Enriched expression of serotonin 1B and 2A receptor genes in macaque visual cortex and their bidirectional modulatory effects on neuronal responses.

Abstract

To study the molecular mechanism how cortical areas are specialized in adult primates, we searched for area-specific genes in macaque monkeys and found striking enrichment of serotonin (5-hydroxytryptamine, 5-HT) 1B receptor mRNA, and to a lesser extent, of 5-HT2A receptor mRNA, in the primary visual area (V1). In situ hybridization analyses revealed that both mRNA species were highly concentrated in the geniculorecipient layers IVA and IVC, where they were coexpressed in the same neurons. Monocular inactivation by tetrodotoxin injection resulted in a strong and rapid (<3 h) downregulation of these mRNAs, suggesting the retinal activity dependency of their expression. Consistent with the high expression level in V1, clear modulatory effects of 5-HT1B and 5-HT2A receptor agonists on the responses of V1 neurons were observed in in vivo electrophysiological experiments. The modulatory effect of the 5-HT1B agonist was dependent on the firing rate of the recorded neurons: The effect tended to be facilitative for neurons with a high firing rate, and suppressive for those with a low firing rate. The 5-HT2A agonist showed opposite effects. These results suggest that this serotonergic system controls the visual response in V1 for optimization of information processing toward the incoming visual inputs.

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Cerebral Cortex August 2009;19:1915--1928
doi:10.1093/cercor/bhn219
Advance Access publication December 4, 2008
Enriched Expression of Serotonin 1B and
2A Receptor Genes in Macaque Visual
Cortex and their Bidirectional Modulatory
Effects on Neuronal Responses
Akiya Watakabe1,2, Yusuke Komatsu1, Osamu Sadakane3,
Satoshi Shimegi3, Toru Takahata1, Noriyuki Higo4,
Shiro Tochitani1,2,7, Tsutomu Hashikawa5, Tomoyuki Naito3,
Hironobu Osaki3, Hiroshi Sakamoto3, Masahiro Okamoto3,
Ayako Ishikawa3, Shin-ichiro Hara3, Takafumi Akasaki3,
Hiromichi Sato3and Tetsuo Yamamori1,2,6
1Division of Brain Biology, National Institute for Basic Biology,
38NishigonakaMyodaiji,Okazaki444-8585,Japan,2Department
of Basic Biology, Graduate University for Advanced Studies, 38
Nishigonaka Myodaiji, Okazaki 444-8585, Japan,3Laboratory of
Cognitive and Behavioral Neuroscience, Graduate School of
Medicine, Osaka University, Toyonaka 560-0043, Japan,4System
Neuroscience Group, Neuroscience ResearchInstitute, National
Institute of Advanced Industrial Science and Technology,
Umezono 1-1-1, Tsukuba 305-8568, Japan,5Laboratory for Neural
Architecture, Brain Science Institute, RIKEN, Wako 351-0198,
Japan and6National Institute for Physiological Sciences, 38
Nishigonaka Myodaiji, Okazaki 444-8585, Japan
7Current address: Anatomy and Developmental Neurobiology,
Institute of Health Biosciences, University of Tokushima
Graduate School, 3-18-15 Kuramotochou, Tokushima
770-8503, Japan.
Akiya Watakabe, Yusuke Komatsu, and Osamu Sadakane
contributed equally to this work.
To study the molecular mechanism how cortical areas are
specialized in adult primates, we searched for area-specific genes
in macaque monkeys and found striking enrichment of serotonin (5-
hydroxytryptamine, 5-HT) 1B receptor mRNA, and to a lesser
extent, of 5-HT2A receptor mRNA, in the primary visual area (V1). In
situ hybridization analyses revealed that both mRNA species were
highly concentrated in the geniculorecipient layers IVA and IVC,
where they were coexpressed in the same neurons. Monocular
inactivation by tetrodotoxin injection resulted in a strong and rapid
(<3 h) downregulation of these mRNAs, suggesting the retinal
activity dependency of their expression. Consistent with the high
expression level in V1, clear modulatory effects of 5-HT1B and 5-
HT2A receptor agonists on the responses of V1 neurons were
observed in in vivo electrophysiological experiments. The modula-
tory effect of the 5-HT1B agonist was dependent on the firing rate
of the recorded neurons: The effect tended to be facilitative for
neurons with a high firing rate, and suppressive for those with
a low firing rate. The 5-HT2A agonist showed opposite effects.
These results suggest that this serotonergic system controls the
visual response in V1 for optimization of information processing
toward the incoming visual inputs.
Keywords: 5-HT, activity-dependent, area-specific, monocular deprivation,
primate, visual cortex
Introduction
Primates have highly complex cerebral cortices compared with
other mammals. One characteristic feature of the primate
cortex is the highly developed visual system. For example, at
least 32 visual areas have been distinguished in the neocortex
of primates (Felleman and Van Essen 1991). Among the visual
areas, V1 (primary visual area) is considered to be one of
? 2008 The Authors
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the earliest functional subdivisions that existed in the cortex of
the ancestral mammals (Northcutt and Kaas 1995). During the
course of evolution, however, there appear to have emerged
considerable differences in the functional architecture and
intracortical connectivity of V1 across species (see e.g.,
Zarrinpar and Callaway 2006; Van Hooser 2007). The monkey
V1 also has come to exhibit a highly laminated structure (Lund
1988), which reflects the structural separation of the different
types of visual information (e.g., Sincich and Horton 2005). In
our previous study, we identified occ1/frp mRNA as a molecule
that is highly enriched in the macaque V1 (Tochitani et al.
2001; Yamamori and Rockland 2006). Interestingly, the area-
specific expression patterns of the occ1 mRNA were observed
in macaques and marmosets but not in the other species
examined (Takahata et al. 2006). On the basis of the existence
of such a gene, we hypothesized that there may be a set of
genes that contribute to the structural and functional special-
izations of the primate visual system. If such genes do exist, the
identification and characterization of their functions in V1 will
markedly contribute to our understanding of the mechanisms
of primate vision.
To examine this possibility, we first carried out a new round
of screening in search of genes with V1-enriched expression
among macaque neocortical areas and found that the 5-HT (5-
hydroxytryptamine, serotonin) 1B receptor mRNA is highly
enriched in V1. In addition, we also found that the 5-HT2A
receptor mRNA exhibits an area/lamina pattern very similar to
that of the 5-HT1B receptor mRNA at the V1/V2 border, which
is consistent with previous reports (Burnet et al. 1995; Lope ´ z-
Gime ´ nez et al. 2001). 5-HT1B and 5-HT2A receptors belong to
G-protein--coupled receptors and activate a cascade of in-
tracellular signaling events (reviewed in Baumgarten and
Gothert 1997; Barnes and Sharp 1999; Sari 2004). They are
involved in neuropsychiatric disorders, as well as in a wide

