross the midline in the spinal
cord (caudal to the hemisection) were found in all cell
groups described to give rise to ipsilaterally descending
pathways (Fig. 10). These neurons were less numerous
than the neurons giving rise to ipsilaterally descending and
projecting axons.
3.2.4. Segmental differences
Injections at different levels of the spinal cord resulted for
the most part in labeling of neurons in the cell groups

Fig. 11. Photomicrographs of retrogradely labeled neurons in the brainstem after spinal WGA–HRP injections. Except for A, C, and F, the tracer injections were
preceded by a hemisection contralateral to the injection. Note that the different panels represent parts of brainstem levels either ipsilateral or contralateral to the
injection. A: tectospinal neurons in the superior colliculus (SC; upper cervical cord case 28); B: rubrospinal neurons in the magnocellular division of the red
nucleus (case 67); C: labeled neurons in the lateral PAG and the Edinger–Westphal region (EW; arrow heads; case 69); D and E: lateral pontine tegmentum
contralateral (D) and ipsilateral (E) to the injection (case 37); F: labeled neurons in the LVe (case 51); G and H: labeled neurons in the dorsolateral pons; I: labeled
neurons in the SpVe (case 37); J and K: labeled neurons at the level of the superior olivary complex (SO) and facial nerve (7n) contralateral (J) and ipsilateral (K) to
the injection (case 37); L: labeled neurons in the Sol (case 37); M–O: labeled neurons in the medullary medial tegmentum from rostral (M) to caudal (O) in the GiA,
GiV, ROb, RPa, LPGi (case 37). Note that labeled neurons in the A5 and A7 regions are more numerous contralaterally than ipsilaterally, whereas labeled neurons
in the Sol are more numerous ipsilaterally than contralaterally. Also, Barrington’s nucleus only contains labeled neurons ipsilateral to the injection site, whereas
labeled neurons are present bilaterally in the locus coeruleus and locus coeruleus pars alpha. Bar = 100 mm. See figure on previous page.
24 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36

Fig. 12. Photomicrographs that show double labeling for 5HT and TH (A, B, D and F) and for 5HT and CTB (C and E). A: TH-IR neurons (red) form a shell
around 5HT-IR neurons in the DR-d; B, D, and F: 5HT- and TH-IR neurons are intermingled in the A1 cell group and LPGi; C and E: spinal cord projecting
neurons (red) and 5HT-IR neurons (green) with partially overlapping distributions in the caudal medulla oblongata. Note double-labeled neurons (yellow) were
present ipsilateral to the injection (C; case 107), but not contralaterally (E; case 107). Also note the shift in position of TH-IR cells relative to the Amb from
rostral to caudal (B to F). Bar = 100 mm.
 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36 25

Fig. 13. Photomicrographs that show double labeling for CTB (after spinal cord injections) and TH. In the lateral pontine tegmentum (A7 region), double-
labeled neurons are more numerous contralateral (A) and ipsilateral (B) to the injection site. Rostrally in the LC (C), double-labeled neurons are mainly present
ventrally in the locus coeruleus pars alpha. Caudally (D), the LC contains hardly any double-labeled neurons in the LC. Note the spatial relation between
Barrington’s nucleus (Barr) and the LC. Medial to the facial nerve (E; A5 cell group), a small portion of the TH-IR neurons is double labeled. In the caudal
medulla oblongata, TH-IR in the A1 cell group are distinct from spinal cord projecting neurons in the caudal NRA (F) and no double-labeled neurons were
present in the Sol (G). Bar in F = 100 mm.
26 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36
described above, with quantitative differences that are
beyond the scope of this report (for the medulla oblongata,
see VanderHorst, 2005). However, the following segment-
specific populations of labeled neurons were observed.
Labeled neurons were found in the deep layers of the sup-
erior colliculus (levels 3 to 6) contralateral to the injection
site (tectospinal system) after injections involving the upper
cervical cord (Fig. 11A), but not after injections into the C6–
7 or more caudal segments. In the ipsilateral PAG (Fig. 10,
levels 3–5), retrogradely labeled neurons were present after
injections into the thoracic cord and especially the cervical
cord, but not after lumbar cord injections. In Barrington’s
nucleus in the dorsolateral pons, labeled neurons were
absent after cervical cord injections, present after injections
involving the thoracic and upper lumbar cord, and most
numerous after injections into the lumbosacral segments. In
the medulla oblongata, at level 10, i.e. immediately caudal
to the compact formation of the nucleus ambiguus, labeled
neurons were present after thoracic and cervical cord
injections, but not after injections only involving more
caudal levels of the spinal cord (see VanderHorst, 2005).
