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The Journal of Neuroscience, June 1, 2002, 22(11):4550-4562
Overexpression of 5-HT1B Receptor in Dorsal Raphe
Nucleus Using Herpes Simplex Virus Gene Transfer Increases Anxiety
Behavior after Inescapable Stress
Michael S.
Clark,
Timothy J.
Sexton,
Molly
McClain,
Daniel
Root,
Ruth
Kohen, and
John F.
Neumaier
Department of Psychiatry and Behavioral Sciences and Harborview
Medical Center, University of Washington, Seattle, Washington 98195
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ABSTRACT |
5-HT1B autoreceptors have been implicated in
animal models of stress and are regulated selectively by
serotonin-selective reuptake inhibitors such as fluoxetine. These
terminal autoreceptors regulate serotonin release from dorsal raphe
nucleus (DRN) projections throughout rat forebrain. However, it has not
been previously possible to manipulate 5-HT1B autoreceptor
activity selectively without also changing 5-HT1B activity
in other neurons mediating different behavioral responses. Therefore,
we have developed a viral-mediated gene transfer strategy to express
hemagglutinin-tagged 5-HT1B and manipulate these
autoreceptors in DRN. Green fluorescent protein (GFP) was coexpressed
from a separate transcriptional unit on the same amplicon to assist in
monitoring infection and expression. We confirmed the expression and
biological activity of both transgenic proteins in
vitro. When injected directly into DRN using stereotaxic
procedure, HA-5-HT1B receptors were expressed in
serotonergic neurons and translocated to the forebrain. The effect of
DRN expression of HA-5-HT1B on stress-induced behaviors was
compared with control rats that received GFP-only amplicons. There was
no change in immobility in the forced swim test. However, HA-5-HT1B expression significantly reduced entrances into
the central region of an open-field arena after water-restraint stress without altering overall locomotor activity, but not in the absence of
stress exposure. HA-5-HT1B expression also reduced entries into the open arms of the elevated plus maze after water restraint. Because these tests are sensitive to increases in anxiety-like behavior, our results suggest that overactivity of 5-HT1B
autoreceptors in DRN neurons may be an important mediator of
pathological responses to stressful events.
Key words:
herpes simplex virus; HSV; dorsal raphe nucleus; autoreceptor; hemagglutinin; forced swim test; open-field test; elevated plus maze
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INTRODUCTION |
5-HT1B
autoreceptors are localized in the terminals of serotonergic axonal
projections from midbrain raphe nuclei throughout the rat forebrain
(Jacobs and Azmitia, 1992 ). These inhibitory autoreceptors respond to
extracellular serotonin (5-HT) by reducing release of 5-HT from axonal
terminals acutely and by reducing the amount of 5-HT synthesized over
time (Hoyer and Middlemiss, 1989; Hjorth et al., 1995). Therefore,
5-HT1B autoreceptors constitute a negative
feedback system that regulates 5-HT neurotransmission on the basis of
local conditions at the site of release.
5-HT1B receptors are also synthesized in many
other neurons widely distributed throughout the forebrain, where they
inhibit the release of various other neurotransmitters such as
acetylcholine, glutamate, and GABA (Barnes and Sharp, 1999). Because
there are only ~20,000 serotonergic neurons in rat brain (Wiklund et
al., 1981), the proportion of 5-HT1B receptors in
rat CNS that are autoreceptors in serotonergic neurons is very small.
Indeed, chemical lesioning of the serotonergic system has small and
inconsistent effects on total 5-HT1B binding
density (Verge et al., 1986; Offord et al., 1988; Sexton et al.,
1999).
Pharmacological and null mutation ("knock-out") strategies have
implicated 5-HT1B receptors in a number of
physiological processes and complex behaviors (Hen et al., 1993).
However, in most cases it has not been possible to ascribe the
5-HT1B effects to a particular population of
neurons, because 5-HT1B receptors on various
neuronal terminals are intermingled in practically all forebrain areas. This has made it difficult to study the specific cellular mechanisms by
which 5-HT1B receptors are involved in brain functions.
