 |
Previous Article | Next Article 
Volume 17, Number 7,
Issue of April 1, 1997
pp. 2295-2313
Copyright ©1997 Society for Neuroscience
Distribution of Brain-Derived Neurotrophic Factor (BDNF) Protein
and mRNA in the Normal Adult Rat CNS: Evidence for Anterograde Axonal
Transport
James M. Conner1,
Julie
C. Lauterborn2,
Qiao Yan3,
Christine M. Gall2, and
Silvio Varon1
1 Department of Biology, 0506, University of
California, San Diego, La Jolla, California 92093, 2 Department of Anatomy and Neurobiology, University of
California, Irvine, California 92697, and 3 Amgen Center,
Thousand Oaks, California 91320
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES
ABSTRACT
A sensitive immunohistochemical technique was used, along with
highly specific affinity-purified antibodies to brain-derived neurotrophic factor (BDNF), to generate a detailed mapping of BDNF
immunoreactivity (BDNF-ir) throughout the adult rat CNS. A parallel
analysis of sites of BDNF synthesis was performed with in
situ hybridization techniques using a cRNA probe to the exon encoding mature rat BDNF protein. These combined data revealed (1)
groups of cell bodies containing diffuse BDNF-ir throughout the CNS
that were strongly correlated with fields of cells containing BDNF
mRNA; (2) varying degrees of BDNF-ir outside of cell bodies, in what
appeared to be fibers and/or terminals; and (3) many regions containing
extremely heavy BDNF-immunoreactive fiber/terminal labeling that lacked
BDNF mRNA (e.g., medial habenula, central nucleus of the amygdala, bed
nucleus of stria terminalis, lateral septum, and spinal cord). The
latter observation suggested that in these regions BDNF was derived
from anterograde axonal transport by afferent systems. In the two cases
in which this hypothesis was tested by the elimination of select
afferents, BDNF immunostaining was completely eliminated. These data,
along with the observation that BDNF-ir was rarely found within
dendrites or fibers en passage, suggest that BDNF
protein produced in adult CNS neurons is polarized primarily along
axonal processes and is preferentially stored in terminals within the
innervation target.
Key words:
neurotrophin;
immunohistochemistry;
in situ
hybridization;
neurotrophic factor;
BDNF;
anterograde axonal
transport
INTRODUCTION
The neurotrophins are a related family of growth
factors presently consisting of nerve growth factor (NGF),
brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),
NT-4/5, and NT-6 (Berkemeir et al., 1991; Narhi et al., 1993 ; Gotz et
al., 1994 ; Ibáñez, 1994). Although much evidence suggests
that these factors play an important role in regulating the survival,
growth, and differentiation of select populations of peripheral
neurons, their physiological role in the developing and adult CNS is
less well defined. Achieving an understanding of neurotrophin function in the CNS will likely require several experimental approaches, including elucidation of sites of synthesis and storage for each neurotrophin.
To date, many investigators have reported on the localization of mRNAs
for the various neurotrophins, although few of these studies have
provided a detailed mapping throughout the CNS. NGF expression has been
localized primarily to the hippocampal formation, olfactory bulb, and
cortex all target regions of basal forebrain cholinergic neurons
(Korsching et al., 1985 ; Shelton and Reichardt, 1986 ; Whittemore et
al., 1986 ; Ernfors et al., 1990 ; Guthrie and Gall, 1991 ). Additional
studies have indicated that high levels of NGF mRNA are found within
the basal forebrain (Lauterborn et al., 1991 , 1995 ), hypothalamus
(Spillantini et al., 1989 , Ceccatelli et al., 1991 ), and brainstem
(Ceccatelli et al., 1991 ). BDNF mRNA seems to be broadly distributed
throughout the CNS (Ernfors et al., 1990 ; Phillips et al., 1990 ;
Ceccatelli et al., 1991 ; Friedman et al., 1991; Guthrie and Gall, 1991 ;
Gall et al., 1992 ; Castrén et al., 1995 ). In contrast, NT-3 mRNA
is the most narrowly distributed, with high levels of expression
restricted to hippocampal CA2 stratum pyramidale, the dentate gyrus
granule cells, and the cerebellar granule cells (Ernfors et al., 1990 ;
Maisonpierre et al., 1990 ; Ceccatelli et al., 1991 ) and lower levels of
NT-3 mRNA in substantia nigra, midline and intralaminar thalamus, and
posterior amygdala (Seroogy and Gall, 1993 ; Lauterborn and Gall, 1994 ).
NT-4/5 expression in the CNS seems to be very low (Timmusk et al.,
1993 ), and its in situ distribution has not been determined.
The recently discovered NT-6 is restricted to nonmammalian species, and
its distribution remains largely unknown (Gotz et al., 1994 ).
The cellular localization of neurotrophin proteins in the CNS has
proceeded much more slowly, probably reflecting the technical limitations inherent in the immunohistochemical approach. Recently, we
have generated antibodies and developed fixation and
immunohistochemical protocols permitting the visualization of NGF
protein in the normal adult rat brain (Conner et al., 1992 ). This
protocol also has been used successfully to localize NGF in a wide
variety of circumstances, including (1) the normal distribution of NGF
in human and nonhuman primates (Mufson et al., 1994 ), (2) changes in
NGF accumulation by basal forebrain cholinergic neurons in human
Alzheimer's tissue (Mufson et al., 1995 ), (3) the distribution of NGF
protein in transgenic mouse lines with NGF gene alterations (Carlson et
al., 1995 ; Ma et al., 1995 ), and (4) changes in the in situ
distribution of NGF protein after various experimental manipulations
(Conner and Varon, 1992 , 1995 ; Conner et al., 1994 ; Holtzman and
Lowenstein, 1995 ).
In the present investigation, we have used the sensitive
immunohistochemical protocol developed for NGF, along with a well characterized preparation of affinity-purified polyclonal antibodies to
BDNF (Yan et al., 1997 ), to generate a detailed mapping of BDNF protein
in adult rat brain and spinal cord. A parallel analysis of BDNF mRNA
distribution was also carried out. These combined data revealed several
regions containing high densities of BDNF-immunoreactive processes but
no detectable mRNA, thereby suggesting that BDNF may have been
distributed to these regions by way of anterograde axonal transport.
This hypothesis was tested in two different systems using lesion
paradigms to destroy specific cell populations supplying afferent
innervation to regions containing abundant BDNF protein and no
detectable mRNA.
