DISTRIBUTION OF NOREPINEPHRINE TRANSPORTERS IN THE
NON-HUMAN PRIMATE BRAIN
H. R. SMITH, T. J. R. BEVERIDGE AND L. J. PORRINO*
Department of Physiology and Pharmacology, Wake Forest University
School of Medicine, Medical Center Boulevard, Winston-Salem, NC
Abstract—Noradrenergic terminals in the central nervous sys-
tem are widespread; as such this system plays a role in varying
functions such as stress responses, sympathetic regulation,
attention, and memory processing, and its dysregulation has
been linked to several pathologies. In particular, the norepi-
nephrine transporter is a target in the brain of many therapeutic
and abused drugs. We used the selective ligand [3H]nisoxetine,
therefore, to describe autoradiographically the normal regional
distribution of the norepinephrine transporter in the non-human
primate central nervous system, thereby providing a baseline to
which alterations due to pathological conditions can be com-
pared. The norepinephrine transporter in the monkey brain
was distributed heterogeneously, with highest levels occur-
ring in the locus coeruleus complex and raphe nuclei, and
moderate binding density in the hypothalamus, midline tha-
lamic nuclei, bed nucleus of the stria terminalis, central nu-
cleus of the amygdala, and brainstem nuclei such as the
dorsal motor nucleus of the vagus and nucleus of the solitary
tract. Low levels of binding to the norepinephrine transporter
were measured in basolateral amygdala and cortical, hip-
pocampal, and striatal regions. The distribution of the nor-
epinephrine transporter in the non-human primate brain was
comparable overall to that described in other species, how-
ever disparities exist between the rodent and the monkey in
brain regions that play a role in such critical processes as
memory and learning. The differences in such areas point to
the possibility of important functional differences in norad-
renergic information processing across species, and suggest
the use of caution in applying findings made in the rodent to
the human condition. © 2005 Published by Elsevier Ltd on
behalf of IBRO.
Key words: norepinephrine transporter, [3H]nisoxetine, rhe-
sus monkey, autoradiography.
Studies of the central noradrenergic system have demon-
strated its broad involvement in widely varying functions
such as responses to stress and sympathetic regulation,
as well as higher cognitive processing such as attention
and memory retrieval. In addition it has been shown that
dysregulation of this system may in part underlie such
pathological states as depression and anxiety, and studies
have demonstrated an emerging role for the norepineph-
rine (NE) system in the pathogenesis of drug addiction and
withdrawal (Farfel et al., 1992; Harris and Aston-Jones,
1993; McDougle et al., 1994; Aston-Jones et al., 1999;
Delfs et al., 2000; Macey et al., 2003; Mash et al., 2005).
The norepinephrine transporter (NET) is a member of
the Na?/Cl?-dependent neurotransmitter transporter gene
family, and is critical for limiting extracellular NE concen-
trations. The NET is a molecular target site of many current
anti-depressant and anti-anxiety therapies (Zhu and Ordway,
1997; Zavosh et al., 1999; Owens et al., 2000; Rubin, 2000;
Roubert et al., 2001; Weinshenker et al., 2002; Richelson,
2003), as well as the abused stimulants cocaine and
amphetamine (Koe, 1976; Heikkila et al., 1979; Reith et al.,
1983; Ritz et al., 1990; Florin et al., 1994; Pifl et al., 1999;
Haughey et al., 2000). In addition, genetic studies in hu-
man populations have linked specific polymorphisms of the
NET gene to such pathologies as depression and anorexia
nervosa (Urwin et al., 2002; Inoue et al., 2004; Ryu et al.,
2004) and to sensitivity to pharmacotherapies targeted to
the monoamine transporters (Tellioglu and Robertson,
2001; Yoshida et al., 2004). Consequently, the NET has
been the focus of countless molecular studies which have
described in detail its structural and functional character-
istics (for reviews see Blakely et al., 1994; Olivier et al.,
2000; Norregaard and Gether, 2001; Gainetdinov and Ca-
ron, 2003; Glatt and Reus, 2003; Torres et al., 2003).
Despite the diverse functions of the NE system in both
normal and pathological processes, the widespread clini-
cal use of NET blockers to treat mood-related disorders,
and recent interest in the development of NET ligands for
positron emission tomography imaging (Ding et al., 2003;
Schou et al., 2004), our understanding of the normal dis-
tribution of the primate NET remains far from complete.
