Download Projection patterns from the amygdaloid nuclear complex to

BR A I N R ES E A RC H 1 1 4 3 ( 2 00 7 ) 1 1 6 –12 5
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Projection patterns from the amygdaloid nuclear complex to
subdivisions of the dorsal raphe nucleus in the rat
Hyun S. Leea,⁎, Yun J. Euma , Seung M. Job , Barry D. Waterhouse c
a
Department of Anatomy, College of Medicine, Konkuk University, Chungju, Chungbuk 380-701, South Korea
Department of Anatomy, College of Medicine, Gachon University, Incheon, Kyunggi 405-220, South Korea
c
Department of Neurobiology and Anatomy, College of Medicine, Drexel University, 2900 Queen Lane, Philadelphia, PA 19129, USA
b
A R T I C LE I N FO
AB S T R A C T
Article history:
The goal of the present study was to identify the projection from the subdivisions of the
Accepted 17 January 2007
amygdaloid nuclear complex to specified subregions of the dorsal raphe (DR) nucleus and to
Available online 28 January 2007
attempt to compare the density of amygdaloid input to the DR with that of inputs from other
limbic structures. Use of a retrograde tracer, gold-conjugated and inactivated wheatgerm
Keywords:
agglutinin-horseradish peroxidase (WGA-apo-HRP-gold), demonstrated that amygdaloid
Amygdala
input to midline DR subdivision originates mainly from the medial portion of the medial
Dorsal raphe
amygdaloid nucleus, whereas that to lateral wing subdivision derives from the region
Tract tracing
extending from the lateral portion of the medial amygdaloid nucleus to the commissural
Fluorogold
stria terminalis. Use of the retrograde tracer Fluorogold (FG) produced relatively large but
WGA-apo-HRP-gold
circumscribed injection sites comprising midline DR as well as portions of lateral wing
subdivision and confirmed that the medial amygdaloid nucleus provides the major input to
the DR. We also demonstrated that although amygdaloid input was not as extensive as
inputs from other limbic structures such as the medial prefrontal cortex or the lateral
habenular nucleus, it was comparable to input from the lateral septal nucleus. Based on
these observations, we suggest that the medial amygdaloid nucleus provides substantial
input to the DR and may contribute an emotional influence on sleep–wakefulness cycle or
pain–stress modulation. Furthermore, it seems that the medial amygdaloid–DR projection
might be anatomically and functionally distinct from the well-characterized central
amygdaloid–periaqeductal gray (PAG) circuit which is essential for conditioned fear.
© 2007 Elsevier B.V. All rights reserved.
1.
Introduction
The amygdaloid nuclear complex is an almond-shaped brain
region lying between the external capsule and the hypothalamus. It is divided into two main nuclear masses: (1) a
centromedial nuclear group and (2) a corticobasal group. It is
well known that the central amygdaloid nucleus within the
former group projects to a variety of brainstem targets that
mediate specific signs of fear and anxiety (Davis, 1994). The
central and medial nuclei within the same group receive, in
return, serotonergic or noradrenergic innervation from the
dorsal raphe (DR) or the locus coeruleus (LC) (Amaral and
Insausti, 1992; Sadikot and Parent, 1990).
The DR is reciprocally connected to several limbic structures (Koehler and Steinbusch, 1982; Peyron et al., 1998) and
changes in serotonin (5-hydroxytryptamine, 5-HT) function
⁎ Corresponding author. Fax: +82 43 851 9329.
E-mail address: [email protected] (H.S. Lee).
0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2007.01.081
BR A I N R ES E A RC H 1 1 4 3 ( 2 00 7 ) 1 1 6 –1 25
are closely associated with emotional behaviors (Blier and de
Montigny, 1994; Pineyro and Blier, 1999). For example, the
medial prefrontal cortex, whose dysfunction is closely associated with affective disorders such as schizophrenia and
depressive illnesses, provides extensive input to the DR (Hajos
et al., 1998; Lee et al., 2003). The lateral habenular nucleus as
well as the lateral septal nucleus also contributes massive
input to the DR (Peyron et al., 1998). Differential roles for
various limbic structures including the hippocampus, the
amygdala, and the DR in regulating feeding, memory retention, and anxiety have been reported (Carlini et al., 2004).
Recent anatomical and electrophysiological studies reported
that the stress-related neuropeptide corticotrophin-releasing
factor (CRF) modulates the activity of serotonergic neurons in
the rat DR and that the central amygdaloid nucleus contains
numerous CRF-immunoreactive neurons (Lowry et al., 2000;
Valentino et al., 2001; van Bockstaele et al., 1998). Although
morphological studies have provided evidence that CRFcontaining amygdaloid neurons target LC dendrites (van
Bockstaele et al., 1998), the projection from the amygdaloid
complex to the DR has not been studied extensively (Peyron
et al., 1998).
