nuclear organization of cholinergic, putative catecholaminergic and serotonergic systems in the
TRANSCRIPT
Journal of Chemical Neuroanatomy 40 (2010) 177–195
Nuclear organization of cholinergic, putative catecholaminergic and serotonergicsystems in the brains of two megachiropteran species
Leigh-Anne Dell a, Jean-Leigh Kruger a, Adhil Bhagwandin a, Ngalla E. Jillani a,John D. Pettigrew b, Paul R. Manger a,*a School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, Johannesburg, South Africab Queensland Brain Institute, University of Queensland 4072, Australia
A R T I C L E I N F O
Article history:
Received 26 March 2010
Received in revised form 28 May 2010
Accepted 28 May 2010
Available online 4 June 2010
Keywords:
Megabat
Chiroptera
Neuromodulatory systems
Diphyly
Evolution
Mammalia
A B S T R A C T
The nuclear organization of the cholinergic, putative catecholaminergic and serotonergic systems within
the brains of the megachiropteran straw-coloured fruit bat (Eidolon helvum) and Wahlberg’s epauletted
fruit bat (Epomophorus wahlbergi) were identified following immunohistochemistry for cholineacetyl-
transferase, tyrosine hydroxylase and serotonin. The aim of the present study was to investigate possible
differences in the nuclear complement of the neuromodulatory systems of these species in comparison
to previous studies on megachiropterans, microchiropterans and other mammals. The nuclear
organization of these systems is identical to that described previously for megachiropterans and
shows many similarities to other mammalian species, especially primates; for example, the putative
catecholaminergic system in both species presented a very compact nucleus within the locus coeruleus
(A6c) which is found only in megachiropterans and primates. A cladistic analysis of 38 mammalian
species and 82 characters from these systems show that megachiropterans form a sister group with
primates to the exclusion of other mammals, including microchiropterans. Moreover, the results
indicate that megachiropterans and microchiropterans have no clear phylogenetic relationship to each
other, as the microchiropteran systems are most closely associated with insectivores. Thus a diphyletic
origin of Chiroptera is supported by the present neural findings.
� 2010 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Journal of Chemical Neuroanatomy
journal homepage: www.e lsev ier .com/ locate / jchemneu
1. Introduction
The order chiroptera is divided into two suborders, namelyMicrochiroptera (or microbats) and Megachiroptera (or megabats).Although these suborders appear superficially similar morpholog-
Abbreviations: III, oculomotor nucleus; IV, trochlear nucleus; Vmot, motor division of tr
VIIv, facial nerve nucleus, ventral division; X, dorsal motor vagus nucleus; XII, hypoglo
medullary tegmental nucleus; A2, caudal dorsomedial medullary nucleus; A4, dorsal m
locus coeruleus; A6d, diffuse portion of locus coeruleus; A7d, nucleus subcoeruleus, diff
A9l, substantia nigra, lateral; A9m, substantia nigra, medial; A9pc, substantia nigra, pars
area; A10c, ventral tegmental area, central; A10d, ventral tegmental area, dorsal; A10dc,
cell group; A13, zona incerta; A14, rostral periventricular nucleus; A15d, anterior hypoth
A16, catecholaminergic neurons of the olfactory bulb; AP, area postrema; B9, supralemn
tegmental group; C2, rostral dorsomedial medullary nucleus; ca, cerebral aqueduct; Cb
nucleus; CO, cochlear nucleus; CVL, caudal ventrolateral serotonergic group; Diag.B, dia
nucleus, dorsal division; DRif, dorsal raphe nucleus, interfascicular division; DRl, dorsal
dorsal raphe nucleus, ventral division; EW, Edinger–Westphal nucleus; GC, periaqued
nucleus; Is.Call., Islands of Calleja; LDT, laterodorsal tegmental nucleus; LV, lateral ven
median raphe nucleus; N.Acc, nucleus accumbens; N.Amb, nucleus ambiguus; N.Bas, nuc
superior salivatory nucleus or facial nerve; pIX, preganglionic motor neurons of the in
piriform cortex; PPT, pedunculopontine nucleus; py, pyramidal tract; Rmc, red nucleus,
RPa, raphe pallidus nucleus; RVL, rostral ventrolateral serotonergic group; SC, superio
olfactory tubercle; tri, internal olfactory tract; trl, lateral olfactory tract; VPO, ventral p
* Corresponding author. Tel.: +27 11 717 2497; fax: +27 11 717 2422.
E-mail address: [email protected] (P.R. Manger).
0891-0618/$ – see front matter � 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchemneu.2010.05.008
ically, megachiropterans encompass a single family known asPteropodidae, whereas microchiropterans are comprised of 17different families (Mindell et al., 1991). Megachiropterans aregenerally larger in size, frugivorous and only found in Old Worldregions, while microchiropterans are smaller, primarily insectivo-
igeminal nucleus; VI, abducens nucleus; VIId, facial nerve nucleus, dorsal division;
ssal nucleus; 3n, oculomotor nerve; 4V, fourth ventricle; A1, caudal ventrolateral
edial division of locus coeruleus; A5, fifth arcuate nucleus; A6c, compact portion of
use portion; A7sc, nucleus subcoeruleus, compact portion; A8, retrorubral nucleus;
compacta; A9v, substantia nigra, ventral or pars reticulata; A10, ventral tegmental
ventral tegmental area, dorsal caudal; A11, caudal diencephalic group; A12, tuberal
alamic group, dorsal division; A15v, anterior hypothalamic group, ventral division;
iscal serotonergic nucleus; C, caudate nucleus; C1, rostral ventrolateral medullary
, cerebellum; cc, corpus callosum; CGM, medial geniculate body; CLi, caudal linear
gonal band of Broca; DRc, dorsal raphe nucleus, caudal division; DRd, dorsal raphe
raphe nucleus, lateral division; DRp, dorsal raphe nucleus, peripheral division; DRv,
uctal grey matter; GP, globus pallidus; IC, inferior colliculus; IP, interpeduncular
tricle; mcp, middle cerebellar peduncle; mlf, medial longitudinal fasciculus; MnR,
leus basalis; NEO, neocortex; P, putamen; pVII, preganglionic motor neurons of the
ferior salivatory nucleus; PBg, parabigeminal nucleus; PC, cerebral peduncle; PIR,
magnocellular division; RMg, raphe magnus nucleus; ROb, raphe obscurus nucleus;
r colliculus; scp, superior cerebellar peduncle; Sep.M, medial septal nucleus; TOL,
ontine nucleus.
Ta
ble
1S
pe
cie
sa
nd
cho
lin
erg
icn
ucl
ei
use
dfo
rcl
ad
isti
ca
na
lysi
s.T
he
nu
cle
ih
av
eb
ee
nn
ote
da
sp
rese
nt
(+),
ab
sen
t(�
),o
rin
cip
ien
t(+
/�).
For
sco
rin
go
fth
ese
nu
cle
iin
the
Ma
cCla
de
pro
gra
m,t
he
da
taw
as
en
tere
da
sp
rese
nt
(2),
ab
sen
t(0
),
inci
pie
nt
(1).
A‘‘?
’’re
pre
sen
tsa
bse
nt
da
tafo
rth
at
nu
cle
us
or
spe
cie
sa
nd
wa
sn
ot
sco
red
,no
ru
sed
inth
ea
na
lysi
s.T
he
da
tafo
rth
ista
ble
wa
sd
eri
ve
dfr
om
:P
laty
pu
s–
Ma
ng
er
et
al.
(20
02
a);
Ech
idn
a–
Ma
ng
er
et
al.
(20
02
a);
Lab
shre
w
–K
ara
saw
ae
ta
l.(2
00
3);
He
dg
eh
og
–D
ino
po
ulo
se
ta
l.(1
98
8);
Sch
rie
be
r’s
lon
g-fi
ng
ere
db
at
–M
ase
ko
an
dM
an
ge
r(2
00
7);
Litt
lefr
ee
tail
ed
ba
t–
Kru
ge
re
ta
l.(s
ub
mit
ted
for
pu
bli
cati
on
);C
om
me
rso
n’s
lea
f-n
ose
db
at
–K
rug
er
et
al.
(su
bm
itte
dfo
rp
ub
lica
tio
n);
He
art
-no
sed
ba
t–
Kru
ge
re
ta
l.(s
ub
mit
ted
for
pu
bli
cati
on
);A
fric
an
she
ath
-ta
ile
db
at
–K
rug
er
et
al.
(su
bm
itte
dfo
rp
ub
lica
tio
n);
Pe
rsia
ntr
ide
nt
ba
t–
Kru
ge
re
ta
l.(s
ub
mit
ted
for
pu
bli
cati
on
);sh
ee
p–
Ferr
eir
ae
ta
l.(2
00
1);
gir
aff
e–
Bu
xe
ta
l.(2
01
0);
rat
–A
rmst
ron
ge
ta
l.(1
98
3),
Ta
go
et
al.
(19
89
);m
ou
se–
Ka
lesn
yk
as
et
al.
(20
04
),V
an
de
rHo
rst
an
dU
lfh
ak
e(2
00
6);
Hig
hv
eld
mo
lera
t–
Da
Sil
va
et
al.
(20
06
),B
ha
gw
an
din
et
al.
(20
08
);
Ca
pe
du
ne
mo
lera
t–
Bh
ag
wa
nd
ine
ta
l.(2
00
8);
Afr
ica
np
orc
up
ine
–Li
ma
che
re
ta
l.(2
00
8);
ele
ph
an
tsh
rew
–P
iete
rse
ta
l.(2
01
0);
rock
hy
rax
–G
rav
ett
et
al.
(20
09
);ca
t–
Kim
ura
et
al.
(19
81
),V
ince
nt
an
dR
ein
er
(19
87
),S
hir
om
an
ie
t
al.
(19
88
);fe
rre
t–
He
nd
ers
on
(19
87
);d
og
–T
aft
iet
al.
(19
97
),S
t-Ja
cqu
es
et
al.
(19
96
);tr
ee
shre
w–
Fitz
pa
tric
ke
ta
l.(1
98
8);
Eg
yp
tia
nro
use
tte
–M
ase
ko
et
al.
(20
07
);S
tra
w-c
olo
ure
dfr
uit
ba
t–th
isst
ud
y;
Wa
hlb
erg
’se
pa
ule
tte
dfr
uit
ba
t–
this
stu
dy
;co
mm
on
ma
rmo
set
–E
ve
ritt
et
al.
(19
88
);sq
uir
rel
mo
nk
ey
–La
vo
iea
nd
Pa
ren
t(1
99
4);
ma
caq
ue
mo
nk
ey
–M
esu
lam
et
al.
(19
84
),K
us
et
al.
(20
03
);b
ab
oo
n–
Sa
toh
an
dFi
big
er
(19
85
);h
um
an
–M
izu
ka
wa
et
al.
(19
86
),M
esu
lam
et
al.
(19
89
).
Sp
eci
es
Orn
ith
orh
yn
chu
s
an
ati
nu
s
Ta
chy
glo
ssu
s
acu
lea
tus
Did
elp
his
vir
gin
ian
a
Ma
cro
pu
s
eug
enii
Sun
cus
mu
rin
us
Eri
na
ceu
s
euro
pa
eus
Min
iop
teru
s
sch
reib
ersi
i
Ch
aer
op
ho
n
pu
mil
is
Hip
po
sid
ero
s
com
mer
son
i
Ca
dri
od
erm
a
cor
Co
leu
ra
afr
a
Tri
aen
op
s
per
sicu
s
Sus
scro
fa
Ov
is
ari
es
Gir
aff
a
cam
elo
pa
rda
lis
Tu
rsio
ps
tru
nca
tus
Ra
ttu
s
no
rveg
icu
s
Mu
s
mu
scu
lus
Th
ryo
no
my
s
swin
der
ian
us
Co
mm
on
na
me
s
Pla
typ
us
Ech
idn
aO
po
ssu
mW
all
ab
yLa
b
shre
w
He
dg
eh
og
Sch
reib
er’
s
lon
g
fin
ge
red
ba
t
Litt
le
fre
e-
tail
ed
ba
t
Co
mm
ers
on
’s
lea
f-n
ose
d
ba
t
He
art
-
no
sed
ba
t
Afr
ica
n
she
ath
-
tail
ed
ba
t
Pe
rsia
n
trid
en
t
ba
t
Pig
Sh
ee
pG
ira
ffe
Bo
ttle
no
se
do
lph
in
Ra
tM
ou
seG
rea
ter
Ca
ne
rat
Ch
oli
ne
rgic
Co
rtic
al
cho
lin
erg
ic
inte
rne
uro
ns
��
??
�?
��
��
��
?�
??
++
�
Isla
nd
so
fC
all
eja
++
??
+?
++
++
++
?+
??
++
?
Olf
act
ory
tub
erc
le+
+?
?+
?+
++
++
+?
+?
?+
+?
Nu
cle
us
acc
um
be
ns
++
??
+?
++
++
++
?+
??
++
?
Ca
ud
ate
/Pu
tam
en
++
??
+?
++
++
++
?+
+?
++
?
Glo
bu
sp
all
idu
s+
+?
?+
?+
++
++
+?
+?
?+
+?
Me
dia
lse
pta
ln
ucl
eu
s+
+?
?+
?+
++
++
+?
+?
?+
+?
Dia
go
na
lb
an
d
of
Bro
ca
++
??
++
++
++
++
?+
??
++
?
Nu
cle
us
ba
sali
s+
+?
?+
++
++
++
+?
++
?+
+?
Do
rsa
lh
yp
oth
ala
mic
��
??
+?
++
++
++
?+
+?
++
?
Ve
ntr
al
hy
po
tha
lam
ic�
�?
?+
?+
++
++
+?
++
?+
+?
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195178
rous and occupy both New World and Old World regions (Mindellet al., 1991). Megachiropterans have highly developed visual andolfactory senses for foraging and obstacle avoidance; whereasmicrochiropterans have an advanced auditory sense and useultrasonic laryngeal emissions for echolocation (Pettigrew et al.,1989).