MATERIALS AND METHODS
BDNF antibodies and cDNA probes. Affinity-purified
rabbit polyclonal antibodies to BDNF used in this investigation were
generated and characterized as described previously (Conner et al.,
1996 ; Yan et al., 1997 ). This antibody preparation was shown to be
specific for BDNF by several criteria. (1) In a Western blot, the
antibody was capable of recognizing as little as 0.1 ng of BDNF per
lane but did not cross-react with NGF, NT-3, or NT-4/5 at
concentrations of even 100-fold greater; (2) in a chick dorsal root
ganglion bioassay, the antibody specifically interfered with the
survival-promoting activity of BDNF but was not capable of inhibiting
the actions of either NGF or NT-3; and (3) the specific pattern of
BDNF-ir observed in CNS tissues with this antibody was not detected in mice in which the BDNF gene was deleted but was present in mice with a
deletion of the NGF gene (J. Conner, unpublished observations). Antisense BDNF mRNA was generated from the rat recombinant plasmid pR1112-8 with T3 TNA polymerase after digestion with PvuII.
This cRNA probe contains 384 bases complementary to the mRNA encoding mature rat BDNF protein (Isackson et al., 1991 ). Radiolabeled sense
mRNA was generated from the plasmid DNA as described previously (Gall
and Isackson, 1989 ).
BDNF immunohistochemistry. Adult male and female Sprague
Dawley rats (Simonsen, Gilroy, CA, and Harlan, San Diego, CA) were used
(n = 5 for normal distribution; n = 14 for analysis of lesion effects). All animals were perfused under deep
anesthesia with ~50 ml PBS followed with ~250 ml 2%
paraformaldehyde + 0.2% parabenzoquinone in 0.07 M
phosphate buffer, pH 7.2. Brains were removed, post-fixed for 2 hr in
the same fixative, and cryoprotected overnight in 30% sucrose in 0.1 M phosphate buffer (all solutions at 4°C). Coronal
sections (40 µm) were cut on a sliding microtome and stored in
Millonig's buffer until they were processed for BDNF-ir as has been
described (Conner et al., 1996 ). In brief, sections (taken every 240 µm) were washed in 0.1 M Tris-buffered saline (TBS), pH
7.4, incubated in TBS containing 0.25% Triton X-100, and incubated in
TBS + 2% BSA + 5% normal goat serum. Staining was performed by
incubating sections with primary antibodies (50 ng/ml anti-BDNF) for 48 hr at 4°C, with secondary antibodies (1.5 µg/ml biotinylated goat
anti-rabbit; Vector Labs, Burlingame, CA) for 3 hr at room temperature,
and with an avidin-biotin-peroxidase reagent (1:250 dilution, ABC
Elite; Vector Labs) for 90 min at room temperature. Sections were then
reacted with a solution containing 0.04% diaminobenzidine tetrahydrochloride, 0.06% nickel chloride, and 0.06%
H2O2 in Tris-HCl buffer.
In situ hybridization for BDNF mRNA. Sprague Dawley
rats (Simonsen) (n = 5) were anesthetized by
intraperitoneal injection with sodium pentobarbital and killed by
perfusion with 50 ml 0.9% saline followed by 350 ml 4%
paraformaldehyde in 0.1 M phosphate buffer (PPB). Brains
were removed from the cranium and post-fixed in PPB for 24 hr at 4°,
cryoprotected in 20% sucrose in PPB for 24-48 hr, and then sectioned
on a freezing microtome at a thickness of 25 µm in the coronal plane.
Tissue sections were processed free-floating as described previously
(Lauterborn et al., 1991 ). Briefly, coronal sections were permeabilized
with proteinase K, treated with acetic anhydride, and then hybridized
with cRNA probe (1 × 104 cpm/µl) at 60°C for
24-36 hr. Hybridization was followed by two rinses in 4× SSC at
60°C, treatment with ribonuclease A (1.2 kU/ml) (Sigma, St. Louis,
MO) for 30 min at 45°C, and washes through descending concentrations
of SSC to a final stringency of 0.1× SSC at 60°C. Dithiothreitol was
added to all washes at a final concentration of 5 mM.
Tissue was then mounted onto gelatin-coated slides and air-dried. For
emulsion autoradiography, slides were dehydrated in ethanol and
defatted in chloroform. Hybridization was visualized by both film
(Amersham -max) and emulsion (Kodak NTB2) autoradiography, with
exposure times of 3-4 d at room temperature and 4-6 weeks at 4°C,
respectively. After autoradiographic development, emulsion-coated
slides were counterstained with cresyl violet, coverslipped with
Permount, and analyzed by bright- and dark-field microscopy.
Lesions. Unilateral (n = 4) or bilateral
(n = 4) aspirative lesions of the septofimbrial and
triangular septal nuclei were accomplished by removing a 3 × 3 mm
area of skull on either side of Bregma and immediately lateral to the
sagittal sinus. After the dura was opened, successive aspiration of
part of the parietal and cingulate cortices, the supracallosal stria,
and the corpus callosum exposed the fimbria and supracommissural septal
nuclei. Aspiration of the fimbria and supracommissural septal nuclei
was verified visually, and the resulting gap was filled with Gelfoam soaked in sterile PBS. Unilateral (n = 4) or bilateral
(n = 2) lesions of the pontine parabrachial nuclei were
accomplished by passing a 1 mA current for 20 sec through a tungsten
electrode placed 11.5 mm caudal to bregma, 1.9 mm lateral from the
midline, and 7.6 mm down from the skull surface (with the electrode
carrier angled 20° anterior). For both lesion paradigms, animals
survived for 2 weeks after surgery and were then perfused under deep
anesthesia. The precise location of all lesions and the extent of the
damage was assessed histologically.
RESULTS
Immunostaining for BDNF revealed specific labeling throughout the
full extent of the CNS that was confined to select populations of cells
or fibers/terminals in well defined regions (for an overview, see Fig.
1). The anatomical nomenclature used
throughout this description was taken from Paxinos and Watson (1988).
Results presented in the tables are a summary of data obtained by the microscopic observation (made independently by two different
investigators) of histological slides from many animals, and they may
differ slightly from what appears in any single photograph. BDNF
cellular staining always appeared as a diffuse reaction product
distributed throughout the perikaryal cytoplasm and occasionally
extended into the most proximal processes, but it did not occupy the
cell nucleus (Fig. 2A). The assessment
of fiber/terminal staining was made on the basis of high-magnification
examination of the tissue and was characterized as (1) coarse, with
distinctly defined individual fibers (e.g., spinal cord and spinal
trigeminal nucleus) (Fig. 2B), (2) diffuse and finely
punctate with individual fibers not distinguishable (e.g.,
hypothalamus) (Fig. 2C), or (3) heavy punctate, in what
appeared to be noded processes that formed pericellular baskets around
unstained neuronal cell bodies (e.g., bed nucleus of the stria
terminalis, lateral septum, central nucleus of amygdala) (Fig.