One of the goals of the present study was to determine
which nuclear group within the amygdaloid complex provides
117
the major input to the DR. Since sub-regions of the
amygdaloid nuclear complex have been functionally differentiated, the projection pattern from these areas to each DR
subdivision could ascribe functional roles to specified
components of the amygdaloid–DR projection. A second
goal was to determine if the amygdaloid nuclear complex
provides as massive an input to the DR as other limbic
structures such as the medial prefrontal cortex or the lateral
habenular nucleus. We employed two retrograde tracers,
gold-conjugated and inactivated wheatgerm agglutininhorseradish peroxidase (WGA-apo-HRP-gold, WG) and Fluorogold (FG), in order to investigate the distribution and the
density of projections from the amygdaloid nuclear complex
to subdivisions of the DR in the rat.
2.
Results
In the first series of experiments, a retrograde tracer, WGAapo-HRP-gold (WG), was injected into each DR subdivision
(Fig. 1). Although injections of variable size within a specific
DR subdivision have exhibited corresponding variation in the
number of retrogradely labeled cells in the amygdaloid
nuclear complex, only the cases with a confined injection
Fig. 1 – WGA-apo-HRP-gold was injected within the midline DR (A, R119; and B, R123) including both dorsomedial (DRdm) and
ventromedial (DRvm) subdivisions of the nucleus or unilaterally into the lateral wing (DRlw) subdivision of the nucleus
(C, R106; D, R127). Note that the injection in A is displaced laterally. Immunostaining reveals clusters of 5-HT cells that help
define the boundaries of the nucleus. Aq, cerebral aqueduct; arrowheads, midline; mlf, medial longitudinal fasciculus;
Scale bar = 200 μm.
118
BR A I N R ES E A RC H 1 1 4 3 ( 2 00 7 ) 1 1 6 –12 5
within each DR subdivision (medio-lateral dimension, 200 μm)
based on 5-HT immunostaining were utilized in the present
analysis. Midline injection was made either unilaterally (Fig.
1A, R119) or in the median plane (Fig. 1B, R123) at rostral,
intermediate, and caudal DR levels. The tracer was also
injected into the lateral wing subdivision (Fig. 1C, R106;
Fig. 1D, R127) at intermediate levels of the DR.
Individual cases representing midline (Fig. 2A, R119) or
lateral wing (Fig. 2B, R127) injections are depicted to show the
distribution of retrogradely labeled cells along the rostrocaudal extent of amygdaloid nuclear complex (Fig. 2C, sections
1–4). The labeled cells at the ipsilateral amygdaloid nuclear
complex were predominant, while ones at the contralateral
side were meager. For midline injections, the majority of
labeled cells were located in the medial portion of the medial
amygdaloid nucleus (Fig. 2C, asterisks). On the other hand, for
lateral wing injections, labeled neurons were observed at the
region extending from the lateral portion of the medial
amygdaloid nucleus to the commissural stria terminalis
(Fig. 2C, open circles). Labeling at other regions of the amygdala
including basomedial, basolateral, and cortical nuclei was
minimal both for midline and for lateral wing injections.
An example of retrogradely-labeled neurons in the amygdaloid nuclear complex following WG injection into midline
(Figs. 3A–C, R119) or lateral wing DR (Figs. 3D–F, R127) is
depicted in Fig. 3. As described, the majority of labeled cells
were mainly located in the medial amygdaloid nucleus (Figs.
3A–C) following midline DR injection (R119) and in the region
extending from the lateral portion of the medial amygdaloid
nucleus to the commissural stria terminalis (Figs. 3D–F)
following lateral wing injection (R127).
The number of retrogradely labeled neurons within each
amygdaloid nucleus was collected from 7–9 amygdaloid
sections for various injection cases (Table 1). Following midline injection at the intermediate level of DR, the majority of
labeled cells was observed at the medial portion of the medial
amygdaloid nucleus, while only a few cells were found at
other regions including central, basomedial, basolateral, and
cortical nuclei (Table 1, R119, R120, R122, R123, R125, and
R126). Following the tracer injection in the lateral wing
subdivision, the majority of labeled neurons was observed
at the lateral portion of the medial amygdaloid nucleus as
well as the region surrounding the commissural stria
terminalis within the central amygdaloid nucleus (Table 1,
R106, R108, R109, R127, R124, and R130). Injection at caudal
midline of DR produced a pattern of labeling similar to
intermediate midline injection cases, although the number of
labeled cells in the former cases was less than that in the
latter (Table 1, R114, R111, R113, R128, R112, and R118). The
total number of labeled cells following tracer injection in the
rostral midline was extremely limited (Table 1, R103, R104,
R105, R115, R107, and R110).
In the second series of experiments, a retrograde tracer,
Fluorogold, was injected into the midline DR. Iontophoretic
injection of FG produced a relatively large, but confined
(mediolateral dimension, 800 μm) injection site, which
involved the dorsomedial and ventromedial regions of the
midline DR as well as portions of lateral wing subdivision
(Fig. 4A, R129). Although the tracer is taken up by axon
terminals (Fig. 4A; inset, open arrows) and transported to the
Fig. 2 – Following unilateral WG injection into midline
(A, R119) or lateral wing (B, R127) subregions of DR, the
location of retrogradely-labeled cells was plotted on a
rostro-caudal series (C, sections 1–4) of ipsilateral
amygdaloid sections. Each asterisk or open circle represents
one retrogradely-labeled cell body. Arrowheads, external
capsule; BL, basolateral nucleus; BM, basomedial nucleus;
Ce, central nucleus; Co, cortical amygdaloid nucleus; cst,
commissural stria terminalis; ec, external capsule; f, fornix;
ic, internal capsule; LH, lateral hypothalamus; Me, medial
amygdaloid nucleus; opt, optic tract; st, stria terminalis.
afferent sites, the extent of the injection was delineated by the
cluster of FG-immunostained somata (Fig. 4A; inset, asterisks).