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variety of physiological functions in different systems. The 5-
HT1B receptor, in particular, has been reported to modulate
neurotransmission in various pathways, such as the retinocol-
licular (Mooney et al. 1994), retino-suprachiasmic nuclear
(Pickard et al. 1999), and thalamocortical (Laurent et al. 2002)
pathways. The monkey V1 is densely innervated by serotonergic
terminals (de Lima et al. 1988) and possesses abundant 5-HT
radioligandbinding sites (Rakic etal. 1988; Parkinson et al. 1989).
Considering the enhanced expressions of 5-HT1B and 5-HT2A
receptor genes in V1, it is likely that the two 5-HT receptors play
important roles in modulating neurotransmission in V1.
The macaque V1 has been an excellent model system for
studying the functional organization of the primate cortex, in
which stimulus-evoked responses can be manipulated by
controlling external stimuli. A wealth of information on the
basic electrophysiological properties (Hubel and Wiesel 1977)
and anatomical circuitry of V1 neurons is available (Lund 1988;
Callaway 1998) and accumulating to date. Supported by these
previous studies, we wanted to understand the functional
meaning of V1-specific expression of the two 5-HT receptor
genes. First, we wanted to determine what anatomical
structures are associated with the expressions of the 5-HT1B
and 5-HT2A receptor mRNAs. Results of the in situ hybridiza-
tion (ISH) study showed that these mRNAs are highly enriched
in the excitatory neurons in geniculorecipient layers IVA and
IVC. Interestingly, we found that activity-driven regulation
plays a key role in this lamina specificity, as is found for
cytochrome oxidase (CO) (Wong-Riley 1994) or occ1 mRNA
(Tochitani et al. 2001) expressions. Second, we wanted to
determine the functional significance of this enriched expres-
sion using electrophysiological and pharmacological techni-
ques (Sato et al. 1996; Ozeki et al. 2004). Here, we provide
evidence that 5-HT1B and 5-HT2A receptor agonists modulate
neuronal responses in V1. Altogether, our results suggest the
importance of the serotonergic system in modulating the
behavior of V1 neurons, and that the V1-enriched expression of
the two 5-HT receptor genes should provide a basis for the
functional specialization of the primate V1.
Materials and methods
Anatomy
Experimental Animals
For restriction landmark cDNA scanning (RLCS) and semiquantitative
reverse transcription--PCR (RT-PCR) analysis, postmortem brain tissues
of African green monkeys (Cercopithecus aethiops) were obtainedfrom
the Japan Poliomyelitis Research Institute, as previously described
(Tochitanietal.2001;Watakabeetal.2001;Komatsuetal.2005).ForISH
experiments, the brains of 8 adult macaques, 7 Japanese monkeys
(Macaca fuscata) and 1 crab-eating monkey (Macaca fascicularis),
were used. These monkeys were perfused through the hearts with 4%
paraformaldehyde under anesthesia, and thus considered to be a
minimum postmortem period before tissue fixation. Monocular in-
activation for 21 days was performed as previously described (Tochitani
et al. 2001). Four nontreated monkeys were used for ISH experiments
and 4 monkeys were used for tetrodotoxin injection; 1 monkey for each
experiment of 21 days, 1 day, 6 and 3 h monocular inactivation. Part of
sections from the same monkey brains (2 nontreated monkeys and one
with tetrodotoxin injection for 21 days) used in previous studies
(Tochitani et al. 2001; Takahata et al. 2006) was used. All the
experiments described here were performed in compliance with the
guidelines for animal experiments of the National Institutes of Natural
Sciences, Japan and the National Institutes of Health, USA.
RLCS and Semiquantitative RT-PCR Analyses
RLCS was carried out essentially the same as described previously
(Suzuki et al. 1996; Shintani et al. 2004) using poly (A)+ RNA purified
from 4 (frontal, motor, temporal, and visual) areas depicted in Figure
1A. In this analysis, mRNAs from these areas were converted to double-
stranded cDNA by reverse transcription and digested with BclI (first
dimension) and HinfI (second dimension) for 2-dimensional gel
electrophoresis (Fig. 1B). The 4 cDNA samples were electrophoresed
simultaneously in a single run and the patterns of spots were compared
visually by 3 independent persons. Five candidates were chosen for
gene identification and RT-PCR analyses. One spot, which we identified
as the 3#-untranslated region (UTR) of 5-HT1B receptor gene (see
below for detail), showed the largest area differences among the 5
spots and used for subsequent analyses. Detailed description of the
method and the genes obtained by this screening will be published
elsewhere (Y. K. and T. Y., unpublished data).
Identification of 5-HT1B Receptor in RLCS Spots
The cDNA spot cloned from the RLCS analyses was used to screen
a cDNA library constructed using the poly (A)+ RNA purified from the
monkey visual cortex. All the positive clones obtained by this screening
matched the human genome sequence downstream of the 5-HT1B
receptor gene locus. The longest clone, RC15i, contained the sequence
spanning position 2764--4260 (coding sequence [CDS] = pos. 1--1173).
Because 5-HT1B receptor mRNA is longer than 28S ribosomal RNA (4.7
kb) (Jin et al. 1992), it was likely that the RLCS spot corresponds to the
5-HT1B receptor. Therefore, we conducted semiquantitative RT-PCR
using the primer sets within the cDNA retrieved from the RLCS gel
(data not shown) and within the coding sequence for the 5-HT1B
receptor (Fig. 1A). RT-PCR with both primers showed the same visual
area--specific pattern. Furthermore, ISH was carried out using 3
nonoverlapping probes: RC15i, containing the putative 3#-UTR region
(pos. 2764--4260), the coding sequence of the 5-HT1B receptor gene
(pos. 150--571), and a different region of the coding sequence with
3#-UTR (pos. 616--1599). All 3 probes showed the same ISH patterns
(data not shown). From these results, we concluded that this spot
identified by the RLCS analysis is the product of the 5-HT1B receptor
gene.
Monocular Inactivation
Monocular inactivation for shorter time periods (3--24 h) was
performed with slight modification in anesthesia as follows. Briefly,
monkeys were anesthetized with ketamine (6 mg/kg) and medeto-
midine (0.25 mg/kg) by intramuscular injection (i.m.). Tetrodotoxin
(15 lg dissolved in 10 lL of saline) was injected manually into the
vitreous cavity of the left eye using a Hamilton syringe over 5 min and
the syringe was left in position for further 15 min. After injection, the
loss of the pupillary reflex in the injected eye was confirmed. The
effect of medetomidine was then reversed by intramuscular injection
of atipamezole (1.25 mg/kg). The reversal effect was very quick and
the monkey was almost fully recovered after 10 min of atipamezole
injection, suggesting that the effect of ketamine had been already
gone at that time point. After 2.5 h of atipamezole injection
(approximately 3 h after tetrodotoxin injection), the monkey was
administered with ketamine (6 mg/kg, i.m.) followed by an overdose