2D). Throughout the CNS, the cellular localization of
BDNF immunostaining and mRNA was restricted to cells possessing a
neuronal morphology. In all cases, the specificity of the
immunostaining was confirmed by omitting the primary antibody or
through the use of preadsorption controls (Fig. 3).
Fig. 1.
Distribution of BDNF-ir in the adult rat brain.
Both plates show at low magnification a rostrocaudal series of sections
(left to right) that were obtained from two female rats
immunostained using affinity-purified antibodies specific for BDNF
(bright-field illumination). BDNF immunostaining in male rats (not
shown) was indistinguishable from that seen in females. Scale bar, 3 mm.
[View Larger Versions of these Images (93 + 79K GIF file)]
Fig. 2.
Characteristic patterns of BDNF immunostaining in
the rat CNS. Panels show examples of BDNF immunostaining in basolateral amygdaloid nucleus (A), spinal cord (B),
hypothalamus (C), and lateral septum (D)
that are representative of the quality of immunolabeling seen
throughout the CNS (bright-field illumination). As shown in
A, perikaryal labeling was characterized by a diffuse
reaction product distributed throughout the cell cytoplasm but
excluding the cell nucleus. B-D, Various types of
fiber/terminal staining. As seen in spinal cord lamina 1 (B,
small arrow) and laminae 3 (B, open arrows),
numerous individual fibers were immunolabeled; immunoreactive fibers
were also concentrated in lamina 2 (B, large arrow).
Other fields contained punctate immunostaining that appeared either
diffuse or clustered around unlabeled cell bodies (C,
arrows), or well defined, with discrete pericellular baskets
around unlabeled cells (D, arrow). Scale bars (shown in
B): A, B, 100 µm; (shown in
D) C, D, 50 µm.
[View Larger Version of this Image (123K GIF file)]
Fig. 3.
Specificity of BDNF immunostaining.
A-C, Tissue sections processed for immunohistochemistry
with either the BDNF antibody omitted (A), the BDNF
antibody solution preadsorbed against purified recombinant human BDNF
(B), or the BDNF antibody solution preadsorbed against
mouse -NGF (C) (bright-field illumination). As seen
in A and B, when the BDNF antibody was
omitted or preadsorbed with BDNF protein, no specific staining was
present. In C, preadsorbing against mouse -NGF
revealed a pattern of immunostaining that was identical to sections
reacted with the BDNF antibody alone (see Fig. 1). Scale
bar, 2 mm.
[View Larger Version of this Image (65K GIF file)]
In the following paragraphs the distributions of both BDNF-ir and BDNF
cRNA hybridization throughout the CNS will be described. Although
tissue from separate sets of rats was processed for the two techniques,
these data will be presented together for the sake of comparison within
a field.
The distribution of BDNF protein and mRNA in the olfactory bulb,
cortex, and hippocampal formation is summarized in Table 1. In the main olfactory bulb, a few periglomerular
cells were lightly labeled for both BDNF mRNA and protein. In the
mitral cell layer, a few cells were lightly labeled for BDNF-ir but not for mRNA, and in the granule cell layer, a faint haze of
autoradiographic grains, indicative of low mRNA content, was
distributed over the entire region, but no BDNF-ir was detected. In
contrast, granule cells in the accessory olfactory bulb were lightly
immunostained but not labeled by the cRNA. In the anterior olfactory
nucleus (Fig. 4A-C), all subregions
contained numerous cells that were well labeled for BDNF mRNA and
moderately labeled for BDNF-ir. Moderate BDNF immunostaining and
hybridization were detected in nearly all cells within the rostral
portion of the tenia tecta but were nearly absent from more caudal
sections. In contrast, many cells containing BDNF-ir and BDNF mRNA were
distributed within the intermediate lateral septal nucleus adjacent to
the tenia tecta at this level (Fig. 4D-F).
Throughout the rostrocaudal extent of the claustrum and endopiriform
nucleus, many cells were heavily labeled for BDNF mRNA and moderately
heavily labeled for BDNF-ir (Fig. 4G,H).
Table 1.
BDNF in the olfactory bulb, cortex, and hippocampal
formation
|
BDNF-ir fibers |
BDNF-ir
cells |
BDNF mRNA |
|
| Mitral cell
layer |
 |
2/l |
0 |
| Internal granule
layer |
 |
0 |
4/l |
| Glomerular layer
(periglomerular) |
 |
1/l |
2/l |
| External plexiform/tufted
cells |
 |
0 |
0 |
| Granule cell layer of
AOB |
± |
2/l-m |
0 |
| Anterior olfactory
nucleus |
+ |
3/m |
3-4/h |
| Orbital
cortex |
 |
3/l |
2-3/m |
| Insular cortex (granular + agranular) |
 |
3/l-m |
3/m-h |
| Frontal
cortex |
 |
3/m |
3/m |
| Cingulate
cortex |
 |
3/l-m |
3-4/h |
| Piriform
cortex |
 |
3/m |
3-4/h |
| Parietal
cortex |
 |
3/l |
2-3/m |
| Perirhinal
cortex |
 |
3/l-m |
3/m-h |
| Retrosplenial
cortex |
 |
3/l |
3/m |
| Occipital
cortex |
 |
2-3/l |
2-3/m |
| Temporal
cortex |
 |
3/l |
2-3/m |
| Entorhinal
cortex |
 |
3/m-h |
3/m-h |
| Claustrum |
± |
3-4/h |
3-4/h |
| Endopiriform
nucleus (dorsal/ventral) |
 |
2-3/m |
3/h |
| Tenia tecta (rostral
part) |
 |
3/m |
4/h |
| Tenia tecta (caudal
part) |
 |
0 |
4/l |
| Indusium
griseum |
 |
2/l-m |
1/m |
| CA1-stratum
oriens |
++ |
1/l |
0 |
| CA1-stratum
pyramidale |
 |
2/l-m |
3-4/m |
| CA1-stratum
radiatum |
++ |
0 |
0 |
| CA1-stratum lacunosum
moleculare |
++ |
0 |
0 |
| CA2-stratum
oriens |
++ |
1/m |
0 |
| CA2-stratum
pyramidale |
 |
3/l-m |
4/h |
| CA2-stratum
lucidum |
+++++ |
0 |
0 |
| CA3-stratum
oriens |
++ |
1/m |
0 |
| CA3-stratum
pyramidale |
 |
4/m-h |
4/h |
| CA3-stratum
lucidum |
+++++ |
0 |
0 |
| Dentate gyrus-polymorph
layer/hilus |
+++++ |
** |
1-2/h |
| Dentate gyrus-granule cell
layer |
 |
4/l-m |
4/m |
| Dentate gyrus-inner molecular
layer |
++++ |
0-1/m |
0 |
| Dentate gyrus-middle molecular
layer |
+ |
0 |
0 |
| Dentate gyrus-outer molecular
layer |
 |
0 |
0 |
| Subiculum |
± |
2/m |
3-4/m |
| Presubiculum |
 |
0 |
2/l |
| Parasubiculum |
 |
0 |
2/l |
|
Fibers: , none; +, very light staining; ++, light staining;
+++, moderate staining; ++++, heavy staining; +++++, extremely heavy
staining. Cells: 0, no cells observed; 1, occasional cells; 2, few
scattered cells; 3, moderate number of cells; 4, densely packed cells;
**
, could not be determined. l, Lightly stained; m, moderately stained;
h, heavily stained.