Divergence of these two suborders has been estimated at 58mya (Nikaido et al., 2000); but the debate of a monophyletic versusa diphyletic origin of the chiropterans remains unresolved – i.e. arethese two suborders really related and share a common flyingancestor, or are they more distantly related and did flight evolvetwice in mammals? In 1986, Pettigrew proposed the ‘‘FlyingPrimate’’ hypothesis whereby he stated that megachiropterans aremore closely related to primates than to microchiropterans andthus a diphyletic origin for chiroptera exists. This conclusion wasbased on the analysis and comparison of connection patternsbetween the retina and midbrain of primates and megachiropter-ans, which share a hemifield arrangement so that each midbrain isconnected to the contralateral nasal hemiretina and the ipsilateraltemporal hemiretina. This makes the retinotectal system ofprimates and megachiropterans like the hemidecussated genicu-lostriate pathway of all mammals, but unlike the retinotectalsystem of all other mammals, where the midbrain on each side hasa complete representation of the retina (Pettigrew, 1986). Thisfinding in the visual sytem of megachiropterans was in accord withprevious studies that linked primates and megachiropterans,including those of Linnaeus who noted the anatomical similarities,and Smith and Madkour (1980) who found similarities in thestructure of the glans penis of primates and megachiropterans thatwere not found in other eutherians. There are many features thatare uniquely shared between primates and megabats. These aredetailed in Table 1 of Pettigrew et al. (1989) and include: 1.Decussation of the retinotectal pathway; 2. Lamina versus eyearrangement in the lateral geniculate nucleus of the dorsalthalamus; 3. Motor pathways; 4. Metacarpo–phalangeal ratio; 5.Reproductive characters; 6. Protein sequence data (from FoxP2,opsin, globin, prestin); and 7. Cranial characters.
On the other hand, the flying primate hypothesis was opposedby a large number of morphological features that were shared byboth kinds of chiropterans (Nikaido et al., 2000), almost all of thesein the hand wing, but also in other flight-related aspects. Forexample, neonates of both microchiropterans and megachiropter-ans have hooked milk teeth, that might be shared-derivedcharacters linking both kinds of bats, but might equally well havebeen acquired independently to aid purchase by the neonate on ahighly mobile mother (Neuweiler, 2000). Similarly, the strongmorphological similarities between microchiropteran and mega-chiropteran wings might have arisen independently in the twolineages of chiropterans because of the considerable selectionpressures that flight brings to bear upon both morphology andmetabolism. While the morphological features of forelimbs inchiroptera as related to flight appear to suggest a sister relationshipfor chiropterans and thus monophyly, one measure of themetacarpals and phalanges of the handwing shows completeindependence of microchiropteran and megachiropteran wings, inkeeping with the possibility that the wings evolved separately(Pettigrew et al., 1989); moreover, shared features of neuralpathways related to vision unite megachiropterans and primates,to the exclusion of all other mammals and indicate a diphyleticorigin (Pettigrew, 1986; Pettigrew et al., 1989, 2008; Mindell et al.,1991). If megachiropterans and microchiropterans do indeed havea common flying ancestor then the brains of megachiropterans andprimates would have had to converge in many details, bothmacroscopic and microscopic (Pettigrew et al., 1989), in order to beso similar. If, however, megachiropterans share a more recentcommon ancestor with primates than with microchiropterans,
Late
ral
hy
po
tha
lam
ic�
�?
?+
?+
++
++
+?
++
?+
+?
Me
dia
lh
ab
en
ula
r+
+?
?+
?+
++
++
+?
++
?+
+?
An
teri
or
nu
cle
i,
do
rsa
lth
ala
mu
s
��
??
�?
��
��
��
?�
�?
��
?
Pa
rab
ige
min
al
nu
cle
us
(PB
g)
��
??
�?
��
�+
+�
??
+?
++
?
PP
Nm
c
(pe
du
ncu
lop
on
tin
e,
ma
gn
oce
llu
lar)
++
??
+?
++
++
++
??
+?
++
?
PP
Np
c
(pe
du
ncu
lop
on
tin
e,
pa
rvo
cell
ula
r
��
??
�?
��
��
��
??
�?
��
?
LDT
mc
(la
tero
do
rsa
l
teg
me
nta
l,
ma
gn
oce
llu
lar)
++
??
+?
++
++
++
??
+?
++
?
LDT
pc
(la
tero
do
rsa
l
teg
me
nta
l,
pa
rvo
cell
ula
r)
��
??
�?
��
��
��
??
�?
��
?
Su
pe
rio
rco
llic
ula
r
inte
rne
uro
ns
��
??
�?
��
��
��
??
�?
++
?
Infe
rio
rco
llic
ula
r
inte
rne
uro
ns
��
??
�?
��
��
��
??
�?
��
?
Ed
ing
er–
We
stp
ha
l
nu
cle
us
��
??
�?
�+
++
++
??
+?
++
?
III
(ocu
lom
oto
r
nu
cle
us)
++
??
+?
++
++
++
??
+?
++
?
IV(t
roch
lea
rn
ucl
eu
s)+
+?
?+
?+
++
++
+?
?+
?+
+?
Vm
ot
(tri
ge
min
al)
++
??
+?
++
++
++
??
+?
++
?
VI
(ab
du
cen
sn
erv
e
nu
cle
us)
++
??
+?
++
++
++
??
+?
++
?
VII
do
rs+
+?
?+
?+
++
++
+?
?+
?+
+?
VII
ve
nt
++
??
+?
++
++
++
??
+?
++
?
Nu
cle
us
am
big
uu
s+
+?
?+
?+
++
++
+?
??
?+
+?
X(v
ag
us
ne
rve
nu
cle
us)
++
??
+?
++
++
++
??
??
++
?
XII
(hy
po
glo
ssa
l
nu
cle
us)
++
??
+?
++
++
++
??
??
++
?
Sp
ina
lco
rd,v
en
tra
l
ho
rn
++
??
+?
++
++
++
??
??
++
?
Co
chle
ar
nu
cle
us
��
??
�?
��
��
��
??
??
+�
?
pV
II,
pre
ga
ng
lio
nic
sali
va
tory
nu
cle
us
��
??
??
�+
++
++
??
??
++
?
pIX
,p
reg
an
gli
on
ic
infe
rio
rsa
liv
ato
ry
nu
cle
us
��
??
??
�+
++
++
??
??
++
?
Me
du
lla
ry
teg
me
nta
lfi
eld
++
??
??
��
��
�?
??
??
++
?
Ta
tera
bra
nts
ii
Cry
pto
my
s
ho
tten
totu
s
Ba
thy
erg
us
suil
lus
Hy
stri
x
Afr
ica
eau
stra
lis
Ele
ph
an
tulu
s
my
uru
s
Pro
cav
ia
cap
ensi
s
Ory
cto
lag
us
cun
icu
lus
Feli
s
catt
us
Mu
stel
a
pu
tori
ou
s
Ca
nis
fam
ilia
ris
Tu
pa
ia
gli
s
Ro
use
ttu
s
aeg
yp
tia
cus
Eid
olo
n
hel
vu
m
Ep
om
op
ho
rus
wa
hlb
erg
ii
Ceb
uel
la
py
gm
aea
Ca
llit
hri
x
jacc
hu
s
Saim
iri
sciu
eru
s
Ma
caca
sp.
Pa
pio
pa
pio
Ho
mo
sap
ien
s
Hig
hv
eld
ge
rbil
Hig
hv
eld
mo
lera
t
Ca
pe
du
ne
mo
lera
t
Afr
ica
n
po
rcu
pin
e
Ele
ph
an
t
shre
w
Ro
ck
hy
rax
Ra
bb
itC
at
Ferr
et
Do
gT
ree
shre
w
Eg
yp
tia
n
Ro
use
tte
Str
aw
colo
ure
d
fru
itb
at
Wa
hlb
erg
’s
ep
au
lett
ed
fru
itb
at
Py
gm
y
Ma
rmo
set
Co
mm
on
Ma
rmo
set
Sq
uir
rel
mo
nk
ey
Ma
caq
ue
mo
nk
ey
Ba
bo
on
Hu
ma
n
��
��
��
??
??
?�
��
??
??
??
?+
++
++
?+
?+
?+
++
?+
?+
+?
?+
++
++
?+
?+
?+
++
?+
?+
+?
?+
++
++
?+
?+
?+
++
?+
?+
++
?+
++
++
?+
?+
?+
++
?+
?+
++
?+
++
++
?+
?+
?+
++
?+
?+
++
?+
++
++
?+
?+
?+
++
?+
?+
++
?+
++
++
?+
?+
?+
++
?+
?+
++
?+
++
++
?+
?+
?+
++
?+
?+
++
?+
++
++
?+
?+
?+
++
?+
?+
++
?+
++
++
?+
?+
?+
++
?+
?+
�+
?+
++
++
?+
?+
?+
++
?+
?+
++
?+
++
++
?+
?+
?+
++
?+
?+
++
?�
��
�+
?�
?�
?�
��
?�
?�
��
?+
++
++
?+
+?
++
++
?+
++
++
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195 179
Ta
ble
1(C
on
tin
ued
)
Ta
tera
bra
nts
ii
Cry
pto
my
s
ho
tten
totu
s
Ba
thy
erg
us
suil
lus
Hy
stri
x
Afr
ica
eau
stra
lis
Ele
ph
an
tulu
s
my
uru
s
Pro
cav
ia
cap
ensi
s
Ory
cto
lag
us
cun
icu
lus
Feli
s
catt
us
Mu
stel
a
pu
tori
ou
s
Ca
nis
fam
ilia
ris
Tu
pa
ia
gli
s
Ro
use
ttu
s
aeg
yp
tia
cus
Eid
olo
n
hel
vu
m
Ep
om
op
ho
rus
wa
hlb
erg
ii
Ceb
uel
la
py
gm
aea
Ca
llit
hri
x
jacc
hu
s
Saim
iri
sciu
eru
s
Ma
caca
sp.
Pa
pio
pa
pio
Ho
mo
sap
ien
s
Hig
hv
eld
ge
rbil
Hig
hv
eld
mo
lera
t
Ca
pe
du
ne
mo
lera
t
Afr
ica
n
po
rcu
pin
e
Ele
ph
an
t
shre
w
Ro
ck
hy
rax
Ra
bb
itC
at
Ferr
et
Do
gT
ree
shre
w
Eg
yp
tia
n
Ro
use
tte
Str
aw
colo
ure
d
fru
itb
at
Wa
hlb
erg
’s
ep
au
lett
ed
fru
itb
at
Py
gm
y
Ma
rmo
set
Co
mm
on
Ma
rmo
set
Sq
uir
rel
mo
nk
ey
Ma
caq
ue
mo
nk
ey
Ba
bo
on
Hu
ma
n
?+
++
++
?+
++
++
++
?+
++
++
?�
��
�+
?�
��
��
��
?�
��
��
?+
++
++
?+
++
++
++
?+
++
++
?�
��
�+
?�
��
��
��
?�
��
��
?�
��
+�
?�
�?
+�
��
?�
��
��
?�
��
+�
?�
�?
��
��
?�
��
��
?+
++
++
?+
+?
?+
++
?+
++
++
?+
++
++
?+
+?
?+
++
?+
++
++
?+
++
++
?+
+?
?+
++
?+
++
++
?+
++
++
?+
+?
?+
++
?+
?+
++
?+
++
++
?+
+?
?+
++
?+
?+
++
?+
++
++
?+
+?
?+
++
?+
?+
++
?+
++
++
?+
+?
?+
++
?+
?+
++
?+
++
++
?+
+?
?+
++
?+
?+
++
?+
++
++
?+
+?
?+
++
?+
?+
++
?+
++
++
?+
+?
?+
++
?+
?+
++
?+
++
++
?+
+?
?+
++
?+
?+
++
?�
��
+�
?�
�?
?�
��
?�
?�
��
?+
++
++
?+
+?
?+
++
?+
?+
++
?+
++
++
++
?+
+?
?+
++
?+
?+
++
?+
++
��
?+
+?
?�
��
?�
?�
��
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195180
then the numerous flight-associated musculoskeletal and molec-ular features may have been derived independently and conver-gently in the two suborders of chiroptera (Pettigrew and Kirsch,1998).
A consensus has arisen supporting monophyly on the basis ofthe results of numerous DNA sequencing studies on bothmitochondrial genes and at least eight nuclear genes (opsin,rhodopsin, prestin, FoxP2, BRCA1, BRCA2, vWF, A2AB) (Teelinget al., 2002, 2005). Since DNA sequence structure is not immunefrom processes leading to convergent evolution, it is possible thatthese results also reflect convergent evolutionary pressures thathave arisen as a result of the high metabolic rate associated withflight (Pettigrew and Kirsch, 1998). The DNA results have producedsome unlikely alliances that have never been suggested before,such as the joining between megachiropterans and rhinolophoidmicrochiropterans to the exclusion of all other microchiropterans.This anomaly might reflect a bona fide new association betweentwo groups of chiropterans that otherwise could not be furtherapart in morphology, physiology and behaviour, but it is alsoreadily explained by the fact that most megachiropterans andrhinolophoids lack torpor and would therefore be subject to moreintense selection pressure from temperature than the other groupsof microchiropterans. These other microbat families have a loweraverage body temperature because of torpor and may not havejoined megachiropterans and rhinolophoids because their DNA isless modified in consequence (Pettigrew and Kirsch, 1998). Studieshave recently appeared using DNA sequences that question themegachiropteran–rhinolophoid link (Shen et al., 2010; Li et al.,2008). Moreover, protein sequence data from the same genes usedfor DNA studies also fail to support the megabat–rhinolophoidassociation and instead provide evidence in favour of the flyingprimate hypothesis (e.g. FoxP2, Li et al., 2007, opsin, Shen et al.,2010). So DNA sequence results in bats may have to be acceptedwith caution in view of the possibility that DNA is perhaps assusceptible to convergent evolution as morphological systems,particularly when the selection pressure is far-reaching, like bodytemperature.
There are a number of other examples where convergenceappears to have resulted in DNA giving inarguably the wrongphylogeny. These include Dictyostelium, a eucaryote which DNAanalysis places with the procaryotes (Loomis and Smith, 1990),Amphioxus, a cephalochordate that is placed outside the echino-derms by DNA analysis (Naylor and Brown, 1998), and mega-chiropterans and rhinolophoids which spuriously associate unlesstheir DNA is corrected for a base compositional bias resulting fromhigh metabolic rate (Pettigrew and Kirsch, 1998). In theseexamples, DNA convergence was detected and corrected usingconflict between the DNA tree and the protein tree of the samegene, a common occurrence in bats compared to other mammals(Shen et al., 2010; Li et al., 2007). In view of the claims forconvergence on both sides of the debate about the phylogeny ofchiropterans, an overall contextual view seems necessary, asopposed to one based upon a single approach. The multiplicity ofcharacters available from neural systems has helped resolvephylogenetic uncertainties previously, and might do so again in thepresent case.
