Neuroscience 226 (2012) 324–347

INTERCALATED AND PARACAPSULAR CELL ISLANDS OF THE

ADULT RAT AMYGDALA: A COMBINED RAPID-GOLGI,
ULTRASTRUCTURAL, AND IMMUNOHISTOCHEMICAL ACCOUNT
D. MARCELLINO, a  M. FRANKOWSKA, a,c L. AGNATI, d These results indicate a short distance and a rapid extrasy-
M. PEREZ DE LA MORA, e V. VARGAS-BARROSO, b naptic form of dopamine volume transmission mediated via
K. FUXE a AND J. LARRIVA-SAHD b*  D1 receptors in the AIC and PIC which may enhance fear and
a
Department of Neuroscience, Karolinska Institutet, 17177 anxiety by suppressing feed-forward inhibition in the baso-
Stockholm, Sweden lateral and central amygdaloid nuclei. The strong sugges-
b tion for a commissural axon projection to the AIC
Instituto de Neurobiologı´a, Universidad Nacional Autónoma de
México, Campus Juriquilla, Querétaro CP 76230, Mexico documented here, coupled with the previous evidences
c
indicting an isocortical and amygdalar contributions to the
Department of Pharmacology, Institute of Pharmacology,
anterior commissure, opens the possibility that the AIC
Polish Academy of Sciences, Smetna 12, 31-343 Kraków, Poland
may be involved in decoding nerve impulses arising from
d
IRRCS, Lido Venice, Italy both the ipsi- and contra-lateral forebrain to, in turn, modu-
e
Division of Neurosciences, Instituto de Fisiologı´a Celular, late the homolateral amygdala. Ó 2012 IBRO. Published by
Universidad Nacional Autónoma de México, CP 04510 México Elsevier Ltd. All rights reserved.
City, Mexico

Key words: anterior commissure, dopamine, volume trans-

Abstract—The anterior and rostral paracapsular intercalated
islands (AIC and PIC, respectively) were studied in the con-
text of the amygdaloid modulation of fear/anxiety using
horizontal sections. The structural analysis carried out
using silver-impregnated specimens revealed that the AIC
is composed of tightly packed, medium-sized spiny neurons
with distinct dendritic and axonal patterns that send project-
ing axons to the central nucleus of the amygdala. The AIC
occupies a strategic position between the basolateral amyg-
daloid complex and the caudal limb of the anterior commis-
sure from which it receives fibers en passage and axon
terminals. Electron microscopic observation of terminal
(i.e., synaptic) degeneration 72 h after the surgical interrup-
tion of the anterior commissure, confirms the synaptic inter-
action between the latter and the AIC neurons. These
observations suggest that these islands may gate the activ-
ity of neurons from the contralateral basal forebrain and
synchronize the anxiogenic output of both amygdalae.
Immunohistochemical analysis indicated that, within the
AIC and rostral PIC, the distance between tyrosine hydroxy-
lase-immunoreactive terminals and the punctate dopamine
D1 receptor immunoreactivity, was in the micrometer range.
*Corresponding author. Address: Department of Developmental
Neurobiology, Instituto de Neurobiologı́a, UNAM, Campus Juriquilla,
3001 Juriquilla Boulevard, CP 76230, Querétaro, Qro., Mexico.
Tel: +52-5-623-4030; fax: +52-5-623-4042.
E-mail address: jlsneuro@unam.mx (J. Larriva-Sahd).
D.M. and J.L.-S. contributed equally to this manuscript.
Abbreviations: ACc, anterior commissure pars caudalis; ACr, anterior
commissure pars rostralis; AIC, anterior intercalated cell mass; AxSh,
axo-shaft synapse; AxSp, axo-spinous synapse; BLA, basolateral
nucleus of the amygdale, anterior part; Ce, central nucleus of the
amygdala; D1-I, immunoreactivity to dopamine 1 receptor; DBH,
dopamine beta hydroxylase; DBH-I, immunoreactivity to dopamine
beta hydroxylase; DTs, degenerating terminals; ICM, intercalated cell
masses, islands, or nuclei; MSN, medium-sized spiny neuron; PIC,
paracapsular intercalated islands; TH, tyrosine hydroxylase enzyme;
TH-I, immunoreactivity to tyrosine hydroxylase enzyme.
mission, anxiety, tyrosine hydroxylase, dopamine D1 recep-
tor.
INTRODUCTION
The amygdala seems to be of paramount importance for
the modulation of fear and anxiety (for reviews, see
LeDoux, 2000; Everitt et al., 2003; Engin and Treit,
2008; Perez de la Mora et al., 2008). It comprises a
number of nuclei, and an array of afferent, efferent, and
interconnecting fibers (Nauta, 1962; Swanson and
Petrovich, 1998; McDonald, 2003) occupying a large part
of the mammalian medial temporal lobe. On the basis of
cytoarchitectonic, histochemical, and connectional
criteria (Cassell et al., 1986; Swanson and Petrovich,
1998; McDonald, 2003) it is considered that the medial
and central nuclei (Ce) are related to the striatum
whereas the basolateral, lateral, and basomedial nuclei
(i.e., basolateral complex) are related to the cerebral
cortex. Among the major nuclei and fiber systems
associated with it, are several distinct clusters of
neurons composed of medium- to small-sized spiny
cells. These neuron-groups have collectively been
termed intercalated cell masses, islands, or nuclei of the
amygdala (ICM) (de Olmos et al., 1985). While the
precise boundaries for each ICM may not be sharp, they
have been classified according to their relative size or
position with respect to the external capsule (see de
Olmos et al., 1985). Developmental studies have
indicated that although both the paracapsular
intercalated islands (PICs) and the Ce have a subpallial
origin, the PIC and at least a part of the Ce derive from
progenitors located in the dorsolateral and ventrolateral

