Download Brain-Derived Neurotrophic Factor mRNA Expression in the Brain of the Teleost

Original Paper
Brain Behav Evol 2009;73:43–58
DOI: 10.1159/000204962
Received: January 29, 2008
Returned for revision: February 27, 2008
Accepted after second revision: November 3, 2008
Published online: February 27, 2009
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Brain-Derived Neurotrophic Factor mRNA
Expression in the Brain of the Teleost
Fish, Anguilla anguilla, the European Eel
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granted a, b
Victoria S.
Suzanne M.
Paula Murphy uteBarry
L. beRoberts
against payment of a pera
mission fee, which
based and
Department of Zoology, Trinity College, University of Dublin, Dublin, Ireland; b Department
of isCellular
the numberIll.of, accesses
Molecular Pharmacology, Chicago Medical School at Rosalind Franklin University,onChicago,
USA
required. Please contact
[email protected]
Dalton a
Borich a
Key Words
Brain-derived neurotrophic factor ⴢ Neurotrophins ⴢ Brain ⴢ
Teleost fish ⴢ Regeneration
Abstract
The spatial expression pattern of Brain-Derived Neurotrophic Factor (BDNF) mRNA in the brain of the European eel (Anguilla anguilla) was determined using non-radioactive in situ
hybridization, and mapped and compared to that of other
vertebrates. Riboprobes were prepared based on a partial
cDNA coding sequence for A. anguilla BDNF that was amplified using degenerate primers, cloned and sequenced. As in
other animal groups, in the eel, BDNF mRNA expression was
seen in the telencephalon, hypothalamus, tectum, many primary and secondary sensory centers, and cranial motor nuclei. However, in contrast to mammals, BDNF mRNA expression was observed in some brain stem nuclei, such as the
reticular formation, that contain cell bodies of neurons that
project down the spinal cord. We suggest that these differences might relate to the continual growth of teleost fish.
Copyright © 2009 S. Karger AG, Basel
Introduction
The neurotrophin Brain-Derived Neurotrophic Factor (BDNF) plays crucial roles in both the developing and
mature mammalian nervous system. It is important dur© 2009 S. Karger AG, Basel
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ing development in regulating neuronal survival, promoting axonal pathfinding and elongation towards targets, and in supporting synaptic plasticity [McAllister et
al., 1999; Huang and Reichardt, 2001; Gillespie, 2003; Cohen-Cory and Lom, 2004] and is involved in neuronal
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plasticity and survival in the adult nervous system [ConDISTRIBUTION 1998;
OF THIS ARTICLE
WITHOUT WRITTEN
FROM S. KARGER AG, BASEL I
nor and ANY
Dragunow,
McAllister
et al., CONSENT
1999; SchinWritten permission to distribute the PDF will be granted against payment of a permission fee, wh
der and Poo, 2000]. Because these are functions that are
likely to be required by neurons recovering from injury,
neurotrophins have also been implicated in regeneration,
and an insufficiency of such growth-enhancing molecules in the injured mammalian nervous system has been
thought to contribute to its limited regeneration ability
[reviewed in Schwab and Bartholdi, 1996].
The overall expression pattern of BDNF mRNA has
been described from in situ hybridization studies of the
rat and pig brain [Wetmore et al., 1990; Castrén et al. 1995;
Conner et al., 1997] and immunohistochemical studies
have revealed the distribution of BDNF protein in the rat
[Conner et al., 1997; Yan et al., 1997; Friedman et al., 1998;
Furukawa et al., 1998] and human [Murer et al., 1999], but
to date comprehensive descriptions of BDNF mRNA and
protein expression patterns have not been published for
the avian brain or for the brain of any anamniote. The
presence of BDNF mRNA in specific brain regions has
been noted though: for example, BDNF mRNA is expressed in the mesencephalic tectum of chick [Herzog and
von Bartheld, 1998] and frog [Duprey-Díaz et al., 2002]
and BDNF protein expression has been reported in pigeon
Victoria S. Dalton
Radiopharmaceuticals Research Institute
ANSTO, PMB 1 Menai, Sydney, NSW 2234 (Australia)
Tel. +61 2 9717 9879, Fax +61 2 9717 9262
E-Mail: [email protected] or [email protected]
Abbreviations used in this paper
A
AP
Cb
CbSg
CbSm
cc
CP
Dc
Dd
Dl
Dlp
Dm
DO
DP
dV
E
EC
flt
GL
GS
HAd
HAv
HC
HL
HV
IC
IRF
LC
LL
LLp
LV
LVII
LX
M
MLF
MLFd
MO
MRF
nCC
ndil
anterior thalamic nucleus
area postrema
cerebellum
granular layer of cerebellum
molecular layer of cerebellum
cerebellar crest
nucleus pretectalis centralis
central zone of the area dorsalis
dorsal zone of the area dorsalis
lateral zone of the area dorsalis
posterior portion of lateral zone of the area dorsalis
medial zone of the area dorsalis
descending octavolateralis nucleus
dorsal posterior thalamic nucleus
descending tract of trigeminal nerve
entopeduncular nucleus
external cell layer
telencephalic lateral longitudinal fasciculus
glomerulus (olfactory bulb)
secondary gustatory nucleus
dorsal habenular nucleus
ventral habenular nucleus
caudal zone of periventricular hypothalamus
inferior lobe of hypothalamus
ventral zone of periventricular nucleus hypothalamus
internal cell layer
inferior reticular formation
locus coeruleus
lateral lemniscus
posterior lateral line nerve
nucleus lateralis valvulae
facial lobe
vagal lobe
Mauthner-like cell
medial longitudinal fasciculus
dorsal subdivision of medial longitudinal fasciculus
magnocellular octavolateralis nucleus
medial reticular formation
commissural nucleus of Cajal
diffuse nucleus of inferior lobe
[Theiss and Güntürkün, 2001] and frog [Duprey-Díaz et
al., 2002] tectum and in the hypothalamo-hypophyseal
system of Xenopus [Wang et al., 2005]. With regard to teleost fish, BDNF expression has been confirmed in the
brain of the southern platyfish [Götz et al., 1992], in the
developing zebrafish brain and retina [Hashimoto and
Heinrich, 1997; Lum et al., 2001], and in the tench retina
[Caminos et al., 1999]. Five neurotrophin receptors have
been described in zebrafish [Martin et al., 1995], two of
which have been reported to be isoforms of the Trk B receptor and their mRNA is widely distributed throughout
the brain and spinal cord of larval zebrafish [Martin et al.,
1995]. Hannestad et al. [2000] observed immunoreactivity for Trk B in the adenohypophysis, striatum and retina
during development of Dicentrarchus.