The retrogradely labeled neurons were observed at several
BR A I N R ES E A RC H 1 1 4 3 ( 2 00 7 ) 1 1 6 –1 25
119
Fig. 3 – The majority of retrogradely-labeled cells were observed at the medial portion of the medial amygdaloid nucleus
following midline DR injection (A–C, R119), whereas neurons were observed in the region extending from the lateral portion of
the medial amygdaloid nucleus to the commissural stria terminalis following lateral wing injection (D–F, R127). B/C and
E/F represent higher magnification views of WG-labeled neurons (curved, open arrows) in A and D, respectively. Ce, central
amygdaloid nucleus; cst, commissural stria terminalis; Me, medial amygdaloid nucleus; opt, optic tract. Scale bars in A and D
represent 50 μm, whereas those in B, C, E, and F signify 10 μm.
limbic structures including the medial prefrontal cortex (Fig.
4B), the lateral habenular nucleus (Fig. 4C), and the lateral
septal nucleus (Fig. 4D). The positive immunostaining at these
afferent sites was distinct from the surrounding regions which
exhibited none of the neuronal labeling (Figs. 4B–D).
An example of retrogradely-labeled neurons within the
amygdaloid nuclear complex following FG injection into the
midline DR is depicted in Fig. 5. The majority of FG-labeled
cells were observed at rostral (Figs. 5A–C, R129) and intermediate (Figs. 5D–F, R134) levels of the medial amygdaloid
nucleus. Higher magnification views of multipolar neurons
often exhibited distinct dendritic morphology, which
extended up to 100 μm from the somata (Figs. 5C and F).
The total number of retrogradely labeled neurons in the
medial amygdaloid nucleus following FG injection (Table 2)
was larger than that of neurons following WG injection (Table
1); probably due to the more extensive injection site observed
with FG. Thus, the number of retrogradely-labeled cells within
the medial amygdaloid nucleus in the former cases was
compared with that of labeled neurons located at several
limbic structures such as the medial prefrontal cortex, the
lateral habenular nucleus, and the lateral septal nucleus
(Table 2). The total number of labeled cells in the medial
amygdaloid nucleus was approximately one-fourth to one-
fifth of that observed in the medial prefrontal cortex or in the
lateral habenular nucleus, while it was a little less, but
comparable to that of neurons in the lateral septal nucleus
(Table 2).
3.
Discussion
Based on WGA-apo-HRP-gold tracing, we demonstrated that
the input to the midline subdivision of the DR originated
mainly from the medial portion of the medial amygdaloid
nucleus, whereas that to the lateral wings derived from the
lateral portion of the medial amygdaloid nucleus as well as the
region surrounding the commissural stria terminalis within
the central amygdaloid nucleus (see summary diagram—Fig.
6); the labeling was predominantly ipsilateral with only a few
cells at the contralateral side. Tracing with Fluorogold
confirmed that relative to other subdivisions of the amygdaloid nuclear complex, the medial amygdaloid nucleus provided the major input to the DR. We also demonstrated that
although amygdaloid input was not as extensive as inputs
from other limbic structures such as the medial prefrontal
cortex or the lateral habenular nucleus, it was comparable to
input from the lateral septal nucleus.
120
BR A I N R ES E A RC H 1 1 4 3 ( 2 00 7 ) 1 1 6 –12 5
Table 1 – Retrogradely-labeled cells were observed at various regions of the amygdaloid nuclear complex following WGAapo-HRP-gold injection into each subdivision of the dorsal raphe nucleus
DR injection sites
Intermediate midline
Lateral wing
Caudal midline
Rostral midline
3.1.
Cases (sex)
R119 (♂)
R120 (♂)
R122 (♂)
R123 (♀)
R125 (♀)
R126 (♀)
R106 (♂)
R108 (♂)
R109 (♂)
R127 (♀)
R124 (♀)
R130 (♀)
R114 (♂)
R111 (♂)
R113 (♂)
R128 (♀)
R112 (♀)
R118 (♀)
R103 (♂)
R104 (♂)
R105 (♂)
R115 (♀)
R107 (♀)
R110 (♀)
Distribution of labeled cells at each amygdaloid nucleus
Medial
Central
Basomedial
Basolateral
Cortical
25
35
19
22
14
18
13
15
18
15
20
17
11
9
7
10
8
9
2
3
1
3
2
1
5
6
3
4
3
5
8
10
12
9
11
10
2
3
3
3
2
4
1
0
0
2
0
0
3
4
2
2
3
2
0
1
2
1
2
2
2
1
0
0
1
0
1
0
1
0
1
1
2
1
1
0
1
0
1
0
1
2
0
1
0
0
1
2
0
1
0
0
1
1
0
1
2
0
1
0
1
1
0
0
2
0
1
1
1
0
0
2
0
0
1
1
0
2
0
1
Technical considerations
Compared with other tracers (such as cholera-toxin B subunit)
used to investigate various inputs to the DR (Peyron et al.,
1998), WG produced a smaller, well-circumscribed injection
site, where diffusion of the tracer into adjacent subdivisions of
the nuclei was negligible. Several precautions were taken to
minimize leakage of the tracer along the injection pathway
from the dural surface. First, pipette tip diameters were kept
small (10–15 μm) so as to minimize unwanted release. Second,
we injected tracer substances in small increments over
30 min, since retrograde labeling with WGA-HRP or WG is
most successful when tracer substances are injected slowly
over extended periods.