Numerous anatomical studies have described the brain-
stem noradrenergic cell groups and their projection fields in
rodents (Loughlin et al., 1982; McKellar and Loewy, 1982;
Foote et al., 1983; Loughlin et al., 1986a,b; Woulfe et al.,
1988, 1990; Grzanna and Fritschy, 1991; Terenzi and In-
et al., 1978; Felten and Sladek, 1983). In addition the distri-
bution of the NET protein has been depicted autoradio-
graphically in rodents (Biegon and Rainbow, 1983; Javitch
et al., 1985; Tejani-Butt, 1992; Schroeter et al., 2000; Kung
et al., 2004; Ghose et al., 2005; Sanders et al., 2005), cats
(Charnay et al., 1995), and humans (Gross-Isseroff et al.,
1988; Donnan et al., 1991; Tejani-Butt et al., 1993; Ordway
et al., 1997), however prior to the characterization by
Tejani-Butt (1992) of [3H]nisoxetine as a selective ligand
for autoradiographic description of the NET, such studies
utilized relatively non-selective ligands such as [3H]mazin-
dol and [3H]desmethylimipramine. The reports by Ordway
*Corresponding author. Tel: ?1-336-716-8575; fax: ?1-336-716-8689.
E-mail address: email@example.com (L. J. Porrino).
Abbreviations: BNST, bed nucleus of the stria terminalis; DR, dorsal
raphe; LC, locus coeruleus; NAcc, nucleus accumbens; NE, norepi-
nephrine; NET, norepinephrine transporter.
Neuroscience 138 (2006) 703–714
0306-4522/06$30.00?0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO.
et al. (1997) and Tejani-Butt et al. (1993) are the only
anatomical descriptions of [3H]nisoxetine binding in the
human brain, and are restricted to the locus coeruleus (LC)
and raphe nuclei. To date there has been no comprehen-
sive description of the normal topography of [3H]nisoxetine
binding sites in the non-human primate brain.
Given the paucity of information regarding the distribu-
tion of the NET in the primate brain despite the increased
use of non-human primates to model human disease
states, the aim of this study was to describe autoradio-
graphically the normal distribution of the NET in the non-
human primate brain, thereby providing a baseline from
which to quantify any changes that may be associated with
pathological conditions or chronic drug exposure. The con-
trolled experimental conditions used for this study permit
the careful evaluation of the distribution of NE transporters
in a primate model which is not subject to the many con-
founds inherent in studies using postmortem human
brains, while the use of the autoradiographic technique
provides a detailed, quantitative representation of the an-
atomical distribution of NET binding.
Subjects used in this study were six adult male rhesus monkeys
(Macaca mulatta), all of which also served as an untreated control
group for a metabolic mapping study of cocaine self-administration
(Letchworth et al., 2001; Nader et al., 2002; Porrino et al., 2002,
2004; Macey et al., 2003; Beveridge et al., 2004). Monkeys were
6–13 years old at the start of the experiment and weighed between
7.5 and 13.0 kg under free-feeding conditions. Their body weights
were maintained at approximately 90–95% of free-feeding weights.
All procedures were performed in accordance with established prac-
tices as described in the National Institutes of Health Guide for Care
and Use of Laboratory Animals, and all efforts were made to mini-
mize the number of animals used for the experiments and their
suffering. In addition, all protocols were reviewed and approved by
the Animal Care and Use Committee of Wake Forest University.
As part of the previously mentioned metabolic study, monkeys
received injections of the radiotracer 2-[14C]deoxyglucose. Im-
mediately after the 45 min experimental procedure, animals
were killed with sodium pentobarbital (100 mg/kg, i.v.). Brains
were immediately removed, blocked at approximately Bregma
6.5 and ?20.5 mm, frozen in isopentane at ?45 °C, and then
stored at ?80 °C. Coronal sections (20 ?m) were cut on a Leica
CM3050 cryostat (Vashaw Scientific, Norcross, GA, USA). Four
of every 20 sections cut were thaw-mounted onto 48?60 mm
coverslips (Brain Research Laboratories, Newton, MA, USA)
for 2-[14C]deoxyglucose autoradiography. Sixteen sections of
every (striatum through amygdala) or every other (remainder of
brain) cycle of 20 were collected for receptor autoradiography
or in situ hybridization histochemistry and were thaw-mounted
onto 50?75 mm chrome-alum/gelatin-subbed or electrostati-
cally charged slides (Brain Research Laboratories).