of Nembutal (at least 100 mg/kg body weight) and perfused
intracardially with 4% paraformaldehyde in 0.1 M phosphate buffer
(pH 7.4).
In Situ Hybridization
The probes used in the ISH experiments were cloned by RT-PCR using
RNA from a rhesus monkey (Supporting Table S1). For the 5-HT1B
receptor, we prepared the 3 different probes described above and used
either mixed or separately. We also conducted ISH for the sense probes
of each gene to confirm the specificity of hybridization (data not
shown).
The tissue sections were made on freezing microtome (15-lm
thickness for double ISH, and 35- to 40-lm thickness for single ISH and
histochemical staining). CO staining was performed as previously
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described(Wong-Riley1994).ISHwas carriedoutasdescribedpreviously
with minor modifications (Tochitani et al. 2001). Free-floating sections
were treated with 10 lg/mL proteinase K for 30 min at 37 ?C. After
acetylation, the sections were incubated in a hybridization buffer
containing 0.5--1.0 lg/mL digoxigenin (DIG)--labeled probes at 60 ?C.
Hybridized sections were first washed in 23 SSC/50% formamide/0.1%
N-lauroylsarcosineat50?CandtreatedwithRNaseA.AfterRNasetreatment,
the sectionswerefurtherwashedin23 SSC/0.1%N-lauroylsarcosineandin
0.23 SSC/0.1% N-lauroylsarcosine at 37 ?C. Hybridization signals were
visualized by alkaline phosphatase immunohistochemistry followed by
nitro blue tetrazolium/5-Bromo-4-chloro-3-indolyl phosphate (NBT/BCIP)
detection (Roche Diagnostics, Tokyo, Japan).
For the double ISH, DIG-, and fluorescein isothiocyanate (FITC)--
labeled antisense probes were prepared and used for ISH. Hybridization
was carried out as described above in the presence of both DIG- and
FITC-labeled riboprobes. The fluorescence detection of the hybridiza-
tion signals was performed as described previously (Komatsu et al.
2005; Watakabe et al. 2007). Cell counting in Figure 4 and supporting
Table S2 was performed as described previously (Watakabe et al. 2007)
in a column of 172 lm (400 pixel) taken from double-stained tissue
sections (15 lm thickness) of V1 from 3 different monkeys. Layers
were determined on the basis of morphology and density of VGluT1-
mRNA--positive neurons.
Electrophysiology
Experimental Animals
Two adult macaques, M. fuscata, were used. Details of the experimen-
tal preparation are previously described (Sato et al. 1996; Ozeki et al.
2004). The animals were anesthetized with ketamine (10 mg/kg, i.m.)
followed by a mixture of isoflurane (2.5--3.5%) and N2O--O2(2:1). The
trachea of each animal was intubated and a catheter was placed in the
femoral vein. The animals were then placed in a stereotaxic head
holder, continuously paralyzed with pancuronium bromide (0.1 mg/kg/
h, i.v.) to minimize eye movements, and maintained under artificial
ventilation. During the recording of neuronal activity, isoflurane dose
was reduced to 0.3--0.7% in N2O:O2 (2:1), and fentanyl citrate
(Fentanest, Sankyo, Tokyo, Japan; 10 lg/kg/h, i.v.) and droperidol
(Droleptan, Sankyo, Tokyo, Japan; 125 lg/kg/h, i.v.) were continuously
infused. This treatment induced a state of neuroleptoanalgesia in the
animals. It enabled us to record visual responses of nearly normal
cortical activity and minimized the effect on mRNA expression which is
regulated in an activity-dependent manner. Only when the heart rate of
the animals exceeded more than 200 beats per min, sodium
pentobarbital (2--5 mg/kg/h, i.v.) was also added to the infusion
solution. A local anesthetic, lidocaine, was administered at pressure
points and around surgical incisions. Rectal temperature and the end-
tidal CO2level were adjusted to 37--38 ?C and 3.5--4%, respectively. All
electrophysiological procedures were carried out in accordance with
the guidelines for animal experiments of the Osaka University School of
Medicine, Japan and the National Institute of Health, USA.
Microiontophoresis
Drugs were administered iontophoretically via multibarreled glass
electrodes attached to a recording pipette. Triple- or 4-barreled glass
micropipettes were used for extracellular single-neuron recording and
the iontophoretic administration of the 5-HT1B receptor-specific
agonist CP93129 dihydrochloride (Tocris, Bristol, UK; 100 mM, pH
3.6), the 5-HT1B receptor-specific antagonist SB216641 hydrochloride
(Tocris; 20 mM, pH 4.0), 5-HT2A receptor-agonist DOI (Sigma-Aldrich,
St Louis, MO; 100 mM, pH 5.5), 5-HT2A receptor-specific antagonist
ketanserin (Tocris, Ellisville, MO; 10 mM, pH 5.5), and the Ringer’s
solution to the neurons under study (Sato et al. 1996). The tip of the
recording electrode protruded by 10--30 lm from the tip of the drug
pipettes. The strength of the ejection currents of drugs and the Ringer’s
solution was between +1 and +60 nA. The retaining current was
between –5 and –15 nA. No cells showed changes in spike size and firing
frequency during the iontophoretic administration of the Ringer’s
solution. We recorded neuronal responses to stimulation with
sinusoidal-grating patch with optimal stimulus parameters (Akasaki
et al. 2002). Peristimulus time histograms (PSTHs) of spike responses
were constructed during 6--10 times of stimulus presentations before
(control condition), during (drug condition) and after (recovery
condition) the drug administration. Only neurons whose response
level was recovered to the control level after cessation of drug
administration (P >0.05, Mann--Whitney’s U test) were included in our
results. In further analysis, the responses in the control and recovery
conditions were averaged and, then, the average was compared with
the responses during drug administration by Mann--Whitney’s U test.
Statistically significant increment and decrement of responses were
classified as response facilitation and suppression, respectively. The
Figure 1. Identification and confirmation of 5-HT1B receptor as V1-specific gene. (A)
Monkey neocortical areas used in this study are shown. The left hemisphere of the
cerebral cortex is shown. Anterior is to the left and posterior to the right. Major sulci
are shown by lowercase letters: p, principal sulcus; as, arcuate sulcus; ce, central
sulcus; ip, intraparietal sulcus; ts, superior temporal sulcus; and l, lunate sulcus. (B)
RLCS analysis of the monkey neocortex. Four distinct regions of the monkey
neocortex depicted in panel A were analyzed. The left panel shows an example of
a 2-dimensional gel of RLCS analysis. The area corresponding to the white box
contained a spot present only in the visual area (shown by arrowhead) but not in
temporal, motor and frontal areas. (C) Semiquantitave RT-PCR demonstrated that the
5-HT1B receptor mRNA expression level was high in the visual (vis) cortex, and very
low in the temporal (temp), somatosensory (som), motor (mot) or frontal (fro) areas.
The same specificity for the visual cortex was observed in 4 different individual
monkeys.The glyceraldehyde-3-phosphate
expressed across areas was used as control.
dehydrogenasegene ubiquitously
Cerebral Cortex August 2009, V 19 N 8 1917