0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neuroscience.2012.08.067
324
 D. Marcellino et al. / Neuroscience 226 (2012) 324–347
ganglionic eminence, respectively (Waclaw et al., 2010).
The basolateral complex, including the anterior part of
the basolateral nucleus (BLA) and the Ce are the main
input and output stations of the amygdala, respectively.
PICs (Millhouse, 1986) are clusters of GABAergic
neurons (Nitecka and Ben-Ari, 1987; McDonald and
Augustine, 1993; Paré and Smith, 1993a,b), which have
been proposed as cellular interfaces that control the
trafficking of nerve impulses to and within the amygdala.
Thus, the main ICM, located ventrally to BLA, and the
medial PICs which lie between the BLA and Ce seem to
modulate the traffic of impulses between these latter
nuclei (Royer et al., 1999). The lateral PICs, located
along the fibers of the external capsule, have been
implicated in controlling the flow of information from the
medial prefrontal cortex to BLA and Ce (Marowsky
et al., 2005; Pape, 2005). In addition to these neuron
clusters, there is a third neuron group, termed the
anterior intercalated cell mass (AIC), lying between
the rostral aspect of the BLA and the distal bundles of
the caudal component of the anterior commissure
(ACc). Although the AIC has been considered to be an
extension of the amygdala (Alheid et al., 2006), its
internal organization, neurotransmitters and putative
interactions with the adjacent structures remain to be
defined (Millhouse, 1986). It is thus far assumed that all
cellular clusters or cell masses exhibit similar
histological appearance and comparable cell types;
however, no systematic search for possible differences
among them have been carried out. Although important
differences in fibrillar (i.e., Timm), histochemical (i.e.,
AChE) (de Olmos et al., 1985), and connectional (Paré
and Smith, 1993a) characteristics have been noted. On
cytological grounds it has been implicit that ICMs are
composed of two major cell types; a medium-sized,
spiny neuron and a large neuron; the former being the
most prevalent throughout the ICMs. Giant neurons (i.e.,
60 lm) are rarely seen (Millhouse, 1986). Association of
ICM with the adjacent tracts is also stressed by the
latter author, although the contribution of them to local
afferences is largely unknown (see Paré and Smith
1993a). Hence, the establishment of the cytological
organization, the neurotransmitters, and local
connectivity of the AIC is regarded as a first, necessary
step, for the understanding of its role in the amygdala
and its interactions with other regions of the basal
forebrain (Paré and Smith, 1994).
The importance of PICs in the amygdaloid circuits for
fear and anxiety has been demonstrated (Paré et al.,
2004; Ehrlich et al., 2009; Amir et al., 2011). Relevant
sensory information converging in the isocortex reaches
the BLA and particularly the lateral nuclei, and after its
local processing it is relayed to the Ce where
appropriate anxiogenic responses are implemented. By
gating the trafficking of nerve impulses between these
two amygdaloid nuclei (Royer et al., 1999, 2000), medial
PICs play an important role in fear and anxiety.
Furthermore, convincing evidence has accumulated that
the expression of fear extinction requires the medial
PICs since their selective destruction with the toxin
saporine coupled to dermorphine, a selective opioid l-
325
receptor agonist, impairs this process (Likhtik et al.,
2008). A prefrontal cortex-dependent potentiation of
BLA inputs to the medial PIC is associated with fear
extinction (Amano et al., 2010). Lateral PICs in their
turn, via feed-forward inhibition, keep BLA neurons in an
inhibited state in the absence of fear, but their inhibition
by dopamine (vide infra), which is known to be released
within the amygdala under stressful situations (Young
and Rees, 1998; Inglis and Moghaddam, 1999;
Yokoyama et al., 2005), enables BLA projection
neurons to participate in the generation of anxiety
responses (Marowsky et al., 2005; Pape, 2005).
Dopamine D1 receptors, located on intercalated
GABA neurons (Scibilia et al., 1992; Maltais et al.,
2000; Fuxe et al., 2003; Jacobsen et al., 2006; Pinto
and Sesack, 2008) mediate the dopaminergic
attenuation of the feed-forward inhibition in the BLA
and Ce exerted by these GABA neurons following
their activation by prefrontal glutamate afferents (see
Marowsky et al., 2005; Pape, 2005). We have
therefore focused our analysis on the
immunocytochemical detection of tyrosine hydroxylase
(TH), dopamine beta hydroxylase (DBH) and DA D1
receptor (D1) in the AIC and surrounding nuclei,
continuing a previous immunocytochemical analysis of
main and PIC groups as seen in coronal sections
(Fuxe et al., 2003).
As the ICMs are regarded as anatomically and
connectionally distinct entities, the current study was
conducted to characterize the cell types and
interactions within the AIC, PIC, and with the adjacent
fiber systems as visualized in horizontal sections
processed with silver and immunohistochemical
techniques. The contribution of dopaminergic nerve
terminals to the AIC neuropil was investigated in the
context of volume and wiring transmission (Fuxe
et al., 2007, 2010; Agnati et al., 2010). Additionally,
the subcellular organization of both neurons and
neuropil of the AIC was studied with the electron
microscope. Since our initial observations in rapid-
Golgi impregnated sections suggested the existence of
a synaptic interaction between the axon collaterals
from the ACc and the AIC neuropil, a supplemental
group of animals was submitted to surgical interruption
of the ACc to search for anterograde degeneration in
the AIC.
EXPERIMENTAL PROCEDURES
Animals
In all cases adult (i.e., 10-week-old) male Charles River rats were
used. Animal manipulation and sacrifice were performed under
the guidelines and with the approval of the Animal Research
Committee of Instituto de Neurobiologı́a or the Swedish
National Board for Laboratory Animals, and approved by ad
hoc ethics committees on animal experiments. All animals were
killed by decapitation or vascular aldehyde perfusion under
deep anesthesia (i.e., 30 mg/kg pentobarbital) or as separately
described below.
326 D. Marcellino et al. / Neuroscience 226 (2012) 324–347
Rapid-Golgi technique
The brains from 45 animals were obtained from deeply
anesthetized animals (see previous section). The brain of each
rat was removed, and tissue samples consisting of 3- to 4-mm-
thick blocks were obtained in the horizontal plane. The
temporal lobe was cut and sliced with a razor blade into parallel
sections. Each tissue block was left for 20 days in an aqueous
solution containing 3% potassium dichromate and 0.25%
osmium tetroxide and then transferred to a 0.75% silver nitrate
solution for the following 12–20 days. The resulting tissue
blocks were held with an external shell of paraffin, and 120- or
150-lm-thick sections were cut on a sliding microtome. The
sections were then left for 10 min in 70% ethyl alcohol,
dehydrated in graded solutions of propyl alcohol/water, cleared
in terpineol/xylene, mounted, and coverslipped.
Cajal’s reduced silver technique
To define axonal tracts and bundles, eight brains were obtained
as described in previous section and processed according to
Cajal’s reduced silver technique. The tissue blocks were
immersed in a solution containing 30% ethyl alcohol and 1.5%
silver nitrate for 4 days. Then, the specimens were left for 24 h
in an aqueous solution containing 1.8% hydroquinone and 3%
paraformaldehyde. Afterward, tissues were embedded in
nitrocellulose and polymerized with ether vapors. The tissue
blocks were then sectioned at 30-lm intervals with a sliding
microtome operated at room temperature. Finally, the sections
were dehydrated in propyl alcohol, cleared in xylene, and
coverslipped with Entellan resin.
Cresyl Violet staining
Brains were embedded in paraffin and cut horizontally at 10 lm.
Then, the sections were immersed for 20 min in a solution
containing 0.1 g of Cresyl Violet and 0.3 ml glacial acetic acid
dissolved in 100 ml of distilled water. Following staining
sections were dehydrated in graded ethanol/water solutions,
cleared in xylene and coverslipped with Entellan.
Electron microscopy
Six rats (n = 6) were utilized in this part of the study. Deeply
anesthetized animals (see Section ‘Animals’) were perfused
through the left ventricle with 250 ml of 4% paraformaldehyde–
2.5% glutaraldehyde in 0.15 M phosphate buffer (PB), pH 7.3.
Following dissection, the brain was immersed in additional
fixative and stored overnight at 4 °C. After 24 h, half- to one-
millimeter horizontal slices through the base of the temporal
lobe were obtained. Areas of interest were sampled under a
dissecting microscope, and the tissues were postfixed for 1 h in
1% osmium tetroxide dissolved in the same buffer as the
aldehydes, dehydrated in graded acetone, and flat embedded
in epoxy resins. Half- to one-micrometer-thin sections were
obtained from the tissue blocks with a Leica ultramicrotome
equipped with glass knives (Leica Microsystems, Mannheim,
Germany). The sections were stained with Toluidine Blue and
coverslipped. From the exposed surface of these trimmed
blocks, ultrathin sections ranging from 80 to 90 nm were
obtained with a diamond knife and mounted on 100-mesh
copper grids. The sections were sequentially stained with
aqueous solutions of uranium acetate and lead citrate and then
studied in a JEOL 1010 electron microscope. Micrographs from
serial sections were merged with the Reconstruct software
(Fiala, 2005).
Surgical interruption of the anterior commissure
To define a possible synaptic interaction between the anterior
commissure and the anterior intercalated cell masses, six adult
rats were included; from these animals, four of them received a
complete section of commissural axons, and two of them were
sham lesioned according to the following procedure. Under
barbiturate anesthesia each animal was held by the auditory
meatuses in a sterotaxic apparatus. A small 3  4-mm window
was drilled and the bony shell removed. Then, an L-shaped
knife measuring 0.7 mm in length (i.e. anteroposteriorly) was
aligned to the sagittal plane. The posterior part of the knife (i.e.
stem) was positioned 0.8 mm caudal from bregma and the
blade itself aligned parallel to the dorsal aspect of the sagittal
superior vein. Then the blade was lowered 8.8 mm, and
removed. Into two sham-lesioned animals, the same
maneuvers were performed but the knife was lowered only
7.0 mm. All animals were left undisturbed until spontaneous
recovery ensued. Three days after the operations animals were
perfused through the left ventricle, and specimens processed
as described in section ‘Cajal’s reduced silver technique’.
Immunohistochemistry
Three rats were perfused under deep anesthesia (see
Section ‘Animals’) using 4% paraformaldehyde and 0.2% picric
acid in 0.1 M sodium phosphate buffer (pH 6.9). The brains
were removed, post fixed for 2 h in the perfusion solution at
4 °C, and rinsed for 48 h in 10% sucrose in 0.1 M phosphate
containing 0.01% w/v sodium azide. The brains were frozen in
a jet stream of CO2 gas and sectioned onto slides using a
cryostat. Ten-micrometer horizontal (Bregma 7.6 to 9.6 mm)
serial sections were incubated overnight at 4° C with primary
antibodies: rat anti-D1 receptor (Sigma Aldrich, St. Louis, MO,
USA) at a concentration of 5 lg/mL, 1:500 dilution of rabbit
anti-TH (Millipore, Temecula CA, USA) and with a 1:1000
dilution of mouse anti-DBH (Millipore, Temecula CA, USA) for
confocal microscopy analysis. Coronal sections (Bregma 2.3
to 2.6) were occasionally utilized (e.g., Fig. 1).
Antibody characterization. Table 1 summarizes the details of
the primary antibodies used in this study:
1. Dopamine D1 receptor antibody. The D1R monoclonal anti-
body (Sigma Aldrich) is raised against the human dopamine
receptor using the 97-amino-acid C-terminus fused to GST
fusion proteins and then derived from spleen cells of rats that
contained the hybridoma cell line reactive with D1 (Levey et al.,
1993; Hersch et al., 1995). The specificity of the D1 antibody
has been demonstrated using cloned D1 receptors in transfec-
ted COS-7 cells with Western blots showing reactivity for only
the D1 receptor with corresponding bands at 40–45 kDa and
65–75 kDa (Levey et al., 1993; Hersch et al., 1995). The spec-
ificity was also demonstrated in isolated rat membranes from
the striatum, which showed a band at 65–75 kDa, a mobility
similar to the cloned receptor (Hersch et al., 1995). This anti-
body was first described by Mitrano DA, Smith Y (2007) J
Comp Neurol 500:788–806. Sigma Aldrich now sells this anti-
body under exclusive license from Emory University. In our
study, it was possible to confirm the distribution of nerve cells
and nerve terminal populations expressing dopamine D1
receptor in different parts of the forebrain including dorsal stri-
atum, intercalated islands (Fuxe et al., 2003), and entopedun-
cular nucleus, respectively (see Fig. 1E).
2. Tyrosine hydroxylase polyclonal antibody (Millipore, Temecu-
la, CA, USA). The rabbit anti-TH polyclonal antiserum
(AB152) was produced using denatured, full-length TH from
rat pheochromocytoma (denatured by sodium dodecyl sul-
fate) and does not react with other closely related catechol-
amine (CA) enzymes, including dopamine b-hydroxylase
 D. Marcellino et al. / Neuroscience 226 (2012) 324–347 327

Fig. 1. Survey micrographs of the amygdala and surrounding regions. (A) Survey light micrograph of the amygdala in a coronal section. The boxed
area defines the high-magnification micrograph reproduced in ‘‘B.’’ (B) Position of the anterior intercalated islands (arrows) with respect to the
amygdaloid nuclei. Note that the anterior intercalated cell masses split around the anterior aspect of the basolateral nucleus (BLA). The major
amygdaloid nuclei in the dorsal part and surrounding areas are indicated. (C). Micrograph from a horizontal section of the left side of the forebrain.
An anterior intercalated cell mass (arrowheads) is bounded by the distal axonal bundles (asterisks) of the caudal component of the anterior
commissure (ACc). Posteriorly, the anterior aspect of the basolateral (BLA) (outlined in blue) and central amygdaloid (Ce, outlined in white) nuclei
are found. (D–E) Coronal section (Bregma level 2.3 to 2.5) immunostained for tyrosine hydroxylase (TH; in red) and dopamine D1 receptor (D1R;
in green) and observed using Odyssey Infrared imaging. (F) Pseudocolored overlay of the two staining patterns with TH (red) and D1R (green)
immunoreactivity. Adult rat specimens. A–C, Cajal’s reduced silver. Scale bars = 100 lm in A–C, and 2 mm in D–F. ACc = anterior commissure,
caudal part; BLA = anterior aspect of the basolateral amygdaloid nucleus; Ce = central amygdaloid nucleus; D1R = dopamine D1 receptor;
EC = external capsule; EP = entopeduncular nucleus; La = lateral amygdaloid nucleus; FStr = fundus striati, St = stria terminalis; TH = tyro-
sine hydroxylase.