Manger (2005) hypothesized that changes in the complexity ofneural systems do not occur during speciation within an order,indicating rather that the number of subdivisions within a systemshould remain constant within species of a specific order. Thishypothesis, initially based on observations in monotremes(Manger et al., 2002a,b,c) and cetaceans (Manger et al., 2003,2004), has since been supported by numerous studies of specieswithin and between mammalian orders (Da Silva et al., 2006;Badlangana et al., 2007; Moon et al., 2007; Bhagwandin et al.,2008; Dwarika et al., 2008; Limacher et al., 2008; Gravett et al.,
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195 181
2009; Pieters et al., 2010; Bux et al., 2010). Thus, the subdivisions,or nuclei, of specific neural systems within the order Chiropterashould be constant and there should be no differences presentwhen comparing microchiropterans and megachiropterans if thetwo suborders are monophyletic. Maseko et al. (2007) performed astudy in which they investigated three neural systems inmegachiropterans (the cholinergic, catecholaminergic and seroto-nergic systems) and found 11 discrete nuclei that were not presentin microchiropterans (Maseko and Manger, 2007). Moreover, thesenuclei, that were present in the megachiropterans, aligned themwith the primates to the exclusion of other mammals, while thelack of these nuclei aligned the microchiropterans with thephylogenetically ambiguous insectivores. These observations thusconfirm that significant and distinct neural differences existbetween the two suborders and support a diphyletic origin ofchiroptera as determined by Pettigrew (1986); however, only onespecies of megachiropteran and microchiropteran have beenexamined to date with the nuclei of the neuromodulatory systemsin mind (but see Kruger et al., 2010).
The aim of this current study is to expand the study performedby Maseko et al. (2007) to test the validity of the previousobservations and to include further species of megachiropteran forcomparison. The current study details the nuclear organization ofthe cholinergic, putative catecholaminergic and serotonergicsystems in two megachiropteran species that have not previouslybeen studied. It is important to the interpretation of this study tonote that the vast majority of the nuclei of the neural systemsdescribed are not involved in any specialized functions generallyassociated with bats, such as echolocation, vision, olfaction orflight. Thus, conclusions from this study should yield unbiasedresults. The results of this study and previous studies (Maseko andManger, 2007; Maseko et al., 2007; Kruger et al., 2010) areanalyzed using cladistic methods.
2. Materials and methods
Three brains of each of two megachiropteran species were used in this study:
Eidolon helvum, the straw-coloured fruit bat (average body mass = 262 g; average
brain mass = 4.3 g); and Epomophorus wahlbergi, Wahlberg’s epauletted fruit Bat
(average body mass = 74 g; average brain mass = 1.8 g). All animals were captured
from wild populations in Kenya and were treated and used according to the
guidelines of the University of the Witwatersrand Animal Ethics Committee, the
Kenya National Museums and the Kenyan Wildlife Services. The animals were
euthanazed (Euthanaze, 1 ml/kg, i.p.), and then perfused intracardially upon
respiratory cessation. The perfusion was initially done with a rinse of 0.9% saline
solution at 4 8C followed by a solution of 4% paraformaldehyde in 0.1 M phosphate
buffer (PB) at 4 8C (approximately 1 l/kg of each solution). The brains were then
removed from the skull and post-fixed overnight in 4% paraformaldehyde in 0.1 M
PB at 4 8C and then allowed to equilibrate in 30% sucrose in 0.1 M PB at 4 8C. Two
brains from each species were frozen in crushed dry ice and sectioned into 50 mm
thick serial coronal sections while the third was sectioned in a sagittal plane.
A one in five series of sections were stained for Nissl, myelin, choline
acetyltransferase (ChAT), tyrosine hydroxylase (TH), and serotonin (5-HT). Nissl
sections were mounted on 0.5% gelatine coated glass slides and then cleared in a
solution of 1:1 chloroform and 100% alcohol overnight, after which the sections
were then stained with 1% cresyl violet. The sections for the myelin series were
refrigerated for two weeks in 5% formalin, mounted on 1% gelatine coated slides and
stained with a modified silver stain (Gallyas, 1979). The immunohistochemistry
sections were initially treated for 30 min with an endogenous peroxidase inhibitor
(49.2% methanol: 49.2% 0.1 M PB: 1.6% of 30% H2O2), followed by three 10 min
rinses in 0.1 M PB. The sections were then preincubated at room temperature for 2 h
in a blocking buffer solution containing 3% normal serum (Chemicon: normal rabbit
serum (NRS) for ChAT sections and normal goat serum (NGS) for TH and 5-HT
sections), 2% bovine serum albumin (Sigma) and 0.25% Triton X-100 (Merck) in
0.1 M PB. The sections were then placed in a primary antibody solution (blocking
buffer with correctly diluted primary antibody) and incubated at 4 8C for 48 h under
gentle shaking. To reveal cholinergic neurons, anti-cholineacetyltransferase (ChAT)
(AB144P, Chemicon, raised in goat) at a dilution of 1:3000 was used. To reveal
putative catecholaminergic neurons, anti-tyrosine hydroxylase (TH) (AB151,
Chemicon, raised in rabbit) was used at a dilution of 1:7500. To reveal serotonergic
neurons, anti-serotonin (AB938, Chemicon, raised in rabbit) at a dilution of 1:10000
was used. This incubation was followed by three 10 min rinses in 0.1 M PB, after
which the sections were incubated in a secondary antibody solution for 2 h at room
temperature. The secondary antibody solution contained a 1:750 dilution of
biotinylated anti-goat IgG (BA-5000, Vector labs, for ChAT sections) or biotinylated
anti-rabbit IgG (BA-1000, Vector Labs, for TH and 5-HT sections) in a solution
containing 3% NGS (or 3% NRS for the ChAT sections), and 2% BSA in 0.1 M PB. This
was followed by three 10 min rinses in 0.1 M PB, after which the sections were
incubated in AB solution (Vector Labs) for 1 h at room temperature. After three
further 10 min rinses in 0.1 M PB, the sections were placed in a solution of 0.05
diaminobenzidine in 0.1 M PB for 5 min (1 ml/section), followed by the addition of
3 ml of 30% H2O2 to each 1 ml of solution in which each section was immersed.
Chromatic precipitation of the sections was monitored visually under a low power
stereomicroscope. This process was allowed to continue until the background
staining of the sections was strong enough to assist with architectonic
reconstruction but not obscure any immunopositive neurons. The precipitation
process was stopped by immersing the sections in 0.1 M PB and then rinsing them
twice more in 0.1 M PB.
The controls employed in this experiment included the omission of the primary
antibody and the omission of the secondary antibody in selected sections. As a
further control for the cholinergic immunhistochemistry, we used choline
acetyltransferase (AG220, Millipore) at a dilution of 5 mg/ml in the primary
antibody solution (see above) as preabsorption assay. This solution was incubated
for 3 h at 4 8C prior to being used on the sections. We also reacted adjacent sections
that were not inhibited. In the sections where the primary antibody had been
inhibited, no staining was evident. The immunohistochemically stained sections
were mounted on 0.5% gelatine coated slides and left to dry overnight. The sections
were then dehydrated in a graded series of alcohols, cleared in xylene and cover
slipped with Depex. All sections were examined under low power using a
stereomicroscope and the architectonic borders of the sections were traced
according to the Nissl and myelin stained sections using a camera lucida. The
immunostained sections were then matched to the traced drawings, adjusted
slightly for any differential shrinkage of the stained sections and immunopositive
neurons were marked. The drawings were then scanned and redrawn using the
Canvas 8TM (Deneba) drawing program. Digital photomicrographs were captured
using a Zeiss Axioskop and the Axiovision software. No adjustments of pixels, or
manipulation of the captured images were undertaken, except for the adjustment of
contrast, brightness, and levels using Adobe Photoshop 7. All architectonic
nomenclature was taken from the atlas of a Megachiropteran brain (Schneider,
1966), while the nomenclature used to describe the immunohistochemically
revealed systems was based on Dahlstrom and Fuxe (1964), Hokfelt et al. (1984),
Tork (1990), Woolf (1991), Smeets and Gonzalez (2000), Manger et al. (2002a,b,c),
Maseko and Manger (2007), Maseko et al. (2007), Moon et al. (2007), Dwarika et al.
(2008), Limacher et al. (2008), Bhagwandin et al. (2008), Gravett et al. (2009) and
Pieters et al. (2010). A cladistic analysis of the data regarding nuclear organization
of the cholinergic, catecholaminergic and serotonergic systems generated in this
study and several previous studies were performed using the commercially
available MacClade program (http://macclade.org/macclade.html). In our tables
(Tables 1–3) presenting all the data used, the nuclei have been noted as present (+),
absent (�), or incipient (+/�). For scoring of these nuclei in the MacClade program,
the data was entered as present (2), absent (0), incipient (1). The classification into
these three grouping was based on a comparative qualitative assessment of the
expression of the nuclei across all species examined.
3. Results
The present study of the cholinergic, putative catecholaminer-gic and serotonergic systems in the brains of two megachiropteranspecies were visualized by means of immunohistochemicalmethods. The major immunohistochemically identifiable groupsof these neural systems within the brains of the two speciesstudied were found to be similar to the general patterns observedin mammals; i.e. no major deviations from the mammalian normswere observed. All the cholinergic, putative catecholaminergic andserotonergic nuclei were found to be consistent with the previousstudy performed by Maseko et al. (2007) on the Egyptian rousette(Rousettus aegyptiacus). The descriptions below of the neuralsystems apply to both E. helvum and E. wahlbergi unless specifiedotherwise.
3.1. Cholinergic neurons
The mammalian cholinergic system is typically divided into thefollowing groups: striatal, basal forebrain, diencephalic, ponto-mesencephalic and cranial nerve nuclei (e.g. Woolf, 1991; Mangeret al., 2002a; Maseko and Manger, 2007; Maseko et al., 2007;Limacher et al., 2008; Bhagwandin et al., 2008). The megachir-opterans studied here showed the same organization of nuclear
Table 2Species and catecholaminergic nuclei used for cladistic analysis. Conventions as per Table 1. The data for this table was derived from: Platypus – Manger et al. (2002b); Echidna – Manger et al. (2002b); Opossum – Crutcher and
Humbertson (1978); Hedgehog – Michaloudi and Papadopoulos (1996); Schrieber’s long-fingered bat – Maseko and Manger (2007); Little free tailed bat – Kruger et al. (2010); Commerson’s leaf-nosed bat – Kruger et al. (2010);
Heart-nosed bat – Kruger et al. (2010); African sheath-tailed bat – Kruger et al. (2010); Persian trident bat – Kruger et al. (2010); pig – Østergaard et al. (1992), Ruggiero et al. (1992), Leshin et al. (1995); sheep – Tillet and Thibault
(1989), Tillet and Kitahama (1998); giraffe – Badlangana et al. (2007), Bux et al. (2010); bottlenose dolphin – Manger et al. (2003, 2004); rat – Dahlstrom and Fuxe (1964), Armstrong et al. (1982), Hokfelt et al. (1984); mouse –
Ruggerio et al. (1984), Satoh et al. (1991), VanderHorst and Ulfhake (2006); greater canerat – Dwarika et al. (2008); Highveld gerbil – Moon et al. (2007); Highveld molerat – Da Silva et al. (2006), Bhagwandin et al. (2008); Cape dune
molerat – Bhagwandin et al. (2008); African porcupine – Limacher et al. (2008); elephant shrew – Pieters et al. (2010); rock hyrax – Gravett et al. (2009); rabbit – Blessing et al. (1978); cat – Cheung and Sladek (1975), Poitras and
Parent (1978), Reiner and Vincent (1987), Kitahama et al. (1990); ferret – Henderson (1987); dog – Dormer et al. (1993), Tafti et al. (1997); tree shrew – Murray et al. (1982); Egyptian rousette – Maseko et al. (2007); Straw-coloured
fruit bat – this study; Wahlberg’s epauletted fruit bat – this study; pygmy marmoset – Jacobowitz and MacLean (1978); squirrel monkey – Felten et al. (1974), Hubbard and Di Carlo (1974a), Lavoie and Parent (1994); macaque
monkey – Garver and Sladek (1975), Schofield and Everitt (1981); baboon – Satoh and Fibiger (1985); human – Bogerts (1981); Pearson et al. (1983); Halliday et al. (1988); Kitahama et al. (1996).
Species Ornithorhynchus
anatinus
Tachyglossus
aculeatus
Didelphis
virginiana
Macropus
eugenii
Suncus
murinus
Erinaceus
europaeus
Miniopterus
schreibersii
Chaerophon
pumilis
Hipposideros
commersoni
Cardioderma
cor
Coleura
afra
Triaenops
persicus
Sus
scrofa
Ovis
aries
Giraffa
camelopardalis
Tursiops
truncatus
Rattus
norvegicus
Mus
musculus
Thryonomys
swinderianus
Common names Platypus Echidna Opossum Wallaby Lab
shrew
Hedgehog Schreiber’s
long fingered
bat
Little
free-tailed
bat
Commerson’s
leaf-nosed
bat
Heart-nosed
bat
African
sheath-
tailed bat
Persian
trident bat
Pig Sheep Giraffe Bottlenose
dolphin
Rat Mouse Greater
Canerat
Catecholaminergic
Spinomedullary
junction
+ + ? ? ? ? ? ? ? ? ? ? ? ? � ? + + +
A1 caudal
ventrolateral
medulla
+ + + ? ? + + + + + + + + + + ? + + +
A2 caudal
dorsomedial
group
+ + + ? ? + + + + + + + + + + ? + + +
C1 rostral
ventrolateral
medulla
+ + ? ? ? ? + + + + + + + � + ? + + +
C2 rostral
dorsomedial
group
+ + ? ? ? ? + + + + + + + + + ? + + +
C3 � � ? ? ? ? � � � � � � � � � ? + + +
Area postrema + + ? ? ? ? + + + + + + + + + ? + + +
A4 � � � ? ? � � � � � � � � � � � � � �A5 + + ? ? ? + + + + + + + + + + + + + +
A6 locus coeruleus
diffuse
+ + + ? ? + + + + + + + + + + + � � +
A6 locus coeruleus
compact
� � � ? ? � � � � � � � � � � � + + �
Subcoerleus
compact
+ + + ? ? + + + + + + + + + + + + + +
Subcoeruleus
diffuse
+ + + ? ? + + + + + + + + + + + + + +
A8 retrorubral area + + + ? ? + + + + + + + + + + + + + +
A9 pars compacta + + + ? ? + + + + + + + + + + + + + +
A9 medial + + + ? ? + + + + + + + + + + + + + +
A9 ventral
reticulata
+ + + ? ? +/� � +/� +/� +/� +/� +/� + + + + + + +
A9 lateral or
pars lateralis
+ + + ? ? + + + + + + + + + + + + + +
A10 ventral
tegmental
area (VTA)
+ + + ? ? + + + + + + + + + + + + + +
A10c + + + ? ? + + + + + + + + + + + + + +
A10dc + + + ? ? + � � � +/� +/� � + + + + + + +
A10 dorsal + + + ? ? + � + + + + + + + + + + + +
A10 dorso-lateral � � � ? ? � � � � � � � � � � + � � �RMR rostral
mesencephalic
raphe cluster
� � � ? ? � � � � � � � � � � + � � �
VL PAG ventral
lateral
periaqueductal
gray cluster
� � � ? ? � � � � � � � � � � + � � �
A11 caudal
diencephalic
+ + + ? ? + + + + + + + + + + + + + +
L.-A.