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recording pipette was filled with 0.5 M sodium acetate containing 4%
Pontamine Sky Blue. At the end of each penetration, dye marks were
produced by passing tip-negative DC current (intensity: 8--10 lA,
duration: 1 s at 0.5 Hz, 100 pulses) and recovered in histological
sections (see below). After recording, the animals were deeply
anesthetized with sodium pentobarbital (50 mg/kg, i.v.) and perfused
transcardially with buffered saline followed by 4% paraformaldehyde in
saline. Thin cortical sections were sliced at 60-lm thickness in the
parasagittal plane and stained with cresyl violet, and the locations of the
recorded sites were identified by microscopic observations. In 1 case,
the cortical sections obtained from 1 of the experimental monkeys
were used to perform ISH of 5-HT1B and 5-HT2A genes, which showed
that their mRNAs were not significantly downregulated during the
recording periods.
Visual Stimulation
When a neuron was encountered to respond to visual stimuli, the
positions and sizes of the receptive fields of the recorded cells were
manually plotted using a hand-held projector on a tangent screen
placed 57 cm in front of the eyes of the animal. Subsequently, the
receptive field properties, such as dominant eye, optimal orientation
and direction of stimulus, spatial frequency, ON/OFF characteristics
with respect to a stationary flashed bar, and tuning to stimulus length
and velocity were assessed with the hand-held projector. Then
a computer-generated visual stimulus was presented on a CRT monitor
(CPD-G500J, SONY; Tokyo, Japan; mean luminance 40 cd/m2; screen
size, 40 3 30 cm2; resolution, 1024 3 768 pixels; and refresh rate, 100
Hz), which was placed 57 cm from the animal. The visual stimuli were
generated by a visual stimulation system VSG 2/3 (Cambridge Research
System, Rochester, UK) and controlled by an IBM-PC/AT compatible
computer using custom-made software. The visual stimulus consisted
of a drifting circular grating patch with optimal orientation, spatial
frequency, and velocity, which covered a cell’s receptive field. The
effects of drugs were tested for either the visual responses or
spontaneous firings. Each stimulus was drifted for 2 s and interleaved
for 1 s with a blank screen of the same mean luminance (40 cd/m2) as
that of the gratings. Either complex or simple cell was classified
according to the ratio of the first harmonic (F1) to the mean (F0) of the
response to a drifting grating stimulus (F1/F0 >1, simple cell; F1/F0 <
1, complex cell) (Skottun et al. 1991).
Results
Identification of 5-HT1B Receptor as V1-Specific Gene
To screen area-specific molecules systematically in the monkey
neocortex, we carried out a new round of screening by the
RLCS method (Suzuki et al. 1996; Shintani et al. 2004). In this
analysis, mRNAs were purified from 4 distinct cortical areas
(Fig. 1A), converted to cDNA by reverse transcription and
digested with a pair of restriction enzymes for 2-dimensional
analysis (Fig. 1B). Among the spots that showed area difference,
we cloned a gene that is specifically expressed in the visual area
(Fig. 1B), which turned out to be the 5-HT1B receptor gene (see
Materials and Methods). Semiquantitative RT-PCR analysis using
primers for the coding sequence of the 5-HT1B receptor gene
confirmed its specific expression in the visual area (Fig. 1C).
To examine the distribution of 5-HT1B receptor mRNA in
the monkey brain in more detail, we carried out ISH analyses.
The expression was strikingly high in V1 and the lateral
geniculate nucleus (LGN) (Fig. 2C,E). Because the mRNA
expression was low in the extrastriate cortex, the abrupt
change in the intensity of mRNA staining was observed at the
border between V1 and V2 (Fig. 2F). Within V1, the expression
of 5-HT1B receptor mRNA was mostly confined to layers IVA
and IVC, the major geniculocortical input layers, and was
particularly strong in the lower part of layer IVCbeta (Fig. 2H).
In addition, the expression was also observed at lower intensity
in layers II/III and VI of V1, which also receive geniculocortical
inputs to a lesser extent. In the LGN, the strong mRNA
expression was observed in all 6 layers, and there was no
significant difference in staining intensity between the magno-
cellular and parvocellular layers (Supporting Figs S1 and S3).
Compared with the strong expression in V1, the expression
of 5-HT1B receptor mRNA in other primary sensory areas was
much weaker. Nonetheless, the ISH signals were localized in
the middle layers (likely to be layer IV) as in V1 and
significantly higher than the surrounding areas (Fig. 2C, A1
and S1 for auditory and somatosensory cortices, respectively;
see also Fig. 3 and supporting Fig. S1O). There were also
expressions at lower levels throughout the neocortex. The
expression was generally restricted to the upper layers. In
addition, in the motor and insular cortices, layer 5 pyramidal
neurons also expressed 5-HT1B receptor mRNA at a low level
(Fig. 3, M1 and IPro). In the entorhinal cortex, 5-HT1B receptor
mRNA was expressed in layers V and VI (Fig. 3, Ent).
In addition, we observed the 5-HT1B receptor mRNA
expression in various subcortical structures, such as the non-
visual thalamus, parabigeminal nucleus, and ventral striatum
(Fig. 2C,D, Th, Pul, and PBG; see supporting material for
detailed account of the 5-HT1B receptor mRNA distribution).
Overall, however, the 5-HT1B receptor mRNA expression was
by far the most conspicuous in V1, followed by that in the LGN.
To date, 14 genes encoding different 5-HT receptors have been
identified in the human genome (Hoyer et al. 2002). To test
whether other 5-HT receptors exhibit an expression pattern
similar to that of 5-HT1B receptor mRNA, we cloned the cDNA
of each 5-HT receptor in macaques and carried out ISH. Table 1
shows the summary of the result for thirteen 5-HT receptor
genes, for which we obtained the reliable ISH data. Among the
13 genes, we detected the expressions of 5-HT1A, 5-HT1E,
5-HT2A, 5-HT2C, 5-HT3A, and 5-HT6 receptor mRNAs in V1 in
addition to 5-HT1B receptor mRNA. Among these genes, 5-HT2A
receptor mRNA exhibited area and lamina preferences similar to
those of 5-HT1B receptor mRNA (Table 1, Figs 4 and 5),
although its expression was moderate all across areas.
Coexpression of 5-HT1B and 5-HT2A Receptor mRNAs in
the Excitatory Neurons in Layer IVC of V1
The dense distribution of both 5-HT1B and 5-HT2A ISH signals
in layer IVC of V1 raised a question as to whether these genes
are coexpressed in the same neurons. To check this point
directly, we performed fluorescence double ISH of 5-HT1B and
5-HT2A receptor genes (Fig. 4). This experiment revealed that
these genes are strongly expressed in geniculorecipient layers
IVA and IVC (Fig. 4B). 5-HT1B receptor mRNA was mostly
confined to these layers, whereas 5-HT2A receptor mRNA was
more widely distributed across layers. Therefore, many neurons
expressed only the 5-HT2A receptor mRNA outside layers IVA
and IVC, whereas both mRNAs were coexpressed within the
same neurons in layers IVA and IVC.
We next asked which types of neurons expressed these
5-HT mRNAs, by performing the double ISH using VGluT1 for
excitatory neurons (Bellocchio et al. 2000; Takamori et al.
2000; Komatsu et al. 2005) and GAD67 for inhibitory ones
(Hendrickson et al. 1994). We found that the vast majority of
the neurons that expressed 5-HT1B or 5-HT2A receptor
mRNAs were the VGluT1 mRNA-positive excitatory neurons
(Fig. 5, supporting Table S2), whereas a small population of
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5-HT mRNA-positive neurons were VGluT1-negative inhibitory
neurons (Fig. 5, white arrows).
Next, we examined the percentages of VGluT1-positive
neurons that coexpress 5-HT1B or 5-HT2A receptor mRNAs
for each layer. The neurons with 5-HT1B receptor mRNA were
mostly restricted to layers IVA, IVCalpha, and IVCbeta, where
they constituted approximately 22.8± 3.5, 75.9± 3.5, and 70.4±
0.6% (mean ± SEM; n = 3) of the VGluT1-positive excitatory
neurons, respectively (Fig. 5A). In layers II, III, V, and VI, 5-HT1B
receptor mRNA was expressed in less than 5% of the excitatory
neurons.
Compared with the 5-HT1B receptor mRNA, the 5-HT2A
receptor mRNA was more widely expressed across layers. In
layer IVC, the 5-HT2A receptor mRNA was expressed in
approximately 80% of the excitatory neurons, which ratio is
similar to the 5-HT1B mRNA. The percentage of the excitatory
neurons expressing the 5-HT2A receptor mRNA decreased to
45--50% in layers III and V (Fig. 5B). The mRNA level per cell
also appeared to be decreased in these layers. Thus, the layer
IVC--enriched expression of the 5-HT2A receptor mRNA is
considered to be due to the higher proportion of positive cells
and the high level of mRNA expression within each cell. 5-
HT2A receptor ISH signals were barely detectable in the
excitatory neurons in layer VI.
5HT1B and 5HT2A receptor mRNAs were also detected in
GAD67 mRNA-positive inhibitory neurons (Supporting Fig. S2).
But the ratio of 5-HT1B and 5-HT2A--positive neurons among
the GAD67-positive cells were quite low, being 5% and 11%, in
our preliminary counting, respectively.
Activity-Dependent Regulation of 5-HT1B and 5-HT2A
Receptors
The enriched expression of 5-HT1B and 5-HT2A receptor
mRNAs in the geniculorecipient layers raises a possibility that
their expressions are controlled by the activity transmitted
through the retino-geniculate pathway, as is known for high
enzymatic activity of CO, an indicator of neuronal activity
(Wong-Riley 1994), and for occ1 mRNA that shows similar layer
specificity and activity dependency for the expression in V1
(Tochitani et al. 2001). To directly test this, we injected
tetrodotoxin into 1 eye of a monkey to deprive retinal activity
(Wong-Riley 1994) and examined whether the mRNA expres-
sions of the two 5-HT receptors are affected. As Figure 6 shows,
monocular inactivation caused a marked change in the
expression of the two 5-HT receptor mRNAs. After 21 days of
monocular inactivation, the expressions of both 5-HT1B (Fig.
6C) and 5-HT2A (Fig. 6D) receptor mRNAs were strongly
downregulated in the column in layer IV that corresponded to
Figure 2. ISH analysis of 5-HT1B receptor mRNA. (A--E) Coronal sections of an adult monkey brain were prepared from the positions as depicted. Bar: 1 cm. Several areas are
shown. Fro, frontal area; temp, temporal area; S1, primary somatosensory area; A1, primary auditory area. PBG, parabigeminal nucleus; Pul, pulvinar nucleus; St, striatum. Major
sulci are shown by lowercase letters: p, principal sulcus; cal, calcarin sulcus; ce, central sulcus; lf, lateral fissure; ts, superior temporal sulcus. (F) 5-HT1B receptor mRNA
expression at the V1/V2 border (shown by the black arrow). Bar: 500 lm. (G) The adjacent Nissl-stained section. (H) 5-HT1B receptor mRNA expression in V1. Bar: 200 lm.
Cerebral Cortex August 2009, V 19 N 8 1919