|
|
Fig. 4.
BDNF-ir and mRNA in forebrain regions.
A-J, Sections through the anterior olfactory nucleus
(A-C), caudal portion of the tenia tecta
(D-F), claustrum and piriform cortex
(G, H), and septal region (I,
J) processed for immunohistochemistry (C, F, H,
J; bright field) and in situ hybridization
(B, E, G, I; dark field). Photomicrographs in
A and D are of Nissl-stained tissue.
Patterns of BDNF-ir and cRNA labeling were very similar in the anterior olfactory nucleus and rostral tenia tecta (TT)
(A-C) and, more caudally, in the claustrum
(Cl), endopiriform nucleus (DEn),
and piriform cortex (Pir) (G, H).
In the caudal portion of the tenia tecta, very light hybridization but
no immunostaining were observed (D-F);
notably heavy hybridization and immunostaining were present in the
adjacent intermediate lateral septum (LSI)
(D-F). In septal regions, cRNA-labeled cells
were seen only in the medial septum (MS), whereas
intense BDNF-ir was localized mostly to fibers/terminals in the lateral
septum (I, J). Scale bars (shown in
C): A-C, 500 µm; (shown in
F): D-F, 200 µm; (shown in
H): G, H, 500 µm; (shown in
J): I, J, 500 µm.
ac, Anterior commissure; AI, agranular
insular cortex; AOD, anterior olfactory nucleus, dorsal
part; AOP, anterior olfactory nucleus, posterior part;
E/OV, ependymal layer/olfactory ventricle;
gcc, genu of the corpus callosum; HDB,
horizontal limb of the diagonal band; LSD, lateral
septum, dorsal part; LSV, lateral septum, ventral part;
lv, lateral ventricle; VDB, vertical limb of the diagonal band.
[View Larger Version of this Image (153K GIF file)]
In cortex, BDNF-ir and cRNA-labeled cells were distributed throughout
all rostrocaudal levels with both hybridization and immunostaining
densities varying markedly across cortical regions (Fig.
5, Table 1). In nearly all cortical regions, immuno- and hybridization-labeling were observed in layers II, III, V, and VI, with
only a few lightly labeled cells in layer IV. The most robust
neocortical labeling was often seen deep in layer VI adjacent to the
corpus callosum, especially in parietal cortex (Fig. 5A-C). By comparison, fewer labeled cells were present in layer VI of temporal
cortex (Fig. 5D-F). Within a given field, the
laminar patterns of hybridization and immunolabeling were generally the same, as shown for entorhinal cortex in Figure 5G-I. Layer
I of all cortical regions was completely lacking BDNF
immunostaining or cRNA hybridization.
Fig. 5.
Comparison of the distributions of BDNF-ir and
mRNA in cortical fields. Sections through parietal
(A-C), temporal (D-F),
and entorhinal (G-I) cortices were processed for
in situ hybridization to localize BDNF mRNA (B,
E, H; dark field) or immunohistochemistry to localize BDNF-ir
(C, F, I; bright field). Sections through similar planes
were Nissl-stained (A, D, G; bright field). In parietal
cortex (A-C), the BDNF cRNA heavily labeled
neurons in layer VI, moderately in layers
II/III, and lightly in layer V; BDNF-ir
was very heavy in deep layer VI, lighter in layers
II, III, V, and superficial layer VI, and
almost undetectable in layer IV. In temporal cortex
(D-F), BDNF mRNA was moderately labeled in layers II/III and V/VI, with lighter
labeling in layer IV. BDNF-ir in temporal cortex was
heaviest in layers II/III, moderate in layers
V/VI, and light in layer IV. In
entorhinal cortex (G-I), the patterns of BDNF
cRNA hybridization and immunoreactivity were similar. Scale bars (shown
in A): A-C, 500 µm; (shown in
D): D-F, 500 µm; (shown in
G): G-I, 1 mm. cc, Corpus
callosum.
[View Larger Version of this Image (138K GIF file)]
In the hippocampus, BDNF immunostaining was detected in both cell
bodies and fibers/terminals (Fig. 6). In the pyramidal
cell layer, the intensity of cRNA hybridization and immunostaining and
the number of cells labeled varied across subfields (Fig. 6B,C). In CA1, a few scattered cells were
cRNA-labeled with a moderate density of autoradiographic grains, and a
few cells had detectable immunoreactivity. In CA2, most cells had
moderate to heavy cRNA labeling and light to moderate levels of
immunostaining. In CA3, both cRNA labeling and BDNF-ir were relatively
dense in nearly all cells (although it was often difficult to
distinguish immunolabeled CA3 pyramidal cells because of the intense
BDNF immunostaining within the mossy fiber zone). Only an occasional immunolabeled cell was detected in stratum oriens of CA1-CA3, and no
mRNA-positive cells were observed in this layer. Immunoreactive fibers
were distributed throughout regions CA1-CA3. As illustrated for region
CA1 in Figure 6D, the density of fiber staining
varied across the hippocampal laminae, with greater immunostaining in strata oriens and radiatum and less BDNF-ir in strata lacunosum moleculare and pyramidale. In the dentate gyrus, nearly all cells in
stratum granulosum were mRNA-positive and BDNF-immunoreactive (Fig.