Dell
eta
l./Jou
rna
lo
fC
hem
ical
Neu
roa
na
tom
y4
0(2
01
0)
17
7–
19
51
82
A1
2tu
be
ral
cell
gro
up
++
??
?+
++
++
++
++
++
++
+
A1
3zo
na
ince
rta
++
??
?+
++
++
++
++
+�
++
+
A1
4ro
stra
l
pe
riv
en
tric
ula
r
++
??
?+
++
++
++
++
++
++
+
A1
5d
ors
al
++
??
?�
��
��
��
��
�?
++
+
A1
5v
en
tra
l+
+?
??
+�
��
+/�
+/�
�+
++
?+
++
A1
6(o
lfa
cto
ryb
ulb
)+
+?
??
++
++
++
+?
??
�+
++
Ta
tera
bra
nts
ii
Cry
pto
my
s
ho
tten
totu
s
Ba
thy
erg
us
suil
lus
Hy
stri
x
afr
ica
eau
stra
lis
Ele
ph
an
tulu
s
my
uru
s
Pro
cav
ia
cap
ensi
s
Ory
cto
lag
us
cun
icu
lus
Feli
s
catt
us
Mu
stel
a
pu
tori
ou
s
Ca
nis
fam
ilia
ris
Tu
pa
ia
gli
s
Ro
use
ttu
s
aeg
yp
tia
cus
Eid
olo
n
hel
vu
m
Ep
om
op
ho
rus
wa
hlb
erg
ii
Ceb
uel
la
py
gm
aea
Ca
llit
hri
x
jacc
hu
s
Saim
iri
sciu
eru
s
Ma
caca
sp.
Pa
pio
pa
pio
Ho
mo
sap
ien
s
Hig
hv
eld
ge
rbil
Hig
hv
eld
mo
lera
t
Ca
pe
-du
ne
mo
lera
t
Afr
ica
n
pro
cup
ins
Ele
ph
an
t
shre
w
Ro
ck
hy
rax
Ra
bb
itC
at
Ferr
et
Do
gT
ree
shre
w
Eg
yp
tia
n
Ro
use
tte
Str
aw
colo
ure
d
fru
itb
at
Wa
hlb
erg
’s
ep
ale
tte
d
fru
itb
at
Py
gm
y
Ma
rmo
set
Co
mm
on
Ma
rmo
set
Sq
uir
rel
mo
nk
ey
Ma
caq
ue
mo
nk
ey
Ba
bo
on
Hu
ma
n
++
++
??
??
??
+?
??
??
??
?+
++
++
++
++
?+
++
++
+?
++
++
++
++
++
++
?+
++
++
+?
++
++
++
++
++
++
?+
++
++
??
++
++
++
++
++
++
?+
++
++
??
++
?+
++
++
��
��
?�
��
��
�?
��
��
++
++
++
++
?+
++
++
+?
++
++
��
��
++
++
��
++
++
+?
++
++
++
++
++
++
++
++
++
+?
++
++
++
++
++
++
++
++
++
+?
++
++
��
��
��
+�
��
++
++
+?
++
++
++
++
++
++
++
++
++
+?
++
++
++
++
++
++
++
++
++
+?
++
++
++
++
++
++
++
++
++
+?
++
++
++
++
++
++
++
++
++
+?
++
++
++
++
++
++
++
++
++
+?
++
++
++
++
++
�+
++
++
++
+?
++
++
++
++
++
++
++
++
++
+?
++
++
++
++
++
++
++
++
++
+?
++
++
++
++
++
++
++
++
++
+?
++
++
++
++
++
++
++
++
++
+?
++
++
++
++
++
++
++
++
++
+?
++
++
��
��
��
��
��
��
��
�?
��
��
��
��
��
��
��
��
��
�?
��
��
��
��
��
��
��
��
��
�?
��
��
++
++
++
++
??
++
++
+?
++
++
++
++
++
++
??
++
++
+?
++
++
++
++
++
++
??
++
++
+?
++
++
++
++
++
++
??
�+
++
+?
++
?+
++
++
�+
++
??
�+
++
??
++
?+
++
++
++
�+
??
�+
++
??
++
?+
++
++
++
?+
??
?+
++
??
??
??
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195 183
Table 3Species and serotonergic nuclei used for cladistic analysis. Conventions as per Table 1. The data for this table was derived from: Platypus – Manger et al. (2002c); Echidna – Manger et al. (2002c); Opossum – Crutcher and Humbertson
(1978); Wallaby – Ferguson et al. (1999); Hedgehog – Michaloudi and Papadopoulos (1995); Schrieber’s long-fingered bat Maseko and Manger (2007); Little free tailed bat – Kruger et al. (2010); Commerson’s leaf-nosed bat – Kruger
et al. (2010); Heart-nosed bat – Kruger et al. (2010); African sheath-tailed bat – Kruger et al. (2010); Persian trident bat – Kruger et al. (2010); sheep – Tillet (1987); giraffe – Badlangana et al. (2007), Bux et al. (2010); rat – Dahlstrom
and Fuxe (1964), Tork (1990); mouse – Steinbusch (1981); greater canerat – Dwarika et al. (2008); Highveld gerbil – Moon et al. (2007); Highveld molerat – Da Silva et al. (2006), Bhagwandin et al. (2008); Cape dune molerat –
Bhagwandin et al. (2008); African porcupine – Limacher et al. (2008); elephant shrew – Pieters et al. (2010); rock hyrax – Gravett et al. (2009); rabbit – Bjarkam et al. (1997); cat – Poitras and Parent (1978), Leger et al. (2001); dog –
Kojima et al. (1983); Egyptian rousette – Maseko et al. (2007); Straw-coloured fruit bat – this study; Wahlberg’s epauletted fruit bat – this study; pygmy marmoset – Jacobowitz and MacLean (1978); common marmoset – Hornung
and Fritschy (1988); squirrel monkey – Felten et al. (1974), Hubbard and Di Carlo (1974b), Lavoie and Parent (1994); macaque monkey – Charara and Parent (1998), Takeuchi et al. (1982); human – Baker et al. (1990).
Species Ornithorhynchus
anatinus
Tachyglossus
aculeatus
Didelphis
virginiana
Macropus
eugenii
Suncus
murinus
Erinaceus
europaeus
Miniopterus
schreibersii
Chaerophon
pumilis
Hipposideros
commersoni
Cardioderma
cor
Coleura
afra
Triaenops
persicus
Sus
scrofa
Ovis
aries
Giraffa
camelopardalis
Tursiops
truncatus
Rattus
norvegicus
Mus
musculus
Thryonomys
swinderianus
Common
names
Platypus Echidna Opossum Wallaby Lab
shrew
Hedgehog Schreiber’s
long
fingered
bat
Little
free-tailed
bat
Commerson’s
leaf-nosed
bat
Heart-nosed
bat
African
sheath-
taied
bat
Persian
trident
bat
Pig Sheep Giraffe Bottlenose
dolphin
Rat Mouse Greater
Canerat
Serotonergic
Periventricular
organ
+ + � � ? � � � � � � � ? � � ? � � �
Caudal linear
nucleus (CLi)
+ + + + ? + + + + + + + ? + + ? + + +
Supralemniscal (B9) + + + + ? + + + + + + + ? + + ? + + +
Median raphe
nucleus (MnR)
+ + + + ? + + + + + + + ? + + ? + + +
DR lateral (DRL) + + + + ? + + + + + + + ? + + ? + + +
DR ventral (DRV) + + + + ? + + + + + + + ? + + ? + + +
DR dorsal (DRd) + + + + ? + + + + + + + ? + + ? + + +
DR interfascicular
(DRif)
+ + + + ? + + + + + + + ? + + ? + + +
DR peripheral
(DRp)
+ + + + ? + + + + + + + ? + + ? + + +
DR caudal (B6) � � + + ? + + + + + + + ? + + ? + + +
Raphe
magnus (RMg)
+ + + + ? + + + + + + + ? + + ? + + +
Raphe
pallidus (RPa)
+ + + + ? + + + + + + + ? + + ? + + +
RVL rostral
ventrolateral
+ + + + ? + + + + + + + ? + + ? + + +
CVL caudal
ventrolateral
� � � + ? + + + + + + + ? + + ? + + +
Raphe
obscurus (ROb)
+ + + + ? + + + + + + + ? + + ? + + +
Tatera
brantsii
Cryptomys
hottentotus
Bathyergus
suillus
Hystrix
africaeaustralis
Elephantulus
myurus
Procavia
capensis
Oryctolagus
cuniculus
Felis
cattus
Mustela
putorious
Canis
familiaris
Tupaia
glis
Rousettus
aegyptiacus
Eidolon
helvum
Epomophorus
wahlbergii
Cebuella
pygmaea
Callithrix
jacchus
Saimiri
sciuerus
Macaca
sp.
Papio
papio
Homo
sapiens
Highveld
gerbil
Highveld
molerat
Cape
dune
molerat
African
porcupine
Elephant
shrew
Rock
hyrax
Rabbit Cat Ferret Dog Tree
shrew
Egyptian
Rousette
Straw
coloured
fruit bat
Wahlberg’s
epaletted
fruit bat
Pygmy
Marmoset
Common
Marmoset
Squirrel
monkey
Macaque
monkey
Baboon Human
� � � � � � � � ? � ? � � � � � � � ? �+ + + + + + + + ? + ? + + + + + + + ? +
+ + + + + + + + ? + ? + + + + + + + ? +
+ + + + + + + + ? + ? + + + + + + + ? +
+ + + + + + + + ? + ? + + + + + + + ? +
+ + + + + + + + ? + ? + + + + + + + ? +
+ + + + + + + + ? + ? + + + + + + + ? +
+ + + + + + + + ? + ? + + + + + + + ? +
+ + + + + + + + ? + ? + + + + + + + ? +
+ + + + + + + + ? + ? + + + + + + + ? +
+ + + + + + + + ? + ? + + + + + + + ? +
+ + + + + + + + ? + ? + + + + + + + ? +
+ + + + + + + + ? + ? + + + + + + + ? +
+ + + + + + + + ? + ? + + + + + + + ? +
+ + + + + + + + ? + ? + + + + + + + ? +
L.-A.
Dell
eta
l./Jou
rna
lo
fC
hem
ical
Neu
roa
na
tom
y4
0(2
01
0)
17
7–
19
51
84
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195 185
complexes. The results yielded for the cholinergic nuclei in the twomegachiropteran brains studied are congruent with the study byMaseko et al. (2007) in the Egyptian roussette, and as with thatspecies, no cortical cholinergic neurons were observed.
3.1.1. Striatal cholinergic interneurons
For both species the nucleus accumbens, caudate, putamen,globus pallidus, islands of Calleja and olfactory tubercle contained
[(Fig._1)TD$FIG]
Fig. 1. Drawings of sections through one half of the brains of both Eidolon helvum and Ep
represents a single neuron) and nuclear organization of the cholinergic system
pontomesencephalon (middle figurines) and some cranial nerve nuclei (lower figurines)
abbreviations.
cholineacetyltransferase immunoreactive (ChAT+) neurons(Fig. 1). The anterior border of nucleus accumbens (N.Acc)appeared rostral to the level of the anterior commissure andwas observed ventral to the dorsal striatopallidal complex anddorsal to the olfactory tubercle. The caudate (C) and putamen (P)nuclei were readily identified as distinct entities as their mutualborders were demarcated by a strongly coalesced internal capsule.Both nuclei were found rostral in the cerebral hemisphere, lateral
omophorus wahlbergi depicting the ChAT immunoreactive neurons (each black dot
in three different coronal planes, namely basal forebrain (upper figurines),
. Note the compact and localized ChAT immunoreactive neurons in PBg. See list for
[(Fig._2)TD$FIG]
Fig. 2. Photomicrographs showing the pedunculopontine (PPT), laterodorsal
tegmental (LDT), parabigeminal (PBg) and trochlear (IV) cholinergic nuclei in
megachiroptera. (A) Low power photomicrograph of all four nuclei in Epomophorus
wahlbergi. Scale = 500 mm. (B) Higher power showing just LDT and PPT in Eidolon
helvum. Scale = 500 mm.
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195186
to the lateral ventricle and observed to taper caudally to reach themid-diencephalic level. At a location posterior and medial to theputamen nucleus, several cholinergic neurons were identifiedwithin the globus pallidus (GP). On the floor of the anterior portionof the cerebral hemisphere, the islands of Calleja (Is. Call) and theolfactory tubercle (TOL) were identified in a position ventral to thenucleus accumbens and rostral to the anterior commissure.
3.1.2. Cholinergic nuclei of the basal forebrain
Within the basal forebrain the medial septal nucleus, diagonalband of Broca and nucleus basalis were observed to contain ChAT+neurons (Fig. 1). The medial septal nucleus (Sep.M) was found to belocated in the rostral lower half of the medial wall of the cerebralhemisphere, between the rostrum of the corpus callosum and thefloor of the cerebral hemisphere, medial to the dorsal striatopalli-dal complex and rostro-dorsal to the hypothalamus. The diagonalband of Broca (Diag. B) was identified on the ventromedial cornerof the cerebral hemisphere, anterior to the hypothalamus. Nucleusbasalis (N.Bas) was located ventral to the anterior commissure,lateral to the hypothalamus between it and the globus pallidus.