Page 6

the inactivated eye in a similar manner to that observed in CO
staining (Wong-Riley 1994) (Fig. 6A) or in the occ1 mRNA
distribution (Fig. 6B). Activity-dependent expression of 5-HT1B
receptor mRNA was also observed in the LGN (Supporting Fig.
S3). To determine time required for the monocular inactivation
to take effect, we examined shorter periods of monocular
inactivation (1 day, 6 and 3 h). The downregulation of 5-HT1B
and 5-HT2A receptor mRNAs in V1 occurred even after only
3 h of monocular inactivation (Fig. 6E,F). These results suggest
that the V1-specific expression patterns of 5-HT1B and 5-HT2A
receptor mRNAs are sustained by the ongoing visual activity.
Effects of 5-HT1B- and 5-HT2A Agonist/Antagonist on
Visual Responses in V1
To directly examine the functional roles of 5-HT1B and 5-HT2A
receptors in vivo, we conducted single-unit recordings of V1
neurons of anesthetized and paralyzed adult macaques, in
conjunction with the microiontophoretic administration of
specific agonists and antagonists of 5-HT1B and 5-HT2A
receptors (5-HT1B agonist, CP93129; 5-HT1B antagonist,
SB216641; 5-HT2 agonist, DOI; 5-HT2A antagonist, Ketanserin).
The results of CP93129 (5-HT1B agonist) in 45 neurons and
DOI (5-HT2A agonist) in 44 neurons are summarized in Figures
7 and 8, respectively.
Figure 7A shows the magnitude of visual responses of V1
neurons to a drifting sinusoidal-grating patch in which
responses duringiontophoresis
CP93129, were plotted against control responses. Of the 45
neurons recorded, 33 showed significant changes in firing rates
during the CP93129 administration (P < 0.05, Mann--Whitney’s
U test), and in most of them (25 of 33), the effect of CP93129
was facilitative (Fig. 7A). Eight neurons showed suppressive
of the5-HT1B agonist,
Figure 3. 5-HT1B receptor mRNA expression in various cortical areas. The expression of 5-HT1B receptor mRNA was examined in various cortical areas. The asterisks indicate
the presumptive layer IV. V2; secondary visual area, V4; area V4 on the prelunate gyrus, TE; area TE in the inferior temporal gyrus, PO; area PO, PE; area PE (area 7) on the
superior parietal lobule, A1; primary auditory area, S1; primary somatosensory area (area 3b) facing the central sulcus, S2; secondary somatosensory area, M1; primary motor
area, 6DC; area 6DC or premotor area, FEF; frontal eye field (area 8), 46; area 46 on the bank of principle sulcus, Cing; cingular cortex (area 23), Ent; entorhinal cortex, IPro; insular
proisocortex, DI; dysgranular insular cortex. Scale Bar: 200 lm.
1920
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Page 7