(DBH), phenylalanine hydroxylase, tryptophan hydroxylase, donkey anti-mouse (DyLight–633) secondary antibodies
dihydropteridine reductase or phenylethanolamine-N-methyl- (Jackson ImmunoResearch Europe Ltd. Newmarket, Suffolk,
transferase using Western blot methods (manufacturer’s UK) at a dilution of 1:200. Tissue sections were cover-slipped
technical information). The specificity of the antibodies was using Fluoroshield with DAPI (Sigma Aldrich) and kept at 4° C
assessed by documenting the localizations of immunopositive until imaged. Confocal microscopy was performed using a
cells and terminal networks throughout the serial sections of Leica SP2 AOBS confocal microscope (Leica Microsystems,
the rat brain and comparing them with those described in Mannheim, Germany) equipped with a 4-laser configuration to
the literature (Rivera et al., 2008). This antibody was recently a Leica DM IRE2 inverted microscope.
described by Fetissov et al., 2009 J Comp Neurol 513:1–20.
3. Dopamine b-hydroxylase antibody. The DBH monoclonal anti-
OdysseyÒ Infrared Imaging. Following primary antibody
body (Millipore) was raised against purified bovine adrenal
incubation, sections were incubated for 1 h at RT with goat
DBH (manufacturer’s technical information). The specificity
anti-rat (IRDyeÒ 800CW) and goat anti-rabbit (IRDyeÒ 680CW)
of this antibody was confirmed with the immunohistochemical
secondary antibodies (Li-COR Biosciences, Cambridge, UK) at
labeling of brainstem noradrenergic (NA) cell groups and their
a dilution of 1:2000. Fluorescence was detected using the
NA terminal networks throughout the central nervous system,
OdysseyÒ Infrared Imaging System (21 lm resolution, 1.2 mm
and is extensively documented in the literature and accepted
offset with highest quality). Channel sensitivity was optimized
by the scientific community (Rivera et al., 2008). Perikaryal
for each set of stained sections and maintained for that group
axon, and axonal terminal immunolabeling was absent when
of samples. The relative location of the slice and identification
this antibody was preadsorbed with a 10-fold excess of bovine
of brain regions were determined by comparison to images in
adrenal DBH (Sigma Aldrich) (Rinaman, 2001). This antibody
Paxinos and Watson Rat Brain Atlas (1986).
was recently described by Lee SB, et al. (2002) J Comp Neu-
rol 514:387–402.
Data analysis

Confocal microscopy. Following primary antibody incubation, Neurons, fiber bundles, axons, and their collaterals were
sections were incubated for 2 h at room temperature with donkey reproduced with a camera lucida adapted to a Zeiss Axioplan 2
anti-rat (DyLight–546), donkey anti-rabbit (DyLight–488) and microscope, utilizing 10, 40, or 100 objectives (numerical
328 D. Marcellino et al. / Neuroscience 226 (2012) 324–347

Table 1. Antisera

Antigen Immunogen Source of antibody Dilution used

Dopamine D1 receptor Recombinant fusion protein containing the C-terminal Sigma Aldrich, rat monoclonal, #D2944, 5 lg/ml
97 amino acid of human D1 dopamine receptor lot 128K4770
Tyrosine hydroxylase Denatured tyrosine hydroxylase from rat Millipore, rabbit polyclonal, #AB152, lot 1:500
NG1723896
Dopamine b-hydroxylase Purified bovine DBH (from adrenal medulla) Millipore, mouse monoclonal clone 1:500
4F10.2, #MAB308, lot LV1696521

aperture = 0.3, 1.0, and 1.4, respectively). Drawings from forty,

fully impregnated neurons of each type were included in the
morphometric analysis (vide infra). The drawings were
scanned, digitized, and measured with a personal computer
aided by Kontron 400 software. Measurements included the
largest and transverse axes of the soma. Drawings presented
in Figs. 3–5 and 7 were obtained from horizontal sections,
Interaural 1.4 and Bregma 8.6 mm determined from
Paxinos and Watson Rat Brain Atlas (Paxinos and Watson,
1986). A set of 50 medium-sized spiny neurons (MSNs; see
Section ‘Cytological organization of the anterior intercalated
islands’), chosen on the basis of the quality of the impregnation
and completeness of their processes, was utilized to define
axonal direction (i.e., projection). Synapses were counted in
digital electron micrographs obtained from a minimum of three
sets of ultrathin sections, from three different animals. At an
initial magnification of 12,000, a total area of 10,000 lm2
per animal was photographed. The numbers of axo-spinous
synapse (AxSp) (see Section ‘Interactions of AICs with BLA
and Ce neurons’) and axo-shaft synapse (AxSh) were
determined, and the resulting numerical densities were
averaged and expressed as percent of total synapses.

RESULTS
Cytological organization of the anterior intercalated
islands
In coronal sections impregnated with Cajal’s reduced
silver method or double immunolabeled for TH and D1
immunoreactivity (Fig. 1) the major neuronal groups of
the amygdala are indicated as well as amygdaloid nuclei
(Fig. 1A and B) showing codistribution of TH and D1
immunoreactivities can be visualized (Fig. 1D–F). The
nerve cell clusters of the AIC can be visualized in
aniline-stained sections (Figs. 2A–C and 3A–C). The
Fig. 2. Light photomicrographs from horizontal sections in plastic-
AIC lies between the axon bundles of the caudal
embedded specimens. (A) Low-power view showing the caudal limb
component of the anterior commissure (ACc), facing the of the anterior commissure (ACc) surrounding (asterisks) two neuro-
BLA and Ce caudally (Fig. 1C). Serial sections disclose nal clusters (arrows) of the anterior intercalated cell masses. (B) An
the intimate association of the ACc fascicles with the intercalated cell mass containing several medium-sized spiny neu-
AIC (Figs. 4 and 5D). Although the neuron clusters rons (black arrows) and a large-sized neuron (white arrow). Note the
nuclear capsule composed of myelinated axons. (C) High-power
described here have been considered a rostral part of micrograph showing a medium-sized (black arrow) and a large
the main intercalated nucleus, the term AIC is preferred; neuron containing a large invaginated nucleus (white arrow) sur-
largely because serial, horizontal sections reveal that rounded by Nissl substance (arrowheads). Asterisks = myelinated
the two limbs organizing the AIC are united ventro- axons surrounding the nucleus. (D) Sagittal knife-cut through the
anterior commissure (AC); note the extensive vacuolization of that
caudally. Perhaps more important is that both AIC
area of the anterior commissure adjacent to the knife cut (hollow
neuronal clusters are completely encased by arrows). BNST = bed nuclei of the stria terminalis, III = third ventri-
commissural fibers and, therefore, separated from the cle. Adult rat brain, one-micrometer-thick sections. Scale
main intercalated nucleus. bars = 100 lm in A; 20 in B; 10 in C, and 200 in D.
The general organization of the AIC and PIC and their
interaction with neighboring structures as revealed by
silver impregnations are presented in camera lucida composed of numerous, MSN and occasional large, non-
drawings (Figs. 5–9). As demonstrated earlier by spiny, neurons. Survey views of the AIC show that MSN
Millhouse (1986) the cell clusters of the AIC are primarily somata and processes are tightly packaged within the
 D. Marcellino et al. / Neuroscience 226 (2012) 324–347 329

Fig. 3. Representative composite photomicrographs from a horizontal section of the left temporal lobe. (A) Relationships of the anterior intercalated
(AIC), and paracapsular intercalated islands (PIC) with respect to the anterior basolateral (BLA) and lateral (La) nuclei of the amygdala (dashed
lines). Photomicrographs at high magnifications showing AIC (B and C) and LPIC (C). Sections have been stained with 0.5% Cresyl Violet
and correspond to Bregma level 8.2 to 8.3 from the Paxinos and Watson stereotaxical atlas (1986). Abbreviations: AIC, anterior intercalated
islands; BLA, anterior aspect of the basolateral nucleus; Ce, central amygdaloid nucleus; La, lateral nucleus; LPIC, lateral paracapsular intercalated
islands. Nissl staining, adult rat brain. Scale bars = 250 lm in A; 100 lm in B and C.