3.1.3. Diencephalic cholinergic nuclei
Within the diencephalon, ChAT+ neurons were found withinthe medial habenular nucleus and were seen to form three distinctnuclei within the dorsal, lateral and ventral portions of thehypothalamus. The medial habenular nucleus was located in thedorsal medial aspect of the diencephalon adjacent to the thirdventricle in a position typical of that reported for mammals. Thedorsal hypothalamic group was observed near the third ventricle ina dorsal and medial location within the hypothalamus, with mostneurons occuring at the middle anterioposterior level of thehypothalamus. The ChAT+ neurons forming the lateral hypotha-lamic lateral group were located in the dorsal lateral aspect of thehypothalamus, intermingled with the catecholaminergic A13nucleus near the zona incerta (see below). The ChAT+ neuronsforming the ventral hypothalamic group were found in theventromedial aspect of the hypothalamus, adjacent to the thirdventricle and floor of the brain and caudal in the hypothalamus.
3.1.4. Pontomesencephalic cholinergic nuclei
Within the pontomesencephalon ChAT+ neurons were ob-served within the parabigeminal nucleus (PBg), the pedunculo-pontine tegmental nucleus (PPT) and the laterodorsal tegmentalnucleus (LDT) (Figs. 1 and 2). The parabigeminal nucleus was foundto be located at the lateral wall of the midbrain tegmentum, ventralto the inferior colliculus and at the approximate rostro-caudal levelof the oculomotor nucleus. The cholinergic neurons forming thepedunculopontine tegmental nucleus were situated dorsallywithin the pontine tegmentum, surrounding the superior cerebel-lar peduncle (scp) and extended from the level of the trochlearnucleus to the trigeminal motor nucleus. ChAT+ neurons located inthe lateral ventral periventricular grey matter in the pons formedthe laterodorsal tegmental nucleus (LDT). Some of the LDT neuronswere seen to intermingle with those of noradrenergic neurons ofthe locus coeruleus (A6c and A6d, see below). These pontome-sencephalic cholinergic nuclei were positioned in locations similarto those previously observed in other mammals.
3.1.5. Cholinergic cranial nerve nuclei
The following ChAT+ nuclei were identified in positions that aretypical for mammals: Edinger–Westphal (EW), third (III, oculomo-tor), fourth (IV, trochlear), fifth motor (Vmot, motor trigeminal),sixth (VI, abducens), seventh dorsal and ventral (VIId and VIIv,facial), ambiguus (N.Amb.), 10th (X, dorsal motor vagus) and 12th(XII, hypoglossal) (Figs. 1 and 2). ChAT immunoreactivity was alsofound in the preganglionic motor neurons of both the superior
salivatory (pVII) and inferior salivatory (pIX) nuclei which werelocated dorsal and lateral to the dorsal division of the facial nucleusand nucleus ambiguus (pVII), and anterior to the anterior poles ofthe hypoglossal and dorsal motor vagal nuclei (pIX). In E. helvum
the abducens nucleus (VI) and preganglionic motor neurons of theinferior salivatory nucleus (pIX) were more strongly represented innumber than in E. wahlbergi.
3.2. Putative catecholaminergic neurons
Tyrosine hydroxylase immunoreactive neurons (TH+), classi-fied in this study as putative catecholaminergic neurons, formed arange of identifiable nuclear complexes found throughout thebrain extending from the olfactory bulb to the spinomedullaryjunction. The locations of these nuclear complexes were typical ofthe pattern seen in other mammals. The catecholaminergic nucleiidentified in this study were identical to those described byMaseko et al. (2007), and thus no catecholaminergic nuclei werefound outside the bounds of classically defined nuclei asoccasionally described for other mammals (Smeets and Gonzalez,2000).
3.2.1. Olfactory bulb neurons (A16)
These small, triangular shaped TH+ neurons were foundthroughout the stratum granulosum, with their dendrites forminga mesh around the glomeruli. This appearance and location istypical for all mammals studied to date (Smeets and Gonzalez,2000; Pieters et al., 2010).
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195 187
3.2.2. Diencephalic nuclei (A15-A11)
Within the hypothalamus six nuclei containing TH+ neuronswere observed, these being the A15d, A15v, A14, A13, A12 and A11nuclei. The dorsal division of the anterior hypothalamic group(A15d) was located in the dorsal anterior aspect of the hypothala-mus, immediately ventral to the anterior commissure, in a site that issimilar to that observed in rodents and the Egyptian rousette(Hokfelt et al., 1984; Maseko et al., 2007). The ventral division of theanterior hypothalamic group (A15v) was identified in the ventrallateral part of the hypothalamus at the level of the optic chiasm. In E.
wahlbergi the A15d and A15v nuclei the cell numbers are notstrongly expressed, they are in the typical mammalian location, buthave a cell population that is lower in density in comparison to thealready low density cell population of A15d as described by Masekoet al. (2007) for the Egyptian rousette. The TH+ neurons that make upthe rostral periventricular group (A14) were situated at thehypothalamic periventricular zone, adjacent to the third ventricleand formed a dorsoventral column closely apposed to theventricular wall. Extending laterally from the dorsal hypothalamusthe zona incerta group (A13) was identified, while the tuberal cellgroup (A12) was located in the ventral medial hypothalamusbetween the inferior margin of the third ventricle and the opticchiasm in the region of the arcuate nucleus and infundibulum.Finally, the caudal diencephalic nucleus (A11) was located in themost caudal part of the hypothalamus, surrounding the caudal andinferior parts of the third ventricle. In E. wahlbergi, relatively largeneurons were present in A11, but this large size of the A11 neuronswas not observed in E. helvum.
[(Fig._3)TD$FIG]
Fig. 3. Drawings of sections through one half of the brainstem of both Eidolon helvum and
represents a single neuron) and nuclear organization of the catecholaminergic system in
figurines). Note how localized and compact A6c is in both species, being surrounded b
megachiropterans share exclusively with primates. See list for abbreviations.
3.2.3. Midbrain nuclei (A10-A8)
The midbrain nuclei consisted of three main divisions: theventral tegmental nuclei (VTA, A10 complex), the substantia nigra(A9 complex) and the retrorubral nucleus (A8) (Figs. 3 and 4). Theneurons forming the specific A10 nucleus of the A10 nuclearcomplex were located dorsolateral to the interpeduncular nucleusbetween the interpeduncular nucleus and the exit of theoculomotor nerve. The A10 central (A10c) nucleus was situatedat the midline, immediately dorsal and anterior to the inter-peduncular nucleus. A10 dorsal (A10d) was found to lie around themidline immediately ventral to oculomotor nucleus and dorsal toA10c. The A10 dorsocaudal nucleus (A10dc) was located within theperiaqueductal grey matter, between the cerebral aqueduct andoculomotor nucleus. The A10dc nucleus appeared slightly differentbetween species. In E. wahlbergi the TH+ cells forming the A10dcnucleus are more numerous than in E. helvum; but in E. helvum theA10dc nucleus is relatively large and long in an anteroposteriordirection with a low cell density, which is not the case for E.
wahlbergi. The neurons forming the A9 pars compacta (A9pc)nucleus of the substantia nigra were found immediately dorsal tothe cerebral peduncle, whereas the neurons forming the A9 ventralnucleus (A9v) were intermingled with the fibres that form thecerebral peduncle and were situated ventral to A9pc. At the mostlateral end of A9pc, on the ventral lateral edge of the midbraintegmentum, the A9 lateral (A9l) nucleus was observed. Theneurons of the A9 medial nucleus (A9m) were located lateral toA10 and the exiting oculomotor nerve but medial to the neuronsthat coalesce to form A9pc. The retrorubral nucleus (A8) was
Epomophorus wahlbergi depicting the TH immunoreactive neurons (each black dot
two different coronal planes, namely midbrain (upper figurines), and pons (lower
y a more diffuse aggregation of TH immunoreactive neurons (A6d), a feature the
[(Fig._4)TD$FIG]
Fig. 4. Photomicrographic montage of the TH immunoreactive neurons forming
catecholaminergic nuclei in the midbrain of Epomophorus wahlbergi. Scale = 1 mm.
See list for abbreviations.
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195188
situated in the midbrain tegmentum, dorsal to A9pc, anddorsocaudal and lateral to the magnocellular division of the rednucleus.
3.2.4. Pontine nuclei (A7-A4)
The TH+ neurons within the pons formed several distinct nucleithat combined form the locus coeruleus nuclear complex (Figs. 3and 5). Within the pontine tegmentum dorsal and anterior to thetrigeminal motor nucleus, and in a position adjacent to theperiventricular grey matter, a densely packed cluster of TH+neurons was observed to form the the A7 compact portion (A7sc,subcoeruleus compact) of the subcoeruleus. This nucleus repre-sents that originally described as the subcoerleus by Dahlstromand Fuxe (1964), but is herein classified as part of a larger A7cluster composed of both a compact (A7sc) and a diffuse (A7d)nucleus (Maseko et al., 2007). The A7 diffuse nucleus (A7d,subcoeruleus diffuse) was located in the lateral pontine tegmen-tum, anterior, lateral and dorsal to the trigeminal motor nucleus,and medial to the superior cerebellar peduncle. The TH+ neuronsforming the A6 compact nucleus (A6c, locus coeruleus compactportion) were situated in the most lateral pontine periventriculargrey matter, bordering the dorsal pontine tegmentum, whereasthose neurons forming the A6 diffuse nucleus (A6d, locus coeruleusdiffuse portion) are located in the ventral lateral periventricular
[(Fig._5)TD$FIG]Fig. 5. Low power photomicrograph of the locus coeruleus complex in Epomophorus
wahlbergi. Note that the TH immunoreactive neurons forming A6c are highly
compact, a feature only seen in megachiroptera and primates. Scale = 1 mm. See list
for abbreviations.
gray matter, in an area that is similar to that of the cholinergiclateral dorsal tegmental nucleus (LDT, see above). In both speciesthe A6c nucleus was identified as being very similar to that seen inprimates. The fifth arcuate nucleus (A5) was found in theventrolateral pontine tegmentum, lateral to the superior olivarynucleus (OLS) and the facial nerve nucleus. Within the dorsomedialperiventricular gray matter, adjacent to the ventricular wall, thedorsal medial division of locus coeruleus (A4) was observed. In E.
helvum A4 appears relatively small and is located higher in thepervientricular grey matter in comparison to E. wahlbergi.
3.2.5. Medullary nuclei (A2, A1, C2, C1, area postrema)
Within the medulla of the two megachiropteran speciesstudied, TH+ neurons formed five distinct catecholaminergicnuclei, as is seen in most other mammals studied (Maseko et al.,2007; Pieters et al., 2010). The TH+ neurons that form the rostralventrolateral medullary tegmental group (C1) were located withinthe rostral ventrolateral medullary tegmentum between the levelof the nucleus ambiguus and hypoglossal nucleus, and extendedinto the tegmentum from the ventrolateral medullary edge. Therostral dorsomedial medullary group (C2) was situated dorsal tothe motor vagus nucleus and near the floor of the fourth ventricle.In the caudal ventrolateral medullary tegmentum, lateral to thelateral reticular nucleus, TH+ neurons assigned to the caudalventrolateral medullary tegmental group (A1) were identified,while neurons of the caudal dorsomedial medullary group (A2)were found between the dorsal motor vagus and hypoglossalnuclei, extending into the dorsal caudal medullary tegmentum.The final medullary nucleus, area postrema (AP), was situated inthe most caudal portion of the dorsal medulla, dorsal and medial tothe dorsal motor vagus and hypoglossal nuclei. It must be notedthat AP is more strongly represented in E. wahlbergi than E. helvum.
3.3. Serotonergic neurons
The serotonergic nuclei of the megachiropterans studied werefound from the level of the oculomotor nucleus through to thespinomedullary junction, as observed in most mammals previous-ly studied (Maseko et al., 2007). In mammals the serotonergicsystem is divided into two main clusters, namely the rostral andcaudal clusters (Tork, 1990; Bjarkam et al., 1997), and this has beenapplied to the two megachiropteran species studied. Theindividual nuclei observed were identical to those described byMaseko et al. (2007) and thus were all typical of observations in alleutherian mammals.
3.3.1. Rostral cluster
Within the rostral cluster several distinct nuclei were observed,that include the caudal linear nucleus (CLi), the supralemniscalgroup (B9), the dorsal raphe nuclear cluster (with six distinctnuclei) and the median raphe nucleus (MnR) (Figs. 6–8). The caudallinear nucleus was situated on either side of the midline, anteriorand inferior to the decussation of the superior cerebellar peduncleand dorsal to the interpeduncular nucleus. The supralemniscalserotonergic nucleus was identified in the ventrolateral midbrain,dorsal to the lemniscal pathways and appeared to be a lateralcontinuation of the ventral portion of the caudal linear nucleus. Oneither side of the midline two distinct columns of serotonergicneurons, located caudal to the decussation of the superiorcerebellar peduncle and ventral to oculomotor and trochlearnuclei, continuing caudally to the most anterior level of thetrigeminal motor nucleus were assigned to the median raphe(MnR). The dorsal raphe (DR) was observed to consist of six distinctnuclei, mostly situated within the periaqueductal and periven-tricular grey matter, from the level of the trochlear nucleus to themost anterior level of the trigeminal motor nucleus. The dorsal
[(Fig._6)TD$FIG]
Fig. 6. Drawings of a section through one half of the brainstem of both Eidolon helvum and Epomophorus wahlbergi depicting the 5-HT immunoreactive neurons (each black dot
represents a single neuron) and nuclear organization of the rostral cluster of the serotonergic system at the coronal plane of the midbrain. See list for abbreviations.
[(Fig._7)TD$FIG]
Fig. 7. Low power photomicrographs of 5-HT immunoreactive neurons showing the organization of the rostral clsuter of serotonergic nuclei at different coronal levels,
focussing on the dorsal raphe complex in (A) Eidolon helvum and (B) Epomophorus wahlbergi. Scale in B = 500 mm and applies to both. See list for abbreviations.
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195 189
[(Fig._8)TD$FIG]
Fig. 8. Photomicrographs of 5-HT neurons forming various nuclei in the rostral
serotonergic cluster of megachiroptera. (A) The supralemniscal serotonergic
nucleus (B9) in Eidolon helvum. Scale = 500 mm. (B) The caudal division of the
dorsal raphe complex (DRc) and the median raphe nucleus (MnR) in Eidolon helvum.
Scale = 1 mm. (C) The caudal division of the dorsal raphe complex (DRc) and the
median raphe nucleus (MnR) in Epomophorus wahlbergi. Scale = 500 mm. See list for
abbreviations.