6B,C,E). In the dentate molecular layer, a
distinctive laminar pattern of BDNF-ir was seen (Fig.
6E). Immunostaining was moderate in the inner
molecular layer, light in the middle molecular layer, and not
detectable in the outer molecular layer. In the hilus, some cells were
heavily labeled by the cRNA, and a few immunoreactive perikarya were
visible; however, the dense immunohistochemical labeling of fibers in
the deeper hilus prevented identification of immunoreactive cells in
this field (Fig. 6C). The field of fiber labeling in the
deep hilus was the most dense and well stained in the entire CNS and
corresponded to the location of the mossy fiber axons of the granule
cells.
Fig. 6.
Distribution of BDNF-ir and mRNA in hippocampus.
Low-magnification (A-C) and high-magnification
(D, E) photomicrographs of sections through hippocampus
that were either Nissl-stained (A, bright field) or
processed for in situ hybridization (B,
dark field) or immunohistochemistry (C-E, bright
field). In hippocampus, the BDNF cRNA densely labeled pyramidal cells
in CA3, CA2, and the hilus, and less densely the CA1 pyramidal cells
and the dentate gyrus granule cells (GrDG)
(B). BDNF immunostaining densely labeled processes
within the mossy fiber system (C). At higher
magnification, one could see that in region CA1 BDNF immunostaining was
diffuse in strata oriens (Or) and radiatum
(Rad) and weaker in stratum lacunosum moleculare
(LMol) (D). Scattered neurons in
the CA1 pyramidal cell layer (Py) were immunolabeled
(D). In the dentate gyrus (DG) molecular
layer, a trilaminar pattern of BDNF-ir was seen, with moderately dense
labeling in the inner molecular layer (iml),
light labeling in the middle molecular layer
(mml), and no detectable staining in the outer
molecular layer (oml). Scale bars (shown in C):
A-C, 500 µm; (shown in D): D,
E, 100 µm and 75 µm, respectively. LHb,
Lateral habenular nucleus.
[View Larger Version of this Image (69K GIF file)]
The distributions of BDNF-ir and mRNA within the basal forebrain and
basal ganglia are summarized in Table 2. In the basal forebrain, many densely immunostained and BDNF mRNA-positive cells were
distributed in the rostral part of the intermediate lateral septum
(Fig. 4D-F). In other basal forebrain
regions, such as medial septal nucleus (Fig. 4I),
vertical limb of the diagonal band (Fig. 4I),
magnocellular preoptic nucleus, medial preoptic area, and
septohypothalamic nucleus, there were a few BDNF mRNA-positive cells.
In these regions, only an occasional BDNF-immunoreactive cell was seen
(Fig. 4J). In addition, many lightly immunostained cells were observed in the triangular septal nucleus and the
septofimbrial nucleus (not shown). Immunoreactive fibers also were
detected in many basal forebrain regions. In the dorsal and ventral
portions of the lateral septum, fibers were well labeled and often
appeared to form pericellular baskets around unstained perikarya (Figs. 2D, 4J). In contrast, the medial
septum was relatively devoid of fiber staining (Fig.
4J). Moderate BDNF-immunoreactive fiber labeling also
was observed in the septohypothalamic nucleus and the subfornical
organ. In the basal ganglia, no BDNF mRNA and little BDNF-ir was
detected in either cells or fibers. Single, well labeled
BDNF-immunoreactive cells with a neuronal morphology were only
occasionally observed in the dorsal most aspect of the striatum and in
the adjacent corpus callosum.
Table 2.
BDNF in the basal forebrain, amygdala, and basal
ganglia
|
BDNF-ir fibers |
BDNF-ir
cells |
BDNF mRNA |
|
| Lateral septal nucleus,
intermediate |
++ |
3 /m-h |
3/m-h |
| Lateral
septal nucleus, dorsal/ventral |
+++ |
0 |
0 |
| Medial septal
nucleus |
+ |
1/m |
2-3/l-m |
| Diagonal band nucleus, horizontal
limb |
+/++ |
1/m |
0 |
| Diagonal band nucleus, vertical
limb |
+/++ |
1/m |
2/l-m |
| Nucleus basalis of
Meynert |
++ |
0 |
0 |
| Ventral pallidum |
 |
0 |
0 |
| Islands
of Calleja |
 |
0 |
0 |
| Lateral preoptic
nucleus |
++ |
1/l |
0 |
| Magnocellular preoptic
nucleus |
 |
1/l |
2/l |
| Medial preoptic
area |
++ |
1/l |
2-3/m |
| Medial preoptic
nucleus |
+++ |
2/h |
2-3/m |
| Substantia
inominata |
+ |
1/l-m |
0 |
| Bed nucleus of the stria terminalis,
medial/lateral/ventral/intermediate |
++/+++ |
0 |
0 |
| Bed nucleus
of the stria terminalis, juxtacapsular/lateral
dorsal |
++++ |
0 |
0 |
| Septohypothalamic
nucleus |
+++ |
1/m |
2/m |
| Olfactory
tubercle |
+ |
0 |
0 |
| Optic
chiasm |
 |
0 |
0 |
| Suprachiasmatic
nucleus |
+ |
0 |
0 |
| Triangular septal
nucleus |
± |
3/l |
0 |
| Septofimbrial
nucleus |
++ |
3/l |
3-4/m |
| Subfornical
organ |
++/+++ |
0 |
0 |
| Corpus
callosum |
 |
1/m |
0 |
| Caudate
putamen |
± |
1/m |
0 |
| Globus
pallidus |
 |
0 |
0 |
| Accumbens nucleus |
+ |
0 |
0 |
| Nucleus
of the lateral olfactory tract, part 1 |
 |
0 |
0 |
| Nucleus of
the lateral olfactory tract, part 2 |
 |
2-3/l |
3/l |
| Nucleus
of the lateral olfactory tract, part
3 |
 |
2-3/m |
2/m-h |
| Central amygdaloid nucleus,
lateral |
++++ |
0 |
0 |
| Central amygdaloid nuclues,
medial |
++ |
0 |
0 |
| Basolateral amygdaloid nucleus,
anterior |
 |
3/h |
3-4/h |
| Basolateral amygdaloid nucleus,
ventral |
 |
2/m |
2/m-h |
| Basolateral amygdaloid nucleus,
posterior |
 |
2/m-h |
3/h |
| Medial amygdaloid nucleus,
ventral |
+ |
1/l-m |
3/m-h |
| Cortical amygdaloid
nucleus |
+ |
2/l-m |
3/m |
| Basomedial amygdaloid
nucleus |
+ |
1/l-m |
3/l-m |
| Lateral amygdaloid
nucleus |
 |
1/l-m |
2/m-h |
| Anterior amygdaloid
area |
+ |
1/m |
1/l |
| Amygdalohippocampal
area |
+ |
2/m |
2-3/m-h |
| Amygdalostriatal transition
area |
++ |
0 |
0 |
|
Fibers: , none; +, very light staining; ++, light staining;
+++, moderate staining; ++++, heavy staining; +++++, extremely heavy
staining. Cells: 0, no cells observed; 1, occasional cells; 2, few
scattered cells; 3, moderate number of cells; 4, densely packed cells;
**
, could not be determined. l, Lightly stained; m, moderately stained;
h, heavily stained.