Fig. 4. Serial horizontal sections from dorsal (A) to ventral (F) through the distal part of the caudal limb of the anterior commissure of the right
cerebral hemisphere. Note that bundles of the anterior commissure (hollow arrow) proceed laterally to reach the rostral aspect (white arrow) of the
amygdala. Then, axon bundles encase the anterior intercalated cell masses (arrowheads). Cx = cerebral cortex; EC = external capsule). Adult rat
brain, Cajal’s reduced silver nitrate. Scale bar = 100 lm.
330 D. Marcellino et al. / Neuroscience 226 (2012) 324–347

Fig. 5. Camera lucida drawings depicting position and relationships of the intercalated cell masses of the amygdala. (A) The anterior intercalated
cell mass (AIC, black arrows) lies rostral to the basolateral (BLA) and lateral to the central (Ce) nuclei of the amygdala. BM = basal medial nucleus
of the amygdala, St = stria terminalis. (B) Higher magnification view of the anterior intercalated cell mass (AIC). A large, aspiny, neuron (green) is
seen surrounded by numerous medium-sized neurons in the AIC. Adjacent to it is the most rostral island of the medial paracapsular cell mass (open
arrows) that proceeds medially between the central (upper) and basolateral (dotted) amygdaloid nuclei. Note that the external capsule (EC) together
with the caudal part of the anterior commissure (ACc), build-up a fibrous capsule around the AIC. Black arrow = pyramidal cell in the basolateral
complex of the amygdala. i: The large aspiny neuron drawn in B (green) is reproduced at higher magnifications. (C) High-magnification drawing of
the AIC. Note the tight packaging of dendrites distributed in the nuclear neuropil. At the bottom there is a soma of a pyramidal-like cell (dashed) of
the basolateral amygdaloid nucleus (dotted). (D) Distal axonal bundles of the caudal component of the anterior commissure (single arrow head)
diverge and surround an AIC area (asterisk) that lies rostral to the basolateral (double arrow head) and central nuclei (double asterisk) of the
amygdala. Arrow = external capsule. Scale bars = 200 lm in A, 40 lm in B, Bi, C and D. Adult rat specimen, horizontal sections, Golgi–Cox–Nissl
staining. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

nuclear domain (Fig. 5A–C). The MSN soma is oval, and it spine-like out-pocket can be seen (Fig. 6). The overall
averages 16.5 lm in the longest axis. The contour of a structure of an MSN is bipolar since the soma usually
MSN soma is generally smooth, although an occasional gives rise to paired dendrites that diverge in opposite
 D. Marcellino et al. / Neuroscience 226 (2012) 324–347 331

Fig. 6. High-magnification camera lucida drawing from a medium-sized spiny neuron in the anterior intercalated cell mass. The spindle-shaped
soma exhibits two sets of spinous dendrites, one of them pierces the axonal capsule and exhibits large tuberosities (encircled).
Arrowheads = axon; arrow = somatic spine-like. i: Cartoon showing the relative position of the neuron dendrites with respect to the nuclear
capsule. ii: Higher magnification drawing showing a dendritic shaft (boxed) having numerous pediculated spines alternating with a few large, square-
shaped spines (arrows). Rapid-Golgi technique, adult rat brain. Scale bars = 10 lm and 5 in ii.

directions (Figs. 6 and 7A). Each primary dendrite ramifies
once or twice originating relatively long (i.e., >100 lm),
terminal branches. Regardless of order, dendrites are
varicose and exhibit a moderate to high number of spines
(Figs. 5C, 6 and 7). On the basis of their shape and
dimensions, two types of dendritic spines are found. A
first type or fungiform is by and large the most prevalent,
corresponding to typical dendritic spines observed on
neurons in basal ganglia (Wilson and Groves, 1980) and
the BLA (Fig. 7A). Another type of dendritic spine
consists of a roughly squared-shape protuberance
measuring 0.9–1.3 lm and devoid of a pedicle (Figs. 6,
inset ii, and 8). The latter spines are relatively infrequent,
as they average nine to ten percent of the total population
of the MSN dendrite. A set of distal, i.e., terminal,
dendrites exhibits still a third structural differentiation. In
fact, those dendrites piercing the axonal capsule of the
AIC display a series of two to four large (i.e., > 1 lm)
tuberosities (Figs. 6, inset ii, and 9A), that mimic those
described for the so-called heterodendritic neuron in the
core of the oval nucleus of the bed nuclei of the stria
terminalis (Larriva-Sahd, 2006). The MSN axon, like that
of the intercalated neuron (Royer et al., 2000), arises
from the soma (Figs. 7A and 9A), or occasionally, from a
primary dendrite (Fig. 6). Aside from origin, the parent
axon follows a wide undulating trajectory through the AIC
providing one or two thin collaterals that resolve in the
neighboring neuropil. In 50 well-impregnated neurons it is
332 D. Marcellino et al. / Neuroscience 226 (2012) 324–347

Fig. 7. Camera lucida drawings illustrating putative connectivity of the anterior intercalated cell islands. (A) Distal axons from the caudal limb of the
anterior commissure (arrows), together with ascending, spiny, dendrites from pyramidal-like cells (hollow arrows) in the basolateral nucleus of the
amygdala (BLA) bound the medial aspect of the anterior intercalated cell masses (shaded, AIC). Arrowhead = axon of a medium-sized spiny
neuron. (B) Neurons of the anterior intercalated cell masses (dashed) lie between the basolateral nucleus of the amygdala (BLA) and the caudal
limb of the anterior commissure (ACc). Note that axons arising from the anterior commissure (arrowheads) and pyramidal-like cell (black arrows)
originate thin collaterals that appear to contact somata and proximal dendrites of medium-sized spiny neurons (dashed) in the anterior intercalated
cell mass (AIC). Adult rat, rapid-Golgi technique. Scale bars = 20 lm.

found that the MSN parent axon leaves medially (68%)
(Fig. 9A), laterally (22%), or anteriorly (10%) (Fig. 6).
Axons running medially originate successive collaterals
to the latero-capsular and medial part of the Ce before
the axon leaves the plane of sectioning or impregnation
ceases. Axons directed laterally incorporate into the
external capsule, while those traveling rostrally parallel
the ACc fibers.
Only three medium-sized aspiny neurons (Fig. 5B)
were identified in our study. As described earlier by
Millhouse (1986), the soma of an aspiny neuron is
larger (>25 lm) than that from an MSN, ovoid, and
gives rise to long, smooth, dendrites. Accordingly, the
paired dendrites of these cells lack dendritic
appendages and usually extend beyond the nuclear
confines. No axon, other than the initial segment, was
visualized in our sample.
Extrinsic fibers and processes to the AIC
The AIC is intimately associated with axons from the
adjacent ACc and receives both dendritic and axonal
contingents from the BLA.
 D. Marcellino et al. / Neuroscience 226 (2012) 324–347
Fig. 8. Seventy-two exposure photomontage showing axon collater-
als (hollow arrows) arising from commissural axons (white arrows)
that appose proximal dendrites (circles) of the medium-sized neuron
at the upper right. Black arrows = large, non-pediculated, spines.
Adult rat brain, horizontal section, rapid-Golgi technique. Scale
bar = 50 lm.
The first and chief source of extrinsic afferents
appears to be the ACc. As shown in Figs. 1C, 4 and
5D, the AIC is surrounded by fibers from the ACc that
encase it. In fact, as soon as the ACc approaches the
external capsule, a series of six or seven bundles fan-
out (Figs. 1C and 5D). The most medial bundles
accommodate the two limbs that structure the AIC
(Figs. 1B, C and 2A). High-magnification inspection of
silver-impregnated sections shows that commissural
axons give rise to collaterals that approach dendrites of
the MSN of the AIC establishing putative synaptic
contacts with them (Fig. 7B and circles in Fig. 8).
Furthermore, these fibers may proceed further caudally
to other groups of PICs (Fig. 5B) or to distal dendrites
of pyramidal-like neurons from the BLA (Figs. 5A and
7A). A second source of putative axon terminals derive
from BLA neurons. Indeed, a set of axons arising from
pyramidal-like cells in the BLA leaves the nucleus,
penetrating via the caudal part of the AIC. Each axon
provides short collaterals that resolve throughout the
AIC neuropil (Fig. 7B) (see Amir et al., 2011), then the
parent fiber proceeds further rostrally. Lastly, a distinct
contribution to the AIC neuropil is represented by the
distal processes of large pyramidal-like BLA neurons
(Millhouse and DeOlmos, 1983). In fact, numerous thick
dendrites of large pyramidal-like neurons extend
anteriorly, piercing the caudal aspect of the AIC to
penetrate the nucleus proper (Fig. 5B) or, as just
mentioned here, bound it medially (Fig. 7A).
Interactions of AICs with BLA and Ce neurons
As has been reported for other intercalated insular
systems (Millhouse, 1986; Royer et al., 1999, 2000)
including the main intercalated island (Manko et al.,
2011), BLA neurons send afferents to the AIC. The
specimen reproduced in Fig. 7B shows an example of
an ascending axon of a pyramidal-like neuron that
pierces the AIC and distributes within its neuropil.
333
Reciprocally, dendrites from pyramidal-like neurons of
the BLA reach the AIC (vide supra) (Figs. 5B and 7A)
and seem to receive synaptic contacts from the axon
collaterals of medium spiny neurons. Additionally, a set
of AIC axons traveling caudally (Fig. 5A, arrowhead)
penetrate into the BLA neuropil. Lastly, the occasional
dendrites of pyramidal-like cells that pierce successively
the MPIC and Ce, appear to be contacted by the axons
from the MSNs in the former (Fig. 9B). Interestingly,
medium-sized neurons in the MPIC with rostro-caudally
oriented dendrites (Fig. 9B) send axons medially, that
may contact dendrites from both pyramidal-like neurons
in the BLA and neurons in the latero-capsular part of the
Ce.
Immunohistochemistry
D1/TH and TH/DBH double immunolabeling analysis of
the amygdala in horizontal sections. Focus on the
intercalated cell islands. The architecture of the AIC
and PIC can be demonstrated at low magnifications in
horizontal sections through their strong D1 receptor
immunoreactivity (D1-I) (Fig. 10). The AIC as well as
both the medial and lateral PIC show a strong green
D1-I. The tyrosine hydroxylase-immunoreactive (TH-I)
terminals visualized through their strong red labeling,
are seen to substantially innervate the AIC, while their
innervation of the caudal component of medial and
lateral PIC is negligible (Fig. 10). A dense TH-I terminal
network is demonstrated in the latero-anterior and
latero-capsular part of the Ce (Fig. 10B, C). TH-I
terminals are found to a lesser extent in the anterior
BLA but they have no clear association to D1-I.
The relationships between the high density of punctate
D1-I profiles (green), likely spines, and the large number of
TH-I dopamine varicosities (red) in the AIC have been
studied at higher magnifications (Fig. 11A, C, E). As seen
in Fig. 11B, D, F showing TH-I and the enzyme dopamine
beta hydroxylase immunoreactivity (DBH-I) instead, the
vast majority of the TH-I terminals in AICs lack DBH-I
(green) and therefore represent dopamine nerve
terminals. As seen especially in Fig. 11E, the distances
between the punctate D1-I and the TH-I varicose
terminals are in the micrometer range as inferred from
the scale bars with some seemingly direct contacts
between them. The punctate green D1-I profiles are
located between the densely packed nerve cell bodies
with blue nuclei (DAPI stained) and are substantially
smaller in size than the TH-I varicosities. In view of the
dense dendritic network demonstrated in the AIC in
specimens after the rapid-Golgi technique, it likely
represents D1-immunoreactive dendritic profiles with
spines. In the area medial to and within the AIC
(Fig. 11B) a few green DBH-I terminal profiles with slight
yellowish fluorescence (indicated by arrows) display TH-I
in Fig. 11A. However, in the hypothalamus, for
comparison, the vast majority of the DBH-I terminals
(green) develop a distinct yellow fluorescence after co-
labeling for TH (red immunofluorescence, in Fig. 12).
Previous work has also indicated that cortical
noradrenergic terminals, including those in the
334 D. Marcellino et al. / Neuroscience 226 (2012) 324–347

