Cerebral Cortex May 2010;20:1164--1174
Advance Access publication August 24, 2009
Development of Cannabinoid 1 Receptor
Protein and Messenger RNA in Monkey
Dorsolateral Prefrontal Cortex
Stephen M. Eggan1, Yoshito Mizoguchi1, Samuel R. Stoyak2and
David A. Lewis1,2
1Department of Psychiatry, University of Pittsburgh, Pittsburgh,
PA 15213, USA and2Department of Neuroscience, University of
Pittsburgh, Pittsburgh, PA 15260, USA
Stephen M. Eggan and Yoshito Mizoguchi have contributed
equally to this work.
Adolescent cannabis use is associated with an increased risk of
schizophrenia and with impairments in cognitive processes reliant
on the circuitry of the dorsolateral prefrontal cortex (DLPFC).
Additionally, maternal cannabis use is associated with cognitive
dysfunction in offspring. The effects of cannabis are mediated by
the cannabinoid 1 receptor (CB1R), which is present in high density
in the primate DLPFC. In order to determine how developmental
changes in CB1Rs might render DLPFC circuitry vulnerable to
cannabis exposure, we examined the density and innervation
patterns of CB1R-immunoreactive (IR) axons and the expression of
CB1R mRNA in the DLPFC from 81 macaque monkeys, ranging in
age from embryonic 82 days to 18 years. Overall CB1R
immunoreactivity in the gray matter robustly increased during the
perinatal period and achieved adult levels by 1 week postnatal.
However, laminar analyses revealed that CB1R-IR axon density
significantly decreased with age in layers 1--2 but significantly
increased in layer 4, especially during adolescence. In contrast,
CB1R mRNA levels were highest 1 week postnatal, declined over
the next 2 months, and then remained unchanged into adulthood.
These findings provide a potential substrate for discrete, age-
dependent effects of cannabis exposure on the maturation of
primate DLPFC circuitry.
Keywords: cholecystokinin, GABA, interneurons, parvalbumin,
Cannabis use predominates in adolescents (Gruber and Pope
2002) and current data document a new trend for continuing
cannabis use through the childbearing years (Sundram 2006).
These patterns of use suggest that D9-tetrahydrocannabidiol
(D9-THC), the principal psychoactive ingredient in cannabis,
could interfere with the proper maturation of brain circuitry
during adolescence in users and/or during the pre- and
perinatal periods in their offspring (Viveros et al. 2005).
Indeed, the children of cannabis-smoking mothers exhibit
cognitive dysfunction (Fride 2004), and cannabis use during
adolescence is associated with later impairments in certain
cognitive functions (Viveros et al. 2005; Sundram 2006), such
as working memory, that are mediated by the dorsolateral
prefrontal cortex (DLPFC) (Egerton et al. 2006). Furthermore,
cannabis use, especially before 15 years of age, is associated
with an increased risk of schizophrenia (Moore et al. 2007;
Murray et al. 2007), a disorder characterized by both
dysfunction of the DLPFC and impaired working memory
(Lewis et al. 2005).
These developmental consequences of cannabis use might
reflect the effects of D9-THC on endocannabinoid signaling
mediated by the cannabinoid 1 receptor (CB1R) (Freund et al.
2003), which is highly expressed in the mature primate DLPFC
(Eggan and Lewis 2007). In the neocortex, CB1Rs are heavily
localized to the terminals of a subset of c-aminobutyric acidergic
(GABAergic) basket interneurons that contain the neuropeptide
cholecystokinin (CCK) and that target the perisomatic domain
(i.e., cell body and proximal dendrites) of pyramidal cells
(Marsicano and Lutz 1999; Bodor et al. 2005). In the rodent
neocortex and hippocampus, CB1R-immunoreactive (IR) cells
and axons undergo distinct developmental refinements in
relative density and laminar distribution, and in the hippocam-
pus, these refinements parallel those of CCK-IR cells and axons
(Morozov and Freund 2003a, 2003b; Deshmukh et al. 2007).
CCK-IR structures also undergo distinct changes in relative
density and laminar distribution during perinatal and postnatal
development in the macaque monkey DLPFC (Oeth and Lewis
1993). However, thedevelopmental
innervation density and laminar distribution pattern of CB1R-IR
structures have not been determined in the primate DLPFC.
The maturation of primate DLPFC circuitry is associated
with substantial changes in other sources of perisomatic
GABAergic inputs to pyramidal neurons during both the
perinatal and adolescent periods of development. For example,
both pre- and postsynaptic markers of inputs from parvalbumin
(PV)-IR basket and chandelier neurons to the cell bodies and
axon initial segments, respectively, of pyramidal neurons
exhibit marked developmental refinements (Anderson et al.
1995; Erickson and Lewis 2002; Cruz et al. 2003 2009). In
addition, the maturation of the axon arbors and perisomatic
synapses from PV-IR basket neurons during adolescence is
shaped, at least in part, by GABA signaling (Chattopadhyaya
et al. 2007). Interestingly, cannabinoid activation of CB1Rs
suppresses the release of GABA from terminals and completely
abolishes inhibitory postsynaptic currents elicited by stimula-
tion of CCK-IR basket cells (Galarreta et al. 2004; Trettel et al.