Procedures for [3H]nisoxetine autoradiography were adapted for
the non-human primate from those of Tejani-Butt (1992). Tissue
sections were preincubated at room temperature in buffer (50 mM
Tris, 300 mM NaCl, 5 mM KCl, pH 7.4) for 20 min to remove any
residual 2-[14C]deoxyglucose. This procedure has been shown
to remove residual [14C] remaining in the tissue (Moore et al.,
1998a,b; Letchworth et al., 2001; Nader et al., 2002). Sections
were then incubated for 4 h at 4 °C in buffer containing 3 nM
[3H]nisoxetine (80 Ci/mmol; Perkin-Elmer, Boston, MA, USA) in
the presence (non-specific binding) or absence (total binding) of
1 ?M mazindol. Sections were rinsed three times (5 min each) in
buffer at 4 °C, with a final 10 s rinse in ice-cold deionized water.
Sections were immediately dried under a stream of cold air and
placed on Hyperfilm-3H (Amersham, Arlington Heights, IL, USA) for
two (LC) or 6 weeks in the presence of [3H] standards (Amersham).
After appropriate exposure times, films were developed with GBX
developer (Eastman Kodak, Rochester, NY, USA), fixed and rinsed.
Densitometry and data analysis
Analysis of autoradiograms was conducted by quantitative densi-
tometry with a computerized image processing system (MCID,
Imaging Research, St. Catherines, Ontario, Canada). Tissue
equivalent values (fmol/mg wet weight tissue) were determined
from optical densities and from a calibration curve obtained by
densitometric analysis of the autoradiograms of
Measurements of NET binding sites were made in 57 brain re-
gions which ranged across 12 levels throughout the rostro-caudal
extent of the monkey brain. Specific binding was determined by
subtracting non-specific binding values (measured in adjacent
sections) from the total binding values.
[3H]nisoxetine binding in the rhesus monkey brain
The regional distribution of [3H]nisoxetine binding to the
NET was assessed at multiple levels throughout the rostro-
caudal extent of the normal rhesus monkey brain (see
Table 1, Fig. 1). Cytoarchitectural criteria for each brain
region were identified using Nissl-stained sections adja-
cent to those used for autoradiography. Nomenclature and
identification of structures were determined according to
the atlas of Paxinos and colleagues (2000). While the
overall pattern of NET binding was consistent across ani-
mals, there was a tendency for certain regions, such as the
nucleus accumbens (NAcc) shell and the dorsal raphe
(DR), to exhibit a high degree of between-subject variabil-
ity in binding density. Areas displaying the greatest levels
of [3H]nisoxetine binding included the LC and components
of the raphe complex, while more moderate levels were
observed in the hypothalamus, midline thalamic nuclei,
bed nucleus of the stria terminalis (BNST) and various
brainstem nuclei. The lowest levels of binding were mea-
sured in cortical, hippocampal, and striatal regions. Non-
specific binding levels (see Fig. 2) averaged 7.48 fmol/mg
wet weight tissue (19% of total binding).
Both across and within the subdivisions of the cortex cho-
sen for quantification there was little variability in the levels
of NET binding sites. With the exception of Area 25 (medial
wall of prefrontal cortex) and Area 24 (cingulate gyrus),
most cortical areas exhibited uniformly low levels of signal
(Table 1). Area 25 exhibited the greatest intensity of signal
measured in the cortex, particularly at its caudal extent,
where anterior striatum was present (Fig. 1B). More ros-
H. R. Smith et al. / Neuroscience 138 (2006) 703–714 704
trally, the binding in this cortical area was similar to that of
the other regions measured (Fig. 1A). While the pattern of
number of NET sites in the deeper layers of the cortex (layers
5, 6), Area 24 also exhibited a darker band of binding sites
more superficially, corresponding to layers 3 and 4.
The hippocampus exhibited extremely low binding site
densities, with a relatively uniform pattern of distribution
throughout the dentate gyrus and CA3-4 fields, and even
lower levels measured in the CA1 field (Fig. 1H).
Striatum and extended amygdala
[3H]nisoxetine binding to the NET in the dorsal striatum
was the lowest measured in the brain. Measurements were
made at two levels of the precommissural striatum: an
anterior level at which the NAcc was not differentiated into
core and shell (Fig. 1B), and a more posterior level at
which these subdivisions were clearly discernible (Fig.
1C). With the exception of the NAcc and closely adjacent
ventral caudate, the striatum was uniformly devoid of bind-
ing signal. In contrast, the shell of the NAcc displayed a
heterogeneous pattern of binding, with the densest label-
ing at its most dorsal point, and decreasing levels of signal
more ventrally and laterally (Fig. 1C). This gradient was
particularly discernable at the most caudal extent of the
shell, while more rostrally the very dense binding at the
dorsal tip of the structure was considerably less apparent.