Fig. 9. Axonal interactions of medium-sized spiny neurons of the intercalated cell masses with the adjacent amygdaloid nuclei. (A) Axons (blue and
green) from two medium-sized neurons give rise to successive axon collaterals to the intercalated cell mass proper (asterisk) as well as to the
lateral- (CeLC, hollow arrows), and medial-capsular parts (CeM, black arrows) of the central amygdaloid nucleus. Note that incoming commissural
fibers (soft pencil) send axon collaterals (double arrowheads) that distribute in the neuropil adjacent to the medium-sized spiny neurons. Distal
dendrites, impinging commissural axons acquire a tuberous appearance (arrowheads). (B) The axon (green) of a spiny neuron (black) in the medial
paracapsular intercalated cell mass (MPIC) runs horizontally and appears to terminate successively on distal dendrites of a pyramidal-like neuron
(gray) and in neurons in the lateral part of the central nucleus of the amygdala (CeLC). AIC = anterior intercalated cell mass; BLA = basolateral
amygdaloid nucleus; EC = external capsule. Rapid-Golgi technique, adult rat brain. Scale bars = 20 lm. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)

amygdaloid cortex, show reduced TH-I compared to
dopamine terminals (Hokfelt et al., 1977).
The relationships between the high density of
punctate D1-I profiles (green) and the moderate number
of varicose TH-I nerve terminals (red) in medial and
lateral PICs have also been visualized at higher
magnifications (Fig. 13A, C). TH-I and DBH-I in
Fig. 13B, D demonstrate that the vast majority of the
TH-I terminals in medial and lateral PICs lack DBH-I
(green) and therefore represent dopamine nerve
terminals. In view of the lower density of TH-I terminals
compared with the AIC the distances of the TH-I
terminals to the punctate D1-I profiles of the LPIC
become substantially larger. In areas surrounding the
lateral and medial main intercalated cell islands a
substantial number of TH-I terminals (red) are also
found that lack DBH-I, giving evidence for the existence
of dopamine terminals also in these regions (Fig. 13B,
D). A similar density of DBH-I terminals appeared to
exist in these nuclei as well, and in several cases they
could be found to contain a red TH-I fluorescence
(arrows in Fig. 13B, D; see above) leading to yellowish
fluorescence in the merged image.
As demonstrated in Fig. 14A, a very dense TH-I nerve
terminal plexus lacking DBH-I (Fig. 14B) exists in the
latero-anterior subnucleus of the Ce, thus representing a
very rich dopamine nerve terminal plexus. This dopamine
terminal plexus is, however, not associated with D1-I,
which is the case for AIC and PICs (Figs. 11, 13A, C and
15A). This appears to be the case also for the moderately
dense plexus of TH-I- and DBH-negative nerve terminals
in the BLA (Figs. 12B, D and 13B). It should be noted
however that a weak D1-I might exist in parts of BLA
(Fig. 13A). Moving caudally in BLA toward BL, DBH-I
(green) terminals of low density are found representing
noradrenergic terminals (Fig. 12B, D) in which the TH-I
 D. Marcellino et al. / Neuroscience 226 (2012) 324–347
Fig. 10. Rostro-caudal distribution of tyrosine hydroxylase (TH)- and
dopamine D1 receptor (D1R) immunoreactivities in horizontal sections
through the left side amygdala. The brain area found in the rectangle
of panel A is found at higher magnifications in panels B and C. Partial
codistribution of TH (red) and D1R (green) immunofluorescence
(yellowish) is mainly observed in the anterior intercalated cell islands
(AIC), lateral and medial paracapsular intercalated islands in the
rostral part (LPIC and MPIC, respectively) mainly show up with a
weakly yellowish D1R immunofluorescence. The caudal component
of LPIC and MPIC shows a green fluorescence since hardly any TH
immunofluorescence exists within them. A strong TH immunofluo-
rescence due to densely packed TH-positive nerve terminal networks
was observed in the central nucleus (Ce) while TH immunofluores-
cence of a medium intensity is found in the anterior basolateral
amygdaloid nucleus (BLA). Only low immunofluorescence was
observed in the lateral amygdaloid nucleus (La). Horizontal sections
were taken at Bregma level 8.2 to 8.3 from the Paxinos and
Watson stereotaxical atlas (1986). Lateral (L), medial (M), dorsal (D)
and caudal (C) directions are indicated in B. Scale bars = 2 mm (A),
300 lm (B) and 150 lm (C). Abbreviations: AIC, anterior intercalated
islands; BLA, basolateral nucleus; Ce, central amygdaloid nucleus;
CeLA, central–anterolateral subdivision; CeLC, centro-laterocapsular
subdivision; D1R, dopamine D1 receptor; La, lateral nucleus; FStr,
fundus striatum; LPIC, lateral paracapsular islands; MPIC, medial
paracapsular islands; LV, lateral ventricle; PRh, perirhinal cortex; TH,
tyrosine hydroxylase. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this
article.)
(red) is difficult to visualize at least at the magnification
used and for the same reasons discussed above.
Ultrastructure of the AIC neurons and neuropil
Because terminal fibrils and boutons en passage are
strongly suggestive (vide supra), but not conclusive, for
335
a synaptic interaction, our initial interpretation that the
ACc axons terminate on AIC neurons was further
evaluated in a group of adult animals. Information
regarding the ultrastructural characteristics of both AIC
neurons and neuropil in adult mammals is not available;
therefore, a general description will precede information
concerning experimental animals.
Neurons. In both aniline and epon-embedded
specimens the AIC is a well-defined, capsulated
nucleus, containing neurons that, on the basis of
somatic location and size, as well as the content of
cytoplasmic vesicles, may be classified into two types
(Figs. 2B, C, 16 and 17). The first and most common
neuron corresponds to the MSN that exhibits the
following profile. The soma of an MSN is elliptical and
contains a smooth, oval, nucleus that is usually centrally
located. The latter is composed of a coarsely granular
material with discreet heterochromatic clumps (Fig. 16A)
in which one or two small nucleoli are embedded
(Fig. 2C). The cytoplasm contains numerous
mitochondria and rough endoplasmic reticulum (RER)
that is composed of short tubular cisternae, scattered
throughout the perikaryon. Electron-dense granules, if
any, are rare (vide infra). Small neurotubular aggregates
are commonly seen at the root of proximal dendrites at
either neuronal pole (Fig. 17A). Occasional spine-like
structures protrude from the somatic cell membrane
(Figs. 6 and 18D). One of eight AIC neurons
corresponds to a large or type II neuron that bears the
following peculiarities. In addition to its larger size, a
type II neuron occurs chiefly at the nuclear periphery
(Figs. 2C and 16B). Although the cell nucleus is
generally smooth, one or two conspicuous invaginations
give it a dentate appearance (Figs. 2C and 16B). The
biosynthetic machinery of a type II neuron is well
developed. Thus, the Nissl substance occupies a
substantial part of the cytoplasmic area, being
composed of large, stacked RER cisternae (Figs. 2B, C,
16B and 17B). Numerous rounded mitochondria lie
between the RER cisternae. Adjacent to the nucleus,
one or two well-developed Golgi areas can be seen
associated with numerous, prominent lysosome-like
granules.
Neuropil. The AIC stands out for its homogeneous
aspect containing occasional small bundles of thin
myelinated fibers and four major types of synapses
(Fig. 18). By and large the most common synaptic type
observed is axo-spinous (AxSp), accounting for eight of
every ten boutons (i.e., 82%) in the AIC neuropil.
However, two sub-types can be identified based on the
structure of the postsynaptic element. The first consists
of a synaptic bouton containing numerous small,
agranular vesicles with occasional dense-cored vesicles
that are larger and establish asymmetrical contacts with
either small (<0.3/0.4 lm) pediculated spines or large
(i.e., <1/1.4 lm W/L) spines. The relative frequency of
the former AxSp terminal is double that of that AxSp
targeting a large spine. AxSp targeting small spines are
similar to those observed elsewhere in the basal ganglia
336 D. Marcellino et al. / Neuroscience 226 (2012) 324–347