44
Brain Behav Evol 2009;73:43–58
nI
nIV
nIXm
nMLF
nTL
nVIIm
nVm
nVs
nXm
OEN
ON
PPd
PGm
PMg
PMm
PMp
PPp
PT
PVO
Rinf
SC
SCO
sgt
SO
SPV
SRF
sv
T
TO
TP
TPp
TS
VCb
Vd
Vl
VL
VM
Vp
VST
Vv
nucleus isthmi
trochlear nucleus
glossopharyngeal motor nucleus
nucleus of medial longitudinal fasciculus
nucleus lateralis tuberis
facial motor nucleus
trigeminal motor nucleus
trigeminal sensory nucleus
vagal motor nucleus
octavolateralis efferent nucleus
optic nerve
nucleus pretectalis periventricularis, pars dorsalis
preglomerular nuclear complex
nucleus preopticus magnocellularis, pars gigantocellularis
nucleus preopticus magnocellularis, pars magnocellularis
nucleus preopticus magnocellularis, pars parvocellularis
nucleus preopticus parvocellularis posterior
posterior thalamic nucleus
paraventricular organ
inferior raphe nucleus
suprachiasmatic nucleus
subcommissural organ
secondary gustatory tract
superior olive
stratum periventriculare
superior reticular formation
saccus vasculosus
tectum
tangential octavolateralis nucleus
nucleus posterior tuberis
periventricular nucleus of posterior tuberculum
torus semicircularis
valvula cerebelli
dorsal nucleus of the area ventralis
lateral nucleus of the area ventralis
ventrolateral nucleus of the thalamus
ventromedial nucleus of the thalamus
posterior nucleus of the area ventralis
vestibulospinal tract
ventral nucleus of the area ventralis
The present study is based on the European eel, Anguilla anguilla, a teleost fish whose rapid morphological
and functional regeneration after a complete spinal cord
transection has now been studied by us in some detail
[Doyle et al., 2001; Dervan and Roberts 2003a, b; Doyle
and Roberts, 2004 a, b, 2006]. Here we aimed to: (1) isolate
and characterize a cDNA fragment complementary to the
mRNA sequence which encodes the mature form of eel
BDNF protein molecule and (2) examine BDNF mRNA
expression throughout the eel brain using in situ hybridization analysis. This report provides the background to
a subsequent study on the eel in which we shall examine
how BDNF mRNA expression in the eel central nervous
system might be affected by spinal cord transection and
its subsequent repair.
Dalton /Borich /Murphy /Roberts
Materials and Methods
For the present study, comprehensive sets of eel brain sections
were hybridized so that the pattern of BDNF expression throughout the whole brain could be charted.
Animals and Tissue Preparation
The brains were obtained from European eels, A. anguilla L. (n
= 30), of unknown sex in a non-migratory, immature stage of the
life cycle caught in the wild in Counties Mayo and Westmeath,
Ireland. The eels ranged in length from 300 to 500 mm and were
kept in aquaria with recirculating water at 25 ° C. The procedures
were approved by a local committee and were carried out in accordance with the European Communities Council Directive
(86/609/EEC). All chemicals were from Sigma, UK, unless stated.
For tissue extraction, eels were sedated by immersion in tricaine methane sulfonate (MS 222, 0.4 g/l H2O, pH 7.4) and then
injected subcutaneously with alphaxalone anaesthetic (0.4 ml
‘Saffan’, Schering-Plough Animal Health, IE). For RNA extraction, fresh brain tissue was dissected, snap frozen in liquid nitrogen and stored at –80 ° C. For histology, each anaesthetized eel was
perfused through the heart with phosphate-buffered saline solution (PBS; 0.1 M, pH 7.4) containing procaine (1 g/l) followed by a
fixative solution of 4% paraformaldehyde (PFA) in PBS. The brain
was removed and postfixed in 4% PFA/PBS for 24 h.
After postfixation, the brains for in situ hybridization were
mounted in 5% agar/PBS and completely sectioned (100 ␮m) serially in the horizontal or transverse planes with a vibrating microtome (VT1000S, Leica Microsystems, Germany). The sections
were then dehydrated through a series of methanol/PBT (PBT =
0.1% Triton X-100 in PBS; 25, 50, 75%; 1 ! 5 min) washes, followed by 2 ! 5 min 100% methanol washes and stored at –20 ° C.