3.3. Transmitter phenotype of spinal cord projecting
neurons in the brainstem
3.3.1. Retrogradely labeled neurons immunoreactive for
ChAT
After CTB injections into the thoracic and upper lumbar
cord, a few dispersed CTB-labeled neurons were found close
to ChAT-IR neurons in the PPTg and LDTg in the
mesopontine tegmentum. In the medulla oblongata, a few
were present caudal to the compact formation of the Amb in
the rostral and caudal NRA. None of the CTB-labeled
neurons were double labeled for ChAT.
3.3.2. Retrogradely labeled neurons immunoreactive for
5HT
In the midbrain and pons, no overlapping distributions
were found between retrogradely labeled neurons and 5HT-
IR neurons. However, the distribution of spinal cord
projecting neurons and 5HT-IR neurons overlapped exten-
sively in the medulla oblongata, i.e. RMg, RPa, ROb, LPGi,
and the ventrolateral medulla at the level of the xCST. All
these regions, but especially the LPGi, contained neurons
double labeled for CTB and 5-HT (Fig. 12C). These double-
labeled neurons were present bilaterally in the non-
hemisected cases. In the hemisected cases, double-labeled
neurons were only found ipsilaterally to the injection (and
contralateral to the hemisection; Fig. 12E).
3.3.3. Retrogradely labeled neurons immunoreactive for
TH
The distributions of TH-IR and CTB slightly overlapped in
the ventral PAG (level 6), the dorsolateral pons (LC and LC
pars alpha) and throughout the lateral pontine tegemtum. At
caudal medullary levels, a few TH-IR neurons were found
among bulbospinal neurons in the Sol and ventrolateral
medulla.
In some of these regions, neurons double-labeled for CTB
and TH were found. An occasional double-labeled neuron
was found in the ventral rostral PAG (Edinger–Westphal
region). Significant populations of double-labeled neurons
were present in the Kölliker–Fuse region (Fig. 13A) and the
LC region (pars alpha; Fig. 13C). CTB-labeled neurons in
Barrington’s nucleus formed a cluster that was clearly
distinct from TH-IR neurons in the LC region (Fig. 13D). A
few double-labeled neurons were found more caudally in the
lateral pontine tegmentum (A5 region; levels 2 to 3;
Fig. 13E). The populations of double-labeled neurons were
found both ipsilaterally and contralaterally to the injection
sites, but especially in the lateral pontine tegmentum (A5
and A7 cell groups) they were more prominent contral-
aterally (compare Fig. 13A and B).
No double-labeled neurons were present in the Sol
(Fig. 13G) or ventrolateral caudal medulla (Fig. 13F). In the
Sol, TH-IR neurons were located mainly medially to CTB-
labeled neurons. In the ventrolateral medulla at the level of
the xCST, the TH-IR cell group (A1 group) was located
ventrolateral to the group of contralaterally projecting CTB
labeled neurons in the caudal NRA.
3.4. Spatial relationships between 5HT- and TH-IR
neurons
In a number of regions, 5HT- and TH-IR neurons had
partially overlapping distributions. In the DR, TH-IR
neurons form a shell around 5HT-IR neurons in the dorsal
subdivision (Fig. 12A). At medullary levels, TH-IR neurons
and 5HT-IR neurons formed a mixed population in the A1/
C1 region and the LPGi (Fig. 12B–F). No double-labeled
neurons were found in these regions.
3.5. Effects of strain, gender and endocrine status
The majority of the immunohistochemical experiments
was performed in ovariectomized female C57Bl/6 mice.
Although the present series of experiments was not set up to
study quantitative differences related to strain, gender or
endocrine status, control experiments were added to exclude
any major qualitative differences, i.e. differences in location
or absence of certain neuronal populations. No such
differences were found between (inbred) C57Bl/6 mice
and outbred CD-1 mice, between ovariectomized and
estrogen treated females, or between males and females.
4. Discussion
4.1. Technical considerations and nomenclature
The tracers wheat germ agglutinin–horseradish perox-
idase (WGA–HRP; Sigma, St. Louis, MO) and cholera toxin
 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36
subunit B (CTB; List Biological Laboratories, Campbell,
CA) were used to label cell bodies of spinal cord-projecting
neurons. WGA–HRP has the advantage that it is transported
via fast axonal transport, in the mouse allowing survival times
of less than 24 h. In addition, WGA–HRP is more sensitive,
and smaller injections result in larger numbers of retrogradely
labeled neurons compared to CTB (VanderHorst et al., 2004).
WGA–HRP is also less easily taken up by fibers of passage
than CTB. For these reasons, in the single labeling part of the
study, WGA–HRP was used. CTB was injected in cases that
were used for the double labeling (immunohistochemical)
part of the study. Immunohistochemistry often requires a
fixative containing paraformaldehyde only and no glutar-
aldehyde. Because paraformaldehyde significantly decreases
the sensitivity of WGA–HRP, this tracer was not the first
choice for immunohistochemical experiments.