Because this steep gradient occurred in such a narrow
rostrocaudal range and it was not possible to be confident
that this range was suitably represented in all animals, the
level of binding reported in Table 1 reflects only measure-
ments obtained for the more ventral portion of the shell,
where binding was more uniform.
As was the case in the NAcc, the topography of
[3H]nisoxetine binding sites in the extended amygdala was
markedly heterogeneous. For example, the BNST dis-
played a non-uniform pattern of binding, with densest lev-
els of NET sites in the lateral dorsal and ventral subdivi-
sions (Fig. 1D). In contrast, the medial and lateral BNST
Table 1. Regional densities of [3H]nisoxetine binding in the rhesus
Brain region[3H]nisoxetine binding
Anterior cingulate (area 24)
Medial PFC (area 25)
Medial PFC (area 32)
Gyrus rectus (area 14)
Orbital PFC (area 13)
Dorsolateral PFC (area 46)
Somatosensory (area 3a)
Somatosensory (area 3b)
Motor (area 4)
Anterior nucleus accumbens
Accumbens shell (ventral)a
Bed nucleus of the striatus terminalisb
Table 1. Continued
Brain region[3H]nisoxetine binding
Dorsal tegmental area
A1 noradrenergic cell group
Dorsal motor n. of the vagus
Nucleus tractus solitarius
Inferior olivary nucleus
Spinal trigeminal nucleus
Cerebellum (molecular layer)
Data shown are specific binding levels (total minus non-specific) and
represent fmol/mg wet weight tissue. Values are expressed as
aThe value shown for the NAcc shell does not include measurements
taken for the caudal dorsomedial cap of the structure, which was not
adequately represented across all animals.
bValues for the BNST were previously reported in Macey et al. (2003).
H. R. Smith et al. / Neuroscience 138 (2006) 703–714705
Fig. 1. Representative autoradiograms of [3H]nisoxetine binding to the NET in 12 rostral–caudal levels of the rhesus (Macaca mulatta) monkey brain.
Levels portrayed are: A, prefrontal cortex; B, anterior striatum; C, striatum at the level of the NAcc shell; D, BNST; E, anterior hypothalamus; F,
posterior hypothalamus and amygdala; G, anterior thalamus; H, posterior thalamus and hippocampus; I, raphe complex; J, LC; K, rostral brainstem;
L, caudal brainstem. Abbreviations used: A1, A1 noradrenergic cell group; ACaud, anterior caudate; ACing (24), anterior cingulate, Brodmann area
24; ANA, anterior nucleus accumbens; APut, anterior putamen; Arc, arcuate nucleus; BLA, basolateral amygdala; BNSTl, bed nucleus of the stria
terminalis, lateral; BNSTld, bed nucleus of the stria terminalis, lateral dorsal; BNSTm, bed nucleus of the stria terminalis, medial; BNSTv, bed nucleus
of the stria terminalis, ventral; CA, CA fields of hippocampus; CeA, central amygdala; CL, caudalinear nucleus; CM, centromedial thalamus; Crb,
cerebellum; DG, dentate gyrus; DLPFC (46), dorsolateral PFC, Brodmann area 46; DMH, dorsomedial hypothalamus; DMV, dorsal motor nucleus of
the vagus; DRc, dorsal raphe, caudal; DRd, dorsal raphe, dorsal; DRif, dorsal raphe, intrafascicular; DRv, dorsal raphe, ventral; DT, dorsal tegmental
H. R. Smith et al. / Neuroscience 138 (2006) 703–714706
Fig. 1. (Continued) area; ER, entorhinal cortex; GR (14), gyrus rectus, Brodmann area 14; IMD, intermediodorsal thalamus; IO, inferior olive;
LH, lateral hypothalamus; LP, lateral parabrachial nucleus; LPOA, lateral preoptic area; MDM, medial mediodorsal thalamus; MPFC (25), medial
prefrontal cortex, Brodmann area 25; MPFC (32), medial prefrontal cortex, Brodmann area 32; MPOA, medial preoptic area; MR, median raphe; NAC,
nucleus accumbens core; NASd, nucleus accumbens shell, dorsal part; NASv, nucleus accumbens shell, ventral part; NP, nucleus prepositus; NTS,
nucleus tractus solitarius; OPFC (13), orbital prefrontal cortex, Brodmann area 13; PaVH, paraventricular hypothalamus; PaVT, paraventricular
thalamus; PCaud, posterior caudate; PC/CL, paracentral/centrolateral thalamus; PeVH, periventricular hypothalamus; PPut, posterior putamen; Re,
reunions thalamic nucleus; SC, subicular complex; SO, supraoptic nucleus; SpT, spinal trigeminal nucleus; SubC, sub-coeruleus; VMH, ventromedial
hypothalamus; 12N, hypoglossal nucleus.