Fig. 11. Double immunolabeling is given of tyrosine hydroxylase (TH) and dopamine D1 receptors (A, C, E) and TH and dopamine b-hydroxylase
(B, D, E) in the anterior intercalated cell mass as visualized in adjacent horizontal sections. A clear codistribution of TH and D1-R is demonstrated in
A, C and E around nuclear profiles stained blue with DAPI. Dopamine b-hydroxylase (DBH)-immunoreactive terminals are rare in adjacent sections
(B, D, F) indicating that the punctate TH immunofluorescence observed represents dopamine terminals. In panels B and D arrows point to a few
terminal puncta with a red TH and green DBH immunofluorescence giving a yellow fluorescence. Note that using confocal laser microscopy the
distances between the dotted dopamine D1R immunoreactivity, likely representing the dendritic spines of medium-sized AIC nerve cell bodies, and
the TH-immunoreactive terminals are in the micrometer range as seen from the scale bars likely to representing an extrasynaptic form of volume
transmission (C and D). In A, C, and E the TH immunoreactive area overlaps by about 50% with the D1-immunoreactive area. Thus, a substantial
degree of synaptic D1-mediated DA transmission may also exist in the AIC. Sections correspond to a Bregma level 8.2 to 8.3 from the Paxinos
and Watson stereotaxical atlas (1986). Scale bar = 47 lm (A, B), and 21 lm (B, C) and 8 lm (D, E). Abbreviations: AIC, anterior intercalated cell
mass; DAPI, 40 -6-diamidino-2-phenylindole; DBH, dopamine b-hydroxylase; D1R, dopamine D1 receptor; TH, tyrosine hydroxylase.

or amygdala (Paré et al., 1995). The second type of axon postsynaptic element (Fig. 18A, B). The AxSh synapse
terminal or axo-shaft (AxSh) consists of a presynaptic accounts for one fifth of the total synaptic population.
terminal comparable to the AxSp that furnishes an Like the AxSp the presynaptic terminal contains
asymmetrical synapse with a dendritic shaft, the numerous agranular vesicles, dense-cored vesicles, if
 D. Marcellino et al. / Neuroscience 226 (2012) 324–347
Fig. 12. Coexistence of tyrosine hydroxylase and dopamine
b-hydroxylase immunofluorescence in the periventricular hypotha-
lamic region in a horizontal section. In panel A, a square indicates the
hypothalamic area from where the high magnification in panel B has
been taken. (B) The majority of the TH-immunoreactive terminals
around nuclear profiles stained blue with DAPI, demonstrate a
yellowish fluorescence due to the coexistence of red TH and green
DBH immunofluorescence, which therefore represents noradrenergic
nerve terminals. However, some processes probably dendrites, only
show a red immunofluorescence and probably represent dopamine
processes belonging to the tubero-infundibular dopamine neurons.
Scale bars = 150 lm in A and 47 lm B. Bregma level 9.3 from the
Paxinos and Watson stereotaxical atlas (1986).
any, are sparse. As implicit, AxSh synapses (Fig. 18A, B)
contact a thick dendrite, and establish an asymmetrical
synapse. Although synaptic terminals contacting the
neuron somata were infrequent (<2%) in our sample
(i.e., a minimum of three grids of tissue samples were
337
examined per animal), these consist of slender
terminals that establish symmetrical contacts (Fig. 18C,
D). Still a fourth type of synapse is represented by axon
terminals containing large dense-cored vesicles (DCV)
and a variable proportion of small agranular vesicles
(Fig. 18E). DCV containing boutons are rare (<1%) and
establish asymmetrical interactions with either somata
or proximal dendrites (Fig. 18F).
Neuropil of animals with interruption of fibers of the
anterior commissure. Surgical section of the anterior
commissure 72 h before sacrifice yields to local
(Fig. 2D) preterminal, and terminal (Fig. 19) axonal
degeneration. In fact, massive axonal necrosis was
observed in the stump of the anterior commissure
adjacent to the knife cuts (Fig. 2D). Survey micrographs
at the AIC neuropil reveal numerous terminal boutons
and reactive glial processes (Fig. 19A–E). Alternating
with other structurally normal terminals (vide supra),
there are numerous degenerating terminals (DTs)
having the following characteristics. The profile of a DT
is irregular due to numerous indentations bounding
cytoplasmic protrusions (Fig. 19B). The DT has, as a
rule, a highly electron-dense matrix of uneven
appearance due to presumptive vesicular and
mitochondrial debris. These alterations made it difficult
to define the nature of both vesicular and junctional (i.e.,
postsynaptic density) zones of DTs. However, in a set
of DTs, the postsynaptic element corresponded to a
large spine; furthermore, when the plane of sectioning is
appropriate, a large dendritic spine associated with a DT
may exhibit what appears to correspond to the
postsynaptic density (Fig. 19C, E). Virtually all DTs are
associated with glial processes evidenced by their
electron-lucid cytoplasm (Fig. 19A–E); a complete glial
DT engulfment was occasionally observed. Serial
sections through the AIC neuropil disclose the
tridimensional arrangement DTs confirming that these
terminate on large dendritic spines (Fig. 19E). No DTs
are observed in specimens from sham-lesioned animals.
DISCUSSION
This study gives the first thorough, structural and
neurohistochemical characterization of the anterior
intercalated cell islands (AIC) with the rapid-Golgi
technique in combination with electron microscopy and
analysis of D1-, TH-, and DBH-immunoreactivities with
confocal laser microscopy to study their DA and NA
afferents and their D1 receptors. Our observations in
Golgi-impregnated specimens, revealed that the AIC
occupies a unique position in the amygdala. Further, the
innervation of the AIC neuropil by commissural axons,
opens the possibility that the AIC is involved in decoding
neuronal inputs from the contralateral amygdalar and
isocortical nuclei and areas. Immunohistochemically, the
role of the dopaminergic neurotransmission acting via
D1-mediated volume transmission influencing dendritic
spines of AIC neurons is emphasized. Putative
interactions of the intercalated paracapsular islands with
the neighboring amygdalar nuclei are also described.
338 D. Marcellino et al. / Neuroscience 226 (2012) 324–347

Fig. 13. Distribution pattern of tyrosine hydroxylase (TH, red) and dopamine D1 receptor (D1R, green) immunoreactivities in panels A and C and TH
and dopamine b-hydroxylase (DBH, green) immunoreactivities in panels B and D, within adjacent horizontal sections of the rostral amygdala. The
TH and dopamine D1R immunoreactivities codistribute within AIC (A) but DBH-immunoreactive terminals are not present (B). The medial and lateral
PIC (C, D) show only a weak codistribution TH and D1R immunofluorescent punctate profiles with no DBH immunoreative terminals present. A high
density of TH immunofluorescent terminals is found in CeLA with very few DBH terminals present and absence of D1-immunoreactive profiles (A, B)
giving evidence for a rich DA innervation in this region but not linked to D1 receptors. Within the BLA a moderately dense plexus of TH terminals is
found showing no DBH immunoreativity with only a few exceptions (green) and associated with only a weak D1 immunoreactivity not evenly
distributed (A, B), Thus, only minor or no noradrenergic mechanisms exist in all these regions with a preferential D1R-mediated DA
neurotransmission in the intercalated islands, while D2-like receptor-mediated DA transmission appears to dominate in the CeLA and in the BLA. In
contrast, a low-density plexus of DBH-immunoreactive terminals is apparent within BL and La (C, D) not showing any visible TH immunoreactivity.
Blue profiles in panels A and C represent cell nuclei stained with DAPI. Scale bar = 75 lm. Sections correspond to a Bregma level 8.2 to 8.3
from the Paxinos and Watson stereotaxical atlas (1986). Abbreviations: AIC, anterior intercalated islands; BL, basolateral amygdaloid nucleus; BLA,
anterior aspect of the basolateral amygdaloid nucleus; Ce, central amygdaloid nucleus; CeLA, central–anterolateral subdivision; DAPI, 40 -6-
diamidino-2-phenylindole; D1R, dopamine D1 receptor; La, lateral nucleus; LPIC, lateral paracapsular cell masses; MPIC, medial paracapsular cell
masses; TH, tyrosine hydroxylase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)

Structural characterization of the AIC neurons described in these islands (Brodal, 1947; Hall,
1972; Millhouse and DeOlmos, 1983; Millhouse, 1986).
The present results in Golgi-impregnated sections
MSNs of the AIC correspond to GABAergic cells
validate and extend previous observations on the
(Nitecka and Ben-Ari, 1987; McDonald and Augustine,
cytological organization of the intercalated cell islands.
1993; Paré and Smith, 1993a,b, 1994; Tamamaki et al.,
Application of the Golgi technique and its variants to the
2003; Marowsky et al., 2005; Perez de la Mora et al.,
adult rat brain (see Larriva-Sahd, 2006, 2008) revealed
2008, 2010). Primary dendrites of most AICs branch
novel cytological features and structural organization of
sparsely and, due to their bipolar character, lie along the
the anterior intercalated islands (AICs). In fact,
rostro-caudal axis remaining confined to the nuclear
utilization of long periods of impregnation disclosed that
neuropil. Unlike dendrites, the axon of an MSN leaves
the adult AICs are composed of numerous, tightly
the AIC and often projects to the Ce. This evidence
packaged, MSNs rather than the relatively sparse
confirms the original observations performed by
 D. Marcellino et al. / Neuroscience 226 (2012) 324–347 339