Use of axon transport inhibitors, such as colchicine,
might reveal a more complete visualization of the
monoaminergic neurons when using immunohistochemistry
(Ceccatelli et al., 1989). However, disadvantages of such
treatment include changes in gene expression patterns
(Ceccatelli et al., 1991) and limitation in the use of neuronal
tracers that are transported in axons. Therefore colchicine
was not used in the present study, and while the overall
distribution of monoaminergic neurons is in all likelihood
not affected, immunoreactive monoaminergic neurons in
each group might have been underrepresented. Similarly,
retrogradely labeled neurons that have been visualized using
tracer injections also represent a subpopulation of spinal
cord projecting neurons, because only part of the cord was
injected in each tracing experiment. Combined, the results
represent an underestimate of transmitter-identified projec-
tion neurons and thus have to be interpreted with caution.
Before we discuss each of the cholinergic, monoaminer-
gic and spinal cord projection systems, the following issues
have to be addressed with respect to nomenclature.
Catecholaminergic, serotoninergic, and cholinergic neurons
in CNS of the rat have been divided into groups called B1
(caudal) to B9 (rostral) for the serotoninergic system
(Dahlström and Fuxe, 1964), A1 (caudal) to A12 (rostral)
for the dopaminergic and noradrenergic systems (Dahlström
and Fuxe, 1964; Hökfelt et al., 1984), C1 (caudal) to C4
(rostral) for the adrenergic cell groups (Dahlström and Fuxe,
1964; Hökfelt et al., 1984), and Ch1 (rostral) to Ch6 (caudal)
for the cholinergic system (Mesulam et al., 1983). We will
refer to this nomenclature as well as to the corresponding
anatomical structures (as used in Section 3), because both
reference systems are often used in parallel. We would like to
stress here that each of the cholinergic, serotoninergic or
catecholaminergic regions is more or less heterogeneous
with respect to its cytochemistry and connections as
demonstrated by studies in the rat (Rye et al., 1987; Holets
et al., 1988; Van Bockstaele et al., 1993; Hökfelt et al., 2000;
Kirifides et al., 2001). For example, subpopulations of
serotonergic neurons in the DR project to distinct forebrain
regions (Kirifides et al., 2001), and a subpopulation of
27
medullary serotonergic neurons expresses GABA (reviewed
in Hökfelt et al., 2000).
4.2. Cholinergic system
In the mesopontine tegmentum, cholinergic neurons were
present in the PPTg or so-called Ch5 group (Mesulam et al.,
1983), the LDTgV (Mesulam et al., 1983), and the more
dorsally located LDTg. The location of these groups is
largely in line with studies in the rat (Rye et al., 1987),
although at rostral levels, the PPTg in the rat occupies a more
ventral position. In the mouse atlas of Paxinos and Franklin
(2001), the PPTg extends further dorsally (Fig. 14, level +2).
None of the cholinergic neurons in these regions projected
to the upper lumbar or thoracic cord. This is consistent with
findings in the rat, where a few cholinergic neurons were
found to project to the cervical cord, but not to the thoracic
cord (Rye et al., 1988). Instead, PPTg and LDTg project to
mesolimbic regions and the pontomedullary reticular forma-
tion (Rye et al., 1987, 1988), and are involved in the control of
the sleep–wake cycle, locomotion and motivation.
In addition to these mesopontine cell groups, ChAT or
VAChT-IR cholinergic neurons were found in the cranial
nerve motor nuclei and throughout the spinal ventral horn.
These neurons represent somatic motoneurons. In the atlas
of Paxinos and Franklin (2001), the trigeminal motor
nucleus and the facial nucleus were indicated more rostrally,
whereas no differences were present regarding the oculo-
motor nuclei, the nucleus ambiguus, accessorius and
hypoglossus (Fig. 14, levels +4 and +3, 7 to 9, and 13).
Cholinergic neurons were also found in the dorsal motor
nucleus of the vagal complex and the thoracic IML,
representing parasympathetic and sympathetic preganglionic
neurons, respectively. This is in agreement with studies in the
rat (Barber et al., 1984; Arvidsson et al., 1997; Hellström
et al., 2003). No distinct labeling of neurons (either with the
ChAT or the VAChT antibody) was found in the nucleus of
Edinger–Westphal in the rostral midbrain and the SPN,
respectively, two regions containing preganglionic parasym-
pathetic neurons. It is possible that the levels of ChAT and
VAChT is these latter populations were too low for detection.
These findings are in contrast to rat (Armstrong et al., 1983)
and cat (Kimura et al., 1981) in which parasympathetic
neurons were lightly labeled for ChAT. It cannot be ruled out
that these neurons use another transmitter system as proposed
for parts of the uterine parasympathetic preganglionic neurons
in the rat (Papka et al., 1995).
No cytoplasmic immunoreactivity for ChAT or VAChT
was observed in the spinal dorsal horn. This is in contrast to
reports in the rat in which large numbers of ChAT-IR
neurons were found in spinal laminae III–V (Houser et al.,
1983; Barber et al., 1984; Borges and Iversen, 1986).