H. R. Smith et al. / Neuroscience 138 (2006) 703–714 707
divisions exhibited a more moderate degree of binding,
and were indistinguishable from each other based on bind-
ing patterns. A detailed analysis of [3H]nisoxetine binding
to the NET in the BNST of the same animals used in this
study has been previously reported (Macey et al., 2003).
The amygdala displayed low binding in all divisions with
the exception of the central nucleus, which exhibited a
moderate degree of binding that stood out strikingly com-
pared with the remainder of the structure (Fig. 1F).
Hypothalamus and thalamus
[3H]nisoxetine binding was measured at two levels in the
hypothalamus. At the more rostral level, the most conspic-
uous binding occurred in the supraoptic and paraventricu-
lar nuclei, while the medial and lateral preoptic divisions
displayed low to moderate levels of labeling (Table 1, Fig.
1E). More caudally, the hypothalamus as a whole exhibited
a greater magnitude of signal, with less variability in bind-
ing levels between subdivisions (Fig. 1F). Medial struc-
tures such as the peri- and paraventricular nuclei and the
arcuate nucleus possessed the highest degree of NET
binding, although the dorso- and ventromedial divisions
also displayed relatively robust labeling. At this level, the
lowest level of binding sites in the hypothalamus was
observed in the lateral division.
[3H]nisoxetine binding to the NET in the thalamus was
primarily confined to midline nuclei, with particularly high
levels in the paraventricular nucleus, moderate labeling in the
centromedial, intermediodorsal, and reuniens nuclei, and
lower levels in medial mediodorsal as well as the paracentral
and centrolateral nuclei, which were measured as one struc-
ture (Table 1, Fig. 1F, G, H). It should be noted, however, that
the relatively low binding level reported in Table 1 for the
paracentral/centrolateral nuclei is due to a heterogeneous
distribution of restricted areas of intense labeling disbursed
within a background of appreciably lower density.
Midbrain, brainstem, and cerebellum
Other than the LC complex, the raphe complex exhibited
the greatest binding densities in the brain (Table 1, Fig. 1I).
Fig. 2. Representative autoradiograms of [3H]nisoxetine labeling of the NET in the rhesus monkey brain demonstrating total and non-specific binding
patterns. A (total) and C (non-specific), binding at the level of the amygdala and hypothalamus; B (total) and D (non-specific), binding at the level of the LC.
H. R. Smith et al. / Neuroscience 138 (2006) 703–714 708
[3H]nisoxetine binding in this region was characterized by
a great deal of between-subject variability in density, par-
ticularly in its most dorsal and lateral aspects, where bind-
ing levels between animals ranged from moderate to very
dense. On the whole the ventral and intrafascicular as-
pects of the DR exhibited particularly dense labeling, along
with the caudalinear nucleus. Dense binding was also
observed in the median raphe and the dorsal and ventro-
lateral aspects of the DR.
The LC, the primary contributor to the dorsal noradren-
ergic pathway, possessed the greatest level of NET bind-
ing sites measured in the brain (Table 1, Fig. 1J) and a
high degree of consistency in binding densities between
animals. In addition, very dense binding was measured in
the lateral parabrachial nuclei (Fig. 1J), and dense labeling
was observed in the subcoeruleus (Fig. 1J) and the dorsal
motor nucleus of the vagus (Fig. 1L). Moderate labeling of
the NET was measured in the nucleus tractus solitarius
and A1 noradrenergic cell group, dorsal tegmental area,
spinal trigeminal nucleus, the hypoglossal nucleus, and the
inferior olive (Fig. 1J, K, and L). The lowest level of
binding within the brainstem structures selected for
measurement was in the nucleus prepositus. Finally, low
levels of [3H]nisoxetine binding sites were also observed
in the molecular layer of the cerebellum.
The present data represent a systematic and detailed
description of the distribution of [3H]nisoxetine binding to
the NET across the entire rostrocaudal extent of the pri-
mate brain. The highest densities of NET binding sites were
observed in the LC complex as well as in the DR. Divisions
of the hypothalamus, BNST, central amygdala, and other
NE-containing cell groups and projection sites in the brain-
stem exhibited high to moderate levels of labeling. The
lowest levels of binding were observed in the basal ganglia
and throughout the cortex, including the hippocampus.