effect of CP93129, whereas the remaining 12 neurons, no
effect. To confirm that the effect of CP93129 is receptor-
mediated, we coadministered the 5-HT1B receptor-specific
antagonist SB216641. SB216641 antagonized the effects of
CP93129 in all the 5 neurons tested, including both facilitated
and suppressed neurons (data not shown).
Although CP93129 showed both facilitative and suppress-
ive effects, we noted that all the neurons, except for 1 that
were suppressed by the 5-HT1B agonist had small response
magnitude. The part of the graph corresponding to the
response magnitude less than 50 spikes/s (shown with a dotted
line in Fig. 7A) was enlarged in Figure 7B. In this graph, all the
neurons suppressed by CP93129 exhibited firing rates lower
than 13 spikes/s. To examine whether the incidence of each
type of response modulation depends on the difference of
firing rate of neurons, we classified neurons showing significant
response modulation into 2 groups according to firing rate,
either higher or lower than 13 spikes/s, and performed Fisher’s
exact test. However, the difference was not at statistically
significant level (P = 0.098) in spite of the high incidence of
suppressed neurons in low firing group, which was owing to 1
neuron that showed high firing rates (an average of 82.5 spikes/s)
at control but it showed suppressive modulation. Thus, we
speculate that CP93129 mainly facilitated the visual responses
of V1 neurons but tended to suppress a population of neurons
with low firing rates.
This raised a possibility that the sign of the modulatory
effects of CP93129 is dependent on the neuron’s response
level. To test this hypothesis, we changed the response
magnitude of the recorded neuron by changing contrast of
stimuli presented and examined whether CP93129 has
differential effects on single neurons with different activity
levels. Figure 7C depicts the PSTHs that show visual responses
of a V1 neuron to a drifting sinusoidal-grating patch at 2
stimulus contrasts of 20% (right) and 100% (left) in 3 drug
conditions; control, during iontophoresis of CP93129, and
recovery from the drug effect. In this example, CP93129 clearly
facilitated the responses at high grating contrast (100%) but
suppressed them at low grating contrast (20%). The effects of
CP93129 on the stimulus contrast-response relationship was
tested for the same neuron (Fig. 7D). This example demon-
strates that the activation of 5-HT1B receptors facilitates the
responses to high-contrast (>30%) stimulation, but is suppres-
sive or ineffective for responses to low-contrast stimulation
(<20%). Similar response-dependent effects of CP93129 were
observed for all the 3 neurons tested, including the one shown
in Figure 7C,D.
The firing-rate--dependent effects of CP93129 may improve
the signal-to-noise ratio (S/N ratio) of activity of V1 neurons. To
test this point, we calculated the S/N ratio as the ratio of the
number of spikes in the visual response to spontaneous
discharge which was added by 1 [S/N = (visual response)/
(spontaneous discharge + 1)] because our sample included
neurons without spontaneous discharge (0 spikes/s). The S/N
ratio was calculated for the 24 neurons which were facilitated by
CP93129, and was significantly larger during drug administration
than control (Wilcoxon signed-rank test, P < 0.01). Thus, our
results suggest that the effect of the 5-HT1B receptor agonist is
dependent on the response level of each neuron, and that it
improves S/N ratio of visual input from the LGN to the cortex.
Next, we examined the effects of the 5-HT2A agonist, DOI,
on visual responses. Results were very intriguing because DOI
also exerted bidirectional modulatory effects on the neuron’s
firing rate, but the sign of response modulation was opposite
that of the 5-HT1B receptor agonist CP93129. Figure 8A,B
shows a summary of the modulatory effects of DOI on 44
neurons. Most of the facilitative effects (12/44 cells) were
concentrated in neurons that exhibited firing rates lower than
25 spikes/s. On the other hand, suppressive effects (9/44 cells)
were more often observed in neurons with firing rates higher
than 25 spikes/s. Therefore, to examine the relationship
between the incidence of the type of response modulation by
Table 1
Expression of various 5-HT receptor genes in V1, V2, and LGN
5-HT1A 1B1D1E1F2A 2B 2C 3A3B467
\\V1[[
Layer I
Layer II/III
Layer IVA
Layer IVB
Layer IVCalpha
Layer IVCbeta
Layer V
Layer VI
\\V2[[
Layer I
Layer II/III
Layer IV
Layer V
Layer VI
\\LGN[ [
Magno
Parvo
?
B
?
?
?
?
?
þ
?
þ
þþþ
?
þþ
þþþþ
?
þ/?S
?
?
?
?
?
þ/?
?
?
?
þ
?
?
?
?
þ
þ
?
?
?
?
?
þ/?#
?
?
?
þþ
þþþ
þ
þþþ
þþþþ
þþ
þ
?
?
?
?
?
?
?
?
?
S
?
?
?
?
?
þ
?
?
?
?
?
?
þ
þ/?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
þ/?
?
?
þ
?
?
?
?
þ/?
þ
?
þ/?
?
?
?
?
?
?
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þþ
?
?
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þ
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?
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þ/?#
?
?
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þþ
þþþ
þþ
þ
?
?
?
?
?
?
S
?
þþ
?
?
?
?
þ
þ/?
?
?
?
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?
?
?
?
þ
?
?
þ
?
þ/?
þ
?
þ/?
?
?
?
þ
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þþ
þþ
þ/?
þ/?
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þ
?
?
?
?
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?
?
þ
þ
I
I
Note: ISH analyses for the known 5-HT receptor genes except for the 5-HT5B receptor gene, which is reported to be nonfunctional in humans, are shown. Although we also tested the 5-HT5A receptor
gene, we observed a high background and did not obtain specific hybridization signals. For 5-HT2B and 5-HT3B receptor genes, no specific hybridization signals were observed in the sections tested. For
the other receptors, we observed specific hybridization signals in certain brain regions. The intensities described here were rated from the relative intensity of the signals and were determined on the
basis of the following criteria: ?, not detected at more than the background level; þ/?, expression was observed but close to detection limit; þ, þþ, and þþþ; distinct expression with different
intensities. We often observed ISH signals in sparsely scattered cells (denoted as S). For 5-HT1B and 5-HT2A receptor mRNAs, to represent their very high specificity for layer IVCbeta, þþþþ was
used. For 5-HT1A receptor mRNA, its expression in layers II and III of V1 was observed only at the border between layers I and II (denoted as B). For 5-HT1F receptor mRNA, weak but consistent signals
were observed at the border between layers IVCbeta and VA and we are not certain as to which layer this signal belongs to (denoted as #). For 5-HT7 receptor mRNA, very weak signals were observed
in the LGN in a few scattered cells that appeared to be interneurons (denoted as I).
Cerebral Cortex August 2009, V 19 N 8 1921

Page 8

Figure 4. Double ISH of 5-HT1B and 5-HT2A receptor genes in monkey V1. (A) Double ISH with 5-HT1B (green) and 5-HT2A (red) gene probes. The white boxes in layers II, IVA,
and IV/V are magnified in (B). Bar: 200 lm. (B) A higher magnification view of the white boxes in (A). Note that most of the 5-HT1B--positive neurons coexpress 5-HT2A receptor
mRNA in layers IVA and IVC, but not necessarily so in layer II. Bar: 50 lm.
Figure 5. Double ISH of 5-HT1B/2A receptor and VGluT1 gene probes in monkey V1. (A) Double ISH with 5-HT1B (red) and VGluT1 (green) gene probes. Bar: 100 lm. The white
box in layer II and that at the border between layers IV and V, respectively, are magnified below. White arrows indicate the 5-HT1B receptor mRNA-positive neurons with no
detectable expression of VGluT1 mRNA. Bar: 20 lm. The ratios of the neurons that express 5-HT1B receptor mRNA among the VGluT1-positive excitatory neurons (1B/VG) in
layers II, III, IVA, IVB, IVCalpha, IVCbeta, V, and VI are shown by a bar graph on the right side of panel A. Error bars are SEM (n 5 3). (B) Double ISH with 5-HT2A (red) and VGluT1
(green) gene probes. The red signals represent 5-HT2A receptor mRNA. The other descriptions are the same as in panel A. Note that the ratio of the 5-HT2A--positive neurons in
the bar graph is high in the upper layers as well. In these upper layers, we generally detected a lower level of 5-HT2A receptor mRNA per cell, compared with that in layer IVC.
1922
Serotonin Receptors in Monkey V1
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Page 9