2004; Bodor et al. 2005; Foldy et al. 2006). Together, these data
converge on the idea that the exogenous activation of CB1Rs
during pregnancy or adolescence may alter the balance of
inhibitory inputs to the perisomatic region of DLPFC pyramidal
neurons, disrupt the developmental trajectories of these
GABAergic inputs, and produce persistent alterations in DLPFC
circuitry that could impair cognitive function.
Thus, knowing how CB1R protein and mRNA normally
develop in the monkey DLPFC is essential to understand how
cannabis exposure during sensitive periods may underlie the
associated increased liability for cognitive impairments and
schizophrenia later in life. Consequently, to understand the
involvement of CB1R-mediated signaling in the maturation of
DLPFC circuitry, we used immunocytochemical and in situ
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hybridization techniques to quantify changes across develop-
ment in the density and laminar distribution of CB1R-IR
structures and of CB1R mRNA levels in DLPFC area 46 of
Materials and Methods
Animals and Tissue Preparation
For immunocytochemical studies, 2 cohorts of macaque monkeys were
utilized. The first cohort of animals consisted of 11 pigtail macaque
monkeys (Macaca nemestrina) of both sexes, ranging in age from
embryonic (E)82 days to postnatal (PN)11 months (Supplementary
Table S1) (Oeth and Lewis 1993; Conde ´ et al. 1996). The second cohort
consisted of 40 rhesus monkeys (Macaca mulatta) of both sexes,
ranging in age from PN2 days to 18 years (Supplementary Table S2)
(Erickson and Lewis 2002; Cruz et al. 2003). Animal perfusion, brain
removal and blocking, tissue fixation and tissue storage procedures
have been described in previous studies that utilized tissue from both
monkey cohorts (Oeth and Lewis 1993; Conde ´ et al. 1996; Erickson and
Lewis 2002; Cruz et al. 2003) (see Supplementary Materials and
Methods for details). We have previously shown that our storage
procedure does not affect cortical immunoreactivity for a number of
antigens (Rosenberg and Lewis 1995; Erickson et al. 1998; Pierri et al.
1999; Lewis et al. 2001; Erickson and Lewis 2002; Cruz et al. 2003).
Blocks from the left hemisphere were sectioned coronally (40 lm) and
every 10th section was stained for Nissl substance to identify both the
location of area 46 within the principal sulcus and laminar boundaries
using cytoarchitectonic criteria (Barbas and Pandya 1989).
For in situ hybridization experiments, a third cohort of 30 female
rhesus macaque monkeys ranging in age from PN1 week to 11.5 years
was used (Supplementary Table S3). Sixteen monkeys were deeply
anesthetized and perfused transcardially with ice-cold modified
artificial cerebrospinal fluid (aCSF) (Gonzalez-Burgos et al. 2008). In
4 animals, 2--4 weeks prior to perfusion with aCSF, a small block of
tissue containing dorsolateral area 9 and the medial bank of the
principal sulcus was surgically excised from the rostral third of the
principal sulcus in the left hemisphere for electrophysiology studies.
The remaining 14 animals were unperfused and euthanized with an
overdose of sodium pentobarbital (Supplementary Table S3). Following
extraction, brains were blocked coronally and the right hemisphere
was flash frozen in isopentane on dry ice and stored at –80 ?C. Serial
coronal sections (16 lm) containing DLPFC area 46 were cut from the
caudal third of the principal sulcus in the right hemisphere of each
monkey on a cryostat, thaw mounted on Supra-frost slides (VWR
Scientific, West Chester, PA), and stored at –80 ?C until processed for in
For all 3 cohorts of monkeys, animals were housed as described
previously (Cruz et al. 2003) and experimentally naive at the time of
perfusion except as noted above. All procedures were conducted in
accordance with federal guidelines and with the approval of the
University of Pittsburgh’s Institutional Animal Care and Use Committee.
Free-floating coronal tissue sections containing DLPFC area 46 were
processed for CB1R immunoreactivity as previously described (see
Eggan and Lewis 2007 and Supplementary Materials and Methods) with
an affinity-purified rabbit anti-CB1R antibody raised against the last
15 amino acid residues of the C-terminus of the rat CB1R (anti-CB1R-
L15; diluted 1:7500; generously provided by Dr Ken Mackie, Indiana
University, Bloomington, IN) and CB1R immunoreactivity was visual-
ized using 3,3#-diaminobenzidine (Sigma, St Louis, MO). The specificity
of the primary antibody has been verified by several lines of evidence
(see Eggan and Lewis 2007 and Supplementary Materials and Methods).
One section from every animal was processed in a single immunocy-
tochemistry experiment. Three immunocytochemistry experiments
were performed in order to process a total of three sections for each
animal, and so that any interexperimental differences were perfectly
controlled for across animals.
In Situ Hybridization
Sense and antisense riboprobes against human CB1R mRNA were
synthesized as previously described (Eggan et al. 2008) from a 714-bp
fragment corresponding to bases 435--1148 of the human CNR1 gene
(Genbank accession number NM_033181) and reduced in length to
approximately 100 bp by alkaline hydrolysis to increase the effective-
ness of tissue penetration (Supplementary Materials and Methods). The
specificity of the riboprobe for CB1R mRNA was confirmed as
previously described (see Eggan et al. 2008 and Supplementary
Materials and Methods). One section from each monkey was used to
analyze the expression of CB1R mRNA levels in area 46. Animals were
divided into 2 groups, each containing an equivalent number of animals
from each age group and a total of 2 in situ hybridization runs were
performed (Supplementary Materials Methods).