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195190
raphe interfascicular nucleus (DRif) was identified between themedial longitudinal fasciculi. Immediately dorsal to the DRif, thedorsal raphe ventral (DRv) nucleus was located in the ventrome-dial portion of the periaqueductal grey matter. The dorsal raphedorsal (DRd) was found dorsal to the DRv within the periaque-ductal grey matter, but did not reach the ventral aspect of thecerebral aqueduct. Also within the periaqueductal grey matter butdorsal and lateral to DRv and DRd were the serotonergic neurons ofthe dorsal raphe lateral nucleus (DRl). In E. helvum, the DRl nucleusis clearly expressed and relatively large in terms of neuronalnumber in comparison to that observed in E. wahlbergi. The
neurons forming the dorsal raphe peripheral nucleus (DRp) werelocated in the ventrolateral aspect of the periaqueductal greymatter lateral to both DRd and DRv, with some neurons beingfound in the adjacent pontine tegmentum. The most caudalnucleus in the rostral cluster, the dorsal raphe caudal (DRc), wasidentified within the periventricular grey matter as a caudalcontinuation of DRl.
3.3.2. Caudal cluster
Within the caudal cluster, five distinct serotonergic nuclei wereidentified in the medulla of both species. The raphe magnusnucleus (RMg) was situated in the ventral midline and extendedfrom the level of the trigeminal motor nucleus to the caudal level ofthe facial nerve nucleus. Associated with the pyramidal tracts wasthe raphe pallidus (RPa), which was located at the most ventralmidline of the medulla. The neurons forming part of the rostralventrolateral nucleus (RVL) were identified lateral to the inferiorolive, in the ventrolateral medullary tegmentum and extend fromthe most anterior level of the trigeminal motor nucleus to thetrapezoid body. The caudal ventrolateral nucleus (CVL) forms acontinuation of RVL and was found in the ventrolateral medullategmentum, caudal to the trapezoid body and extending to thespinomedullary junction. Forming two loosely arranged columnseither side of the midline from the level of the nucleus ambiguus tothe spinomedullary junction were the serotonergic neurons of theraphe obscurus nucleus (ROb).
3.4. Phylogenetic analysis
Using MacClade we ran a series of phylogenetic analyses basedon the nuclear organization of the cholinergic, catecholaminergicand serotonergic systems across a range of mammalian species (seeTables 1–3 for the data used in this analysis and Fig. 9 for diagrams ofthe results). This analysis included 38 taxa and 82 characters. Ourfirst step was to allow the program to create the shortest and mostparsimonious tree without additional interpretation. The result ofthis initial analysis (Fig. 9A) demonstrated a diphyletic origin for thechiroptera based on these characters, and placed the megachir-optera as a sister group to primates and the microchiroptera with therepresentative insectivores. The length of this tree was 55 (theminimal number of possible steps for parsimony), with aconsistency index of 0.64, a retention index of 0.79, and a rescaledconsistency index of 0.51. It should be of interest to note that theother relationships formed in this analysis conform strongly topreviously published mammalian phylogenies.
Following this initial analysis we tested the strength of the treeby manipulating the phylogenetic relationships (using toolsavailable in MacClade) to adhere strictly to three recentlypublished mammalian phylogenies, including chiropteran mono-phyly as the major change. The first phylogeny tested was thatproposed by Asher et al. (2009). In this phylogeny, majordifferences to that generated based on our data include themonophyly of the chiroptera and the distinction of the Afrotheria(in this study the Afrotheria are represented by the rock hyrax andelephant shrew) as an early mammalian branch forming theAtlantogenata. Following these manipulations the treelength was65, the consistency index 0.54, the retention index 0.69, and therescaled consistency index 0.38. We then tested the phylogeny ofArnason et al. (2002) where the major differences to the self-generated tree include the monophyly of the chiroptera and thesplitting of the insectvora. Following these manipulations, thetreelength became 62, with a consistency index of 0.57, a retentionindex of 0.72, and a rescaled consistency index of 0.41. The finalphylogeny tested is that proposed by Lee and Camens (2009),where the major differences were the same as that seen in Asheret al. (2009). Following manipulation of the phylogenetic relation-
[(Fig._9)TD$FIG]
Fig. 9. Reconstructions of various phylogenetic trees using the commercially available program MacClade, based on the data provided in Tables 1–3 of the cholinergic,
catecholaminergic and serotonergic systems in 38 taxa (the bottlenose dolphin was eliminated from the analysis due to the lack of data across all systems) and 82 characters.
(A) This tree is based directly in the result provided by MacClade when using the data collected from the three neural systems and represents the most parsimonious result
with only 53 substitutions necessary to form the tree. Note the sister group arrangement of the megachiroptera with the primates and the microchiroptera with the
insectivores. (B) This tree is based on the phylogeny provided by Asher et al. (2009) which forces monophyly upon the two chiropteran groups and increases the number of
substitutions to 63. (C) This tree is based on the phylogeny provided by Arnason et al. (2002), again forcing bat monophyly and separating the insectivores and increases the
number of substitutions to 60. (D) This tree is based on the phylogeny provided by Lee and Camens (2009), again forcing monophyly and increases the number of substitutions
to 63. The phylogenetic analysis of the three neural systems investigated presents bat diphyly, with megachiropterans as a sister group to primates, as the most parsimonious
phylogeny. CI – consistency index; RI – retention index; RCI – recalculated consistency index.
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195 191
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195192
ships, the treelength became 65, with a consistency index of 0.54, aretention index of 0.69, and a rescaled consistency index of 0.38,indicating strong similarity with the results obtained using the treeproposed by Asher et al. (2009).
In all three tests of the self-generated phylogeny, the treelengthincreased substantially, indicating that the number of steps,changes, or substitution of characters needed to obtain the proposedphylogeny increased (from 55 to 62/65, 7 to 10 additional steps). Inall three tests the consistency index decreased (from 0.64 to 0.54/0.57). A consistency index of 1 equals perfect congruency in the data,anything less than 1 indicates a level of homoplasy in somecharacters. The self-generated tree, indicating bat diphyly providesthe highest consistency index, and thus the least need for recourse tohomoplasy as an explanation of the data (in this case the sistergrouping of megachiropterans and primates). In all three tests theretention index decreased from the self-generated tree (from 0.79 to0.72/0.69). This index is a second characteristic of the derived trees,whereby a retention index of 1 equals full parsimony, a retentionindex of 0 being total homoplasy. The self-generated chiropterandiphyly tree again reduces the need to resort for extensivehomoplasy to explain the data. Lastly, the rescaled consistencyindex also decreased in all three tests (from 0.51 to 0.41/0.38)underlining the same features as described above, this being thatassuming chiropteran diphyly lowers the need for extensive use ofhomoplasy as a solution to the longer phylogenetic trees.
4. Discussion
The present study aimed to examine the nuclear organization ofthe cholinergic, putative catecholaminergic and serotonergicsystems in two megachiropteran species and compare these systemsto those of other megachiropterans, microchiropterans and othermammals so that data concerning the debate of the phylogeneticorigins of the two chiropteran suborders could be generated andanalyzed. The megachiroptera studied exhibited an identical nuclearorganization of their cholinergic, putative catecholaminergic,serotonergic systems when compared to the other megachiropteranstudied previously (Maseko et al., 2007) and showed manysimilarities to other mammals, especially the primates (Smeetsand Gonzalez, 2000; Tork, 1990; Woolf, 1991; Maseko et al., 2007;Pieters et al., 2010; see Tables 1–3). Minor intra-nuclear organiza-tional differences were noted between the two megachiropteranspecies studied and these minor variations may reflect subtlefunctional differences. The megachiropterans exhibit numerousnuclei that are also present in rabbits, tree shrews and primates butthat are not present in microchiropterans (Maseko and Manger,2007; Kruger et al., 2010). Thus differences in the nuclearcomplements of these systems indicate a diphyletic origin of theChiroptera, as the nuclear complexity of these systems appears tochange in a predictable manner in the course of evolution, indicatingseparation at the ordinal level (Manger, 2005).
4.1. Neural systems of megachiroptera
The nuclear organization of cholinergic, putative catecholamin-ergic and serotonergic neural systems were identified as beingidentical in both E. helvum and E. wahlbergi and identical to themegachiropteran (Rousettus aegyptiacus) examined in the study byMaseko et al. (2007). This observation indicates that differentmegachiropteran species have the same complement and com-plexity of homologous nuclei and thus no changes in nuclearcomplexity occurred within this suborder during evolution(Manger, 2005). Minor differences in the relative size andrepresentation of a few nuclei in the cholinergic and putativecatecholaminergic and were observed between E. helvum and E.
wahlbergi. These differences may be attributed to slight functional
differences that may exist between the two species. In E. helvum,
the abducens cranial nerve nucleus and the preganglionic motornucleus of the inferior salivatory nerve are more readily apparentthan in E. wahlbergi. The oculomotor nerve not only innervates theextraocular eye musculature and levator palpebrae muscle(Warwick, 1953), but also receives input from the visual cortex(Woolf, 1991). Thus the visual cortex in E. helvum may controlmovement of the eyes in more direct manner than that of E.
wahlbergi. The preganglionic motor nucleus of the superiorsalivatory nerve innervate the laryngeal and pharyngeal muscles(Woolf, 1991), which could be more pronounced in E. helvum formate calling (DeFrees and Wilson, 1988). In the putativecatecholaminergic system, small differences were noted betweenE. helvum and E. wahlbergi in the diencephalic, midbrain andpontine nuclear groups. Within the diencephalon and in compari-son to E. helvum, E. wahlbergi exhibited small A15v and A15d nucleiwith reduced cell size but an A11 nucleus with larger cells.Diencephalic nuclei in general are involved in pituitary secretionand thus reproduction (Smeets and Gonzalez, 2000) but noliterature regarding the specific function of these individual nucleiis currently available, and thus it is difficult to speculate as to whyE. wahlbergi exhibited small A15v and A15d nuclei with reducedcell size but an A11 nucleus with large cells. Within the midbrain ofE. wahlbergi, the A10dc nucleus of the ventral tegmental area ismore well expressed in terms of neuronal number than thehomologous nucleus in E. helvum. Unfortunately, the functionalaspects of this relatively minor nucleus is unknown (Smeets andGonzalez, 2000). Although the individual pontine catecholaminer-gic nuclei also have no specific function, they collectively innervatethe main and accessory olfactory bulbs and are involved incardiovascular function, olfaction, central auditory and beha-vioural states such as vigilance and attention (Smeets andGonzalez, 2000). The A4 division of the locus coeruleus appearssmaller in E. helvum and may be due to E. helvum having a slightlyreduced olfactory system in comparison to E. wahlbergi.
4.2. Similarities of megachiropterans to primates
It is important to compare the neural systems of megachiropterato primates to reiterate that they form a sister group to the primatesas previously concluded by Pettigrew (1986) and more recentlyMaseko et al. (2007). In this current study it was found that thenuclear organization of the cholinergic, putative catecholaminergicand serotonergic neural systems of E. helvum and E. wahlbergi (aswell as R. aegyptiacus, Maseko et al., 2007) was identical to theprimates that have been previously studied (see Tables 1–3). Inparticular the A6 compact portion of the locus coeruleus complexprovides strong evidence that similar nuclear complements existbetween the megachiropterans and primates. In the rostral cluster ofthe serotonergic neural system, the DRl nucleus in E. helvum wasobserved as being large and clearly expressed and this is a prominentfeature that has only been identified previously in primates (Tork,1990). As the nuclear complement of neural systems do not changewithin an order irrespective of brain size, lifestyle and phenotype(Manger, 2005), it can be concluded that E. helvum, E. wahlbergi andother megachiroptera may be closely related to the primates, mostlikely as a sister group. It would be of great interest to examine thesesystems in the brain of a Dermopteran species (the ‘‘flying lemurs’’),as these are a well-accepted sister group to the primates, and are thegroup from which Pettigrew et al. (1989) propose that themegachiropterans evolved.
In previous neural studies showing that megabats and primateshad a number of potentially shared-derived characters, these werelargely confined to the visual system. Martin (1986) thought thatthis might represent functional convergence between twounrelated groups of mammals that each occupies the ‘‘fine branch
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195 193
niche’’. There is no evidence that the role of the binocular,hemidecussated eye–midbrain pathway shared by megabats andprimates is connected to Martin’s (1986) hypothetical niche, so hisproposal is on no firmer ground than the flying primate hypothesisbeing questioned. The present results show a similar set of shared-derived characters between megabats and primates, but in thiscase they come from different neural systems that cannot besubsumed into Martin’s visual hypothesis. The large number ofindependent neural systems linking megabats to primates supporta phylogenetic connection that is difficult to explain by functionalconvergence.
4.3. Differences of megachiroptera to microchiroptera and other
mammals
Studies done by Maseko et al. (2007) and Maseko and Manger(2007), found that numerous differences exist between thecholinergic, putative catecholaminergic and serotonergic neuralsystems of megachiroptera and microchiroptera and that thesedifferences place chiropterans into distinctly different mammalianorders. Simultaneous to the current study, an additional study wasperformed to examine the neural systems of five microchiropteraspecies that have previously not been studied (Kruger et al., 2010).The results of this simultaneous study (provided in Tables 1–3)showed some differences to the results obtained by Maseko andManger (2007); however, these are likely to be the result of the use ofdiffering antibodies for the cholinergic system (Kruger et al., 2010).The cholinergic system of the rock hyrax differs from megachir-opterans and other mammals in the sense that the rock hyrax is theonly mammal that possesses parvocellular nuclei associated withthe more traditional magnocellular cholinergic nuclei of thepontomesemcephalon (Gravett et al., 2009). Within the putativecatecholaminergic system, A4, A6c, and A10dc were observed asbeing absent in microchiropterans yet are present in megachir-opterans, a finding congruent with Maseko et al. (2007) and Masekoand Manger (2007). Two nuclei, A9v and A15v are described asabsent in microchiropterans and the tree shrew (Maseko andManger, 2007) but the concurrent unpublished study (Kruger et al.,2010) found that A9v may be present, as a small nucleus with a fewneurons, as observed in some insectivore species (Maseko et al.,2007). Despite these differences between studies, these nuclei arecommonly found amongst the mammals and do not align the twochiropteran sub-orders more closely. In comparison to previousstudies, the serotonergic system of the five microchiropteransshowed no nuclear differences compared to megachiropterans(Maseko et al., 2007). The nuclear organization of the serotonergicsystem as a whole is not diagnostic for comparison of specificeutherian mammalian orders but it does clearly distinguishmonotremes (prototherians) and marsupials (metatherians) fromthe eutherian mammals as monotremes lack the CVL and DRc nucleiand the marsupials lack the CVL (Crutcher and Humbertson, 1978;Manger et al., 2002c). Despite the many similarities in the nuclearorganization of the systems studied when comparing the Mega- andMicrochiroptera, the similarities are features common to mostmammals previously studied, while the differences are notable inthat they again align the megachiropterans most closely with theprimates as compared to all other mammalian species. The featuresfound in the microchiropterans appear to align them most closelywith the insectivores (Kruger et al., 2010).