However, similar to our results, cholinergic neurons were
absent in the dorsal horn in other studies in primate (Kus
et al., 2003) and rat (Gilmor et al., 1996; Hellström, 2004).
ChAT- and VAChT-IR neurons were also present in lamina
28 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36

Fig. 14. Line drawings of sections taken from the atlas of the mouse brain by Paxinos and Franklin (2001; on the left) and line drawings of sections used in this
overview that have been adjusted in size to fit the outlines of the atlas sections (on the right; for further details see Section 2). Data on the location of
monoaminergic and cholinergic neurons is superimposed on the atlas sections, with red, green and blue dots representing 5HT-IR, ChAT-IR, and TH-IR neurons,
 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36 29

respectively. Level 0 is defined as the level at which the aqueduct of Sylvius opens into the 4th ventricle. IA coordinates represent the distance in mm from the
interaural line, and B coordinates represent the distance in mm from Bregma (from Paxinos and Franklin, 2001). See List of Abbreviation for the abbreviations.
30 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36
X and medial lamina VII, in agreement with studies in the rat
(Barber et al., 1984). These neurons, in all likelihood,
represent propriospinal neurons.
4.3. Serotoninergic system
Serotoninergic cells were present in the DR and the
ventrolaterally adjacent tegmentum. The DR might be
subdivided into lateral, dorsomedian and ventromedian
regions (corresponding to area B7), and an inferior region
(area B6), similar to the rat (Kirifides et al., 2001). However,
the lateral subdivision extended further caudally, dorsal to the
cholinergic LDTg (Fig. 14, levels +1 and 0). More caudally,
5HT-IR neurons formed a group in the PnR (area B5).
Although we found 5HT-IR neurons lining the floor of the 4th
ventricle (area B4), this group was not distinct from the
inferior DR (area B6) and did not extend into the medulla
oblongata. In the ventral mesopontine tegmentum, large
numbers of 5HT-IR neurons were present in the MnR (B8) and
more ventrally and laterally in the RtTg (B9). The B9 region
extended more dorsolaterally than depicted in the mouse brain
atlas (Fig. 14, level +3; Paxinos and Franklin, 2001).
None of the neurons in the above groups was found to
project to the (thoracic and upper lumbar) spinal cord, in line
with a double labeling study in the rat (Skagerberg and
Björklund, 1985). Instead, different subdivisions of these
groups give rise to ascending and descending projections to
distinct regions of the forebrain, midbrain, and brainstem
(Moore et al., 1978; Van Bockstaele et al., 1993; Kirifides
et al., 2001).
At the pontomedullary level, some 5HT-IR neurons were
present in the RMg and adjacent tegmentum (area B3).
Although only few neurons were found in this region, their
location is in line with data obtained in the rat by Dahlström
and Fuxe (1964). The RMg extended rostrally only to the
level of the PnR, clearly more caudally than depicted in the
mouse atlas (Fig. 14, levels +1 to 6; Paxinos and Franklin,
2001). A few 5 HT-IR neurons in our study were found
lateral to the pyramids in the rostral medulla oblongata at the
level of the RMg, but the majority of 5HT-IR neurons in the
LPGi were located at the level of the ROb (B1) and RPa (B2)
as far caudal as the xCST. This is different from data in the
rat of Dahlström and Fuxe (1964), who included the LPGi in
region B3. This is also far more caudally than depicted in the
atlas of the mouse (Fig. 14, levels 3 to 11; Paxinos and
Franklin, 2001). The presence of a scattered group of 5HT-
IR neurons in the ventrolateral medulla at the level of the
xCST and the observation that the ROb and RPa cannot be
clearly separated are in agreement with findings in the rat
(Dahlström and Fuxe, 1964).
Part of the 5HT-IR neurons in the RPa, ROb, RMg and
especially LPGi were found to project to the spinal cord,
consistent with studies in other species (Skagerberg and
Björklund, 1985; Jones and Light, 1990). Spinal cord
projecting 5HT-IR neurons were numerous in the ROb/RPa
and LPGi, but sparse in the RMg (under similar experi-
mental conditions). Because the RPa and ROb preferentially
innervate the ventral horn, whereas the RMg and adjacent
tegmentum project predominantly to the dorsal horn in rat
and cat (Basbaum and Fields, 1979; Skagerberg and
Björklund, 1985), this finding suggests that relatively few
5HT-IR neurons project to the dorsal horn in the mouse.
Indeed, using the same anitsera, the 5HT-IR fiber distribu-
tion in the spinal superficial dorsal horn was less prominent
in our study in the mouse compared to the rat (Johnson et al.,
1993) and cat(Arvidsson et al., 1990b).