Measurement and Analysis of CB1R Immunoreactivity and mRNA
Levels of CB1R immunoreactivity were measured without knowledge
of animal number or age by random coding of slides. Slide-mounted
sections were illuminated under a microscope (Leitz Diaplan, Wild
Leitz GmbH, Wetzlar, Germany) and images of the ventral bank of the
principal sulcus in DLPFC area 46 from each of the 3 sections for each
rhesus monkey were captured at a final magnification of 374 by a video
camera, digitized, and quantified at a final resolution of 4.0 lm/pixel
using a Microcomputer Imaging Device (MCID) system (Imaging
Research Inc, London, ON, Canada). Within each image, relative optical
density (ROD) levels of CB1R immunoreactivity were measured in the
total cortical gray matter by drawing contours of the full cortical
thickness that included areas as large as possible (Eggan and Lewis
2007; Eggan et al. 2008). The mean (±standard deviation, SD) total area
sampled per animal was 11.9 ± 2.6 mm2.
In order to assess laminar patterns of CB1R immunoreactivity, 2--3
cortical traverses ~1 mm wide extending from pial surface to white
matter were arbitrarily placed over the ventral bank of the principal
sulcus where the tissue section was cut perpendicular to the pial
surface and the ROD within each traverse was measured in 2 sections
from each animal (Eggan et al. 2008). Laminar boundaries were
determined by calculating the percent depth of each layer in each
traverse in adjacent Nissl-stained sections using the Neurolucida
program (MicroBrightfield, Inc., Colchester, VT); the mean ROD levels
of CB1R immunoreactivity within each layer were then determined for
Expression levels of CB1R mRNA were quantified using the MCID
system without knowledge of animal number or age by random coding
of film autoradiographs. Film autoradiographs were transilluminated on
a lightbox, and images were captured by a video camera and digitized
(final resolution 18.0 lm/pixel). Images of slide-mounted hybridized
tissue sections were captured and superimposed on corresponding
autoradiographic film images in order to delineate the pial surface and
the gray and white matter borders (Eggan et al. 2008). Contours were
drawn to include the full cortical thickness in the ventral and dorsal
banks of the principal sulcus in DLPFC area 46. Optical density
(OD) was measured in those contours and expressed as nanoCuries/
gram (nCi/g) tissue by reference to radioactive carbon-14 standards
(American Radiolabeled Chemicals, St Louis, MO) exposed on the same
film. The mean (±SD) total area sampled per animal was 21.0 ± 6.7 mm2.
To determine differences in CB1R mRNA expression across lamina,
OD was measured in ~1-mm-wide cortical traverses as described above
with the exception that a traverse was placed over the dorsal bank of
the principal sulcus if quantifiable area was limited in the ventral bank.
Because RNase A treatment (Supplementary Materials and Methods)
destroys Nissl-stainable substances within the cytoplasm precluding the
identification of laminar borders based on cytoarchitectonic criteria,
the OD in each layer within each traverse was determined by dividing
the total cortical thickness into zones of 1--10%, 10--20%, 20--50%,
50--60%, 60--80%, and 80--100% from the pial surface to white matter;
these zones approximate the locations of cortical layers 1--6, re-
spectively, in the monkey (Hashimoto et al. 2009). All cortical and
laminar gray matter OD values were corrected by subtracting
background OD values obtained from the white matter of each animal.
Cerebral Cortex May 2010, V 20 N 5 1165
All images of slides or autoradiograms processed in an experimental
run were acquired in the same session under identical microscope or
lightbox and room illumination and with the same gain and black levels
and flatfield correction.
For cortical density measures, univariate analyses of covariance
(ANCOVA) were conducted to examine differences in the develop-
mental expression levels of CB1R immunoreactivity and CB1R mRNA.
OD was entered as the dependent variable, age group as the main
effect, and freezer storage time and sex (CB1R immunoreactivity
measures only) as covariates. For measures of CB1R mRNA expression,
perfusion and prior biopsy were included as blocking factors. Tissue
storage time did not have a significant effect for either CB1R
immunoreactivity (F1,39= 0.0; P = 0.996) or CB1R mRNA expression
(F1,20= 1.3; P = 0.275). Sex did not have a significant effect for CB1R
immunoreactivity (F1,39= 0.2; P = 0.621). For laminar density measures,
multivariate analyses of variance (MANOVA) were performed, with OD
levels for each layer entered as the dependent variable and age group as
the main effect (perfusion and prior biopsy were included as blocking
factors for measures of CB1R mRNA expression). For all significant
ANCOVAs and MANOVAs, the least significant difference post hoc
test (with a = 0.05) was used to assess the differences between age
Development of CB1R-IR Structures in Pigtail Macaque
In DLPFC area 46 of pigtail macaque monkeys (Supplementary
Table S1), the relative density and laminar distribution of CB1R-
IR structures changed markedly through embryonic and
perinatal development (Fig. 1). The overall level of CB1R-IR
structures was lowest at E82, the earliest age examined (Fig. 1A),
robustly increased from E132 to PN41 days of age (Fig. 1B--E),
and then remained relatively unchanged, except for certain
layer-specific changes (see below), through PN9 months
At E82, thin, vertical CB1R-IR axons were already present in
both the cortical plate and subplate (Fig. 1A). These axons
had few varicosities and gave rise to occasional oblique or
horizontally oriented branches (Fig. 2A). From E132 to E157
days, the number of CB1R-IR axon branches and varicosities
markedly increased in layers 1--4 and 6, whereas the density of
CB1R-IR axons in layer 5 did not appear to change (Fig. 1B,C).