Two additional perfused brains were equilibrated in 30% sucrose/
PBS solution overnight at 4 ° C and frozen in optimal cutting temperature compound (BDH, UK) before being cut on a cryostat
(OTF, Bright, Huntingdon, UK). These sections (30 ␮m) were
then stained with cresyl violet (0.5%) to provide reference series
for cytoarchitectural study.
lows: 95 ° C for 1 min followed by 35 cycles of 95 ° C for 30 s, 62 ° C
for 40 s, 72 ° C for 1 min. PCR products were cloned into the
pCR쏐II vector using the TA Cloning Dual Promoter Kit (Invitrogen, UK) and the insert sequenced on both DNA strands. The
corresponding amino acid sequence was predicted using MacVector TM software (Accelrys, San Diego, Calif., USA).
In order to confirm the identity of the cloned putative eel
BDNF sequence, BLASTN searches [Altschul et al., 1997] of the
NCBI database were carried out. ClustalW alignments [Higgins,
1994] with the eel BDNF cDNA and predicted amino acid sequences were also carried out with neurotrophin sequences retrieved from the NCBI GenBank and GenPept databases.
where S = C and G, R = G and A, Y = C and T. PCR was carried
out using 1% of the sscDNA template, 0.6 ␮ M of each BDNF primer, 2 units Taq DNA polymerase (New England Biolabs, UK) and
0.2 mM dNTPs (Invitrogen, UK). Cycling conditions were as fol-
Probe Synthesis and in situ Hybridization
Plasmid DNA, containing the BDNF insert, was linearized
with appropriate restriction enzymes (i.e., EcoRV and BamHI,
New England Biolabs, UK). Antisense and sense RNA probes
were generated by in vitro transcription with SP6 and T7 RNA
polymerases (Roche, Germany), respectively in the presence of
digoxigenin-labeled ribonucleotides (Roche, Germany) from
1 ␮g of linearized plasmid.
For in situ hybridization, free-floating sections were rehydrated through a series of methanol/PBT (75, 50, 25%; each 1 !
5 min) washes at 4 ° C. After 2 ! 10 min washes in PBT, they were
treated with proteinase K (10 ␮g/ml in 50 mM Tris-HCl pH 7.5,
5 mM EDTA) for 5 min at room temperature, washed twice in PBT
and fixed for 20 min in 0.2% glutaraldehyde/4% PFA in PBT. Fixation was followed by washes in PBT at room temperature (3 !
5 min) and 1 ! 30 min at 55 ° C. The sections were prehybridized
at 55 ° C for 15 min in a hybridization solution containing 2%
blocking reagent (Roche, Germany), 50% formamide, 5! SSC,
0.5% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 500 ␮g/ml Heparin, 1 ␮g/ml yeast tRNA, 0.1%
Tween 20 and 5 mM EDTA and left to prehybridize overnight at
55 ° C in fresh solution.
Probes were denatured at 80 ° C for 3 min and sections were
then incubated at 55 ° C overnight in hybridization solution containing either antisense or sense BDNF probe at a concentration
of 2 ng/␮l. RNase free conditions were maintained throughout all
prehybridization and hybridization steps as advised in Rattray
and Michael [1998]. Post-hybridization washes were carried out
at 60 ° C as follows: 2 ! 10 min in 2! SSC; 3 ! 20 min in 2!
SSC/0.1% CHAPS; 3 ! 20 min in 0.2! SSC/0.1% CHAPS.
The sections were then washed for 2 ! 10 min in TNT
(TNT = 100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Tween 20) at
room temperature and blocked in blocking buffer (0.1 M maleic
acid, 0.15 M NaCl, 3% blocking reagent (Roche, Germany)) for
3 h at 4 ° C. Sections were incubated overnight in fresh blocking
buffer containing a 1:1000 dilution of anti-digoxigenin Fab fragments conjugated with alkaline phosphatase (Roche, Germany) at
4 ° C. The sections were washed (5 ! 1 h) at room temperature in
TNT and rocked in TNT overnight at 4 ° C. On the day the signal
was developed, sections were washed in 3 changes of NMT
(NMT = 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2)
for 15 min each. The chromogenic reaction was carried out in
NMT containing 175 ␮g/ml 4-nitro blue tetrazolium chloride
(NBT; Roche, Germany) and 62.5 ␮g/ml 5-bromo-4-chloro-3-indolyl-phophate (BCIP; Roche, Germany). Sections were developed in the dark at room temperature and then fixed in 4% PFA/
PBS before mounting on slides.
BDNF in Eel Brain
Brain Behav Evol 2009;73:43–58
Cloning of an Anguilla anguilla BDNF cDNA Fragment
Total RNA was extracted from the brain of one animal using
Tri reagent (MRC, Cincinnati, Ohio, USA) and single-stranded
complementary DNA (sscDNA) was synthesized from 1 ␮g of
DNase treated total RNA in the presence of oligo (dT)12–18 primers
(Invitrogen, UK), using PowerScriptTM Reverse Transcriptase (BD
Biosciences, UK) according to the manufacturer’s instructions.
Degenerate PCR primers were designed using MacVectorD
software (Accelrys, San Diego, Calif., USA), based on conserved
coding regions of available BDNF cDNA sequences from four teleost species: carp, platyfish, zebrafish and flounder (GenBank
accession numbers L27171, X59942, NM_131595 and AY074888,
respectively). The resulting degenerate primer sequences were as
follows:
forward: 5ⴕ- CTCRCGGGTGATGATCAGCAACCA-3ⴕ
and reverse: 5ⴕ-TSCCYCTY TTAATGGTCAATGTGCATA-3ⴕ,
45
Fig. 1. Partial 467 bp cDNA sequence for
Anguilla anguilla BDNF (GenBank accession number AY762996) with predicted
amino acid sequence (GenPept accession
number AAV31512). The binding positions of the forward and reverse degenerate primers are located in the boxed regions. Positions of degenerate bases are
marked by the closed arrowheads. Degenerate nucleotide choices are included at the
relevant positions. The open arrowheads
indicate the position of a consensus sequence for an N-glycosylation site. The arrow marks the site at which the precursor
is cleaved to form mature BDNF.