DOI and the class of firing rate demarcated at 25 spikes/s, we
performed Fisher’s exact test and found statistically significant
difference among those factors (P <0.01). This analysis showed
that DOI facilitates visual responses of neurons with a low firing
rate but suppresses those of neurons with a high firing rate.
However, DOI shows an affinity not only for 5-HT2A but also
for 5-HT2B/2C (Barnes and Sharp 1999). To confirm that the
modulatory effect by DOI is mediated by 5-HT2A receptors, we
tested the effect of ketanserin, a highly selective antagonist of
the 5-HT2A receptors (Barnes and Sharp 1999), on endogenous
serotonin (Fig. 8C,D). On the basis of the results of DOI, we
expected that ketanserin would block the facilitative effect of
the endogenous serotonin on neurons with low firing rates.
Indeed, the suppressive effect of ketanserin was mostly
observed in neurons with firing rates lower than 25 spikes/s.
Ketanserin also showed a facilitative effect on 1 neuron with
a high firing rate. Thus, we conclude that 5-HT2A receptors
show response-dependent modulatory effects, but that its
effects are opposite those of 5-HT1B receptors.
Finally, we show the laminar distributions of the effects of
CP93129, DOI and kentanserin (summarized in Table 2). In
contrast to the highly layer-specific distribution of 5-HT1B and
5-HT2A receptor mRNAs, we found no difference in drug effect
between layer IVC and other layers (Fisher’s exact test,
CP93129, P = 0.60; DOI, P = 0.47; ketanserin, P = 1.00).
Although the numbers of sampled cells are relatively small, the
facilitative and suppressive effects of CP93129 and DOI were
evenly distributed across layers.
Discussion
In this study, we report that 5-HT1B and 5-HT2A receptor
mRNAs are highly enriched in the geniculorecipient layers in
macaque V1. Studies by us as well as by other researchers
indicate that this expression pattern is not observed in
nonprimate mammals, suggesting species-specific (perhaps
primate-specific) roles of 5-HT1B and 5-HT2A receptors in
visual processing. Our in vivo electrophysiological experiments
demonstrated that the 5-HT1B and 5-HT2A agonists exert
modulatory effects on the responses of V1 neurons in macaque
monkeys. These data suggest a correlation between area-
specific gene expression and functional properties.
Distribution of 5-HT1B Receptor mRNA and Proteins in V1
In contrast to the highly specific localization of the 5-HT1B
receptor mRNA in the geniculorecipient layers, the effects of
receptor agonists were observed throughout all 6 layers in V1
suggesting a wider distribution of the receptor proteins than
that of the mRNA. Consistent with this observation, previous
radioligand binding studies demonstrate wide-spread distribu-
tion of the 5-HT binding sites within the monkey V1 (Rakic
et al. 1988; Parkinson et al. 1989). Although we do not have our
own experimental data, many reports suggest presynaptic
localization of 5-HT1B receptor protein (Mooney et al. 1994;
Ghavami et al. 1999; Riad et al. 2000; Laurent et al. 2002; also
see Sari 2004 for a review). Especially, Varna ¨ s et al. (2005)
found a high level mRNA expression of 5-HT1B receptor gene,
but very low level receptor binding sites in the LGN of humans,
suggesting that the 5-HT1B receptor protein produced in the
LGN may be transported to the presynaptic sites in the visual
cortex. Similarly, 5-HT1B receptor proteins produced in layers
IVA and IVC may well be distributed widely across layers,
because the neurons in these layers participate in interlaminar
processing, both supra- and infragranular (Lund 1988). How-
ever, it is also possible that the accumulation of mRNA is not
proportional to that of the protein because additional
regulation at the level of translation and turnover rates may
be present.
Compared with the 5-HT1B receptor, 5-HT2A receptor
mRNA was detected more widely across layers (Fig. 5B), which
is consistent with the electrophysiological result (Table 2).
Regarding this point, previous reports suggest postsynaptic
localization of the 5-HT2A receptors (Rakic et al. 1988; Lo ´ pe ´ z-
Gime ´ nez et al. 2001; Miner et al. 2003). The subcellular
localizations of the 5-HT1B and 5-HT2A receptor proteins have
an important implication on the differential roles of these
receptors in modulating visual processing in the early visual
system. Further study is needed to understand the subcellular
localization of 5-HT1B receptor proteins and their physiolog-
ical effects in V1.
Activity-Dependent and V1-Specific Expression of 5-HT
Receptor Genes in Primates
Although activity-dependency clearly plays an important role
in determining the distribution patterns of the 5-HT1B and
Figure 6. Expressions of 5-HT1B and 5-HT2A receptor mRNAs in V1 following
monocular inactivation. TTX was injected into 1 eye twice a week for 21 days (A--D),
or 3 h before sacrifice (E,F). These monocularly inactivated monkey brains were
histochemically analyzed. (A) CO staining near V1-V2 border (shown by the black
arrowheads). (B--D) ISH in V1 and V2. (B) occ1, (C) 5-HT1B receptor, (D) 5-HT2A
receptor. Adjacent sections were analyzed in this order. Black circles in panels (A--D)
indicate layers IVA and IVCbeta. (E and F) 5-HT1B and 5-HT2A receptor mRNA
expressions were examined by ISH after 3 h monocular inactivation (MD). These
photos show tangential sections at the level of layer IVC. Note that 5-HT1B and 5-
HT2A receptor mRNAs exhibit almost identical patterns. Scale bars: 500 lm.
Cerebral Cortex August 2009, V 19 N 8 1923

Page 10

5-HT2A receptor mRNAs, ‘‘activity’’ alone is not sufficient to
account for their high area/lamina specificity. Many genes
under activity-dependent regulation have been reported (e.g.,,
GAD67, Gamma-aminobutyric acid (GABA) receptors, CAMKs,
NRF, zif268; Jones 1990; Benson et al. 1991; Huntsman et al.
1994; Chaudhuri et al. 1995; Tighilet et al. 1998; Nie and Wong-
Riley 1999; Guo et al. 2000). Nevertheless, the expression of
these genes is neither lamina nor area specific (e.g., Okuno
et al. 1997). The expression patterns of occ1, 5-HT1B, and 5-
HT2A receptor genes could be explained if their transcriptions
are upregulated only by retinal inputs but not by other factors.
We found that both the 5-HT1B and 5-HT2A receptor mRNAs
exhibit a very rapid response to the loss of retinal activity,
comparable to that of immediate-early genes such as c-fos and
zif268 (Knapska and Kaczmarek 2004). The high turnover rate
of the two 5-HT receptors may be appropriate for adjusting the
visual cortical functions to change of environment, for
example, diurnal rhythm. In addition, however, there may
exist cell-type specific gene regulation to achieve such
a specific expression in V1.
The expression patterns of 5-HT1B receptor mRNA reported
in this study are quite different from those reported for
nonprimate mammals (Boschert et al. 1994; Bruinvels et al.
1994; Bonaventure et al. 1998). We also examined the
expressions of 5-HT1B and 5-HT2A receptor mRNAs in
marmosets (a New World monkey), ferrets and cats, in addition
to rats and mice, and found similar V1 specificity in only
marmosets (data not shown). In postmortem human brains, 5-
HT1B receptor mRNA is enriched in the middle layers of the
occipital cortex (putative visual area) and the upper layers of
most other areas, the lamina pattern of which is reminiscent of
that in monkeys (Varna ¨ s et al. 2005). Although the reported
area difference in humans does not appear to be so
conspicuous compared with that in monkeys, this may be
due to a long postmortem period before fixation (>29 h) or to
age difference, whereas the processing of monkey samples by
trans-cardiac perfusion fixation is very rapid. From these
observations, we consider that the activity-dependent expres-
sions of 5-HT1B and 5-HT2A receptor mRNAs in adult
geniculorecipient layers may be a characteristic feature of the
primate cortex. Further comparative studies would prove this.
It is intriguing that a set of molecules, occ1, 5-HT1B, and
5-HT2A receptor mRNAs show V1-enriched expression and are
under similar activity-dependent regulation. It is quite possible
Figure 7. Effects of 5-HT1B agonist, CP93129, on visual responses of neurons in macaque V1. (A) Effects of CP93129 on firing rates of 45 V1 neurons. Firing rates under drug-
administered condition (ordinate) were plotted against those of control responses (average of responses during the control and recovery conditions) (abscissa). Symbols indicate
effects of CP93129 (open circle, facilitation; cross, no effect; filled triangle, suppression). (B) Part of the graph shown in A (shown by a dotted line in A) is indicated with enlarged
scales. (C) PSTHs of visual responses of a V1 neuron to a drifting sinusoidal-grating patch at 2 stimulus contrasts of 20% (right) and 100% (left) in 3 drug conditions; control,
during iontophoresis of CP93129, and recovery from the drug effect. (D) Effects of CP93129 on contrast-response function of a complex cell in layer 4B. Circles, squares, and
triangles are averaged responses (±SEM) to the visual stimulus at each grating contrast before, during, and after drug administration, respectively.
1924
Serotonin Receptors in Monkey V1
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Page 11