4.4. Catecholaminergic system
4.4.1. Tyrosine hydroxylase immunoreactive neurons in
the midbrain
In the midbrain large populations of TH-IR neurons were
found in the VTA (A10), SNC (A9), RRF (area A8) that
represent dopaminergic neurons. Based on the distribution
of TH-IR neurons, the border between the VTA and the SNC
cannot be drawn clearly. Additionally, smaller groups of TH-
IR neurons were found that have been described as part of
the A10 cell group: the rostral dorsal A10 group (including
neurons lining the 3th ventricle), rostral ventral A10 group
(including Edinger–Westphal nucleus), and the caudal
dorsal A10 group (in the ventral PAG and caudal part of
the caudal linear nucleus (Hökfelt et al., 1984). The
distribution of TH-IR neurons in these regions is comparable
with that described in the rat (Dahlström and Fuxe, 1964;
Hökfelt et al., 1984) and mouse (Zaborszky and Vadasz,
2001), although the latter study did not show the caudal
dorsal A10 group. Area 8 was larger in its dorsoventral
extent and located somewhat more caudally compared to the
C57BL/6 mouse atlas (Fig. 14, level +3; Paxinos and
Franklin, 2001). None of the catecholaminergic neurons in
the above groups were found to project to the spinal cord.
4.4.2. Tyrosine hydroxylase immunreactive neurons in
the mesopontine tegmentum
TH-IR cell groups in the mesopontine and pontine lateral
tegmentum were organized in a rather diffuse way, forming a
more or less continuous column without distinct borders.
Neurons in these regions represent noradrenergic neurons
and have been subdivided into the so-called A7 cell group
rostrally, and the A5 region caudally (Dahlström and Fuxe,
1964). In our overview, however, the A7 group was located
more ventrally than in the C57BL/6 mouse atlas (Fig. 14). In
addition, the A5 group was not very prominent, and at the
level of the facial nerve it was positioned more dorsally than
in the rat or mouse atlas (Fig. 14, levels 1 to 3; Paxinos
and Franklin, 2001).
In the dorsolateral pons, a compact cluster of TH-IR
neurons was present in the LC, called the A6 cell group. TH-
IR neurons had a continuous distribution that extended from
the LC to the more ventrolaterally located subcoeruleus
region and A5/A7 cell groups, similar to the rat (Dahlström
and Fuxe, 1964; Hökfelt et al., 1984).
 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36
Mesopontine TH-IR neurons that project to the spinal
cord were especially numerous in the LC (pars alpha; A6
cell group) and A7 cell group, whereas a smaller number of
double-labeled neurons was found in the A5 cell group.
Although TH-IR spinal cord projecting neurons reached
the spinal cord via both ipsilaterally and contralaterally
descending pathways, the A5 and A7 cell groups
predominantly use the contralateral pathway, whereas the
A6 cell group or LC projects mainly via ipsilaterally
descending axons. The presence of spinal cord projecting
TH-IR neurons in the A5, A6 and A7 cell groups is
consistent with previous observations in the rat, in which
these groups have been described to project predominantly
(but not exclusively) to the deep dorsal horn/intermediate
zone, ventral horn, and superficial dorsal horn, respectively
(Loewy et al., 1979a; Westlund et al., 1981; Clark and
Proudfit, 1991, 1993; Kwiat and Basbaum, 1992; Goodchild
et al., 2001). However, the observation that these neurons
project mainly via a contralaterally descending pathway is
new (Loewy et al., 1979a; Westlund et al., 1981; Clark and
Proudfit, 1991, 1993; Kwiat and Basbaum, 1992; Goodchild
et al., 2001). With respect to the A7 cell group, this opens the
discussion of what the relation is between the A7 cell group
and the Kölliker–Fuse region, a cell group known to give rise
to mainly contralateral projections. Functionally, the A5 and
A7 cell groups are involved in the control of nociception
(Miller and Proudfit, 1990; Yeomans and Proudfit, 1992),
whereas the A5 and A6 groups mediate cardiovascular
(Loewy et al., 1979b) and somatic motor reflexes (Fung
et al., 1991), respectively.
4.4.3. Tyrosine hydroxylase immunoreactive neurons in
the medulla oblongata
At the pontomedullary junction, a small number of TH-
IR neurons was found in the gray matter dorsolateral to the
4th ventricle and in the midline ventral to the 4th ventricle at
the level of the facial nucleus. These small cell groups
represent in all likelihood adrenergic neurons of the C4 and
C3 cell groups, respectively (Hökfelt et al., 1984).