At E157 days, the density of CB1R-IR axons was highest in
layers 1 and 2, slightly lower in layers 3 and 4, intermediate in
layer 6, and lowest in layer 5 (Fig. 1C). From E157 to PN4 days,
the relative density of CB1R-IR axons appeared to decrease in
layer 3 but to increase in layer 4 producing a prominent dense
band (compare Fig. 1C,D). From PN4 days to PN41 days to PN9
months, the density of CB1R-IR axons increased further in layer
4, whereas the density of CB1R-IR axons decreased in layers
1 and 2 (Fig. 1D--F). Thus, during late embryonic and early
postnatal development the predominant laminar location of
CB1R-IR axons shifted from the superficial layers to layer 4 of
area 46. These developmental differences in laminar patterns
of labeled axon distribution were confirmed by ROD measures
(Supplementary Results and Fig. S1).
At E132, many CB1R-IR cells were detectable in layers
2-superficial 3 and the highest density of CB1R-IR cells was
observed at E157 (Fig. 2B). By PN4 days of age, CB1R-IR cell
density had markedly decreased (data not shown). Most CB1R-
IR cells were nonpyramidal, putative interneurons, based on
their small size and round shape (Fig. 2B).
From E132 (Fig. 1B) to E157 (Figs. 1C and 2C), CB1R-IR
fibers were present in high density in the white matter and
then gradually decreased through the peri- and postnatal
periods (Fig. 1D--F). By PN9 months of age (Figs. 1F and 2D),
the density of CB1R-IR fibers in the white matter had markedly
decreased, making the border between layer 6 and white
matter very distinct. Similarly, a high density of CB1R-IR fibers
in the white matter was observed in rhesus monkeys at
PN7 days (Fig. 3A), and by 4.8 months of age, the density of
CB1R-IR fibers in the white matter was markedly decreased
Development of CB1R-IR Structures in Rhesus Monkeys
To confirm the findings from the pigtail macaque monkey
series, to evaluate changes at later stages of development, and
to assess age-related shifts in the laminar distribution of CB1R-
IR axons, we also examined CB1R immunoreactivity levels in
a cohort of rhesus monkeys (Supplementary Table S2). Like the
pigtail monkeys of comparable postnatal ages, the overall level
of CB1R immunoreactivity in area 46 of rhesus monkeys did not
appear to change with age during postnatal development
(Figs. 1D--F and 3). Quantitative assessments of the entire
cortical gray matter demonstrated similar age-related changes
in mean ROD levels of CB1R immunoreactivity for animals of
comparable postnatal ages in both species (Fig. 4). In the
combined cohorts, overall levels of CB1R immunoreactivity in
area 46 robustly increased during the embryonic and perinatal
periods and then did not change from 1 month of age
through adulthood (Fig. 4). The effect of age group on CB1R
immunoreactivity was highly significant (F9,39 = 10.5; P <
0.001), and post hoc analysis revealed significant increases in
CB1R immunoreactivity across the 3 youngest age groups
followed by a plateau during the remainder of postnatal
development (Fig. 4). Similarly, in the rhesus monkeys alone,
mean ROD levels of CB1R immunoreactivity did not differ as
a function of postnatal age group (F7,32= 0.52; P = 0.810).
Although the overall level of CB1R immunoreactivity was
unchanged during postnatal development, laminar-specific
changes in the distribution of CB1R-IR structures occurred
with age, with the density of CB1R immunoreactivity
decreasing in layers 1--2, increasing in layer 4, and not changing
in layers 3, 5, and 6 (Fig. 3; Supplementary Results and Fig. S2).
Pearson correlation analyses revealed that ROD levels of CB1R
immunoreactivity significantly declined with age in layer
1 (r = –0.410; P = 0.009) but significantly increased with age
in layer 4 (r = 0.412; P = 0.008) (Fig. 5A,C). Comparison of
neonatal (<3 months; n = 9), juvenile (5 months to 3 years; n =
20), and postpubertal animals (>3 years; n = 11) revealed
a significant main effect for age group in layers 1 (F2,37= 9.9;
P < 0.001) and 4 (F2,37= 5.4; P = 0.009) (Fig. 5B,D). Post hoc
tests revealed that mean ROD levels of CB1R immunoreactivity
significantly decreased by 14.9% in layer 1 before 5 months of
age but significantly increased by 10.8% in layer 4 after 3 years
of age (Fig. 5B,D).
Development of CB1R mRNA Expression in Rhesus
Levels of CB1R mRNA expression in area 46 appeared to be
highest qualitatively in animals 1 week of age, lower in animals
CB1 Receptor Development in Monkey Prefrontal Cortex
Eggan et al.
1 month of age, and lowest in animals 3 months of age and
older (Fig. 6A--D). Quantitative analysis demonstrated a signif-
icant (F6,23= 4.1; P = 0.008) effect of age group on CB1R mRNA
OD levels, with post hoc comparisons revealing a significant
41% reduction in CB1R mRNA expression across the 1-week to
3-month age groups followed by no significant change in
expression levels across the older age groups (Fig. 7E).