There was still some concern that tracer could leak from
the pipette as it passed through the superior colliculus en
route to DR sub-regions, thus causing false-labeling. The WG,
like WGA-HRP, does not seem to be absorbed and transported
via fibers-of-passage (Basbaum and Menetrey, 1987). Thus, in
cases with midline DR injections, leakage of tracer within the
decussation of the superior colliculus was not a concern. For
the lateral wing injection, further precautions were taken to
minimize leakage of tracer along the pipette track. First, a
small amount of oil was backfilled into the tip of the pipette
after it had been filled with the tracer so that tissue along the
pipette track would not be exposed to the tracer during the
process of pipette insertion. Second, following complete
delivery of the tracer substance, the pipette was lowered to a
point 0.1 mm deep to the original location and a small amount
of extracellular fluid was back-filled into the pipette before it
was withdrawn from the brain. In practice, the major problem
with lateral wing injections was not spillage along the pipette
track, but rather variable diffusion of tracer into the surrounding periaqueductal gray matter (PAG). Thus, the DR injection
sites were processed for 5-HT immunostaining to confirm the
exact location of the tracer infusion within each DR subdivision (Fig. 1). Tracer spillage into the aqueduct or the 4th
ventricle was most likely cleared via cerebrospinal fluid
circulation, since black granules were not detected along the
ependymal layer adjacent to the injection site.
Previously published reports describe only a few retrogradely-labeled cells in the amygdala following substantial
horseradish peroxidase (HRP) or wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) injections in the DR (Aghajanian and Wang, 1977; Kalen et al., 1985). In these previous
studies, the method of approaching the DR obliquely through
the cerebellum was not optimal for involving the entire dorsoventral extent of the midline DR. Such experiments were also
disadvantaged because of the longer distance from the dural
surface to the DR target and possible bleeding caused by
passage of the pipette through the confluence of superior
sagittal and transverse sinuses. We approached the DR
vertically at midbrain and pontine levels by dual ligation and
midline incision of the superior sagittal sinus and thus, were
able to inject the tracer within the narrow confines of each DR
subdivision. Scarcity of retrogradely labeled cells within the
amygdala in the previous studies might also have been caused
by the instability of HRP or WGA-HRP reaction product. The
tracer used in the present study is known to produce a more
stable reaction product due to the inactivated enzyme, WGAapo-HRP (Basbaum and Menetrey, 1987).
The present study further demonstrated that among subdivisions of the amygdaloid nuclear complex, the medial
nucleus provides substantial input to the DR (Tables 1 and 2).
BR A I N R ES E A RC H 1 1 4 3 ( 2 00 7 ) 1 1 6 –1 25
121
Fig. 4 – Iontophoretic injection of Fluorogold (FG) into the midline DR (A, R129) produced an injection site involving dorsomedial
(DRdm) and ventromedial (DRvm) regions as well as portions of lateral wing subdivision (DRlw). Although the retrograde tracer
was taken up by axon terminals (inset in A, open arrows) within the DR, the efficacy of iontophoresis is often correlated with the
number of labeled somata (inset in A, asterisks) which have taken up the tracer during the process. Retrogradely-labeled
neurons were observed at several limbic structures such as the medial prefrontal cortex (B), the lateral habenular nucleus (C),
and the lateral septal nucleus (D). Insets in B–D represent higher magnification views of FG-labeled cells. Aq, cerebral aqueduct;
Arrowheads, midline; DP, dorsal peduncular region of the medial prefrontal cortex; D3V, dorsal third ventricle; fmi, forceps
minor corpus callosum; gcc, genu of corpus callosum; IL, infralimbic region; LHb, lateral habenular nucleus; LS, lateral septal
nucleus; LV, lateral ventricle; MHb, medial habenular nucleus; mlf, medial longitudinal fasciculus; PV, paraventricular thalamic
nucleus; sm, stria medullaris; I–VI, first to sixth cortical layers. Scale bars in A–D represent 100 μm, whereas those at insets in
A–D signify 25 μm.
A previous study using cholera toxin b subunit (CTb) injection,
however, reported that the central nucleus of the amygdala
provided the major input to ventromedial or dorsomedial parts
of the central DR (Peyron et al., 1998). Such discrepancies might
be explained by the fact that CTb injection in the previous study
(cf. Fig. 1 in Peyron et al., 1998) was large enough to involve the
surrounding PAG. Because CTb is a very sensitive tracer, any
spillage of the tracer into the PAG might have caused some
degree of artifact labeling at the central amygdaloid nucleus.