The densest concentration of transporters observed in
this study was in the LC rather than in any of the norad-
renergic terminal fields. Functional dendritic transporters
may account for many of these LC [3H]nisoxetine binding
sites as there is evidence to suggest that NE is released
from LC dendrites (Groves and Wilson, 1980) and mem-
brane-associated NET immunoreactivity has been demon-
strated to occur on LC dendrites rather than cell bodies
(Schroeter et al., 2000). In addition, it has been shown by
several groups that projections from the caudal brainstem
noradrenergic cell groups terminate in the LC (McKellar
and Loewy, 1982; Foote et al., 1983; Van Bockstaele and
Aston-Jones, 1992), and as both pre- and post-synaptic
adrenoreceptors have been localized to the LC (Lee et al.,
1998a,b; Callado and Stamford, 1999) it is likely that trans-
porters associated with both dendritic and axonal sites
contribute to the extremely dense level of binding in this
The DR was also found to contain surprisingly high
levels of [3H]nisoxetine binding sites. In fact the density of
labeling within this structure was similar to that observed in
the LC, a finding which corresponds closely with previous
studies in the human in which binding to the NET in the DR
was shown to be as high or higher than in the LC (Donnan
et al., 1991; Ordway et al., 1997). We found a very high
degree of variability between animals in the DR, particu-
larly in its lateral and dorsal range, which may be due to
gradients in [3H]nisoxetine binding throughout the rostro-
caudal extent of the structure. The present study mea-
sured the binding sites at only one level of the monkey DR,
however Ordway’s group (1997) assessed multiple rostro-
caudal levels throughout the human raphe, and despite a
high degree of between-subject variability in their results,
differences in binding densities were also apparent across
The study by Ordway and colleagues (1997), and an-
other by Tejani-Butt et al. (1993) are to date the only
descriptions of the topography of NET binding in the hu-
man brain using a highly selective ligand. Both reports,
however, were limited to restricted portions of the brain,
encompassing only the raphe nuclei (Ordway et al., 1997)
and LC (Tejani-Butt et al., 1993; Ordway et al., 1997). The
present findings, however, also largely parallel those
reported in a more comprehensive description using
[3H]mazindol, a non-selective ligand which also has a high
affinity for the dopamine transporter (Donnan et al., 1991).
In accordance with the current findings in non-human pri-
mates, Donnan and colleagues (1991) reported the dens-
est concentration of NET binding in humans to be in the
LC, dorsal motor nucleus of the vagus, and DR, with
moderate levels in hypothalamus and thalamus, and neg-
ligible binding levels in hippocampus, amygdala, and stri-
atum. The apparent absence of NET binding in the
amygdala contradicts our finding that the CeA contains
appreciable levels of NETs, however Donnan’s group
(1991) did not divide the amygdala into subdivisions, and
the contribution that the relatively small central nucleus
may have made to the density measurement for the entire
structure may have been insufficient to elevate substan-
tially the overall number.
In contrast, an earlier study of the human forebrain
utilizing [3H]desmethylimipramine (Gross-Isseroff et al.,
1988) reported a somewhat different distribution of binding
sites. As described in this study, binding of [3H]desmethy-
limipramine, another relatively non-selective ligand, was
densest in the basal and central amygdala, as well as in
the dentate gyrus and CA4 field of the hippocampus. Mod-
erate concentrations of binding sites were reported in ad-
ditional amygdaloid and hippocampal regions, portions of
the striatum, hypothalamus, cortical areas, and thalamus.
This study did not extend to brainstem areas, therefore no
numbers were reported for the LC, however the relative
binding densities reported in the forebrain were not con-
sistent with either the present findings, or those of Donnan
et al. (1991). However, as [3H]desmethylimipramine has
since been shown to bind to sites other than the NET in
both rat (Backstrom et al., 1989) and human (Backstrom
and Marcusson, 1990), this lack of specificity may account
for the discordant results.
H. R. Smith et al. / Neuroscience 138 (2006) 703–714 709
In addition to the consistency with previous autora-
diographic accounts, the present results are also com-
patible with anatomical descriptions of the NE system
using biochemical mapping of NE in humans (Farley and
Hornykiewicz, 1977) and histofluorescence, immunolabel-
ing, and tract-tracing techniques in non-human primates
(Felten et al., 1974; Gatter and Powell, 1977; Bowden et
al., 1978; Felten and Sladek, 1983; Foote et al., 1983).