In the medulla oblongata two distinct longitudinal groups
of TH-IR neurons were present that, similar to the rat, are
located dorsomedially (A2 and C2 cell groups) and
ventrolaterally (A1 and C1 cell groups; Dahlström and
Fuxe, 1964; Ritchie et al., 1982; Hökfelt et al., 1984; Kalia
et al., 1985a,b). Based upon information from the rat it
is likely that each of these groups consists of highly
heterogeneous cell populations (Kalia et al., 1985a,b;
Phillips et al., 2001). Dopaminergic neurons are found in
the AP and the motor nucleus of the dorsal vagal complex,
and adrenergic neurons in the Pr and medial Sol, forming the
C2 cell group (Hökfelt et al., 1984; Kalia et al., 1985b). TH-
IR neurons in the ventromedial, intermediate, and commis-
sural nuclei of the Sol, and its caudal extension (lamina X of
the C1–3 spinal cord) represent mainly noradrenergic
neurons and form the A2 cell group (Kalia et al.,
1985a,b; Phillips et al., 2001). Between the xCST and the
31
obex, noradrenergic, adrenergic and dopaminergic cells are
intermingled (Kalia et al., 1985a,b; Phillips et al., 2001). The
position of the A2/C2 cell groups in the current overview is
in line with the atlas of Paxinos and Franklin (Fig. 14, levels
7 to 13; 2001).
Although a few neurons in the Sol projected to the
spinal cord in the mouse, none of the TH-IR neurons in Sol
were double labeled. This is in line with studies in the rat,
in which Sol-spinal projections are also present (Loewy
and Burton, 1978), but in which they are not (Westlund
et al., 1981) or to a very limited extent (McKellar and
Loewy, 1982) derived from catecholaminergic neurons.
None of the TH-IR neurons in the AP and motor nucleus of
the dorsal vagal complex were found to project to the
spinal cord. This is in contrast to a study in the rat in which
neurons in the C2 (and C3) cell group have been reported
to project to spinal cord (Minson et al., 1990). This
discrepancy might be caused by the location of the tracer
injections or the absence of colchicine treatment, as
discussed above.
In the ventrolateral medulla, TH-IR neurons were found
in a longitudinal column extending from a position medial to
the caudal pole of the facial nucleus and into the C3 segment.
The rostral part of this cell group represents adrenergic
neurons (C1 cell group). In contrast to the rat, only few TH-
IR neurons were present rostral to the obex, and hardly any
were found at the level of the facial nucleus. This suggests
that the C1 cell group in the mouse may be less prominent
than in the rat, but this difference might also be caused by
differences in experimental conditions (see above). Com-
pared to the mouse atlas (Paxinos and Franklin, 2001), we
found the C1 cell group at the level of the rostral Amb in a
more medial position, and scattered TH-IR neurons were
present as medially as the LPGi (Fig. 14, levels 7 to 9).
In contrast is to findings in the rat (Ross et al., 1984;
Tucker et al., 1987; Kwiat and Basbaum, 1990; Minson
et al., 1990), no TH-IR spinal cord projecting neurons were
found in the ventrolateral medulla rostral to the obex (in the
adrenergic C1 cell group).
The caudal part of the TH-IR cell group, located at the
level of the xCST and further caudally (extending into the
C1–3 segments), is more compact and represents nora-
drenergic neurons (A1 cell group). A few 5HT-IR neurons
were found in this group, but it is clearly distinct from the
spinal cord-projecting neurons in the caudal NRA, which are
located more dorsomedially. The caudal part of the A1 cell
group was located more ventrolaterally, i.e. dorsolateral to
the LRt, compared to the mouse atlas (Fig. 14, levels 10 to
13; Paxinos and Franklin, 2001).
None of the TH-IR neurons in the A1 cell group was
found to project to the spinal cord in the mouse, largely in
line with studies in the rat (Westlund et al., 1981; McKellar
and Loewy, 1982). It should be noted that adrenergic and
noradrenergic cell groups in all likelihood are partly
intermingled, especially between the rostral border of the
xCST and the obex (Kalia et al., 1985c).
32 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36
4.4.4. Tyrosine hydroxylase immunoreactive neurons in
the spinal cord
Between the C3 and L4 segments, no TH-IR neurons
were present. However, in the L5–S2 segments a distinct
cluster of TH-IR neurons was found in the region of the LCP.
This structure forms a pathway between the dorsal horn and
the SPN in the L6–S1 segments and is likely involved in the
control and modulation of pelvic visceral functions. A
similar group of presumably dopaminergic TH-IR neurons
has been described in the rat (Mouchet et al., 1986, 1992).
4.5. Spinal cord projection systems: ipsilateral and
contralateral pathways
With respect to the projections from the brainstem to the
spinal cord, our data is limited to the location of neurons of
origin. Because our injections involved the entire gray
matter (dorsal horn, ventral horn and intermediate gray), the
experiments do not provide information regarding the areas
of termination of each of these projection systems in the
spinal cord. Such detailed information regarding some
bulbospinal projections is provided in a recent paper
(VanderHorst, 2005).