|
|
As seen in Table 3, BDNF-ir and mRNA were found in many
areas of the thalamus and mesencephalon, with distinctions between regions containing labeled cells and immunoreactive fibers. For example, the parafascicular thalamic nucleus (Fig. 7
G,H) contained numerous mRNA-positive
and immunoreactive perikarya but no immunostained fibers. In contrast,
in the anteroventral and anteromedial nuclei there was moderate fiber
immunostaining, but there was almost no cellular hybridization or
immunoreactivity (Fig. 7A,B). Within the dorsal and ventral
lateral geniculate nuclei many immunoreactive fibers were present, but
immunoreactive or cRNA-labeled cell bodies were not (Fig.
8I,J). In some fields the
distributions of hybridization and immunolabeling were similar. For
example, in the medial geniculate, the dorsal and ventral portions were
devoid of immunolabeling and hybridization, whereas the medial portion
of this nucleus and the suprageniculate nucleus contained moderate
perikaryal hybridization and immunolabeling (Fig.
7M,N). In the superior colliculus, cRNA hybridization
and immunoreactivity also overlapped in layers deep to the superficial
gray layer (Fig. 7I,J).
Table 3.
BDNF in the
thalamus/mesencephalon
|
BDNF-ir fibers |
BDNF-ir
cells |
BDNF mRNA |
|
| Paratenial thalamic
nucleus |
+ |
0 |
0 |
| Paraventricular thalamic
nucleus |
++ |
1-2/l-m |
3/m |
| Central medial thalamic
nucleus |
++ |
3/l-m |
3/l-m |
| Central lateral thalamic
nucleus |
± |
1-2/l |
2/l |
| Intermediodorsal thalamic
nucleus |
++ |
3/m |
3/l-m |
| Interanterodorsal/interanteromedial
thalamic nucleus |
++ |
0 |
Light haze |
| Anterodorsal thalamic
nucleus |
± |
3-4/l |
3-4/l |
| Anteroventral thalamic
nucleus |
++/+++ |
0 |
0 |
| Anteromedial thalamic
nucleus |
++ |
- |
Light haze |
| Mediodorsal thalamic
nucleus |
+ |
1/l-m |
0 |
| Laterodorsal thalamic nucleus,
dorsomedial |
+ |
0 |
2/l |
| Laterodorsal thalamic nucleus,
ventrolateral |
+ |
0 |
0 |
| Ventrolateral/ventromedial thalamic
nucleus |
 |
0 |
0 |
| Ventral
posterior/posterior |
 |
0 |
0 |
| Reticular thalamic
nucleus |
 |
0 |
0 |
| Reuniens thalamic
nucleus |
++ |
0 |
2/m |
| Rhomboid thalamic
nucleus |
+ |
2/l-m |
2/m |
| Zona
incerta |
+/++ |
0 |
0 |
| Parafascicular thalamic
nucleus |
 |
4/l |
3/m |
| Posterior intralaminar thalamic
nucleus |
++ |
2/m |
2/m |
| Lateral posterior thalamic nucleus,
mediocaudal |
++ |
0 |
0 |
| Lateral geniculate nucleus
(dorsal/ventral) |
++/+++ |
0 |
0 |
| Medial geniculate nucleus
(dorsal/ventral) |
 |
0 |
0 |
| Medial geniculate nucleus,
medial |
+ |
3/m |
3/m |
| Suprageniculate thalamic
nucleus |
+ |
3/m |
3/m |
| Subthalamic
nucleus |
 |
3-4/l |
3/l |
| Peripeduncular
nucleus |
++ |
2/m |
2-3/m |
| Medial pretectal
area |
++ |
2/m-h |
2-3/m-h |
| Parabigeminal
nucleus |
 |
3/m |
3/l-m |
| Ventral tegmental
area |
+/++ |
2/l |
3/m-h |
| Substantia nigra,
compacta |
+/++ |
2/l |
2-3/m-h |
| Substantia nigra,
reticular |
 |
0 |
0 |
| Substantia nigra,
lateral |
++ |
2/m-h |
2/m-h |
| Nucleus of the posterior
commissure |
++ |
1/m |
2/m |
| Retroethmoid
nucleus |
++ |
0 |
0 |
| Medial accessory occulomoter
nucleus |
+ |
1/m |
1/m |
| Superior colliculus, superficial gray
layer |
+ |
0 |
0 |
| Superior colliculus, optic nerve
layer |
 |
0 |
3-4/l |
| Superior colliculus, intermediate and
deep gray layer |
+ |
3/l |
0 |
| Precommissural
nucleus |
++ |
0 |
2-3/l-m |
|
Fibers: , none; +, very light staining; ++, light staining;
+++, moderate staining; ++++, heavy staining; +++++, extremely heavy
staining. Cells: 0, no cells observed; 1, occasional cells; 2, few
scattered cells; 3, moderate number of cells; 4, densely packed cells;
**
, could not be determined. l, Lightly stained; m, moderately stained;
h, heavily stained.
|
|
Fig. 7.