3.2.
Functional implications of amygdaloid projections to
the DR
The medial amygdaloid nucleus is a sexually dimorphic area
and a portion of a neural pathway that regulates reproductive
behavior in animals (Rasia-Filho et al., 1999; Stark et al.,
1998). It is also known that the medial amygdala is involved
in agonistic behavior by affecting social learning processes
(Coolen and Wood, 1998; Luiten et al., 1985). The medial
amygdaloid nucleus receives input from olfactory and
vomeronasal systems as well as gonadal hormone inputs
(Gomez and Newman, 1992). From our material it is evident
that the medial amygdaloid–DR projection is not a sexually
dimorphic pathway (Tables 1 and 2). The involvement of the
DR in the production of behavioral and physiological
responses to pain and stress has been suggested, thus
emotional influences on pain and stress might be modulated
via the medial amygdaloid–DR projection system (Kirouac et
al., 2004; Valentino et al., 2001). Further electrophysiological
studies, however, need to be performed to determine whether
medial amygdaloid–DR projection might be involved in a
certain type of mating behavior.
122
BR A I N R ES E A RC H 1 1 4 3 ( 2 00 7 ) 1 1 6 –12 5
Fig. 5 – Labeled cells were observed mainly at the rostral (A–C, R129) and intermediate (D–F, R134) levels of the medial
amygdaloid nucleus (Me) following injection of FG into the midline DR. B/C and E/F represent higher magnification views of
FG-labeled neurons (arrows) in A and D, respectively. LH, lateral hypothalamus; Me, medial amygdaloid nucleus; opt, optic tract.
Scale bars in A, B, D, and E represent 100 μm, whereas those in C and F signify 10 μm.
Previous reports indicated that cells in the medial amygdaloid nucleus are immunoreactive for tyrosine hydroxylase
as well as vasopressin (Asmus and Newman, 1993; Caffe and
van Leeuwen, 1983). Morphologically distinct populations of
neuropeptide Y-immunoreactive neurons are distributed in
the medial amygdaloid nucleus (Gustafson et al., 1986). A large
number of NADPH-diaphorase positive neurons that might
Table 2 – Retrogradely labeled cells were observed at
various limbic structures following Fluorogold injection
into the midline DR
Cases (sex)
Limbic structures
Medial
Medial
Lateral
Lateral
amygdaloid prefrontal habenular septal
nucleus
cortex
nucleus nucleus
R129
R174
R179
R134
R181
R182
(♂)
(♂)
(♂)
(♀)
(♀)
(♀)
47
61
55
52
45
34
196
251
238
243
189
147
204
287
251
224
199
167
65
78
63
67
56
46
exert an influence on the neuroendocrine secretion system
were also found in the medial amygdaloid nucleus (Tanaka
et al., 1997). Establishing the neurochemical identity of
medial amygdaloid–DR projection neurons will be an important step in further characterizing the functional significance
of this system.
Experiments using Fluorogold confirmed that the medial
amygdaloid nucleus provides the major input to the DR (Fig.
5). We also demonstrated that although amygdaloid input
to DR was not as extensive as inputs from other limbic
structures such as the medial prefrontal cortex or the
lateral habenular nucleus, it was comparable to input from
lateral septal nucleus (Table 2). Functional significance
associated with predominance of medial prefronto-cortical
or lateral habenular input to the DR over amygdaloid or
lateral septal one could not be assessed in the present
study.
Another series of experiments, however, were performed in
the present study to investigate the possibility that portions of
the medial amygdaloid–DR projection might be influenced by
the medial prefronto-cortical input to the amygdala. An
anterograde tracer, PHA-L, was injected into the prefrontal
cortex and WG was subsequently injected into the midline DR;
BR A I N R ES E A RC H 1 1 4 3 ( 2 00 7 ) 1 1 6 –1 25
Fig. 6 – Schematic diagram illustrates a crude topographic
projection from the amygdaloid nuclear complex to midline
or lateral wing (lw) subdivisions of the DR. BL, basolateral
nucleus; BM, basomedial nucleus; Ce, central nucleus; Co,
cortical amygdaloid nucleus; cst, commissural stria
terminalis; ec, external capsule; LH, lateral hypothalamus;
Me, medial amygdaloid nucleus; opt, optic tract.
PHA-L-labeled axonal varicosities originating from the prefrontal cortex were sparse in the medial amygdaloid nucleus,
where WG-labeled, DR-projecting cells were observed (unreported observation). Thus, it would appear that the medial
amygdaloid–DR pathway is not heavily influenced by the
medial prefrontal cortex, despite the fact that the medial prefrontal cortex is a major source of afferent inputs to DR (Lee
et al., 2003). Since the entorhinal cortex has significant
projections to the medial amygdala (McDonald and Mascagni, 1997), the influence of entorhinocortical–amygdaloid
projection over the DR system needs to be further evaluated
in future studies.
Based on highly restricted injection of the WG tracer within
the lateral wing subdivision of DR, we demonstrated that
amygdaloid neurons projecting to the lateral wing DR are
located within the region extending from the lateral portion of
the medial amygdaloid nucleus into the commissural stria
terminalis (Fig. 2C, sections 2 and 3). The retrograde labeling
within the major, medial and lateral portions of the central
amygdaloid nucleus, however, was extremely limited. Therefore, the amygdaloid–DR projection seems to be antomically
and functionally distinct from the extensive, central amygdaloid–PAG projection which is involved in conditioned fear
(Davis, 1994).