Neurons with mainly contralaterally descending path-
ways were located in the tectum, red nucleus, the lateral and
ventrolateral pontine tegmentum (including the A5 and A7
regions) and subcoeruleus region (part of the A6 region), the
gigantocellular reticular formation in the rostral medulla, the
spinal vestibular nucleus, and the NRA.
Neurons with exclusively ipsilaterally descending path-
ways are located in the PAG and Barrington’s nucleus,
whereas the rostral pontine lateral tegmentum, LC (part of
the A6 cell group), LC pars alpha (part of the A6 cell group),
caudal pontine ventromedial tegmentum, lateral vestibular
nucleus, dorsomedial lateral medullary tegmentum, Sol,
RMg (B3 cell group) with the adjacent GiA, the ROb/RPa
(B1 and B2), and LPGi with the adjacent GiV give rise to
mainly ipsilaterally descending pathways. Of these path-
ways Sol and subcoeruleus region pars alpha have a
significant number of contralaterally descending axons as
well. Except for the A5 and A7 regions (see above), these
finding are largely in agreement with studies in other
species, e.g., NRA (VanderHorst and Holstege, 1995;
VanderHorst et al., 2000), Sol (Loewy and Burton, 1978),
and red nucleus (Huisman et al., 1981). Although the above
mentioned regions contain neurons which axons descend in
either the ipsilateral or contralateral white matter, via
collaterals in the spinal cord that cross the midline, most
regions project more or less to both sides of the spinal cord.
4.6. Spinal cord projection systems: extent of
projections to various spinal levels
Many of these descending systems projected to all levels
of the spinal cord that were examined in our retrograde
tracing study. However, a number of regions projected
mainly to the rostral part of the spinal cord, such as the
tectum (upper cervical cord), PAG and a small cell group
caudal to the compact formation of the Amb (cervical and
thoracic cord), and NRA (mainly cervical, thoracic, and
upper lumbar cord). In contrast, Barrington’s nucleus
projected mostly to the thoracic and lumbosacral cord
and not to the cervical enlargement.
In comparison to findings in other species, the tectospinal
and NRA-spinal connections are somewhat differently
organized. In the cat, rat and monkey, the NRA projects
heavily to the sacral segments (VanderHorst and Holstege,
1995; VanderHorst et al., 2000), whereas tectospinal
projections in the cat and rat include the lower cervical
segments (Murray and Coulter, 1982). The segmental
preference of PAG-spinal and Barrington-spinal projections
is in line with findings in other species (Mantyh and
Peschanski, 1982; Mouton and Holstege, 1994).
4.7. Spinal cord projection systems: monoaminergic and
cholinergic fibers in the spinal cord
4.7.1. Cholinergic fibers
Using the VAChT antibody, punctate labeling was
observed, with a coarse pattern in spinal somatic (but not
autonomic preganglionic) motoneuronal cell groups, and a
fine pattern that was most distinct in lamina II (outer
subdivision). The VAChT antibody visualizes the vesicular
acetylcholine transporter that is mainly concentrated on
synaptic vesicles (Gilmor et al., 1996). The coarse pattern
surrounding somatic motoneurons in the spinal ventral horn
(and the extra-ocular cranial nerve motor nuclei) might thus
represent large cholinergic terminals of the C-type that
are postsynaptically associated to muscarinic 2 receptors
(Hellström et al., 2003). These C-boutons most likely
originate from local interneurons (McLaughlin, 1972;
Hellström, 2004). The fine VAChT-IR pattern in the dorsal
horn might represent smaller cholinergic terminals that are
involved in antinociception (Pinardi et al., 2003; Wess et al.,
2003).
An extremely strong VAChT-IR signal was observed in
the LCP, an area mediating pelvic visceral sensory
information as it receives primary afferents from pudendal
and especially pelvic nerves (Nadelhaft and Booth, 1984).
This dense pattern suggests that the cholinergic system is not
only important for the motor control of the bladder (i.e.
bladder contraction or detrusor activity), but might also
modulate the sensory loop of the micturition reflex, as well
as the sensory control of other pelvic organs.
None of the cholinergic neurons in the midbrain
maintained direct connections with the spinal cord, and in
the spinal white matter no ChAT of VAChT immunolabeling
was found. This suggests that cholinergic fibers are derived
from intraspinal cholinergic neurons that were found in
laminae VII, IX, X, and the thoracic IML, or from primary
sensory neurons (Dussor et al., 2004). With respect to
cholinergic antinociception, which involves both spinal and
 V.G.J.M. VanderHorst, B. Ulfhake / Journal of Chemical Neuroanatomy 31 (2006) 2–36
supraspinal mechanisms, this in turn would suggest that the
supraspinal component of cholinergic antinociceptive
mechanisms is not directly mediated via cholinergic-spinal
cord projecting neurons, but via an indirect mechanisms,
possibly involving the serotoninergic and adrenergic
descending systems (Cucchiaro and Commons, 2003).