Microscopic Examination, Data Analysis
Light microscopic images were collected using a microscope
(Nikon E600) and a digital camera (Optronics, Goleta, Calif.).
The locations of the labeled cell profiles were charted at 40!
magnification using a camera lucida. Cells were included when
the label was clearly visible above background but absent from
sense controls.
In order to designate the labeled cells, correlations were made
between the cresyl violet-stained and hybridized sections. Standard nomenclature, as used in previous studies on A. anguilla
[Meredith et al., 1987; Meredith and Roberts, 1987; Roberts et al.,
1989; Molist et al., 1993; Kapsimali et al., 2000; Bosch and Roberts, 2001] was followed whenever possible, otherwise the brain
atlas for A. japonica [Mukuda and Ando, 2003], a closely related
species [Minegishi et al., 2005], was utilized.
Results
Molecular Characterization of BDNF cDNA
A 467 bp partial cDNA fragment was amplified, cloned
and sequenced (fig. 1), that was deemed to encode A. anguilla BDNF. This conclusion was based on the results of
46
Brain Behav Evol 2009;73:43–58
pairwise alignments, which revealed a high level of nucleotide (87–88% with other teleost fish) and protein
(ranging from 79 to 97% for all species examined) identity between the cloned sequence and other BDNF sequences from various species but lower identity level
(^58%) with other neurotrophin amino acid sequences.
In a multiple alignment between the predicted and other
neurotrophin amino acid sequences, it was observed that
the cloned fragment encompasses the five variable regions that exist between the mature forms of the peptide
sequences for the different neurotrophin family members
(fig. 2). Furthermore, all results retrieved by a BLASTN
search of the NCBI GenBank database with the 467 bp
cDNA sequence were BDNF sequences from other species. The best sequence match, with an Expect Value of
2e–142, was found to be the BDNF cDNA sequence for the
flounder, Paralichthys olivaceus (GenBank accession
number AY074888), which shared 89% identity over 459
bases with the 467 bp cloned fragment from A. anguilla.
On submission to the NCBI GenBank database, the partial eel BDNF cDNA sequence was assigned the accession
Dalton /Borich /Murphy /Roberts
Fig. 2. Multiple alignment of the predicted amino acid sequence
for Anguilla anguilla BDNF (GenPept accession number
AAV31512) with amino acid sequences for BDNF, nerve growth
factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5)
from other species. NT-6/7 refers to neurotrophin-6 or neurotrophin-7 as they appear to be orthologues of the same gene in
different species [Dethleffsen et al., 2003]. The amino and the carboxyl termini are indicated by the boxes labeled N-term and Cterm, respectively. The variable regions between neurotrophins
defined by Dethleffsen et al. [2003] are boxed and marked by the
roman numerals I–V. For the purposes of diagrammatic clarity
regions of amino acid sequences only relevant to the eel BDNF
sequence are included and, therefore, portions of prepro- and propeptide regions from some species have been excluded. GenPept
accession numbers and percentage identity with the eel BDNF
sequence (AAV31512) were as follows: BDNF: platyfish (PLA):
CAA42567, 97%; zebrafish (ZEB): NP_571670, 95%; rat:
AAH87634, 91%; NGF: zebrafish: AAO31814, 43%; rat: P25427,
46%; NT-3: zebrafish: AAH92731, 46%; rat: NP_112335, 49%; NT6/7: zebrafish: AAO31821, 46%; platyfish: AA61923, 47%; NT-4/5:
rat: AAA41728, 53%. The sequence for Tetraodon (TET) NT-4/5
was taken from Dethleffsen et al. [2003] and shared 43% identity
with the eel BDNF sequence. * = Identical amino acids; R = conservative substitutions.
BDNF in Eel Brain
Brain Behav Evol 2009;73:43–58
47
A
B
D
C
48
E
Brain Behav Evol 2009;73:43–58
Dalton /Borich /Murphy /Roberts
number AY762996. The predicted amino acid sequence
was given the GenPept accession number AAV31512.
Fig. 3. BDNF mRNA transcript location in transverse sections of
the rostral and middle regions of the eel brain. A Caudal portion
of the central zone of the area dorsalis (Dc in fig. 5C). B Rostral
portion of the lateral nucleus of the area ventralis. Regions containing labeled cells are indicated by the arrows in A and B. C The
stratum periventriculare of the tectum (SPV in fig. 6A–D) is denoted by arrowheads. Labeling in the torus semicircularis (TS in
fig. 6B–D) is indicated by arrows. D Labeled cells are depicted in
the anterior (arrowheads; A in fig. 5F) and ventrolateral (VL) nuclei of the thalamus, in the nucleus preopticus magnocellularis,
pars gigantocellularis (PMg), nucleus preopticus parvocellularis
posterior (PPp) and suprachiasmatic nucleus (arrows; SC in
fig. 5F). Labeling was not seen in the rostral portion of the ventromedial nucleus of the thalamus (VM in fig. 5F), the region
marked by *. E nucleus preopticus magnocellularis, pars parvocellularis (PMp in fig. 5C). Scale bars = 200 ␮m for A , C and D and
100 ␮m for B and E .
packed cells in the ventral nucleus of the area ventralis
(Vv) was also apparent at this level, although the lateral
(Vl) and dorsal (Vd) nuclei of the area ventralis were not
labeled (fig. 5B).