Fig. 14. Double immunolabeling of tyrosine hydroxylase (TH, red) and dopamine D1 receptors (D1, green) (A, C) and of TH and dopamine
b-hydroxylase (DBH, green) (B, D) in the lateral and medial intercalated paracapsular islands (LPIC and MPIC) as visualized from adjacent
horizontal sections. A medium density of TH-immunoreactive terminals is found in the LPIC and MPIC (A, C; left panels) among nuclear profiles
stained blue with DAPI (left panels) and lack DBH immunoreactivity (B, D; right panels) and therefore represent dopamine nerve terminals.
Distances between the TH-immunoreactive terminals and the punctate D1-immunoreactive profiles of the PIC are in the micrometer range as seen
from the scale bars. Note, however, that within the surrounding areas a few double-labeled (TH- and DBH-immunoreactive) profiles (arrows) are
seen representing noradrenaline nerve terminals instead (B, D). Horizontal sections were taken at Bregma level 8.2 to 8.3 from the Paxinos and
Watson stereotaxical atlas (1986). Scale bar = 75 lm. Abbreviations: DAPI, 40 -6-diamidino-2-phenylindole; DBH, dopamine b-hydroxylase; D1R,
dopamine D1 receptor; PIC, paracapsular intercalated islands; TH, tyrosine hydroxylase. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)

Millhouse (1986) and those from more recent studies
(Royer et al., 2000; Marowsky et al., 2005; Geracitano
et al., 2007) about the cytological features of both the
AIC and PIC. Although the present observations on the
cytological appearance of the AIC confirms the early
notion that the AIC belongs to the broad group of
intercalated neurons (Manko et al., 2011), it is clear
from a connectional perspective, that the AIC together
with the main intercalated islands may represent a
distinct subclass of amygdaloid intercalated islands.
Our Golgi observations support the early notion that
the AIC, like the intercalated cell masses in general, is
composed of a double population of neurons that is
medium- and large-sized neurons. In fact, correlative
light and electron microscopy disclosed that the two
neuron types exhibit distinct cytological and subcellular
organization. Unfortunately no large neurons (see
Millhouse, 1986), that might counterpart the TII neuron
identified here, were encountered in our Golgi-
impregnated material. At present, the possibility for an
intrinsic difference in neuronal types between the AIC
and other intercalated cell masses should be left as an
open question. That the internal organization of the AIC
neuropil is unique or, at least different from that in other
groups of intercalated cell islands, remains to be
defined. However, our ultrastructural analysis offers a
normative foundation for future comparative studies
aimed at defining subtle differences in synaptic
organization between other intercalated cell islands.
Our study disclosed a hitherto unsuspected
morphological complexity of the internal structure and
connectivity of the PIC with the adjacent anterior
commissure, BLA, and Ce nuclei of the amygdala.
Thus, in addition to the current view that the medial PIC
340 D. Marcellino et al. / Neuroscience 226 (2012) 324–347

Fig. 15. Distribution pattern of tyrosine hydroxylase (TH, red) and dopamine D1 receptor (D1R, green) immunofluorescent profiles (A) and TH and
dopamine b-hydroxylase (DBH, green) immunofluorescent profiles (B) in adjacent horizontal sections of the anterior basolateral nucleus (BLA) and
the lateral and medial intercalated paracapsular islands in horizontal sections of the amygdala. In the left panel (A), a moderate density of
TH-immunoreactive terminals with red immunofluorescence is found in the BLA; these represent dopamine terminals since they fail to show yellow
immunofluorescence and thus lack green DBH immunofluorescence (B). Note that DBH-immunoreactive terminals (green fluorescence in B) are
mainly found outside the BLA (B, arrows), and that only a weak D1R immunoreactivity, unevenly distributed, exists in BLA. Strong D1R
immunoreactivity (green) codistributing with TH-immunoreactive terminals (red) is present in the AIC and PIC (A). Nuclear profiles stained with DAPI
are shown. Bregma level 9.3 from the Paxinos and Watson stereotaxical atlas (1986). Scale bar = 75 lm. Abbreviations: AIC, anterior
intercalated islands; BLA, anterior basolateral nucleus; DAPI, 40 -6-diamidino-2-phenylindole; DBH, dopamine b-hydroxylase; D1R, dopamine D1
receptor; LPIC, lateral paracapsular cell masses; MPIC, medial paracapsular cell masses, TH, tyrosine hydroxylase. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 16. Survey electron micrographs at the same magnification showing two neuron types in the anterior intercalated cell mass. (A) Putative
medium-sized spiny neuron. The cytoplasm contains short cisternae of the rough endoplasmic reticulum (arrowheads). N = Cell nucleus. (B) A
presumptive large spiny cell at the periphery of the nucleus, bounded by myelinated axons (asterisks). The neuron contains a lobulated nucleus (N)
surrounded by a well-developed rough endoplasmic reticulum (rer) with numerous electron-dense granules (arrows). Ol = oligodendrocyte. Adult
rat brain, lead–uranium contrast. Scale bars = 2 lm.

represents an inhibitory interface that controls the neurons may establish axonal contacts with dendrites of
trafficking of impulses between the BLA and Ce, our both BLA pyramidal cells and latero-capsular Ce
evidence further suggests that at least some medial PIC neurons (Fig. 7B). Furthermore, the medial PICs may
 D. Marcellino et al. / Neuroscience 226 (2012) 324–347 341

Fig. 17. Electron micrographs depicting the cytoplasmic contents of two presumptive neuron types in an anterior intercalated cell mass. (A) The
cytoplasm from a medium-sized neuron contains numerous mitochondria (arrowheads) and short, isolated cisterns of the rough endoplasmic
reticulum (arrows). Note the parallel arrangement of neurotubules (encircled) at the base of an emerging dendrite. (B) Large neuron with an
extensive Nissl area (rer) and with numerous lysosomes (double arrowheads). N = Cell nucleus, G = Golgi apparatus. Adult rat brain, lead–
uranium contrast. Scale bars = 0.5 lm.

Fig. 18. Synaptic types in the neuropil of the anterior intercalated cell mass. (A) A terminal axon (Ax) providing successive asymmetrical synapses
to a large dendritic spine (sp) and to a dendritic shaft (sh). (B) Specimen from an adult rat whose anterior commissure was cut 3 days before
sacrifice. Examples of a degenerating (encircled), shaft (asterisk), and spinous (hollow arrow) terminal are shown. Arrow = granular vesicles.
(C) An axo-somatic, symmetrical, terminal (asterisk). S = soma. (D) Synaptic terminal to a spine-like protuberance emerging from the soma (S).
(E) Synaptic bouton containing dense core (arrows) and small agranular vesicles. (F) An axo-somatic and axo-dendritic (encircled) terminal
containing large, dense-core, vesicles (arrow). Adult rat brain, lead–uranium contrast. Scale bars = 1 lm in A–D, and F, and 0.5 lm in E.
342 D. Marcellino et al. / Neuroscience 226 (2012) 324–347

Fig. 19. Terminal degeneration in the neuropil of the anterior intercalated cell mass following interruption of the anterior commissure fibers.
(A) Numerous degenerating terminals (circles) and presumptive non-lesioned terminals apposing denderitic spines (hollow arrows). (B) Numerous
degenerating terminals exhibiting irregular profile and electron-dense matrix, adjacent to a synaptic bouton (upper left) and a dendrite (lower half).
(C) A degenerating bouton contacting a thin dendrite. Note the electron-dense material associated with the postsynaptic specialization (arrows).
(D) A degenerating bouton (db) synapsing a dendritic spine (Sp). Two electron-dense patches (az) are shown within the postsynaptic cytoplasm.
Note the association of an astrocytic process (Ap) with the degenerating bouton. (E) Tri-dimensional reconstruction from nineteen, successive
sections; one of them is shown in panel D. Dendrite and spine (green), postsynaptic electron-density (yellow), degenerating bouton (blue), and glial
process (red). Adult rat brain, lead–uranium contrast. Scale bars = 2 lm in A, 0.5 in B and C, 0.2 in D and E. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)