Although there are relatively few primate studies with
which to compare the present findings, several autora-
diographic descriptions of the distribution of the NET in
rat brain have been published, thus permitting a direct
comparison between species. A cursory comparison of
[3H]nisoxetine binding between the two species reveals
similar overall regional distributions of the NET in the non-
human primate and the rodent, with similar relative binding
densities in such areas as the brainstem noradrenergic
cells, hypothalamus, and BNST. However several poten-
tially important disparities became apparent when the sub-
regional topography of the NET was examined within many
of the terminal field regions of the forebrain.
In the thalamus of the rat, for example, prominent
binding to the NET has been reported in the limbic-related
anteroventral nucleus (Biegon and Rainbow, 1983; Javitch
et al., 1985; Tejani-Butt, 1992; Belej et al., 1996), along
with moderate levels in sensorimotor and associative relay
nuclei such as the ventral posterior, ventromedial, and
lateraldorsal divisions (Benmansour et al., 1992; Tejani-
Butt, 1992; Belej et al., 1996). In contrast, the densest
labeling in the monkey thalamus occurred in “non-specific”
midline and intralaminar structures such as the paraven-
tricular, centromedial, reuniens and paracentral and cen-
trolateral nuclei, which are robustly innervated by arousal-
related brainstem nuclei such as LC and dorsal tegmental
nuclei, as well as visceral-related nuclei such as the para-
brachial nucleus and nucleus tractus solitarius (Krout et al.,
2002; Van der Werf et al., 2002). The conspicuous labeling
in the anteroventral nucleus of the rat thalamus was not
observed in the non-human primate.
Similarly, the relatively dense labeling observed in the
rodent dentate gyrus (Biegon and Rainbow, 1983; Tejani-
Butt, 1992; Belej et al., 1996) was notably absent in the
monkey, where hippocampal formation [3H]nisoxetine
binding was low and was characterized by a rather homo-
geneous subregional distribution. This pattern of subre-
gional differences also extends to the relative levels of
binding within the hypothalamic nuclei. The dorsomedial
nucleus of the rat hypothalamus has consistently been
reported to contain high levels of NET binding sites (Biegon
and Rainbow, 1983; Benmansour et al., 1992; Tejani-Butt,
1992; Belej et al., 1996), whereas in the monkey, binding in
this division was low to moderate. Midline divisions such as
the paraventricular, periventricular, and arcuate nuclei, as
well as the supraoptic nucleus however, displayed much
denser binding, and, in fact, were among the most NET-
rich structures measured in the monkey forebrain.
Further differences between species were noted in the
amygdala. In the rodent, the amygdala exhibits a relatively
homogeneous binding pattern, with low binding throughout
the structure, except in the basolateral division, where
there is a moderate level of labeling (Benmansour et al.,
1992; Tejani-Butt, 1992). In the non-human primate, while
most of the amygdaloid nuclei are labeled uniformly at a
very low level, it is the central nucleus that exhibits a robust
degree of binding to the NET by [3H]nisoxetine. This is
consistent with an early description of the distribution of NE
in the human amygdala, which was found to be much more
abundant in the central division than in the remaining
nuclei (Farley and Hornykiewicz, 1977).
The NAcc is yet another area in which there may be
significant species-related differences in NET sites. The
evidence for the presence of NET binding sites in the
rodent accumbens is somewhat ambiguous. Although sev-
eral groups have reported a lack of NET binding in the
NAcc of the rat (Biegon and Rainbow, 1983; Javitch et al.,
1985; Benmansour et al., 1992; Tejani-Butt, 1992), Berridge
et al. (1997) have demonstrated a moderate to dense
population of dopamine ?-hydroxylase-immunoreactive fi-
bers in the caudal aspect of the rat NAcc shell, particularly
at its septal pole. In addition, Schroeter et al. (2000) re-
ported a moderate density of NET immunoreactivity in the
caudal portion of the shell. Likewise, in the monkey, we
observed very dense binding in the dorsal-most aspect of
the shell which occurred only at the caudal extent of the
accumbens. In fact, individual animals displayed densities
of binding sites as great, or greater, than those measured
in the BNST. Again, this finding is in agreement with Farley
and Hornykiewicz (1977), who reported slightly greater
concentrations of NE in the human NAcc than in the BNST.