The expression of CB1R mRNA also changed within cortical
layers during postnatal development. In animals 1 week of age,
the density of CB1R mRNA-positive neurons was highest in
layers 2-superficial 3, intermediate in layers 4 and 5, and lowest
in deep layer 3 and layer 6 (Fig. 7A). The subsequent decline in
CB1R mRNA levels was associated with a shift in the relative
laminar distribution of the transcript such that in older animals
only layer 2 contained a prominent peak of CB1R mRNA
expression. Quantitative assessments revealed a significant
effect for age group in all cortical layers (all F‘s > 3.0; all P <
0.030) (Fig. 7B). Post hoc comparisons in each layer revealed
significant decreases in CB1R mRNA expression between each
of the age groups from 1 week to 3 months of age in all layers.
No significant difference in CB1R mRNA expression levels
across the older age groups occurred in layers 1--4, but
significant decreases did occur in layers 5 and 6 in the older
age groups (Fig. 7B).
Figure 1. Brightfield photomicrographs of CB1R immunoreactivity in DLPFC area 46 of pigtail macaque monkeys at E82 days (A), E138 (B), E157 (C), PN4 days (D), PN41 days
(E), and PN9 months (F) of age. In each panel, numbers and hash marks to the right indicate the boundaries of the cortical layers and the layer 6-white matter (WM) border.
Marginal zone (MZ), cortical plate (CP), and subplate (SP). Note the overall increase and the shift in laminar distribution of CB1R immunoreactivity with age. Scale bar 5 200 lm
in F (applies to A--F).
Cerebral Cortex May 2010, V 20 N 5 1167
In this study, we sought to understand the involvement of
CB1R-mediated signaling in the maturation of primate DLPFC
circuitry by examining changes in the levels of CB1R
immunoreactivity and mRNA during pre- and postnatal de-
velopment. The overall level of CB1R immunoreactivity
robustly increased during the pre- and perinatal periods and
then remained stable (Fig. 8A). However, embedded within
these findings were laminar-specific developmental changes in
innervation density, with a decrease in layers 1--2 that was most
marked during the first postnatal year and an increase in layer
4 that was prominent during adolescence (Fig. 8B). In contrast,
CB1R mRNA expression was highest at birth, markedly
decreased during the first 3 postnatal months, and then did
not change through development into adulthood (Fig. 8A); in
association with these changes, a distinct peak in CB1R mRNA
expression emerged in layer 2. Thus, the relative levels and
laminar distribution of both CB1R immunoreactivity and mRNA
exhibited distinctive patterns and different rates of change,
eventually achieving peaks of mRNA expression in layer 2 and
of CB1R-IR axons in layer 4. These findings, which do not
appear to be influenced by potential confounding factors such
Figure 3. Brightfield photomicrographs of CB1R immunoreactivity in DLPFC area 46 of rhesus monkeys at PN7 days (A), 4.8 months (B), 3.3 years (C), and 15 years (D) of age. In
each panel, numbers and hash marks to the right indicate the boundaries of the cortical layers and the layer 6-white matter (WM) border. Scale bar 5 200 lm in D (applies to
Figure 2. High-power brightfield photomicrographs in the pigtail macaque monkey illustrating CB1R-IR axons in the cortical plate at E82 days (A), CB1R-IR neurons and axons in
layer 3 at E157 days (B), and CB1R-IR axons at the layer 6-white matter (WM) border at E157 days (C), and at PN9 months (D) of age. Scale bar 5 30 lm in A and 100 lm in
CB1 Receptor Development in Monkey Prefrontal Cortex
Eggan et al.
as sex, levels of sex steroids, or changes in cortical volume (see
Supplementary Results), suggest a shifting role of CB1Rs in
cortical circuitry that may contribute to the functional
maturation of the DLPFC and to age-specific vulnerabilities to
cannabis exposure during both the perinatal and adolescent
periods of development.
Perinatal Development of CB1Rs in Monkey DLPFC
In monkey area 46, the overall levels of CB1R immunoreactivity
and mRNA exhibited opposite developmental trajectories during
perinatal development (Fig. 8A). Consistent with these findings,
other studies using a more limited number of developmental
time points reported that CB1R radioligand binding is higher in
adult compared with embryonic rats, whereas CB1R mRNA
expression is highest at embryonic ages and lower in the adult
(Berrendero et al. 1999). In addition, different developmental
trajectories for receptor proteins and their cognate mRNAs have
been reported for other neurotransmitter systems (Lidow 1995;
Jung and Bennett 1996; Weickert et al. 2007; Hashimoto et al.
2009). The dissociation between the developmental trajectories
for CB1R mRNA and immunoreactivity may reflect age-related
modifications in CB1R protein translation and/or turnover that
require high levels of CB1R mRNA expression in order to
increase and maintain appropriate levels of CB1R protein early in
development but require less mRNA expression later in
During perinatal development, marked changes occurred in
the types and distribution patterns of structures containing
CB1R immunoreactivity or mRNA. In particular, CB1R immu-
noreactivity and mRNA were transiently present in certain
structures that were not labeled later in development. For
instance, CB1R mRNA was expressed heterogeneously across
all cortical layers in the perinatal period but was restricted to
specific layers later in development. In the white matter
adjacent to the DLPFC, a transient, high density of CB1R-IR
axons was observed at E132 that gradually decreased through
the peri- and postnatal periods until 9 months postnatal when
CB1R-IR axons were no longer detectable in the white matter.