not only control the trafficking of impulses from the BLA to
Ce neuropil but also via putative inhibitory inputs to BLA
neurons and neuropil (Figs. 5 and 7B).
AIC connectivity. Relationship of the AIC with the
caudal component of the anterior commissure
Since the intercalated islands are regarded as inhibitory
interfaces controlling the trafficking of nerve impulses to
and within the amygdala (Royer et al., 1999; Marowsky
et al., 2005; Perez de la Mora et al., 2008, 2010) the
knowledge of their afferent and efferent connections is
crucial. In this work, we demonstrate that the neuropil of
the AIC and BLA receives axon collaterals and terminals
from the caudal limb of the anterior commissure.
Although present and earlier (see Alheid et al., 2006)
light microscopic observations defined an intimate
association between the fibers of the anterior
 D. Marcellino et al. / Neuroscience 226 (2012) 324–347
commissure and the AIC, no direct evidence for a
synaptic interaction between them had been provided
(see Kalisman et al., 2005). This prompted us to utilize
a group of animals whose anterior commissure was cut
with a Halasz-type knife. Following short periods of
survival, short enough to prevent collateral innervation
(Raisman and Field, 1973), numerous degenerating
synapses were found in the AIC neuropil, leading us to
conclude that ACc axons do, in fact, innervate the AIC.
This observation is regarded as highly relevant since it
implies that the AIC may not only be providing a link
between the isocortex and the BLA and Ce
homolaterally (Berretta et al., 2005; Pinard et al., 2012),
but also from the contralateral cerebral hemisphere.
Indeed, fibers arising from the amygdalar and isocortical
nuclei and areas, respectively, that decussate via
anterior commissural fibers (Jouandet and Hartenstein,
1983; Condés-Lara et al., 2003; Hoover and Vertes,
2011) may exert a direct (i.e., synaptic) influence in the
AIC. Thus, it is plausible that the AIC represents a
neuron cluster that is distinct from the broad group of
Fig. 20. Diagrammatic representation of putative ipsi- and contra-
lateral circuitry and synaptic organization of the anterior intercalated
cell mass (AIC), as seen from a dorsal approach of the rat brain.
Arrows and arrowheads designate axons and axon terminals,
respectively. ACc = anterior commissure, pars caudalis;
ACr = anterior commissure, pars rostralis; BLA = basolateral com-
plex and central nucleus (Ce) of the amygdala. The inset shows
convergent homolateral (red) and contralateral (i.e., commissural)
amygdaloid afferences to the anterior intercalated cell mass in the
right side. The axon of the medium spiny neuron (black) resolves, in
turn, in the AIC proper, Ce, and BLA neuropil.
343
intercalated cell masses, and is suited for the
synchronic responses of the amygdalae at either side of
the brain. A model gathering the structural basis
supporting the foregoing discussion is presented in
Fig. 20.
Immunohistochemical characterization of the DA and
NA afferents and D1 receptors of AIC and PIC
Since ultrastructural work by Pinto and Sesack (2008) has
revealed that D1-immunoreactive spines and dendrites are
commonly observed in intercalated cell islands, the
punctate D1 immunoreactivity we discovered within the
clusters of AIC and PIC nerve cells are probably located
on dendritic spines. No clear demonstration of the
punctate D1 immunoreactivity in intercalated nerve cell
bodies could be obtained. Previously, only a diffuse D1
immunoreactivity could be found in the intercalated cell
masses with confocal laser microscopy using transverse
sections (Fuxe et al., 2003). Thus, the major target for
dopamine in these intercalated cell masses may be
the dendrites, including both dendritic shafts (Asan,
1997) and spines. However, fiber and terminal D1
immunoreactivity has been observed in rat and primate
intercalated cell masses, but less commonly (Pinto and
Sesack, 2008; Muly et al., 2009).
With the triple immunolabeling procedures
demonstrating the presence of D1-, TH-, and DBH in the
same horizontal section we could confirm previous
evidence in transverse sections that the PICs are mainly
innervated by dopamine terminals. Thus, very few DBH-
immunoreactive terminals were found within the PICs.
We also obtained evidence for a clear rostro-caudal
gradient in the TH-immunoreactive networks innervating
the medial and lateral PICs with a high dopamine
innervation at the rostral level and very few TH-I
terminals remaining at the caudal levels (Fig. 8B, C). In
spite of this, the amount of D1-I in the caudal PIC
appeared to remain the same in line with previous
observations of the existence of a rostro-caudal gradient
of dopamine terminals in the main intercalated island
(Fuxe et al., 2003). In the AIC we also demonstrated a
high density of TH-I terminals in the presence of very
few DBH-immunoreactive terminals, which is evidence
for a rich dopamine innervation of these intercalated cell
masses even higher than the one found in the rostral PIC.
Additional evidence in this study also confirms the
existence of high densities of dopamine nerve terminal
networks in the latero-capsular part of the Ce, while
moderate densities were found in the BLA becoming
clearly reduced in the posterior part of the basolateral
nucleus, extending and validating previous work (Fuxe,
1965; Swanson, 1982; Brinley-Reed and McDonald,
1999; see reviews by Fallon and Ciofi, 1992; Asan,
1998, and Perez de la Mora et al., 2010). Most of the
TH-I terminals in the BLA and in the latero-capsular part
of the Ce lacked DBH-I but in the remaining part of the
basolateral complex a moderate plexus of DBH-I
terminals were observed in line with previous work
(Fuxe, 1965; Fallon and Ciofi, 1992; Asan, 1998; Li
et al., 2002; Fuxe et al., 2003). The DA nerve terminal
networks appear to innervate preferentially parvalbumin-
344 D. Marcellino et al. / Neuroscience 226 (2012) 324–347
positive GABAergic interneurons in the BLA (Pinard et al.,
2008). However, only a weak D1-immunoreactivity was
observed in parts of the BLA and virtually absent within
the lateral amygdaloid nucleus, in agreement with the
results of Pinto and Sesack (2008). In contrast, others
studying the rat BLA observed a widespread distribution
of D1-I (Maltais et al., 2000; Pickel et al., 2006; Muly
et al., 2009) predominantly located in dendritic spines
but also on terminals as shown with immunoelectron
microscopy. The reason for these discrepancies is
unknown but may be related to differences in the
immunohistochemical protocols.
Unlike in the hypothalamus, it was difficult to observe
TH immunoreactivity in many of the DBH-immunoreactive
terminals of the amygdaloid regions analyzed. This may
reflect the relatively low amounts of TH protein in the
cortical noradrenergic terminal networks belonging to
the locus coeruleus noradrenergic system (see Hokfelt
et al., 1977).
Based on the topographical relationship between
D1- and TH-I in the main intercalated island and PIC, it
was suggested that dopamine neurotransmission in
these regions may occur, to a substantial degree, via
volume transmission (Fuxe et al., 2003). The current
results showing that the distances from the TH-immuno-
reactive terminals to the punctate D1-immunoreactive
dendrites found in the AIC and rostral PIC are within the
micrometer range indicate that a rapid, short-range,
extrasynaptic form of dopamine volume transmission
may operate in both the PIC and AIC. However, around
50% of the TH-immunoreactive area in the sampled
field of the AIC appears to overlap with the
D1-immunoreactive area indicating the existence also of
a substantial degree of synaptic DA transmission via
D1 receptors in the AIC. In contrast, a long-distance
volume transmission may exist at the level of the caudal
PIC and caudal main intercalated island, where a
considerable mismatch between D1-immunoreactive and
TH-immunoreactive structures seems to occur (Fuxe
et al., 2003).
Functional considerations
Our structural and neurohistochemical findings underline
the existence of an important D1 receptor mechanism in
the AIC in line with the results obtained in the PIC (Quirk
et al., 2003; Berretta et al., 2005; Marowsky et al.,
2005), and in the main intercalated island (Manko et al.,
2011) as demonstrated with neurophysiological
techniques after activation of their glutamate afferents
from the prefrontal cortex. The output of the lateral and
medial paracapsular intercalated GABA neurons
becomes markedly reduced by this D1 receptor
mechanism operating via volume transmission with loss
of feed-forward inhibition of the BLA and anterior part of
Ce, respectively, and leading to increases in fear and
anxiety (Marowsky et al., 2005; see also Pape, 2005).
This may be true also for the D1 mechanism in the AIC.
The spatial relationship between the glutamate
prefrontal cortex and dopamine terminals in the
intercalated cell masses (Pinto and Sesack, 2008)
indicates that one mechanism for the D1 receptor
modulation of MSNs in the PIC may be the direct
receptor–receptor interactions between extrasynaptic
NMDA receptors (Rumbaugh and Vicini, 1999; Fellin
et al., 2004) and D1 receptors supported by the
existence of D1–NMDA receptor complexes (Lee et al.,
2002; Pickel et al., 2006). In view of the relative lack of
synaptic convergence of glutamate and dopamine
terminals on the intercalated cells (Pinto and Sesack,
2008), the D1 receptors here are likely to be activated
mainly by dopamine, operating via volume transmission.
The major mechanism may, however, be the direct
inhibition of the intercalated GABA neurons via
postjunctional D1 receptors, causing activation of
G-protein-coupled inward rectifier potassium channels
resulting in hyperpolarization and inhibition of impulse
flow (Marowsky et al., 2005). Reciprocal dopamine-
glutamate modulation of release could also be involved
(Morari et al., 1998). In view of the striking D1 receptor/
dopamine terminal mismatch in the caudal medial and
lateral paracapsular intercalated GABA cells, this
extrasynaptic D1 receptor mechanism operating by
volume transmission is likely to be less efficient at these
caudal levels due to the longer distances of diffusion
involved in slow volume transmission (Fuxe et al., 2003),
as indicated above.
The AICs include clusters of GABAergic nerve cell
bodies of the amygdala projecting also to the basal
forebrain, especially to the substantia innominata and
the horizontal limb of the diagonal band (Paré and
Smith, 1993a,b, 1994). Thus, the D1 receptor
mechanism located in the anterior intercalated cell
masses could be of substantial importance by reducing
the activity of amygdaloidfugal outflow of these
intercalated cell masses. In this way, the AIC can
markedly influence the cortical regions via effects on the
activity of, e.g., the large cholinergic cell groups of
the basal forebrain giving rise to major projections to the
cerebral cortex.
It is conceivable that under normal conditions the AIC
and, presumably, the main intercalated island contribute
to keeping a low level of anxiety in the animal by
generating feed-forward inhibition within the ipsilateral
BLA and Ce. Furthermore, the AIC may also have a
role in synchronizing the firing pattern from one
amygdala with that of the contralateral amygdala and
thus the anxiogenic outputs of both amygdalae. The
observation that the AIC receives a robust contingent of
axon collaterals and terminals from the caudal limb of
the anterior commissure, including the commissural
component of the stria terminalis (Jouandet and
Hartenstein, 1983; Condés-Lara et al., 2003) highlights
the AIC as a potential site of synergizing the responses
generated in the amygdaloid nuclei at either side of the
brain (Fig. 20).
CONCLUSION
Our combined Golgi and immunohistochemical study
begins to characterize the AIC and PIC of the rat in
terms of their structure including nerve cell bodies and
dendrites and their putative afferents and efferents
 D. Marcellino et al. / Neuroscience 226 (2012) 324–347 345

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(Accepted 31 August 2012)

(Available online 15 September 2012)