Localization of BDNF-ir and mRNA in thalamic and
brainstem regions. Dark-field and bright-field photomicrographs showing
BDNF cRNA hybridization (A, C, E, G, I, K, M, O, Q) and
BDNF immunolabeling (B, D, F, H, J, L, N, P, R),
respectively, in thalamus (A, B), mammillary region
(C, D), locus coeruleus (E, F),
parafascicular nucleus (PF) (G,
H), superior colliculus (I, J),
inferior olivary complex (K, L), medial geniculate
nucleus (M, N), parabrachial region (O,
P), and the nucleus of the solitary tract (Q,
R). In many thalamic (A, B, G, H, M, N)
and hypothalamic (C, D) areas, the distributions of BDNF
mRNA and perikaryal BDNF-ir overlapped; although in some regions
[e.g., anteroventral thalamic nucleus (AV)] there was heavy
fiber/terminal BDNF-ir in the absence of hybridization (A,
B). In other regions, such as the locus coeruleus (LC) (E, F) and nucleus of the
solitary tract (Sol) (Q, R),
cRNA-labeled cells could be detected, but it was difficult to identify
immunostained cells because of the fiber/terminal immunolabeling. Weak
labeling with both the cRNA and antibody also overlapped in most layers of superior colliculus, excluding the superficial gray layer, which did
not contain cRNA labeling (I, J). In contrast,
heavy hybridization and BDNF-ir were detected throughout the inferior olivary complex (K, L). Moderate to heavy BDNF cRNA
labeling was found in the external portion of the lateral parabrachial
nucleus (LPBE), whereas heavy fiber/terminal BDNF-ir was
present in most subregions of the lateral parabrachial subfield
(LPB) (O, P); neither cRNA nor antibody
labeled the medial portion of the parabrachial nucleus
(MPB) (O, P). Scale bars (shown in
B, D, J, N): A-D, I, J, M, N, 500 µm, respectively; (shown in F): E,
F, 200 µm; (shown in H, P, R): G, H, O,
P-R, 250 µm, respectively. AP, Area postrema; CG, central (periaqueductal) gray; DpG,
superior colliculus, deep gray layer; DpMe, deep
mesencephalic nucleus; DTg, dorsal tegmental nucleus;
fr, fasciculus retroflexus; Gr, gracile
nucleus; IAM, interanteromedial thalamic nucleus;
InG, superior colliculus, intermediate gray layer;
IOA, inferior olive, subnucleus A; IOC, inferior olive, subnucleus C; IOD, inferior olive,
dorsal nucleus; LDVL, laterodorsal thalamic nucleus,
ventrolateral; LH, lateral hypothalamic area;
LM, lateral mammillary nucleus; LRt,
lateral reticular nucleus; MG, medial geniculate
nucleus; MGM, medial geniculate nucleus, medial portion;
MM, medial mammillary nucleus; Op,
superior colliculus, optic nerve layer; PIL, posterior
intralaminar nucleus; PrC, precommissural
nucleus; PP, peripeduncular thalamic nucleus;
PT, paratenial thalamic nucleus; PV,
paraventricular thalamic nucleus; py, pyramidal tract;
scp, superior cerebellar peduncle; SG,
suprageniculate nucleus; SubC, subcoeruleus nucleus; SuG, superior colliculus, superficial gray layer;
SuM, supramammillary nucleus; VL,
ventrolateral thalamic nucleus.
[View Larger Version of this Image (134K GIF file)]
Fig. 8.
Dense fiber/terminal BDNF-ir was present in many
regions lacking BDNF mRNA expression. A-L, Dark-field
and bright-field photomicrographs of sections processed for in
situ hybridization (A, C-E, I, K) and
immunohistochemistry (B, F-H, J, L), respectively,
through the amygdala (A, B), habenula (C,
F), bed nucleus of stria terminalis (D,
G), spinal cord (E, H), lateral
geniculate nucleus (I, J), and dorsal/ventral
tegmental nucleus (K, L). As seen in A
and B, in the basolateral (BLA) and
lateral amygdala (La), many cells were heavily labeled
for both mRNA and BDNF-ir, whereas in the central amygdala many
immunolabeled fibers/terminals were present but cRNA hybridization was
not. Similarly, in the medial habenular nucleus (MHb)
(C, F) subregions of the bed nucleus of stria
terminalis (D, G), the dorsal (DLG) and
ventral regions (VLG) of the lateral geniculate nucleus
(I, J), the intergeniculate leaf
(IGL) (I, J), and ventral
tegmental nucleus (VTg) (K, L), heavy
fiber/terminal BDNF-ir was found without detectable mRNA. In spinal
cord (cervical level shown), heavily immunostained fibers were present
primarily in lamina 2 (H), which lacked BDNF mRNA
(E). Scale bars (shown in B): A,
B, 500 µm; (shown in F): C, D,
F, G, 500 µm; (shown in H): E,
H, 150 µm; (shown in J, L):
I-L, 500 µm. ac, Anterior commissure; BSTLD, bed nucleus of stria
terminalis, lateral dorsal; BSTLJ, bed nucleus of
the stria terminalis, juxtacapsular; BSTM, bed nucleus
of stria terminalis, medial; CeL, central amygdaloid nucleus, lateral division; CeM, central amygdaloid
nucleus, medial division; CPu, caudate putamen
(striatum); DEn, dorsal endopiriform nucleus;
DG, dentate gyrus; DTg, dorsal tegmental
nucleus; HF, hippocampal formation; ic,
internal capsule; LatC, lateral cervical nucleus;
LC, locus coeruleus; LHb, lateral
habenular nucleus; lv, lateral ventricle;
opt, optic tract; Pir, piriform cortex; PV, paraventricular thalamic nucleus;
SHy, septohypothalamic nucleus; SubG,
subgeniculate nucleus; 4V, 4th ventricle.
[View Larger Version of this Image (158K GIF file)]
As illustrated in Figure 8A,B and summarized in Table
2, striking patterns of BDNF mRNA and BDNF-ir were seen in the
amygdaloid complex. The basolateral, medial, and basomedial amygdaloid
nuclei contained many heavily cRNA-labeled and BDNF-immunoreactive cell bodies but very few immunoreactive fibers. In contrast, the central nucleus of the amygdala (especially the lateral subdivision) contained absolutely no BDNF mRNA or BDNF-immunoreactive cells but did contain high densities of immunolabeled fibers, which often formed pericellular baskets around unlabeled perikarya. Within the bed nucleus of the stria
terminalis (Fig. 8D,G), heavy pericellular fiber
labeling was observed in the lateral dorsal and juxtacapsular regions, whereas other aspects of this nucleus had moderate densities of immunoreactive fibers. BDNF mRNA was not detected in any portion of the
bed nucleus of the stria terminalis.