Consistent with these findings, transient localization of CB1R-
IR axons and CB1R radioligand binding in cortical white matter
have been reported during early development in the rat
(Romero et al. 1997; Deshmukh et al. 2007) and human (Mato
et al. 2003) brain. The fleeting presence of CB1R-IR axons in
the white matter and CB1R mRNA expression in the gray
matter could arise from migrating neurons and/or elongating
neuronal axons projecting from cell bodies in the DLPFC that
transiently express CB1R mRNA. Consistent with this idea,
a recent study in the mouse demonstrated that CB1R-IR
neurons born in the caudal ganglionic eminence and destined
for the hippocampus migrate through the cortical plate
(Morozov et al. 2009). Furthermore, CB1Rs regulate cortical
neuron axonal pathfinding and guidance early in development
(Berghuis et al. 2007), and we found a substantial increase in
the branches of CB1R-IR axons and varicosities in the
superficial cortical layers during the perinatal period that
parallels the marked increase in density of synaptic contacts in
monkey DLPFC during this period of development (Bourgeois
et al. 1994).
The presence of CB1R mRNA and protein in the monkey
DLPFC during the perinatal period suggests that endocannabi-
noid signaling plays a functional role in early developmental
processes, which regulate the formation of cortical circuits,
such as neuronal migration, axon growth and pathfinding, and
synaptogenesis. Indeed, CB1Rs are localized on neuronal
growth cones and activation of CB1Rs in immature CB1R/
CCK-IR neurons from the fetal rat cortex regulates the
morphological development, migration, and neurite growth of
these neurons (Berghuis et al. 2005, 2007). CB1Rs are also
located on the membranes of CCK-IR axon terminals where
they can suppress GABA release (Morozov and Freund 2003a),
and in cell bodies and proximal dendrites where they mediate
a form of slow self-inhibition that is generated by Ca2+
dependent autocrine release of endocannabinoids in CCK-IR
neurons (Bacci et al. 2004). Thus, CB1Rs may also play an early
role in the activity-dependent formation of excitatory synapses
by controlling the release of GABA from, and the excitability of,
CCK-IR neurons during a period of development when GABA
generates depolarizing events that cooperate with glutamate to
activate N-methyl-D-aspartate receptors and the subsequent
formation of glutamate synapses (Ben-Ari 2002).
These marked changes in expression and distribution of
CB1R mRNA and protein suggest that perinatal development is
a sensitive period for exposure to exogenous cannabinoids, and
consequently that cannabis use by pregnant or nursing mothers
could disrupt the normal development of DLPFC circuits.
Consistent with this idea, prenatal exposure to cannabi-
noids produces alterations in the development of a number
of neurotransmitter systems, including dopamine and serotonin
(Fernandez-Ruiz et al. 2000). Furthermore, in the rat hippo-
campus, prenatal D9-THC exposure increases the density of
CCK-IR neurons (Berghuis et al. 2005), whereas activation of
CB1Rs during the perinatal period inhibits GABA release and
disrupts neuronal network activity (Bernard et al. 2005), which
could alter the activity-dependent formation of inhibitory and
excitatory synapses. Similar types of effects in the DLPFC could,
Figure 4. Mean (bar) and individual (open triangles, pigtail monkeys; open circles,
rhesus monkeys) ROD levels of CB1R immunoreactivity across all cortical layers in
DLPFC area 46 are shown for each age group. ANCOVA demonstrated a significant
effect of age group on mean ROD levels and post hoc tests revealed significant
increases in mean ROD levels between the embryonic and 2--7 day age groups but no
change across age groups older than 1--4 weeks of age. Numbers in bars indicate the
number of monkeys in each age group. Abbreviations: d, days; E, embryonic;
m, months; w, weeks; and y, years.
Cerebral Cortex May 2010, V 20 N 5 1169
in part, contribute to the impaired cognitive functions that
develop later in life in the offspring of cannabis-using mothers
Development of CB1Rs during Adolescence in Monkey
The differences in amount and distribution of CB1R mRNA and
immunoreactivity in the monkey DLPFC with development
suggest a shift in the functional role of CB1Rs from the early
regulation of cortical circuit formation to a later, more
restricted role in modulating neurotransmission from specific
neuron types. In particular, although a small percentage of
CB1R-IR axons may arise from neurons containing the calcium-
binding protein calbindin (Bodor et al. 2005), the majority of
axons containing CB1R immunoreactivity in the monkey
DLPFC most likely arise from inhibitory interneurons that
contain the neuropeptide CCK, as indicated by studies in the
rodent hippocampus (Morozov and Freund 2003a). For
example, in the cerebral cortex of both rodents and primates,
1) CB1R-IR cells have morphologic features characteristic of
GABA neurons, such as round cell bodies and axon terminals
that form classic symmetric synapses (Bodor et al. 2005; Eggan
and Lewis 2007); 2) CB1Rs are highly expressed by, and
contained in the terminals of, CCK-IR basket neurons
(Marsicano and Lutz 1999; Katona et al. 2000; Bodor et al.
2005); and 3) activation of CB1Rs suppresses both the release
of GABA from CCK-IR neuron axon terminals and inhibitory
postsynaptic currents in pyramidal neurons (Galarreta et al.