More caudally, densely packed cells in the posterior
area of lateral zone of the area dorsalis (Dlp) were moderately labeled (fig. 5C). The medial region of lateral zone
of the area dorsalis (Dl) was not labeled although more
laterally located cells did show a moderate signal. Most of
the cells in the medial (Dm) and central (Dc; fig. 3A) zone
of the area dorsalis were moderately labeled. A few scattered cells lining the ventricle in the posterior nucleus of
the area ventralis (Vp) were lightly labeled. In the most
caudal sections of the telencephalon (fig. 5D), the majority of cells in the area dorsalis were moderately labeled. A
few scattered cells were lightly labeled in the entopeduncular nucleus (E) and the posterior nucleus of the area
ventralis (Vp).
The BDNF mRNA expression pattern for the diencephalon is indicated in table 1 and figures 3, 5 and 6. In
the rostral portion of the nucleus preopticus parvocellularis posterior (PPp), strong labeling of small densely
packed cells was observed (fig. 5D) and more caudally,
densely packed cells were moderately labeled in this nucleus (fig. 3D, 5E, F). Strong to moderate labeling was also
obvious in the magnocellular nuclei of the preoptic area
(PMp: fig. 3E, 5C; PMm: fig. 5D; PMg: fig. 3D, 5F). BDNF
mRNA expression was also evident in the suprachiasmatic nucleus (SC), the cells of which were heavily labeled
in the rostral region of this nucleus (fig. 5D) and moderately labeled more caudally (fig. 3D, 5E, F). In the epithalamus, most of the cells of the dorsal and ventral habenular nuclei (HAd and HAv; fig. 5E, F) were moderately labeled.
Moderate to light labeling of cells was seen in various
thalamic nuclei (fig. 3D, 5E, F, 6A, B), except in the rostral
portion of the ventromedial nucleus where no labeling
was seen (fig. 3D, 5F, 6A). Strong labeling was apparent in
densely packed hypothalamic cells with a periventricular
location such as the nucleus of the lateralis tuberis (nTL;
fig. 6A, B), the caudal (HC) and ventral (HV) zones of the
periventricular hypothalamus (fig. 6B–D). Moderate labeling was also observed in the majority of the cells in the
diffuse nucleus of the inferior lobe (ndil; fig. 6C, D). Light
labeling was seen in the subcommissural organ (SCO;
fig. 6A) in occasional cells. Moderate labeling was seen in
the majority of cells in the nucleus pretectalis periventricularis, pars dorsalis (PPd; fig. 6A) and light labeling
of some scattered cells was observed in the nucleus pretectalis centralis (CP; fig. 6A).
BDNF in Eel Brain
Brain Behav Evol 2009;73:43–58
Spatial Distribution of BDNF mRNA Expression in
the Eel Brain and Rostral Spinal Cord
Expression of BDNF mRNA was detected using antisense RNA probe and was found in localized positions
throughout the brain, most robustly in parts of the diencephalon and mesencephalon. It was also notable that
several cell groups in the rhombencephalon expressed
BDNF. Throughout, the signal was restricted to cells that
had neuronal morphology and was not observed in equivalent sections hybridized to the BDNF sense control
probe (fig. 4).
Charts showing the distribution of the cells within the
brain of one animal, with BDNF transcripts represented
by black dots, are provided in figures 5–8. Illustrative
photomicrographs are provided in figures 3 and 4. In addition, the BDNF mRNA expression patterns, as seen in
all the hybridized brains, are summarized in table 1 in a
semiquantitative manner as described in the table legend
in correspondence with Conner et al. [1997].
The cell groupings of the telencephalon are listed in
table 1. Weak labeling of scattered cells was observed in
all layers of the olfactory bulb (fig. 5A). In the most rostral telencephalic hemispheres, moderate labeling was
seen mainly in the dorsal, medial and lateral zones of the
area dorsalis and the lateral nucleus of the area ventralis
(fig. 3B). More caudally in the telencephalon (fig. 5B),
moderate labeling was still apparent in the area dorsalis;
however, labeling was absent in the central zone of the
area dorsalis (Dc). Moderate labeling of small densely
49
A
50
B
C
D
E
F
Brain Behav Evol 2009;73:43–58
Dalton /Borich /Murphy /Roberts
Nuclei of the posterior tuberculum that showed light
labeling were the preglomerular nuclear complex in scattered cells (PGm; fig. 6A–C) and the periventricular nucleus of the posterior tuberculum in the majority of cells
(TPp; fig. 6A). Moderate labeling of most of the cells was
seen in the paraventricular organ (PVO; fig. 6A) and
strong labeling of densely packed cells in the nucleus posterior tuberis (TP; fig. 6C) was observed.
The expression pattern for BDNF mRNA in the mesencephalon is indicated in table 1 and figure 3 and 6. The
numerous small, densely packed neurons of the stratum
periventriculare, the deepest and only tectal layer to show
BDNF expression, labeled intensely (SPV; fig. 3C, 6A–D).
Densely packed cells in the torus semicircularis were
moderately labeled (TS; fig. 3C, 6B–D). The signal was
strong in neurons in the nucleus lateralis valvulae (LV;
fig. 6D), otherwise cells in other regions of the mesencephalic tegmentum mostly showed moderate expression.
Some cell masses in the mesencephalic tegmentum that
were not labeled included the red, superior raphe, lateral
mesencephalic profundus, and interpeduncular nuclei,
as well as the tegmental lateral group.