Previous immunocytochemical studies demonstrated that
approximately half of the neurons in the medial division of the
central amygdaloid nucleus contained GABA (Jongen-Relo and
Amaral, 1998). CRF-like immunoreactivity has also been
observed in the medial and lateral portions of the central
amygdaloid nucleus (Sakanaka et al., 1986; van Bockstaele et al.,
1998). Neurochemical identification of cells in the CST region
projecting to the lateral wing of the DR will provide further
insight regarding the functional role of this projection.
123
The present study also demonstrated that the medial
amygdaloid nucleus provides less extensive input to the
caudal midline than to the intermediate midline DR (Table
1). Although the direct projection from the amygdaloid
nuclear complex to the caudal DR is not substantial, there
is still a possibility that the caudal midline DR is influenced
by the amygdala via intranuclear interconnections from
lateral wing or intermediate midline DR to caudal DR (Fite
and Janusonis, 2001; Fite et al., 1999; Janusonis and Fite,
2001).
The amygdaloid nuclear complex provides only a few
projections to the rostral midline DR (Table 1). The rostral pole
of the DR at the level of the oculomotor or the trochlear nuclei
is a unique subdivision in that it exhibits the smallest diurnal
variation of c-Fos expression in the Mongolian gerbil (Janusonis and Fite, 2001). The rostral midline subdivision sends
projections mainly to the caudate–putamen and has a
reciprocal connection with the dorsomedial part of the
substantia nigra, suggesting an integrative role for this
subdivision in motor control (Imai et al., 1986; Pasquier et al.,
1977).
Based on these observations, we conclude that relative to
other subdivisions of the amygdaloid nuclear complex, the
medial amygdaloid nucleus provides substantial input to the
DR and may contribute an emotional influence on sleep–
wakefulness cycle and/or pain–stress modulation. Furthermore, it seems that the medial amygdaloid–DR projection
might be anatomically and functionally distinct from the wellcharacterized central amygdaloid–PAG circuit which is essential for conditioned fear.
4.
Experimental procedures
A total of 34 (male and female in equal numbers for injections
at each DR subdivision) Sprague–Dawley rats whose weight
ranged from 300 to 350 g were used in this study. Twenty-four
animals were used for WGA-apo-HRP-gold injections, while
six were for Fluorogold studies. Additional four animals were
utilized for prefrontocortico-amygdalo-DR projection as
described in the Discussion section. Prior to surgery, each rat
was anesthetized with an intraperitoneal injection of chloral
hydrate (3.6% in distilled water, 1 ml/100 g body weight). All
animals used in this study were treated according to guidelines approved by the institutional animal care and use
committee and conformed to the NIH guidelines on care and
use of animals in research.
4.1.
DR injection site
After anesthesia, rats were placed in a stereotaxic frame with
the dorsal surface of the skull adjusted in the horizontal plane.
The skull around bony lambda was removed and the superior
sagittal sinus ligated with surgical sutures rostrally and
caudally. Angiovasectomy was performed between suture
points in order to expose the cerebral fissure at midline. The
retrograde tracer, WGA-apo-HRP-gold, was injected into midline (n = 6) and lateral wing (n = 6) at intermediate DR level as
well as rostral (n = 6) and caudal (n = 6) midline DR, according to
the atlas of Paxinos and Watson (1998). At the rostrocaudal
124
BR A I N R ES E A RC H 1 1 4 3 ( 2 00 7 ) 1 1 6 –12 5
dimension, the DR existed approximately between 0.5 mm
rostral and 1.5 mm caudal to the bony lambda. Thus injection
into midline at intermediate DR level was often targeted at
0.5 mm caudal to the bony lambda at the depth of 5.6–5.7 mm,
whereas the lateral wing was at 0.3 mm lateral from the
midline at the depth of 5.3–5.5 mm at the same rostro-caudal
dimension. The rostral midline DR was approached at 0.3–
0.1 mm rostral to the bony lambda at the depth of 5.3–5.5 mm,
whereas the caudal DR was at 1.1–1.3 mm caudal to the
lambda at the depth of 5.8–6.0 mm.
4.2.
WGA-apo-HRP-gold injection
The WGA-apo-HRP-gold was synthesized using inactivated
wheatgerm agglutinin-horseradish peroxidase (WGA-HRP;
Sigma, L-0390) and 20 nm (Sigma, G1652) colloidal gold
(Basbaum and Menetrey, 1987). The injection apparatus
consisted of a glass micropipette (tip diameter, 15–20 μm)
hydraulically linked to a 2.0 μl Hamilton syringe. A total
volume of 0.2 μl of WGA-apo-HRP-gold was pressure-injected
into a single site within each DR subdivision over a 30-min
period.
4.3.