In the hypothalamus, varying degrees of cellular labeling for
BDNF-ir and mRNA were observed and a low to moderate level of immunoreactive fiber labeling was present in many areas (Table 4, Fig. 1). A few lightly cRNA-labeled cells were
scattered in the lateral and lateroanterior hypothalamic nuclei and
extended into the tuber cinereum area. More heavily cRNA-labeled and
immunoreactive cells were distributed in the ventromedial hypothalamic
nucleus and in the posterior hypothalamic area. Within the
paraventricular hypothalamic nucleus, numerous cells of the medial and
ventral parvocellular region were well labeled with the cRNA, whereas cells in the lateral magnocellular region were not labeled. Within the
mammillary and supramammillary region, many cells were positive for
both mRNA and immunoreactivity (Fig. 7C,D). Interestingly, BDNF-immunoreactive fibers but no immunoreactive or cRNA-labeled cell
bodies were observed in the median eminence and the infundibular stem.
A similar pattern was also seen in the medial habenular nucleus, which
contained a very high density of immunoreactive fibers but lacked
detectable cellular labeling (immunoreactivity or hybridization) (Fig.
8C,F).
Table 4.
BDNF in the hypothalamus and
epithalamus
|
BDNF-ir fibers |
BDNF-ir
cells |
BDNF mRNA |
|
| Medial habenular
neucleus |
+++++ |
0 |
0 |
| Lateral habenular
nucleus |
+ |
1-2/l |
Light haze |
| Anterior hypothalamic area,
anterior |
++ |
1/l |
0 |
| Lateroanterior hypothalamic
nucleus |
++ |
1/l |
2-3/l |
| Lateral hypothalamic
area |
++ |
1/m |
2/l |
| Anterior hypothalamic area,
central |
++ |
0 |
0 |
| Paraventricular hypothalamic nucleus
(parvocellular) |
++/+++ |
1/m |
3/m |
| Paraventricular hypothalamic
nucleus, lateral (magnocellular) |
+ |
0 |
0 |
| Medial tuberal
nucleus |
++ |
1/l-m |
2-3/m |
| Tuber cinereum
area |
++ |
1/l-m |
2-3/l-m |
| Ventromedial hypothalamic
nucleus |
++ |
2/m-h |
3/h |
| Arcuate hypothalamic
nucleus |
+++ |
0 |
0 |
| Median
eminance |
+/++ |
0 |
0 |
| Dorsomedial hypothalamic nucleus,
diffuse |
++ |
1/m |
3/l-m |
| Dorsal hypothalamic
area |
++ |
1/m |
1/l |
| Perifornical
nucleus |
++ |
0 |
1/m |
| Premammillary
nucleus |
++ |
0 |
2-3/m-h |
| Supramammillary
nucleus |
++ |
2/m |
3/h |
| Medial mammillary
nucleus |
 |
4/l-m |
3-4/m-h |
| Lateral mammillary
nucleus |
 |
4/l |
3-4/m |
| Interpeduncular
nucleus |
+ |
0 |
0 |
| Posterior hypothalamic
area |
++ |
2/l-m |
3/m |
| Infundibular
stem |
+/++ |
0 |
0 |
|
Fibers: , none; +, very light staining; ++, light staining;
+++, moderate staining; ++++, heavy staining; +++++, extremely heavy
staining. Cells: 0, no cells observed; 1, occasional cells; 2, few
scattered cells; 3, moderate number of cells; 4, densely packed cells;
**
, could not be determined. l, Lightly stained; m, moderately stained;
h, heavily stained.
|
|
In the brainstem, many BDNF cRNA-labeled cells were observed, and with
a few exceptions the distribution of these cells strongly correlated
with the distribution of immunoreactive cell bodies (Table
5). Some notable exceptions were the lateral
parabrachial nucleus (Fig. 7O,P), locus coeruleus (Fig.
7E,F), and nucleus of the solitary tract (Fig.
7Q,R), which contained densely cRNA-labeled cells but no
detectable immunoreactive perikarya. Fiber immunostaining was present
in all three regions, although in the lateral parabrachial nucleus
(especially within the external region) and nucleus of the solitary
tract, the high density and unique characteristics of the fiber
staining may have obscured immunoreactive cell bodies. Conversely, in
some brainstem nuclei, such as spinal vestibular nucleus, central
nucleus of the inferior colliculus, and gigantocellular reticular
nucleus, cell bodies were lightly immunostained but BDNF mRNA was not
detected. Very heavy fiber immunolabeling was noted in many brainstem
regions, such as the lateral parabrachial nucleus, nucleus of the
solitary tract (Fig. 7R), area postrema, inferior olive
(Fig. 7L), and ventral tegmental nucleus (Fig. 8L). As seen in Table 5, no cRNA hybridization or
immunostaining was detected in the cerebellum.
Table 5.
BDNF in the brainstem, cerebellum, and spinal
cord
|
BDNF-ir fibers |
BDNF-ir
cells |
BDNF mRNA |
|
| Central
gray |
+++ |
l/m |
3/m |
| Dorsal raphe
nucleus |
++ |
1/l |
0 |
| Deep mesencephalic
nucleus |
+ |
2/m |
2/l |
| Pedunculopontine tegmental
nucleus |
 |
3/m |
3/m |
| Pontine
nuclei |
 |
4/l-m |
4/l-m |
| Spinal vestibular
nucleus |
 |
2/m |
0 |
| Ventral tegmental
nucleus |
++++ |
0 |
0 |
| Cuniform
nucleus |
++ |
1/m |
3/m |
| Peritrigeminal
zone |
+ |
2/m |
0 |
| Laterodorsal tegmental
nucleus |
+ |
0 |
0 |
| Laterodorsal tegmental nucleus,
ventral |
+ |
0 |
0 |
| Dorsal tegmental nucleus,
central/pericentral |
+ |
0 |
0 |
| Periolivary nucleus,
medioventral/lateroventral |
+++ |
0 |
0 |
| Inferior colliculus,
nucleus brachium |
+ |
2/l |
1-2/l |
| Inferior colliculus, central
nucleus |
+ |
2-3/l |
0 |
| Inferior colliculus, dorsal
cortex |
+ |
1-2/l |
0 |
| Inferior colliculus, external
cortex |
 |
3/l-m |
2/l |
| Locus
coeruleus |
++ |
0 |
3/l-m |
| Subcoeruleus
nucleus |
+ |
2-3/m |
2-3/l-m |
| Lateral parabrachial nucleus,
internal/dorsal/central |
+++ |
0 |
3/m |
| Lateral parabrachial
nucleus, external |
++++ |
0 |
3/h |
| Medial parabrachial
nucleus |
± |
0 |
0 |
| Principal sensory trigeminal nucleus,
ventrolateral |
 |
2/m |
3/m |
| Nucleus of the solitary
tract |
| |