that these molecules work cooperatively in the monkey V1 in
response to incoming inputs. Although the specific function of
theocc1moleculeisyetunknown,thisgenebelongstoafamilyof
extracellular matrix proteins, some of which are known to affect
ocular dominance plasticity in rodents (Berardi et al. 2003).
5-HT1B and 5-HT2A receptors affect synaptic transmission, as we
have shown in this paper. In addition, they may also affect
synaptic plasticity by regulating downstream signal transduction
pathways. For example, 5-HT1B receptors are considered to be
negatively coupled to adenylate cyclase (reviewed in Sari 2004),
whereas 5-HT2A receptors recruit phospholipase C, pertussis
toxin--sensitive heterotrimeric G(i/o) proteins, and Src signaling
(Gonzalez-Maeso et al. 2007). The enriched expressions of these
molecules in response to visual inputs could cause or modulate
plasticity in the primate V1.
Firing-Rate--Dependent Effect of 5-HT1B/2A Agonists In
Vivo
The electrophysiological study revealed the complexity of the
in vivo effects mediated by the 5-HT1B and 5-HT2A receptors.
We found that each receptor can exert both suppressive and
facilitative effects, depending on the firing rate of the recorded
neurons. Regarding the 5-HT1B receptor, an analogous
context-dependent bidirectional modulation has been reported
in in vitro preparations of brain slices containing the optic tract
and the LGN (Seeburg et al. 2004) or the ventral posteriomedial
nucleus of thalamus and the somatosensory cortex (Laurent
et al. 2002). For example, Seeburg et al. (2004) reported that 5-
HT1B receptor--mediatedserotonergic
responses in LGN neurons to stimulation of optic tract is
modulation of
Figure 8. Effects of 5-HT2A agonist DOI and antagonist ketanserin on visual responses of neurons in macaque V1. (A) Effects of DOI on firing rates of 44 neurons in V1. Firing
rates under drug-administered condition (ordinate) were plotted against those of control responses (average of responses before and after iontophoretic administration)
(abscissa). Symbols indicate effects of DOI (open circle, facilitation; cross, no effect; filled triangle, suppression). (B) Part of the graph shown in (A) (shown by a dotted line in A) is
indicated with enlarged scales. (C) The effects of ketanserin on the firing rate of 44 V1 neurons. Firing rates during drug condition (ordinate) were plotted against that of control
responses (the average of responses before and after iontophoretic administration) (abscissa). Symbols indicate effects of ketanserin (open circle, facilitation; cross, no effect;
filled triangle, suppression). (D) Enlarged part of the graph shown with dotted line in (C).
Table 2
Lamina distribution of recorded neurons in the electrophysiological experiments
Layer
CP93129
2/34B4C 5/6UnknownTotal
Facilitation
Suppression
No effect
Total
DOI
Facilitation
Suppression
No effect
Total
Ketanserin
Facilitation
Suppression
No effect
Total
7
2
3
7
0
0
7
5
2
6
5
2
1
8
1
2
2
5
25 (56%)
8 (17%)
12 (27%)
4512 13
2
3
7
4
2
3
9
2
0
1
3
1
4
8
3
0
4
7
12 (27%)
9 (20%)
23 (52%)
441213
1
3
2
6
0
1
0
1
0
3
2
5
0
5
8
0
3
1
4
1 (3%)
15 (51%)
13 (45%)
2913
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Page 12

dependent on the temporal frequency of the stimulus. The net
effect of the 5-HT1 receptor agonist on retinogeniculate
transmission is suppressive for low-frequency inputs, but
rather ineffective or facilitative for high-frequency inputs. They
suggested that the alleviation of synaptic depression caused by
high-frequency stimulation may underlie such effects of the 5-
HT1B receptors.
The effects of the 5-HT2A receptors, on the other hand,
should be explained by a different mechanism, because 5-HT2A
receptor activation is considered to cause phospholipase C--
mediated synaptic facilitation by reducing outward potassium
current (Barnes and Sharp 1999; Lambe and Aghajanian 2001).
An activation of 5-HT2A receptor has been known to exert
direct facilitative actions on not only pyramidal neurons but
also interneurons (Araneda and Andrade 1991; Tanaka and
North 1993), by which neighboring pyramidal neurons are
indirectly inhibited (Sheldon and Aghajanian 1991; Marek and
Aghajanian 1996; Zhou and Hablitz 1999). Therefore, serotonin
probably has complex influences by controlling the relative
activity of excitatory and inhibitory neurons within local
circuitry via 5-HT1B and 5-HT2A receptors.
Physiological Significance of Serotonergic Modulation in
Visual System
Serotonergic fibers that arise from the dorsal and median raphe
nuclei (Schofield and Everitt 1981; Sladek et al. 1982) innervate
the neocortex including the visual cortex of the primate
(Morrison et al. 1982; Doty 1983; Foote and Morrison 1984; de
Lima et al. 1988; Wilson and Molliver 1991) and the cat
(Jonsson and Kasamatsu 1983). Raphe nuclei in cats or rats
have been shown to change their activity pattern according to
the level of behavioral arousal across the sleep--wake--arousal
cycle (McGinty and Harper 1976; Lydic et al. 1983; Guzman-
Marin et al. 2000). In addition, the 5-HT release in V1 could be
regulated locally by 5-HT1B autoreceptors on the presynaptic
terminals of the raphe nucleus that innervate V1 (for a review,
Sari 2004) or potentially by the activity from the prefrontal
cortex (Celada et al. 2001). The effect of serotonin in the
cortex should, thus, depend on the dynamic regulation of the
level of serotonin and the receptors.
Although the enhanced expression of 5-HT1B receptor
mRNA in the LGN and V1 suggests its primary role in the visual
system, it was also expressed widely in the thalamus (Fig. 2,
supporting Fig. S1). Weak but significant cortical expression
was concentrated in the thalamocortical input layers of the
primary sensory (auditory and somatosensory) areas (Figs 2 and
3, supporting Fig. S1). The roles of serotonin in V1 might thus
be regarded as an enhancement of a general role of serotonin in
the modulation of thalamocortical transmission.
In this study, we demonstrate that the activation of 5-HT1B
receptors in V1 generally facilitates visual responses but tends
to suppress weak responses. This suggests that, in geniculocort-
ical transmission, nonsychronized spontaneous activity (noise)
from the LGN neurons would be reduced by the suppressive
effectof5-HT1Breceptors,butthevisuallyevokedsynchronized
signals would be preserved or efficiently transferred to V1, thus,
enhancing the S/N ratio in input--output relationship. On the
other hand, neurons in the input layers of V1, which abundantly
express the 5-HT2A receptor may act as a gain controller by
enhancing weak signal response and suppressing excessive
response. We therefore suggest that, serotonin release in V1
exerts coordinated modulatory effects through 5-HT1B and 5-
HT2A receptors on the V1 neurons. It is therefore possible that
the serotonin system has contributed to the evolution of the
elaborated function of the primate visual system.
Supplementary Material
Supplementary material can be found at: http://www.cercor.
oxfordjournals.org/
Funding
Grants-in-Aid for Scientific Research (A) on Priority Areas (A)
and (17024055) to T.Y. and on Priority Areas (17022026) to H.S.;
and JSPS grant (KAKENHI19500304 and KAKENHI19530656) to
A.W. and S. S., respectively.
Notes
A.W., Y.K., and O.S. contributed equally to this work. A.W. performed
the histological studies. Y.K. performed RLCS analysis and the initial
characterization of the distribution of 5-HT1B receptor mRNA. O.S.
principally worked on the neurophysiological and neuropharmacolog-
ical experiments. S.S. made a major contribution in the analysis of
physiological data. We thank Drs Hitoshi Horie, Shinobu Abe, and Sou
Hashizume of the Japan Poliomyelitis Research Institute for supplying
the monkey brains. We thank Drs Junichi Yuasa-Kawada and Masaharu
Noda of the National Institute for Basic Biology, and Shingo Akiyoshi of
the JFCR Cancer Institute for help with the RLCS method. We thank
Sonoko Ohsawa, Kaoru Sawada and Kazuhiko Miki for technical
assistance, and Dr Kathleen Rockland of RIKEN Brain Science Institute
for the critical reading of the manuscript and valuable discussion. We
thank Dr Hiroyuki Kida for helping with the electrophysiological
experiments. Conflict of Interest: None declared.
Address correspondence to Tetsuo Yamamori Dr. Sci., Division of
Brain Biology, National Institute for Basic Biology, 38 Nishigonaka
Myodaiji, Okazaki 444-8585, Japan. Email: yamamori@nibb.ac.jp.
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