4.7.2. Serotoninergic fibers
Serotoninergic fibers were found in all laminae of the
spinal cord and at all rostrocaudal levels, as in the rat (Jones
and Light, 1990). This fits the concept that the serotoninergic
system has a general, modulatory role on sensory as well as
motor functions (Mason and Leung, 1996; White et al.,
1996; Millan, 2002; Willis et al., 2003).
In the dorsal horn, fibers were most numerous in lamina I
and the outer subdivision of lamina II. This component of
the serotoninergic system is important for antinociception
(Millan, 2002; Willis et al., 2003).
The most prominent pattern of serotoninergic fibers was
observed in the thoracic intermediolateral and intermedio-
medial cell columns (Anderson et al., 1989). The SPN
contained only a moderate number of serotoninergic fibers,
whereas in contrast to the cholinergic and (nor)adrenergic/
dopaminergic systems, no robust pattern of serotoninergic
fibers was found in the LCP. This suggests that serotoni-
nergic neurons in the mouse, via direct projections to
autonomic preganglionic neurons, have a strong modulatory
effect on autonomic activity, more so on sympathetic than on
parasympathetic mechanisms.
In addition, there was a strong serotoninergic innervation
of the mouse somatic motor nuclei throughout the spinal cord,
similar to that described in the cat (Arvidsson et al., 1990b),
primate (Arvidsson et al., 1990a), and rat (Jones and Light,
1990; Marlier et al., 1991). These 5HT-IR axons mainly
originate from RPa/ROb neurons and modulate the excit-
ability of somatic motoneurons (Hounsgaard et al., 1988).
All serotoninergic neuronal cell groups in the medulla
oblongata, but none at the mesopontine level contained
serotoninergic neurons that project to the spinal cord.
Serotoninergic fibers tracts were present in the spinal white
matter, but no spinal 5HT-IR neurons. This indicates that
serotoninergic fibers in the murine spinal cord are derived
from the medullary serotoninergic system, in line with studies
in other species (Bullitt and Light, 1989; Jones and Light,
1990).
4.7.3. Tyrosine hydroxylase immunoreactive fibers
TH-IR fibers were found in all spinal laminae, and might
represent noradrenergic, adrenergic and/or dopaminergic
fibers. In the ventral horn, these fibers might modulate the
excitability of motoneurons (White et al., 1996). The distinct
fiber patterns in the dorsal horn especially in the lower part
of the spinal cord (involving mainly, although not exclusively,
the inner subdivision of laminae II and III) might, together
with more diffusely distributed TH-IR fibers in the
intermediate zone (laminae V, VII and X), be involved in
33
antinociception (Millan, 2002; Willis et al., 2003). Distinct
labeling patterns were also observed around autonomic
preganglionic neurons in the thoracic IML (as in the rat:
(Anderson et al., 1989) and the SPN, as well as in the sensory
lumbosacral LCP. This distribution pattern suggests that the
catecholaminergic system might exert major modulatory
effects on autonomic activity, via an action on autonomic
motor functions as well as on visceral sensory processing.
Part of the TH-IR fibers in the mouse spinal cord are
derived from (presumably) noradrenergic neurons in the
lateral mesopontine tegmentum (A7), LC and subcoeruleus
pars alpha (A6) and subcoeruleus region (A5), as these
regions contained TH-IR neurons with direct spinal connec-
tions. In addition, TH-IR fibers might be derived from the,
presumably dopaminergic, group of TH-IR neurons in the
lumbosacral LCP, and/or from dopaminergic subpopulations
of primary sensory neurons in the dorsal root ganglia (Price
and Mudge, 1983; Vega et al., 1991). Altogether, these
findings stress the importance of the catecholaminergic
system in the modulation of sensory information.
5. Concluding remarks
This report has provided comprehensive information on
monoaminergic, cholinergic, and projection systems in the
brainstem and spinal cord of the inbred C57BL/6 and
outbred CD-1 mouse. The overall organization of these
systems in the mouse is largely similar to those in the rat,
although some species differences exist. For example, the
A5 and A7 cell groups project to the spinal cord mainly via
contralateral, and not ipsilateral, pathways. Other systems,
such as the TH-IR cell group in L5–S2 spinal segments have
been described, but are relatively unknown.
The data further supplements information on the
brainstem that is provided in the standard brain atlas for
the C57BL/6 mouse (Paxinos and Franklin, 2001),
especially with respect to the size and/or location of the
catecholaminergic RRF (A8), A5, A1, and C1 cell groups,
and the serotonergic B4, RtTg (B9), LPGi, and RMg groups.
Acknowledgements
This work was sponsored by a research fellowship from
the Royal Netherlands Academy of Arts and Sciences
(KNAW); J.C. de Cock-Stichting, Netherlands; Foundation
‘‘de Drie Lichten’’, Netherlands; Medical Research Council
Sweden, #2003-5168 (VGJMV) and #10820 (BU).
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