Fluorogold injection
A solution of 1% Fluorogold (Fluorochrome Inc.) was prepared
in saline, drawn into the tip of a glass micropipette (tip
diameter, 10–15 μm) via capillary action and deposited within
midline (n = 6) at intermediate DR level using a 2 μA alternating
current applied on a 5-s duty cycle for 20 min through a silver
lead wire (Stoelting, 50880) inserted in the pipette.
4.4.
Perfusion-fixation and silver enhancement reaction
After a survival period of 48–72 h following WGA-apo-HRPgold or FG injections, the animals were perfused using 150 ml
of saline followed by 600 ml of fixative containing 4%
paraformaldehyde in 0.01 M phosphate-buffered saline (PBS,
pH 7.4). The perfusion-fixation was completed with 100 ml of
PBS containing 10% sucrose. The brain was then removed and
stored in 30% sucrose solution in PBS overnight. A series of
40 μm sections were made using a cryostat and every 5th
section was collected in a tissue-culture plate. Following
rinses with distilled water, the tracer was detected using a
commercial silver intensification kit (Sigma, SE-100) as
described in Llewellyn-Smith et al. (1992).
4.5.
Fluorogold immunocytochemistry
Sections were washed with 0.02 M potassium phosphatebuffered saline (KPBS, pH 7.4) and incubated in 1:500 dilution
of rabbit anti-Fluorogold (Fluorochrome Inc.) dissolved in
KPBS, which contains 1% normal goat serum, 0.3% Triton,
and 1% bovine serum albumin (BSA) for 48 h (4 °C). Following
rinses with KPBS, sections were then incubated in 1:250
dilution of biotinylated goat anti-rabbit antiserum (Vector,
BA-1000) made with KPBS for an hour. After rinses, sections
were incubated in ABC complex for an hour and then reacted
with 3, 3′-diaminobenzidine (DAB)-H2O2 kit (Vector, SK-4100)
for 1–2 min (at 4 °C).
Acknowledgment
This work was supported by an intramural grant from Konkuk
University to HSL in 2005.
REFERENCES
Aghajanian, G.K., Wang, R.Y., 1977. Habenular and other midbrain
raphe afferents demonstrated by a modified retrograde tracing
technique. Brain Res. 122, 229–242.
Amaral, D.G., Insausti, R., 1992. Retrograde transport of
3
D-[ H]-aspartate injected into the monkey amygdaloid
complex. Exp. Brain Res. 88, 375–388.
Asmus, S.E., Newman, S.W., 1993. Tyrosine-hydroxylase
mRNA-containing neurons in the medial amygdaloid nucleus
and the reticular nucleus of the thalamus in the Syrian
hamster. Brain Res. Mol. Brain Res. 20, 267–273.
Basbaum, A.I., Menetrey, D., 1987. WGA-apo-HRP-gold: A new
retrograde tracer for light- and electron-microscopic
single- and double-label studies. J. Comp. Neurol. 261,
306–318.
Blier, P., de Montigny, C., 1994. Current advances and trends in
the treatment of depression. Trends Pharmacol. Sci. 15,
220–226.
Caffe, A.R., van Leeuwen, F.W., 1983. Vasopressinimmunoreactive cells in the dorsomedial hypothalamic region,
medial amygdaloid nucleus and locus coeruleus of the rat. Cell
Tissue Res. 233, 23–33.
Carlini, V.P., Varas, M.M., Cragnolini, A.B., Schioth, H.B.,
Scimonelli, T.N., de Barioglio, S.R., 2004. Differential role of the
hippocampus, amygdala, and dorsal raphe nucleus in
regulating feeding, memory, and anxiety-like behavioral
responses to ghrelin. Biochem. Biophys. Res. Commun. 313,
635–641.
Coolen, L.M., Wood, R.I., 1998. Bidirectional connections of the
medial amygdaloid nucleus in the Syrian hamster brain:
simultaneous anterograde and retrograde tract tracing.
J. Comp. Neurol. 399, 189–209.
Davis, M., 1994. The role of the amygdala in emotional learning.
Int. Rev. Neurobiol. 36, 225–266.
Fite, K.V., Janusonis, S., 2001. Retinal projection to the dorsal raphe
nucleus in the Chilean degus (Octodon degus). Brain Res. 895,
139–145.
Fite, K.V., Janusonis, S., Foote, W., Bengston, L., 1999. Retinal
afferents to the dorsal raphe nucleus in rats and Mongolian
gerbils. J. Comp. Neurol. 414, 469–484.
Gomez, D.M., Newman, S.W., 1992. Differential projections of the
anterior and posterior regions of the medial amygdaloid
nucleus in the Syrian hamster. J. Comp. Neurol. 317,
195–218.
Gustafson, E.L., Card, J.P., Moore, R.Y., 1986. Neuropeptide Y
localization in the rat amygdaloid complex. J. Comp. Neurol.
251, 349–362.
Hajos, M., Richards, C.D., Szekely, A.D., Sharp, T., 1998. An
electrophysiological and neuroanatomical study of the medial
prefrontal cortical projection to the midbrain raphe nuclei in
the rat. Neuroscience 87, 95–108.
Imai, H., Steindler, D.A., Kitai, S.T., 1986. The organization of
divergent axonal projections from the midbrain raphe nuclei in
the rat. J. Comp. Neurol. 243, 363–380.