2004; Trettel et al. 2004; Bodor et al. 2005). In addition,
comparison of the current findings with previous studies
indicate that CB1R- and CCK-IR structures in macaque monkey
DLPFC exhibit similar overall distribution patterns in the adult
and undergo nearly identical changes in laminar distribution
during postnatal development. In particular, both CB1R-
(Fig. 8B) and CCK-IR (Oeth and Lewis 1993) axons gradually
decrease in superficial layers and increase in layer 4 during
postnatal development. In contrast, this pattern of develop-
mental refinements of CB1R- and CCK-IR axons in the monkey
DLPFC is distinctly different from those exhibited by other
neuronal subpopulations, such as chandelier neurons (Cruz
et al. 2003, 2009), PV-IR basket cells (Erickson and Lewis 2002;
Cruz et al. 2003), dopamine axons (Rosenberg and Lewis
1995), or excitatory inputs to layer 3 pyramidal neurons
(Anderson et al. 1995; Gonzalez-Burgos et al. 2008).
Figure 5. Scatter plots of ROD levels of CB1R immunoreactivity in DLPFC area 46 for individual rhesus monkeys as a function of age in months plotted on a log scale in layers 1
(A) and 4 (C). Pearson correlation analyses revealed a significant decrease and increase in CB1R immunoreactivity with age in layers 1 (A) and 4 (C), respectively. Bar graphs
showing the mean (þSD) ROD levels within layer 1 (B) and layer 4 (D) for each of the 3 indicated age groups of rhesus monkeys. The mean ROD levels significantly decreased in
layer 1 before 3 months of age but increased in layer 4 after 3 years of age.
CB1 Receptor Development in Monkey Prefrontal Cortex
Eggan et al.
CB1R immunoreactivity has also been observed in asymmet-
ric, excitatory synapses in the neocortex (Katona et al. 2006;
Kawamura et al. 2006), and CB1R agonists modulate glutamate
release (Chevaleyre et al. 2006). However, the antibody utilized
in the present study exclusively labels symmetric, inhibitory
synapses by electron microscopy in both the monkey DLPFC
Figure 7. (A) Plots of mean CB1R mRNA OD as a function of cortical layer in DLPFC area 46 of rhesus monkeys in the postnatal 1 week, 3 month, and 3 to 4 year age groups. CB1R
mRNA expression prominently decreased across layers 2--6 with age. (B) Bar graphs of mean (þSD) OD levels of CB1R mRNA expression within each cortical layer for each age
group of rhesus monkeys. Multivariate analysis of variance demonstrated a significant effect of age group on mean OD levels of CB1R mRNA in each cortical layer. Within each panel,
age groups not sharing horizontal lines on the same level are significantly different at P\0.05; months (m), nanoCuries per gram (nCi/g), week (w), white matter (WM), years (y).
Figure 6. Representative film autoradiograms illustrating CB1R mRNA expression in DLPFC area 46 of rhesus monkeys at postnatal 1 week (A), 1 month (B), 3 months (C), and
8.7 years (D) of age. The density of hybridization signal is presented in pseudocolor according to the calibration bars to the left of A (applies to A and B) and C (applies to C and
D). E illustrates mean (bar) and individual (open circles) OD levels of CB1R mRNA expression across all cortical layers in DLPFC area 46 for each age group of rhesus monkeys.
ANCOVA demonstrated a significant effect of age group on mean OD levels and post hoc tests revealed significant decreases in mean OD levels between the 1-week and
3-month age groups but no change across age groups older than 3 months of age; days (d), months (m), nanoCuries per gram (nCi/g), microCuries per gram (lCi/g), years (y).
Cerebral Cortex May 2010, V 20 N 5 1171
(Eggan and Lewis 2007) and rodent hippocampus (Katona et al.
1999; Hajos et al. 2000), probably because the level of CB1Rs in
excitatory terminals is below the threshold of detectability
(Katona et al. 2006; Eggan and Lewis 2007). This interpretation
is supported by findings that 1) CB1R mRNA levels are much
higher in GABA neurons than in pyramidal cells (Marsicano and
Lutz 1999), 2) the density of CB1Rs is 20- to 30-fold higher in
inhibitory terminals than in excitatory terminals (Kawamura
et al. 2006), and 3) a ~30 times higher concentration of CB1R
agonist is necessary for 50% suppression of neurotransmitter
release from excitatory terminals than from GABA terminals
(Ohno-Shosaku et al. 2002). Furthermore, the CB1R antibody
used in Katona et al. (2006) revealed dense axon labeling in the
strata radiatum and oriens of the rodent hippocampus, which
contain the axon arbors of excitatory CA3 pyramidal neurons,
whereas these hippocampal layers contained the lowest
density of axons labeled by the antibody used in the present
study in both the rodent (Katona et al. 1999; Hajos et al. 2000)
and monkey (Eggan and Lewis 2007). Consistent with
a selective labeling of GABAergic axon terminals in the present
study, the observed developmental pattern of CB1R immuno-
reactivity does not match that of excitatory synapse density in
monkey DLPFC (Bourgeois et al. 1994; Anderson et al. 1995).
Thus, the observed changes in CB1R immunoreactivity across
postnatal development in the present study likely reflect
changes in the expression of CB1R protein specifically in
inhibitory neurons and axon terminals.