This dense accumulation of binding sites was present only
at the most caudal level of the septal pole of the NAcc,
therefore it is possible that as this region marks the rostral
margin of the extended amygdala (Alheid and Heimer,
1988; de Olmos and Heimer, 1999) binding in this area
reflects a zone of transition between the accumbens shell
and the rostral-most portion of the BNST. Indeed, many of
the neurochemical and connectional properties of the cau-
dal NAcc shell distinguish it from the remainder of the
striatum. Immunostaining for such markers as acetylcho-
linesterase, substance P, and serotonin, among others,
reveals distinct patterns of immunoreactivity which clearly
distinguish the dorsomedial shell from its more ventral and
lateral portions, as well as the adjacent core (Haber and
McFarland, 1999). In addition we previously reported a
remarkably similar distribution of serotonin transporters in
the monkey NAcc shell (Smith et al., 1999). Interestingly,
this portion of the NAcc shell receives a very restricted
cortical input, originating almost exclusively from Area 25
(Haber et al., 1995), which also contains the densest con-
centration of NET binding of all the areas of cortex mea-
sured in this study.
The distribution pattern of [3H]nisoxetine binding to the
NET in the non-human primate brain, therefore, although
similar overall to that observed in other species, displays
some notable disparities when compared with the rat that
imply functional differences. In the rodent, for example,
there are moderately dense [3H]nisoxetine binding sites in
the dentate gyrus of the hippocampus, and NE in the
H. R. Smith et al. / Neuroscience 138 (2006) 703–714 710
rodent hippocampus has been demonstrated to play a
critical role in learning and memory (cf. Murchison et al.,
2004; Roozendaal et al., 2004; Scheiderer et al., 2004;
Walling and Harley, 2004; Walling et al., 2004). Similarly,
in the basolateral amygdala, another region in which the
relative densities of binding sites differ between species,
NE has been demonstrated to influence memory consoli-
dation in the rat (for review see McGaugh, 2002). In the
monkey neither of these structures exhibited notable levels
of NET labeling, which may indicate that the contribution of
NE to these functions in the primate may not parallel its
role in the rat.
It is also of interest to note that many of the regions in
which these species-related differences occur are highly
interconnected. Amygdalar inputs to the paraventricular
and reuniens nuclei of the thalamus, for example, originate
in the central nucleus (Price and Amaral, 1981; Van der
Werf et al., 2002). In turn the paraventricular projections to
striatal regions are densest in the caudomedial division of
the shell of the accumbens (Jayaraman, 1985; Van der
Werf et al., 2002), while the reuniens is a major source of
thalamic inputs to the hippocampus (Wouterlood et al.,
1990). As all of the aforementioned structures exhibited
marked differences in binding patterns between species
these disparities are likely to have important functional
Divergence between species is not limited to the nor-
adrenergic system. Indeed, numerous autoradiographic
and immunohistochemical studies have reported differ-
ences in receptor populations between the primate and
rodent, including the opioid (Mansour et al., 1988; Voorn et
al., 1996; Daunais et al., 2001; Bridge et al., 2003), cho-
linergic (Breese et al., 1997; Han et al., 2003; Quik et al.,
2005), and glutamatergic (He et al., 2000) systems, to
name a few. While studies that include tissue from both
human and non-human primate are relatively scarce, a
recent immunohistochemical study by Sanchez-Gonzalez
and colleagues (2005) describes a robust dopaminergic
input to the thalamus which was observed in both human
and monkey brains. As dopamine innervation of the rodent
thalamus is thought to be considerably less abundant
(Groenewegen, 1988) this study further illustrates the po-
tential importance of such differences and the need for
careful evaluations and comparisons of receptor distribu-
tions across non-primate and primate species. These dis-
tinctions in the distribution of neural markers across spe-
cies raise the possibility that mechanisms of information
processing may rely to a different degree on a given neu-
rotransmitter system in one species relative to another.
The use of prudence may be warranted, therefore, in ex-
trapolating conclusions based on rodent models across
species to the human brain.
A fuller understanding of the normal state of the pri-
mate NE system is critical. The NET has been suggested
to play a role in numerous disorders such as drug abuse
and withdrawal, depression, schizophrenia, and Alzhei-
mer’s disease; as such, the normal distribution of the pri-
mate NET presented herein may provide a baseline to
which we can compare perturbations in the state of the
transporter associated with these pathological states.
Acknowledgments—The authors thank Dr. Michael Nader, Susan
Nader, Jennifer Sandridge, Clifford Hubbard, and Tonya Moore for
their assistance in conducting this experiment. This research was
supported by NIH/NIDA grant DA09085.
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