The expression pattern of BDNF mRNA in the rhombencephalon is summarized in table 1. Most cell groups
showed BDNF expression, although none was particularly robust (fig. 4, 7, 8). Little labeling was seen in the
cerebellum although a few scattered cells in the Purkinje
cell layer were lightly labeled (fig. 7B). Large cells in the
superior (SRF; fig. 7A), medial (MRF; fig. 4C, E, 7B–D)
and inferior (IRF; fig. 8A–C) divisions of the reticular
formation, including ‘identifiable’ neurons such as the
Mauthner-like cell, were moderately labeled (M; fig. 4F,
7B). This large cell is similar to the Mauthner neuron of
other teleosts in many respects, including a close relationship with the octavus system [Meredith et al., 1987].
In the octavolateral system, moderate labeling of scattered cells was seen in the magnocellular nucleus (MO;
fig. 7B-D) and octavolateral efferent nucleus (OEN;
fig. 8A), and of most cells in the tangential nucleus (TO;
fig. 7D), but only light labeling of occasional cells was observed in the descending nucleus (DO; fig. 8B).
The majority of cells in the motor nuclei of certain cranial nerves (trochlear, facial, glosso-pharyngeal and vagal, nIV, nVIIm, nIXm, nXm; fig. 4A, 6D, 8A–C), and a
few, scattered cells of the trigeminal motor nucleus (nVm;
fig. 7A) were moderately labeled. But labeling (moderate)
was only seen in sensory regions of the trigeminal nerve
(nVs; fig. 7A), the rostral portion of the vagal lobe (LX;
fig. 8B) and occasionally (light) in the facial lobe (LVII;
fig. 7C, D). The commissural nucleus of Cajal was not labeled (nCC; fig. 8C).
In the rostral spinal cord, large cells were labeled
throughout the ventral horn whilst smaller cells showed
BDNF mRNA expression in the dorsal horn.
Discussion
Our survey of BDNF mRNA expression in the eel reveals a pattern similar to that reported for the mammalian [Wetmore et al., 1990; Conner et al., 1997], avian
[Herzog and von Bartheld, 1998] and amphibian brain
[Duprey-Díaz et al., 2002; Wang et al., 2005]. As in these
species, eel expression was seen in portions of all brain
regions. The labeling was particularly strong in the preoptic area, the hypothalamus, the periventricular nucleus
of the posterior tuberis and the nucleus lateralis valvulae.
However, in contrast to what has been reported for mammals, BDNF mRNA expression in the eel was also evident
in the reticular formation and other brain stem nuclei,
some of which project down the spinal cord and are involved in the control of movement.
showing groups of labeled cells (indicated by the arrows) in the
facial motor nucleus. B Adjacent section to that in A hybridized
to the sense probe. C Transverse section at the level of the medial
reticular formation. Arrows indicate examples of regions containing groups of labeled cells. D Corresponding sense control for
C . E Horizontal section showing labeled cells (examples indicated
by arrows) in the medial reticular formation. F Mauthner-like cell
(M in fig. 7B). Scale bars = 200 ␮m for A–E , 100 ␮m for F.
Anguilla anguilla BDNF cDNA
A. anguilla, an elopomorph, is, to date, the most basic
teleost for which BDNF has been sequenced. In pairwise
alignments, the amino acid sequence for eel BDNF displayed only 33% identity with the sequence of the lamprey neurotrophin, lf-nt [Hallböök et al., 1998]. On the
other hand, it showed 79% identity with the BDNF sequence for the ray [Hallböök et al., 1991], a cartilaginous
fish, and 86–97% identity with BDNF sequences from
other teleost fish, Xenopus, chicken, rat, pig and human.
This result might be reflective of the second of two gene
duplication events thought to be responsible for the evolution of the neurotrophin family from a single ancestral
gene in invertebrates [Bothwell, 2006], which occurred
BDNF in Eel Brain
Brain Behav Evol 2009;73:43–58
Fig. 4. Photomicrographs showing sections of the rhombencephalon hybridized to antisense (A , C , E and F) and sense (B and D)
BDNF probes. A Transverse section of the rhombencephalon
51
A
Fig. 5. BDNF expression in rostral regions
of the eel brain. The profiles are merged
camera lucida tracings of two to five successive transverse sections, the approximate level of which is indicated by the vertical lines in the sagittal view of the brain
at the top right. Neuronal groups, the
names for which are given in full in the
Abbreviations list, are outlined on the
right side of each profile; the dots on the
left side represent the distribution of neuronal cell bodies expressing BDNF. For
clarity they are shown as being of the same
size although they are not, and as having
equivalent expression levels. An indication of the differences in expression level
is provided in table 1. A scale bar, which
represents 500 ␮m, is provided in figure 5
and also applies to figures 6, 7 and 8.
B
E
C
D
F
A
C
B
Fig. 6. BDNF expression in middle regions
of the eel brain. See the legend for figure 5
for further explanation.