The postnatal refinements in the laminar distribution of
CB1R-IR axons suggest that CB1Rs may play a role in the
development of cognitive functions reliant on the DLPFC. For
example, the age-related improvements in performance on
working memory tasks (Goldman 1971; Alexander and Gold-
man 1978; Diamond 2002) parallels the increase of CB1R-IR
axons in layer 4. Working memory is thought to be mediated by
recurrent excitatory connections between discrete popula-
tions of DLPFC pyramidal cells in nonadjacent cortical columns
(Goldman-Rakic 1995). Thus, the developmental increase of
CB1R-IR terminals in layer 4, and the associated greater
capacity for depolarized induced suppression of inhibition
(Pitler and Alger 1992; Wilson and Nicoll 2002), could allow for
an activity-dependent disinhibition of pyramidal neurons that
share the same segregated excitatory inputs (Carter and Wang
2007), contributing to the neural substrate for improved
working memory function.
The age-related improvement in working memory perfor-
mance is also associated with the maturation of other
components of GABA circuitry in the DLPFC (Lewis et al.
2004). In particular, pre- and postsynaptic markers of
perisomatic GABAergic inputs to DLPFC pyramidal cells from
PV-IR neurons, which do not express CB1Rs, undergo
considerable changes during postnatal development, with
(Erickson and Lewis 2002; Cruz et al. 2003, 2009). In the
monkey DLPFC, pyramidal neurons receive convergent peri-
somatic input from PV-IR basket and chandelier neurons and
CB1R/CCK-IR basket neurons (Melchitzky et al. 1999; Eggan
and Lewis 2007). These convergent sources of perisomatic
inhibition in the rodent play specific roles in shaping network
activity (Freund 2003). For example, CB1R/CCK-IR and PV-IR
neurons fire at different phases of network oscillations
(Klausberger et al. 2005), generate temporally distinct epochs
of somatic inhibition (Glickfeld and Scanziani 2006), and play
complementary roles in regulating gamma band oscillations
(Hajos et al. 2000). Furthermore, recent evidence suggests that
CB1R/CCK-IR neurons regulate the activity of PV-IR neurons
(Foldy et al. 2007; Karson et al. 2008, 2009). Thus, the
developmental refinements in both CB1R/CCK- and PV-IR
neurons during adolescence may be involved in the maturation
of prefrontal gamma band synchrony (Uhlhaas et al. 2009),
which in humans, increases with, and in proportion to,
increasing working memory load (Howard et al. 2003).
Taken together, these observations suggest a mechanism by
which cannabis exposure during adolescence could serve as
a risk factor for the later development of schizophrenia.
Specifically, because stimulation of CB1Rs strongly suppresses
inhibitory input to pyramidal neurons from CCK-IR basket
neurons, the indiscriminate activation of these receptors
associated with cannabis use during adolescence may alter the
normal balance of CB1R/CCK-IR and PV-IR inhibitory inputs to
the perisomatic region of DLPFC pyramidal neurons. This
imbalance during a sensitive period may disrupt the develop-
mental trajectories of these GABAergic inputs, producing
Figure 8. Schematic summary Figures illustrating trajectories of overall CB1R immunoreactivity and mRNA levels (A) and density of CB1R-IR axons in layers 1 and 4 (B) across
development. Lines were generated by plotting the density as a percent of the maximum density value for individual animals for each marker as a function of age in months after
conception on a log scale, fitted by Loess regression analysis, and smoothed by hand. The shaded area indicates the approximate age range corresponding to adolescence (15--42
months; Plant 1988) in macaque monkey. Note the different developmental time courses in overall CB1R immunoreactivity and mRNA levels (A) and in the laminar distribution of
CB1R immunoreactivity (B).
CB1 Receptor Development in Monkey Prefrontal Cortex
Eggan et al.
persistent circuitry alterations that impair the mechanisms of
neural synchrony required for the maturation of working
memory performance. Consistent with this interpretation,
markers of both PV-IR (Pierri et al. 1999; Hashimoto et al.
2003) and CB1R/CCK-IR (Eggan et al. 2008) neurons are altered
in the DLPFC of subjects with schizophrenia, including in the
middle cortical layers, where the density of CB1R-IR axons
increases during adolescence (Fig. 8B). Thus, the increased risk
of schizophrenia associated with cannabis exposure during
the disruption of the normal refinements in CB1R axon
innervation patterns, and in PV neurons whose activity is
regulated by the output of CB1R-IR axons (Foldy et al. 2007;
Karson et al. 2008), during this sensitive period of development.
Materialcanbe foundat: http://www.cercor.
National Institutes of Health (grant numbers DA023109,
MH051234 to D.A.L.); National Institutes of Mental Health
Undergraduate Fellowship Program in Mental Health Research
grant R-25 (grant number MH54318-13 to S.R.S.).
The authors thank Dr Ken Mackie for kindly donating the CB1 receptor
antibody, Mary Brady for assistance with the graphics, and Jim
Kosakowski for Matlab program development. Conflict of Interest:
David A. Lewis currently receives investigator-initiated research
support from the BMS Foundation, Bristol-Myers Squibb, Curridium
Ltd and Pfizer and in 2007--2009 served as a consultant in the areas
of target identification and validation and new compound development
to AstraZeneca, Bristol-Myers Squibb, Hoffman-Roche, Lilly, Merck, and
Neurogen. All other authors have no conflicts of interest to disclose.
Address correspondence to Dr. Stephen M. Eggan, Department of
Psychiatry, University of Pittsburgh, 3811 O’Hara Street, W1653 BST,
Pittsburgh, PA 15213. Email: email@example.com.
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