4.4. Cladistic analysis
A phylogenetic analysis of the characters derived from thecholinergic, catecholaminergic and serotonergic system from themegachiropteran species studied herein, the results of priorstudies (Maseko et al., 2007; Bhagwandin et al., 2008; Limacher
et al., 2008; Gravett et al., 2009; Bux et al., 2010; Pieters et al.,2010) and the concurrent study of microchiropterans (Kruger et al.,2010) was performed using Mac Clade. The database used in thecurrent analysis encompassed 38 taxa and 82 characters (seeTables 1–3). In terms of the megachiropterans, all three specieswere clustered together and formed an exclusive sister group tothe primates (Fig. 9). This suggests that the features shared bymegachiropterans and primates are found exclusively in theseorders to the exclusion of all other mammals and strongly supportsthe megachiropteran-primate sister group taxonomy. The analysisplaced the microchiropterans together in a cluster and as a sistergroup to the Insectivora. This placement of the microchiroptera iswell supported in the current literature of mammalian phylogeny(e.g. Arnason et al., 2002; Asher et al., 2009; Lee and Camens,2009); however, the placement of the megachiroptera is conten-tious. When bat monophyly was forced upon the data (Fig. 9), alltreelengths were increased and associated statistical reliabilitywas decreased. This again indicates that for the neural charactersanalyzed in the current study, bat diphyly, with megachiropteransbeing a sister group to primates, is the most parsimoniousexplanation of the data. It is of importance to note that our cladisticanalysis of the neural data is in strong agreement with the majorityof modern mammalian phylogenies, and the only significantdifference being the placement of the megachiropterans.
The results of this study, both qualitative and cladistic, showthat megachiroptera are clearly more closely associated with theprimates than any other mammalian order. Moreover, the resultsindicate that the megachiropterans and microchiropterans have noclear phylogenetic relationship. The microchiroptera appear to bemost closely associated with insectivores (Fig. 9; Kruger et al.,2010). Thus a diphyletic origin of Chiroptera is supported(Pettigrew, 1986; Pettigrew et al., 1989, 2008), suggesting thatMegachiroptera and Microchiroptera are derived from differentmammalian orders and that powered flight evolved twice inmammals.
The central tenet of the current study is the variable appearanceof nuclei in the species studied. There are seven particular nucleiobserved within the current study that provide the basis of supportfor the diphyletic result of the cladistic analysis. In the cholinergicsystem the variability in the ChAT immunoreactivity of theseneurons across species separates the microchiropterans from themegachiropterans and does not separate the megachiropteransfrom the primates. The absence of the A4, A6c, A15d, and incipientappearance of the A9v, A10dc and A15v catecholaminergic nucleisupport the separation of the microchiroptera from the mega-chiroptera and does not separate the megachiroptera from theprimates. The A6c specifically groups the megachiroptera withprimates, tree shrews and rabbits, but other features of tree shrews(such as the lack of A14, A15d and A15v) and rabbits (such as lackof A9v and A15v), leave the megachiropterans studied as thespecies with the most similarities to those seen in primates.
Acknowledgments
This work was supported by funding from the South AfricanNational Research Foundation (PRM and JDP, South AfricanBiosystematics Initiative, KFD2008052300005). The authors wishto extend their gratitude to the members of the National Museumsof Kenya, especially Mr. Bernard ‘Risky’ Agwanda, without whomthis work would not have been possible.
References
Armstrong, D.M., Ross, C.A., Pickel, V.M., Joh, T.H., Donald, D.J., 1982. Distribution ofdopamine-, noradrenaline-, and adrenaline-containing cell bodies in the ratmedulla oblongata: demonstrated by the immunocytochemical localization ofcatecholamine biosynthetic enzymes. J. Comp. Neurol. 212, 173–187.
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195194
Armstrong, D.M., Saper, C.B., Levey, A.I., Wainer, H., Terry, R.D., 1983. Distribution ofcholinergic neurons in rat brain: demonstrated by the immunocytochemicallocalization of choline acetyl transferase. J. Comp. Neurol. 216, 53–68.
Arnason, U., Adegoke, J.A., Bodin, K., Born, E.W., Esa, Y.B., Gullberg, A., Nilsson, M.,Short, R.V., Xu, X., Janke, A., 2002. Mammalian mitogenomic relationships andthe root of the eutherian tree. Proc. Natl. Acad. Sci. USA 99, 8151–8156.
Asher, R.J., Bennett, N., Lehmann, T., 2009. The new framework for understandingplacental mammal evolution. Bioessays 31, 853–864.
Badlangana, N.L., Bhagwandin, A., Fuxe, K., Manger, P.R., 2007. Distribution andmorphology of putative catecholaminergic and serotonergic neurons in themedulla oblongata of a sub-adult giraffe, Giraffa camelopardalis. J. Chem. Neu-roanat. 34, 69–79.
Baker, K.G., Halliday, G.M., Tork, I., 1990. Cytoarchitecture of the human dorsalraphe nucleus. J. Comp. Neurol. 301, 147–161.
Bhagwandin, A., Fuxe, K., Bennett, N.C., Manger, P.R., 2008. Nuclear organization andmorphology of cholinergic, putative catecholaminergic and serotonergic neu-rons in the brains of two species of African mole-rat. J. Chem. Neuroanat. 35,371–387.
Bjarkam, C.R., Sorensen, J.C., Geneser, F.A., 1997. Distribution and morphology ofserotonin-immunoreactive neurons in the brainstem of the New Zealand whiterabbit. J. Comp. Neurol. 380, 507–519.
Blessing, W.W., Chalmers, J.P., Howe, P.R.C., 1978. Distribution of catecholamine-containing cell bodies in the rabbit central nervous system. J. Comp. Neurol.179, 407–424.
Bogerts, B., 1981. A brainstem atlas of catecholaminergic neurons in man, usingmelanin as a natural marker. J. Comp. Neurol. 197, 63–80.
Bux, F., Bhagwandin, A., Fuxe, K., Manger, P.R., 2010. Organization of cholinergic,putative catecholaminergic and serotonergic nuclei in the diencephalon, mid-brain and pons of sub-adult male giraffes. J. Chem. Neuroanat. 39, 189–203.
Charara, A., Parent, A., 1998. Chemoarchitecture of the primate dorsal raphenucleus. J. Chem. Neuroanat. 72, 111–127.
Cheung, Y., Sladek, J.R., 1975. Catecholamine distribution in feline hypothalamus. J.Comp. Neurol. 164, 339–360.
Crutcher, K.A., Humbertson, A.O., 1978. The organization of monoamine neuronswithin the brainstem of the North American opossum (Didelphis virginiana). J.Comp. Neurol. 179, 195–222.
Dahlstrom, A., Fuxe, K., 1964. Evidence for the existence of monoamine-containingneurons in the central nervous system. I. Demonstration of monoamine in thecell bodies of brainstem neurons. Acta Physiol. Scand. 62, 1–52.
Da Silva, J.N., Fuxe, K., Manger, P.R., 2006. Nuclear parcellation of certain immu-nohistochemically identifiable neuronal systems in the midbrain and pons ofthe Highveld molerat (Cryptomys hottentotus). J. Chem. Neuroanat. 31, 37–50.
DeFrees, S.L., Wilson, D.E., 1988. Eidolon helvum. Mamm. Species 312, 1–5.Dinopoulos, A., Michaloudi, H., Karamanlidis, A.N., Antonopoulos, J., Parnavelas, J.G.,
1988. Basal forebrain neurons project to the cortical mantle of the Europeanhedgehog (Erinaceus europaeus). Neurosci. Lett. 86, 127–132.
Dormer, K.J., Anwar, M., Ashlock, S.R., Ruggiero, D.A., 1993. Organization of pre-sumptive catecholamine-synthesizing neurons in the canine medulla oblon-gata. Brain Res. 601, 41–64.
Dwarika, S., Maseko, B.C., Ihunwo, A.O., Fuxe, K., Manger, P.R., 2008. Distribution andmorphology of putative catecholaminergic and serotonergic neurons in thebrain of the greater canerat, Thryonomys swinderianus. J. Chem. Neuroanat. 35,108–122.
Everitt, B.J., Sirkia, T.A., Roberts, A.C., Jones, G.H., Robbins, T.W., 1988. Distributionand some projections of the cholinergic neurons in the brain of the commonmarmoset, Callithrix jacchus. J. Comp. Neurol. 271, 533–558.
Felten, D.L., Laties, A.L., Carpenter, M.B., 1974. Monoamine-containing cell bodies inthe squirrel monkey brain. Am. J. Anat. 139, 153–166.
Ferguson, I.A., Hardman, C.D., Marotte, L.R., Salardini, A., Halasz, P., Vu, D., Waite,P.M., 1999. Serotonergic neurons in the brainstem of the wallaby, Macropuseugenii. J. Comp. Neurol. 411, 535–549.
Ferreira, G., Meurisse, M., Tillet, Y., Levy, F., 2001. Distribution and co-localization ofcholine acetyltransferase and P75 neurotrophin receptors in the sheep basalforebrain: implications for the use of a specific cholinergic immunotoxin.Neuroscience 104, 419–439.
Fitzpatrick, D., Conley, M., Luppino, G., Matelli, M., Diamond, I.T., 1988. Cholinergicprojections from the midbrain reticular formation and the parabigeminalnucleus to the lateral geniculate nucleus in the tree shrew. J. Comp. Neurol.272, 43–67.
Gallyas, F., 1979. Silver staining of myelin by means of physical development.Neurolog. Res. 1, 203–209.
Garver, D.L., Sladek, J.R., 1975. Monoamine distribution in primate brain. I. Cate-cholamine-containing perikarya in the brainstem of Macaca speciosa. J. Comp.Neurol. 159, 289–304.
Gravett, N., Bhagwandin, A., Fuxe, K., Manger, P.R., 2009. Nuclear organization andmorphology of cholinergic, putative catecholaminergic and serotonergic neu-rons in the brain of the rock hyrax, Procavia capensis. J. Chem. Neuroanat. 38, 57–74.
Halliday, G.M., Li, Y.W., Joh, T.H., Cotton, R.G.H., Howe, P.R.C., Geffen, L.B., Blessing,W.W., 1988. Distribution of monoamine-synthesizing neurons in the humanmedulla oblongata. J. Comp. Neurol. 273, 301–317.
Henderson, Z., 1987. Overlap in the distribution of cholinergic and catecholamin-ergic neurons in the upper brainstem of the ferret. J. Comp. Neurol. 265,581–592.
Hokfelt, T., Martenson, R., Bjorklund, A., Kleinau, S., Goldstein, M., 1984. Distribu-tional maps of tyrosine-hydroxylase-immunoreactive neurons in the rat brain.
In: Bjorklund, A., Hokfelt, T. (Eds.), Handbook of Chemical Neuroanatomy, vol.2, Classical Neurotransmitters in the CNS, part 1. Elsevier, Amsterdam, pp. 277–379.
Hornung, J.P., Fritschy, J.M., 1988. Serotonergic system in the brainstem of themarmoset: a combined immunocytochemical and three-dimensional recon-struction study. J. Comp. Neurol. 270, 471–487.
Hubbard, J.E., Di Carlo, V., 1974a. Flourescence histochemistry of monoamine-containing cell bodies in the brain stem of the squirrel monkey (Saimirisciureus). II. Catecholamine-containing groups. J. Comp. Neurol. 153, 369–384.
Hubbard, J.E., Di Carlo, V., 1974b. Flourescence histochemistry of monoamine-containing cell bodies in the brain stem of the squirrel monkey (Saimirisciureus). III. Serotonin-containing groups. J. Comp. Neurol. 153, 385–398.
Jacobowitz, D.M., MacLean, P.D., 1978. A brainstem atlas of catecholaminergicneurons and serotonergic perikarya in a pygmy primate (Cebuella pygmaea).J. Comp. Neurol. 177, 397–416.
Kalesnykas, G., Puolivali, J., Sirvio, J., Miettinen, R., 2004. Cholinergic neurons in thebasal forebrain of aged female mice. Brain Res. 1022, 148–156.
Karasawa, N., Takeuchi, T., Yamada, K., Iwasa, M., Isomura, G., 2003. Cholineacetyltransferase positive neurons in the laboratory shrew (Suncus murinus)brain: coexistence of ChAT/5-HT (Raphe dorsalis) and ChAT/TH (Locus ceruleus).Acta Histochem. Cytochem. 36 (4), 399–407.
Kimura, H., McGeer, P.L., Peng, J.H., McGeer, E.G., 1981. The central cholinergicsystem studied by choline acetyltransferase immunohistochemistry in the cat.J. Comp. Neurol. 200, 151–201.
Kitahama, K., Geffard, M., Okamura, H., Nagatsu, I., Mons, N., Jouvet, M., 1990.Dopamine- and dopa-immunoreactive neurons in the cat forebrain with refer-ence to tyrosine hydroxylase-immunohistochemistry. Brain Res. 518, 83–94.
Kitahama, K., Sakamoto, N., Jouvet, A., Nagatsu, I., Pearson, J., 1996. Dopamine-beta-hydroxylase and tyrosine hydroxylase immunoreactive neurons in the humanbrainstem. J. Chem. Neuroanat. 10, 137–146.
Kojima, M., Takeuchi, Y., Goto, M., Sano, Y., 1983. Immunohistochemical study onthe distribution of serotonin-containing cell bodies in the brainstem of the dog.Acta Anat. (Basel) 115, 8–22.
Kruger, J.L., Dell, L.A., Bhagwandin, A., Jillani, N.E., Pettigrew, J.D., Manger, P.R., 2010.Nuclear organization of cholinergic, putative catecholaminergic and serotoner-gic systems in the brains of five microchiropteran species. J. Chem. Neuroanat.,in press.
Kus, L., Borys, E., Chu, Y.P., Ferguson, S.M., Blakely, R.D., Emborg, M.E., Kordower, J.H.,Levey, A.I., Mufson, E.J., 2003. Distribution of high affinity choline transporterimmunoreactivity in the primate central nervous system. J. Comp. Neurol. 463,341–357.