52
Brain Behav Evol 2009;73:43–58
D
Dalton /Borich /Murphy /Roberts
Table 1. Semi-quantitative Assessment of BDNF mRNA Expression
Telencephalon
Olfactory bulb
Internal cell layer
External cell layer
Glomerulus
Telencephalic hemispheres
Area dorsalis
Medial zone
Lateral zone
Lateral zone, posterior area
Dorsal zone
Central zone (rostral)
Central zone (caudal)
Area ventralis
Lateral nucleus (rostral)
Lateral nucleus (middle)
Lateral nucleus (caudal)
Dorsal nucleus (rostral)
Dorsal nucleus (caudal)
Ventral nucleus (rostral)
Ventral nucleus (caudal)
Posterior nucleus
Entopeduncular nucleus
Diencephalon
Nucleus preopticus
Magnocellularis, pars parvocellulis
Magnocellularis, pars magnocellularis (rostral)
Magnocellularis, pars magnocellularis (caudal)
Parvocellularis posterior (rostral)
Parvocellularis posterior (caudal)
Magnocellularis, pars gigantocellularis
Suprachiasmatic nucleus (rostral)
Suprachiasmatic nucleus (caudal)
Dorsal habenular nucleus
Ventral habenular nucleus
Thalamus
Anterior nucleus
Ventrolateral nucleus
Ventromedial nucleus (rostral)
Ventromedial nucleus (caudal)
Dorsal posterior nucleus
Posterior nucleus
Nucleus lateralis tuberis
Diffuse nucleus of inferior lobe
Caudal zone of periventricular hypothalamus
Ventral zone of periventricular hypothalamus
Subcommissural organ
Nucleus pretectalis periventricularis, pars dorsalis
Nucleus pretectalis centralis
Preglomerular nuclear complex
Periventricular nucleus of posterior tuberculum
Paraventricular organ
Nucleus posterior tuberis
BDNF in Eel Brain
+/2
+/2
+/2
++/2-3
++/2-3
++/3
++/3
–
++/3
++/3
–
+/2
–
+/3
–
++/3
+/2
+/2
+++/3
+++/3
++/2
+++/3
++/3
++/3
+++/3
++/3
++/3
++/3
++/2
++/3
–
+/3
++/2
+/3
+++/3
++/3
+++/3
+++/3
+/1
++/3
+/2
+/2
+/3
++/3
+++/3
Mesencephalon
Torus semicircularis
Stratum periventriculare of tectum
Nucleus of the medial longitudinal fasciculus
Red nucleus
Oculomotor nucleus
Trochlear nucleus
Nucleus lateralis valvulae
Secondary gustatory nucleus
Nucleus isthmi
Superior reticular nucleus
++/3
+++/3
++/2
–
++/3
++/3
+++/3
++/3
++/3
++/3
Rhombencephalon
Cerebellum-Purkinje cell layer
Locus coeruleus
Superior olive
Inferior raphe nucleus
Area postrema
Superior reticular formation
Mauthner-like cell
Medial reticular formation
Inferior reticular formation
Magnocellular octavolateral nucleus
Tangential octavolateral nucleus
Octavolateral efferent nucleus
Descending octavolateral nucleus
Sensory nucleus of the trigeminal nerve
Facial lobe
Trigeminal motor nucleus
Facial motor nucleus
Glossopharyngeal motor nucleus
Vagal lobe (rostral)
Vagal lobe (caudal)
Vagal motor nucleus
Commissural nucleus of Cajal
+/2
+/2
++/3
++/2
+/2
++/2
++
++/2
++/3
++/2
++/3
++/2
+/1
++/3
+/2
++/2
++/3
++/3
++/3
–
++/3
–
Rostral spinal cord
Dorsal horn
Ventral horn
++/2
++/2
Expression level: – = none + = weak ++ = moderate +++ =
strong; cells labeled: 1 = very few cells, 2 = some scattered cells,
3 = most cells. Nuclei have been divided into rostral, caudal and/
or middle when labeling differed between levels.
Brain Behav Evol 2009;73:43–58
53
A
C
B
Fig. 7. BDNF expression in more caudal re-
gions of the eel brain. See the legend for
figure 5 for further explanation.
after the emergence of agnathan fish but before the emergence of cartilaginous fish [Hallböök, 1999; Hallböök et
al., 2006].
BDNF mRNA Expression in the Eel Brain
The ventral region of the lateral zone of the area dorsalis of the telencephalon, where moderate labeling of
some cells was seen throughout, has been suggested as
the teleostean equivalent of the mammalian hippocampus [Wullimann and Rink, 2002; Yamamoto et al., 2007].
In the rodent, strong BDNF expression is seen in the hippocampus [Conner et al., 1997] and BDNF has been implicated in hippocampal synaptic plasticity [Patterson et
al., 1996; Kang et al., 1997], and might serve this role in
teleost fishes as well. In the eel brain, labeling was seen in
regions of the medial zone of the area dorsalis and in the
entopeduncular nucleus, regions that have been homolo54
Brain Behav Evol 2009;73:43–58
D
gized with the mammalian amygdala [Braford and
Northcutt, 1974; Reiner and Northcutt, 1992; Wullimann
and Rink, 2002], where BDNF mRNA expression has
been observed [Conner et al., 1997].
More medially in the diencephalon, clusters of neurons expressed BDNF strongly, including the nucleus
posterior tuberis. This zone contains dopaminergic cells
[Roberts et al., 1989; Rink and Wullimann, 2001] and
connects reciprocally with the dorsal and ventral telencephalon [Folgueira et al., 2004a, b; Rink and Wullimann, 2004]. Accordingly, it has been proposed as the
source of the ascending dopaminergic system of the teleostean brain [Rink and Wullimann, 2001; Wullimann
and Rink, 2002]. The equivalent ascending system in other vertebrates originates in the mesencephalic tegmentum, where in teleosts there are no dopaminergic neurons, an observation that leads to questions about the
Dalton /Borich /Murphy /Roberts
A
C
Fig. 8. BDNF expression in the most cau-
dal regions of the eel brain. See the legend
for figure 5 for further explanation.