Janusonis, S., Fite, K.V., 2001. Diurnal variation of c-Fos
expression in subdivisions of the dorsal raphe nucleus of the
Mongolian gerbil (Meriones unguiculatus). J. Comp. Neurol. 440,
31–42.
Jongen-Relo, A.L., Amaral, D.G., 1998. Evidence for a GABAergic
BR A I N R ES E A RC H 1 1 4 3 ( 2 00 7 ) 1 1 6 –1 25
projection from the central nucleus of the amygdala to the
brainstem of the macaque monkey: a combined retrograde
tracing and in situ hybridization study. Eur. J. Neurosci. 10,
2924–2933.
Kalen, P., Karlson, M., Wiklund, L., 1985. Possible excitatory amino
acid afferents to nucleus raphe dorsalis of the rat investigated
with retrograde wheat germ agglutinin and D-[3H]-aspartate
tracing. Brain Res. 360, 285–297.
Kirouac, G.J., Li, S., Mabrouk, G., 2004. GABAergic projection from
the ventral tegmental area and substantia nigra to the
periaqueductal gray region and the dorsal raphe nucleus.
J. Comp. Neurol. 469, 170–184.
Koehler, C., Steinbusch, H., 1982. Identification of serotonin and
non-serotonin containing neurons of the midbrain raphe
projecting to the entorhinal area and the hippocampal
formation. A combined immunohistochemical and
fluorescent retrograde tracing in the rat brain. Neuroscience 7,
951–975.
Lee, H.S., Kim, M.A., Valentino, R.J., Waterhouse, B.D., 2003.
Glutamatergic afferent projections to the dorsal raphe nucleus
of the rat. Brain Res. 963, 57–71.
Llewellyn-Smith, I.J., Pilowsky, P., Minson, J.B., 1992.
Retrograde tracers for light and electron microscopy. In: Bolam,
J.P. (Ed.), Experimental Neuroanatomy. Oxford Univ. Press,
Oxford, pp. 31–59.
Lowry, C.A., Rodda, J.E., Lightman, S.L., Ingram, C.D., 2000.
Corticotropin-releasing factor increases in vitro firing rates of
serotonergic neurons in the rat dorsal raphe nucleus: evidence
for activation of a topographically organized
mesolimbocortical serotonergic system. J. Neurosci. 20,
7728–7736.
Luiten, P.G., Koolhaas, J.M., de Boer, S., Koopmans, S.J., 1985.
The cortico-medial amygdale in the central nervous
system organization of agonistic behavior. Brain Res. 332,
283–297.
McDonald, A.J., Mascagni, F., 1997. Projections of the lateral
entorhinal cortex to the amygdale: a Phaseolus vulgaris
leucoagglutinin study in the rat. Neuroscience 77,
445–459.
Pasquier, D.A., Kemper, T.L., Forbes, W.B., Morgane, P.J., 1977.
125
Dorsal raphe, substantia nigra and locus coeruleus:
interconnections with each other and the neostriatum. Brain
Res. Bull. 2, 323–339.
Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic
Coordinates. Academic Press, New York.
Peyron, C., Petit, J.M., Rampon, C., Jouvet, M., Luppi, P.H., 1998.
Forebrain afferents to the rat dorsal raphe nucleus
demonstrated by retrograde and anterograde tracing methods.
Neuroscience 82, 443–468.
Pineyro, G., Blier, P., 1999. Autoregulation of serotonin neurons:
Role in antidepressant drug action. Pharm. Rev. 51,
533–591.
Rasia-Filho, A.A., Londero, R.G., Achaval, M., 1999. Effects of
gonadal hormones on the morphology of neurons from the
medial amygdaloid nucleus of rats. Brain Res. Bull. 48,
173–183.
Sadikot, A., Parent, A., 1990. The monoaminergic innervation of
the amygdala in the squirrel monkey: an
immunohistochemical study. Neuroscience 36, 431–447.
Sakanaka, M., Shibasaki, T., Lederis, K., 1986. Distribution and
efferent projections of corticotrophin-releasing factor-like
immunoreactivity in the rat amygdaloid complex. Brain Res.
382, 213–238.
Stark, C.P., Alpern, H.P., Fuhrer, J., Trowbridge, M.G., Wimbish, H.,
Smock, T., 1998. The medial amygdaloid nucleus
modifies social behavior in male rats. Physiol. Behav. 63,
253–259.
Tanaka, M., Ikeda, T., Hayashi, S., Iijima, N., Amaya, F., Ibata, Y.,
1997. Nitrergic neurons in the medial amygdale project to the
hypothalamic paraventricular nucleus of the rat. Brain Res.
777, 13–21.
Valentino, R.J., Liouterman, L., Van Bockstaele, E.J., 2001. Evidence
for regional heterogeneity in corticotrophin-releasing factor
interactions in the dorsal raphe nucleus. J. Comp. Neurol. 435,
450–463.
van Bockstaele, E.J., Colago, E.E.O., Valentino, R.J., 1998.
Amygdaloid corticotrophin-releasing factor targets locus
coeruleus dendrites: Substrate for the co-ordination of
emotional and cognitive limbs of the stress response.
J. Neuroendocrinol. 10, 743–757.