Lavoie, B., Parent, A., 1994. Pedunculopontine nucleus in the squirrel monkey:distribution of cholinergic and monoaminergic neurons in the mesopontinetegmentum with evidence for the presence of glutamate in cholinergic neurons.J. Comp. Neurol. 344, 190–209.
Lee, M.S.Y., Camens, A.B., 2009. Strong morphological support for the molecularevolutionary tree of placental mammals. J. Evol. Biol. 22, 2243–2257.
Leger, L., Charnay, Y., Hof, P.R., Bouras, C., Cespuglio, R., 2001. Anatomical distribu-tion of serotonin-containing neurons and axons in the central nervous system ofthe cat. J. Comp. Neurol. 433, 157–182.
Leshin, L.S., Kraeling, R.R., Kineman, R.D., Barb, C.R., Rampacek, G.B., 1995. Immu-nocytochemical distribution of catecholamine-synthesizing neurons in thehypothalamus and pituitary gland of pigs: tyrosine hydroxylase and dopa-mine-b-hydroxylase. J. Comp. Neurol. 364, 151–168.
Li, G., Wang, J., Rossiter, S.J., Jones, G., Cotton, J.A., Zhang, S., 2008. The hearing genePrestin reunites echolocating bats. Proc. Natl. Acad. Sci. USA 105, 13959–13964.
Li, G., Wang, J., Rossiter, S.J., Jones, G., Zhang, S., 2007. Accelerated FoxP2 evolution inEcholocating Bats. PLoS ONE 2 (9), e900.
Limacher, A.M., Bhagwandin, A., Fuxe, K., Manger, P.R., 2008. Nuclear organizationand morphology of cholinergic, putative catecholaminergic and serotonergicneurons in the brain of the Cape porcupine (Hystrix africaeaustralis): increasedbrain size does not lead to increased organizational complexity. J. Chem.Neuroanat. 36, 33–52.
Loomis, W.F., Smith, D.W., 1990. Molecular phylogeny of Dictyostelium discoideumby protien sequence comparion. Proc. Natl. Acad. Sci. USA 87, 9093–9097.
Manger, P.R., 2005. Establishing order at the systems level in mammalian brainevolution. Brain Res. Bull. 66, 282–289.
Manger, P.R., Fahringer, H.M., Pettigrew, J.D., Siegel, J.M., 2002a. Distribution andmorphology of cholinergic neurons in the brain of the monotremes as revealedby ChAT immunohistochemistry. Brain Behav. Evol. 60, 275–297.
Manger, P.R., Fahringer, H.M., Pettigrew, J.D., Siegel, J.M., 2002b. Distribution andmorphology of catecholaminergic neurons in the brain of monotremes asrevealed by tyrosine hydroxylase immunohistochemistry. Brain Behav. Evol.60, 298–314.
Manger, P.R., Fahringer, H.M., Pettigrew, J.D., Siegel, J.M., 2002c. Distribution andmorphology of serotonergic neurons in the brain of the monotremes. BrainBehav. Evol. 60, 315–332.
Manger, P.R., Ridgway, S.H., Siegel, J.M., 2003. The locus coeruleus complex of thebottlenose dolphin (Tursiops truncatus) as revealed by tyrosine hydroxylaseimmunohistochemistry. J. Sleep Res. 12, 149–155.
Manger, P.R., Fuxe, K., Ridgway, S.H., Siegel, J.M., 2004. The distribution andmorphological characteristics of catecholamine cells in the diencephalonand midbrain of the bottlenose dolphin (Tursiops truncatus). Brain Behav.Evol. 64, 42.
Martin, R.D., 1986. Vertebrate phylogeny: are fruit bats primates? Nature 320,482–483.
L.-A. Dell et al. / Journal of Chemical Neuroanatomy 40 (2010) 177–195 195
Maseko, B.C., Manger, P.R., 2007. Distribution and morphology of cholinergic,catecholaminergic and serotonergic neurons in the brain of Schreiber’s long-fingered bat, Miniopterus schreibersii. J. Chem. Neuroanat. 34, 80–94.
Maseko, B.C., Bourne, J.A., Manger, P.R., 2007. Distribution and morphology ofcholinergic, putative catecholaminergic and serotonergic neurons in the brainof the Egyptian Rousette flying fox, Rousettus aegyptiacus. J. Chem. Neuroanat.34, 108–127.
Mesulam, M.M., Mufson, E.J., Levey, A.I., Wainer, B.H., 1984. Atlas of cholinergicneurons in the forebrain and upper brainstem of the macaque based onmonoclonal choline acetyltransferase immunohistochemistry and acetylcho-linesterase histochemistry. Neuroscience 12, 669–686.
Mesulam, M.M., Geula, C., Bothwell, M.A., Hersch, L.B., 1989. Human reticularformation: cholinergic neurons of the pedunculopontine tegmental nucleiand some cytochemical comparisons of forebrain cholinergic neurons. J. Comp.Neurol. 281, 611–633.
Michaloudi, H.C., Papadopoulos, G.C., 1995. Atlas of serotonin-containing cellbodies and fibres in the central nervous system of the hedgehog. J. BrainRes. 1, 77–100.
Michaloudi, H.C., Papadopoulos, G.C., 1996. Noradrenergic and dopaminergic sys-tems in the central nervous system of the hedgehog (Erinaceus europaeus). J.Hirnforsch. 37, 319–350.
Mindell, D.P., Dick, C.W., Baker, R.J., 1991. Phylogenetic relationships amongmegabats, microbats, and primates. Proc. Natl. Acad. Sci. USA 88, 10322–10326.
Mizukawa, K., McGeer, P.L., Tago, H., Peng, J.H., McGeer, E.G., Kimura, H., 1986. Thecholinergic system of the human hindbrain studied by choline acetyltransferaseimmunohistochemistry and acetylcholinesterase histochemistry. Brain Res.379, 39–55.
Moon, D.J., Maseko, B.C., Ihunwo, A., Fuxe, K., Manger, P.R., 2007. Distribution andmorphology of catecholaminergic and serotonergic neurons in the brain of thehighveld gerbil, Tatera brantsii. J. Chem. Neuroanat. 34, 134–144.
Murray, H.M., Dominguez, W.F., Martinez, J.E., 1982. Catecholaminergic neurons inthe brain stem of tree shrew (Tupaia). Brain Res. Bull. 9, 205–215.
Naylor, G.J.P., Brown, W.M., 1998. Amphioxus mitochondrial DNA, chordate phy-logeny, and the limits of inference based on comparisons of sequences. Syst.Biol. 47, 61–76.
Neuweiler, G., 2000. The Biology of Bats. Oxford University Press, Oxford, UK.Nikaido, M., Harada, M., Cao, Y., Hasegawa, M., Okada, M., 2000. Monophyletic origin
of the order chiroptera and its phylogenetic position among mammalia, asinferred from the complete sequence of the mitochondrial DNA of a Japanesemegabat, the Ryukyu flying fox (Pteropus dasymallus). J. Mol. Evol. 51, 318–328.
Østergaard, K., Holm, I.E., Zimmer, J., 1992. Tyrosine hydroxylase and acetylcholin-esterase in the domestic pig mesencephalon: an immunocytochemical andhistochemical study. J. Comp. Neurol. 322, 149–166.
Pearson, J., Goldstein, M., Markey, K., Brandeis, L., 1983. Human brainstem cate-cholamine neuronal anatomy as indicated by immunocytochemistry withantibodies to tyrosine hydroxylase. Neuroscience 8, 3–32.
Pettigrew, J.D., 1986. Flying primates? Megabats have the advanced pathway fromeye to midbrain. Science 231, 1304–1306.
Pettigrew, J.D., Kirsch, A.W., 1998. Base-compositional biases and the bat problem. I.DNA-hybridization melting curves based on AT- and GC-enriched tracers.Philos. Trans. R. Soc. Lond. B 35, 369–379.
Pettigrew, J.D., Jamieson, B.G.M., Robson, S.K., Hall, L.S., McNally, K.I., Cooper, H.M.,1989. Phylogenetic relations between microbats, megabats and primates(Mammalia: Chiroptera and Primates). Philos. Trans. R. Soc. Lond. 325, 489–559.
Pettigrew, J.D., Maseko, B.C., Manger, P.R., 2008. Primate-like retinotectal decussationin an echolocating megabat Rousettus aegyptiacus. Neuroscience 153, 226–231.
Pieters, R.P., Gravett, N., Fuxe, K., Manger, P.R., 2010. Nuclear organization ofcholinergic, putative catecholaminergic and serotonergic nuclei in the brainof the eastern rock elephant shrew, Elephantulus myurus. J. Chem. Neuroanat.39, 175–188.
Poitras, D., Parent, A., 1978. Atlas of the distribution of monoamine-containingnerve cell bodies on the brainstem of the cat. J. Comp. Neurol. 179, 699–718.
Reiner, P.B., Vincent, S.R., 1987. Topographic relations of cholinergic and noradren-ergic neurons in the feline pontomesencephalic tegmentum: an immunohisto-chemical study. Brain Res. Bull. 19, 705–714.
Ruggerio, D.A., Baker, H., Joh, T.H., Reis, D.J., 1984. Distribution of catecholamineneurons in the hypothalamus and preoptic region of mouse. J. Comp. Neurol.223, 556–582.
Ruggiero, D.A., Anwar, M., Gootman, P.M., 1992. Presumptive adrenergic neuronscontaining phenylethanolamine N-methyltransferase immunoreactivity in themedulla oblongata of neonatal swine. Brain Res. 583, 105–119.
Satoh, K., Fibiger, H.C., 1985. Distribution of central cholinergic neurons in thebaboon (Papio papio). I. General morphology. J. Comp. Neurol. 236, 197–214.
Satoh, J., Irino, M., Martin, P.M., Mailman, R.B., Suzuki, K., 1991. Neurochemical andimmunocytochemical studies of catecholamine system in the brindled mouse. J.Neuropathol. Exp. Neurol. 50, 793–808.
Schneider, R., 1966. Das Gehirn von Rousettus aegyptiacus (E. Geoffroy 1810)(Megachiroptera, Chiroptera, Mammalia). Ein mit Hilfe mehrerer Schnittserienerstellter Atlas. Abhandlungen der senckenbergischen Naturforschenden Ge-sellschaft 513, 1–166.
Schofield, S.P.M., Everitt, B.J., 1981. The organization of catecholamine-contain-ing neurons in the brains of the rhesus monkey (Macaca mulatta). J. Anat. 132,391–418.
Shen, Y.-Y., Liu, J., Irwin, D.M., Zhang, Y.-P., 2010. Parallel and convergent evolutionof the dim-light vision gene RH1 in bats (Order: Chiroptera). PLoS ONE 5, e8838.
Shiromani, P.J., Armstrong, D.M., Berkowitz, A., Jeste, D.V., Gillin, J.C., 1988. Distri-bution of choline acetyltransferase immunoreactive somata in the feline brain-stem: implications for REM sleep generation. Sleep 11, 1–16.
Smeets, W.J.A.J., Gonzalez, A., 2000. Catecholamine systems in the brain of verte-brates: new perspectives through a comparative approach. Brain Res. Rev. 33,308–379.
Smith, J.D., Madkour, G., 1980. Penial morphology and the question of chiropteranphylogeny. In: Wilson, D.E., Gardner, A.L. (Eds.), Proceedings Fifth InternationalBat Research Conference. Texas Tech Press, Lubbock, pp. 347–365.
Steinbusch, H.W.M., 1981. Distribution of serotonin-immunoreactivity in thecentral nervous system of the rat – cell bodies and terminals. Neuroscience 6,557–618.
St-Jacques, R., Gorczyca, W., Mohr, G., Schipper, H.M., 1996. Mapping of the basalforebrain cholinergic system of the dog: a choline acetyltransferase immuno-histochemical study. J. Comp. Neurol. 366, 717–725.
Tafti, M., Nishino, S., Liao, W., Dement, W.C., Mignot, E., 1997. Mesopontineorganization of cholinergic and catecholaminergic cell groups in the normaland narcoleptic dog. J. Comp. Neurol. 379, 185–197.
Tago, H., McGeer, P.L., McGeer, E.G., Akiyama, H., Hersch, L.B., 1989. Distribution ofcholine acetyltransferase immunopositive structures in the rat brainstem. BrainRes. 495, 271–297.
Takeuchi, Y., Kimura, H., Matssuura, T., Sano, Y., 1982. Immunohistochemicaldemonstration of the organization of serotonergic neurons in the brain ofthe monkey (Macaca fuscata). Acta Anat. 114, 106–124.
Teeling, E.C., Madsen, O., Van den Bussche, R.A., de Jong, W.W., Stanhope, M.J.,Springer, M.S., 2002. Microbat paraphyly and the convergent evolution of a keyinnovation in Old World rhinolophoid microbats. Proc. Natl. Acad. Sci. USA 99,1431–1436.
Teeling, E.C., Springer, M.S., Madsen, O., Bates, P., O’Brien, S.J., Murphy, W.J., 2005. Amolecular phylogeny for bats illuminates biogeography and the fossil record.Science 307, 580–584.
Tillet, Y., 1987. Immunocytochemical localization of serotonin-containing neuronsin the myelencephalon, brainstem and diencephalon of the sheep. Neuroscience23, 501–527.
Tillet, Y., Thibault, J., 1989. Catecholamine-containing neurons in the sheep brain-stem and diencephalon: immunohistochemical study with tyrosine hydroxy-lase (TH) and dopamine-ß-hydroxylase (DBH) antibodies. J. Comp. Neurol. 290,69–106.
Tillet, Y., Kitahama, K., 1998. Distribution of central catecholaminergic neurons: acomparison between ungulates, humans and other species. Histol. Histopathol.13, 1163–1177.
Tork, I., 1990. Anatomy of the serotonergic system. Ann. N. Y. Acad. Sci. 600, 9–35.VanderHorst, V.G.J.M., Ulfhake, B., 2006. The organization of the brainstem and
spinal cord of the mouse: relationships between monoaminergic, cholinergic,and spinal projection systems. J. Chem. Neuroanat. 31, 2–36.
Vincent, S.R., Reiner, P.B., 1987. The immunohistochemical localization of cholineacetyltransferase in the cat brain. Brain Res. Bull. 18, 371–415.
Warwick, R., 1953. Representation of the extraocular muscles in the oculomotornuclei of the monkey. J. Comp. Neurol. 9, 449–504.
Woolf, N.J., 1991. Cholinergic systems in mammalian brain and spinal cord. Prog.Neurobiol. 37, 475–524.