B
striatal/basal ganglia organization [Meek, 1994]. In this
context, it is perhaps interesting to note that mammalian
mesencephalic dopaminergic neurons do synthesize
BDNF [Seroogy et al., 1994; Conner et al., 1997].
In the eel, as in mammals [Conner et al., 1997], BDNF
expression was observed in sensory regions, and in sensory integrating structures. For example, BDNF mRNA
expression in the eel was seen in nuclei of the lateral line
and octaval systems. Strong BDNF mRNA expression
was observed in the torus semicircularis, which receives
multimodal input from the lateral line and octavus systems and the tectum, and is considered to be a homologue
of the mammalian inferior colliculus [Schellart, 1990].
Multimodal relay centers of the diencephalon, such as the
thalamus and the preglomerular complex, which may be
homologous to the dorsal thalamus in mammals [Yamamoto and Ito, 2005; Ishikawa et al., 2007], also expressed
BDNF mRNA. BDNF mRNA expression has been described in many of the thalamic nuclei of the rat [Conner
et al., 1997]. Strong BDNF mRNA expression was also
seen in the eel in the nucleus lateralis valvulae, a relay station of indirect cerebellar pathways that appears to be
concerned with visual and non-visual sensory information in fish [Meek, 1990; Schellart, 1990; Wullimann et
al., 1996; Yang et al., 2004, 2007]. The nucleus lateralis
valvulae has been compared to the pontine nuclei of the
mammalian brain stem [Yang et al., 2004] in which BDNF
mRNA expression has been described in the rat [Conner
et al., 1997].
A few scattered cells were labeled in the Purkinje cell
layer of the cerebellum, which lies at the border of the
outer molecular layers (CbSm) and the granular layer
(CbSg). Neurogenesis persists in the adult fish brain and
the main site of proliferative activity is the cerebellum,
but 5-bromo-2-deoxyuridine labeled cells have also been
found in regions such as the telencephalon, diencephalon, mesencephalon and rhombencephalon [Zupanc and
Horschke, 1995; Zupanc, 1999, 2001; Zikopoulos et al.,
2000]. BDNF appears to be important in neurogenesis in
the mammalian brain [Rossi et al., 2006; Chen et al.,
2007; Tonchev et al., 2007; Bath et al., 2008] and it may be
the case therefore that BDNF supports neurogenesis in
the cerebellum and other regions of the eel brain mentioned above.
Moderate BDNF mRNA expression was observed in
the eel throughout the motor column (cranial nerves III–
X), the neurons of which contribute to eye, jaw and gill
movements. BDNF mRNA expression in equivalent cell
groups in the mammalian brain is more varied. For example, none has been seen in the dorsal motor nucleus or
nucleus ambiguus of the vagus nerve [Wetmore et al.,
1990; Conner et al., 1997] nor in the trochlear nucleus,
whereas BDNF mRNA is expressed by oculomotor neurons [Conner et al., 1997].
BDNF in Eel Brain
Brain Behav Evol 2009;73:43–58
55
Many other tegmental motor systems in the eel contribute to locomotory control by providing supraspinal
input to spinal neurons; the eel, as other teleosts, has no
direct input equivalent to the mammalian corticospinal
pathway [Bosch and Roberts, 2001]. There was no BDNF
mRNA expression in the red nucleus but moderate labeling for BDNF mRNA was seen in other spinally projecting nuclei such as the inferior raphe nucleus, the Mauthner-like cell, the superior, medial and inferior divisions
of the reticular formation, the nucleus of the medial longitudinal fasciculus, the ventromedial nucleus of the
thalamus and some octavolateral nuclei. Little BDNF expression is seen in homologous adult mammalian brain
stem nuclei although in the rat, cells immunoreactive for
BDNF have been reported in the spinal vestibular, dorsal
raphe and gigantocellular reticular nuclei [Conner et al.,
1997; Furukawa et al., 1998; Murer et al., 1999]. However,
these, and other cell groups of the reticular formation,
lack BDNF mRNA expression [Ceccatelli et al., 1991;
Friedman et al., 1991; Conner et al., 1997] although BDNF
mRNA is expressed transiently in these regions during
development [Friedman et al., 1991].
It is tempting to think that the basal BDNF expression
of some of the eel descending neurons might be a factor
that aids in their regeneration after cordotomy. However,
the correlation between BDNF expression and axon regrowth is not precise, as BDNF mRNA expression was
seen in both rapidly [e.g., inferior reticular nucleus; Becker et al., 1997, 1998; Roberts et al., unpublished observations] and slowly (e.g., Mauthner cell) regenerating systems. More likely perhaps, the difference in BDNF expression might lie in the different growth patterns of fish
and mammals. Eels, as other teleost fish, continue to
grow throughout life and BDNF levels might need to be
sustained as this continual growth impacts on the nervous system. The growth of the body involves an increase
in the size and number of muscle fibers [Willemse and
van den Berg, 1978], the innervating motoneurons of
which progressively grow [Smit et al., 1991]. As these neurons enlarge, the number of synapses they receive from
brain stem neurons also increases [Smit et al., 2001] and
these cells also grow [Bosch and Roberts, 1994]. An ongoing expression of BDNF might be needed, therefore, to
provide the requisite plasticity for these changing neuronal circuits.
Acknowledgements
We thank Damien Murray, Russell Poole and Peter Stafford
for their generous help in obtaining eels, and Ann Butler for
her expert advice and helpful comments on the manuscript.
This work was funded by Enterprise Ireland; Grant number:
SC/2001/417 and Science Foundation Ireland; Grant number: 02/
IN1/B267.
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