5-HT1B autoreceptors in forebrain are coded for
by messenger RNA expressed primarily in dorsal raphe nucleus (DRN)
(Hamblin et al., 1992; Neumaier et al., 1996b, 2000; Roberts et al.,
1998). Although these neurons project very diffusely to forebrain
cortical and subcortical structures, the cell bodies are closely packed in a small, midline nucleus (Kosofsky and Molliver, 1987).
5-HT1B terminal autoreceptors appear to be
involved in the adaptation of DRN neurons to serotonin-selective
antidepressants, which are also effective in many anxiety disorders
(Bergqvist et al., 1999; Sayer et al., 1999).
5-HT1B mRNA is selectively downregulated in DRN
but not in hippocampus, striatum, or frontal cortex by either
fluoxetine or paroxetine in a time-dependent and reversible manner
(Neumaier et al., 1996a; Anthony et al., 2000). Learned helpless rats
(an animal model of depression involving inescapable stress) have a
reversible deficit in 5-HT release in prefrontal cortex (Sherman and
Petty, 1980; Petty et al., 1992) and increased 5-HT1B mRNA in DRN (Neumaier et al., 1997). These
observations suggest that increased 5-HT1B
autoreceptor activity induces depressive and related anxiety symptoms
and that downregulation of DRN 5-HT1B autoreceptors by antidepressants may be important in normalizing serotonergic neurotransmission and relieving symptoms of depression or
anxiety (Briley and Moret, 1993).
We propose to test the hypothesis that 5-HT1B
autoreceptor overactivity alters the behavioral responses to
inescapable stress. In this study we manipulated the
5-HT1B autoreceptors selectively while avoiding
direct effects on the 5-HT1B heteroreceptors
expressed in nonserotonergic neurons throughout the forebrain. To
accomplish this we used replication-deficient herpes simplex virus type
1 (HSV) (Geller et al., 1990; Neve and Geller, 1995; Neve, 1999a) to
overexpress 5-HT1B autoreceptors in DRN neurons.
In this study we describe the development of a dual expression vector
carrying both epitope-tagged 5-HT1B receptors and
green fluorescent protein (GFP) on separate transcriptional cassettes
and have characterized the effects of 5-HT1B gene
transfer into DRN neurons on behavioral responses to inescapable stress
using the forced swim test (FST), open-field test (OFT), and elevated
plus maze (EPM) test.
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MATERIALS AND METHODS |
Plasmid construction
To introduce a hemagglutinin (HA) epitope tag into the N
terminus of the rat 5-HT1B gene, plasmid MGIIB
(Hamblin et al., 1992) was used as a template to PCR clone the rat
5-HT1B full-length sequence using an upstream
primer
(5'-TTCTAGAGCTATGTACCCATATGACGTCCCAGACTACGCCGAGGAGCAGGGTA-3') that
introduced an XbaI site and an in-frame HA epitope. The
downstream primer (5'-GAGATGCATGATGGAAGCAGT-3') corresponded to the
single NsiI site downstream of the translation start
point. The resulting fragment was cloned into pCR-Script AMP SK+
(Stratagene, La Jolla, CA) as described by the manufacturer. This
sequence was confirmed in its entirety by automated DNA sequencing. The
XbaI/NsiI fragment of this plasmid was then
ligated into the XbaI/NsiI fragment of an
intermediate plasmid created by ligation of the HindIII
fragment of the rat 5-HT1B cDNA, MG11B (Hamblin
et al., 1992), into HindIII cut pGEM3Zf+ (Promega, Madison,
WI). The resulting plasmid, pHA1B, was cut with HindIII,
blunted with Klenow fragment, cut with EcoRI, and
ligated into EcoRI/SmaI cut pCI (Promega). This
plasmid, pCI-HA1B, produces hemagglutinin-tagged
5-HT1B under control of the CMV promoter/enhancer. To produce a version of this plasmid that
coexpresses enhanced GFP (EGFP), plasmid pEGFP-C1 (Clontech) was cut
with BamHI/BglII and recircularized to eliminate
most of the polylinker. The resulting plasmid was then cut with
XbaI, blunted with Klenow fragment, and cut with
NheI, and the GFP cDNA was isolated and ligated into
NheI/SmaI cut pCI (Promega). This plasmid,
pCMV-GFP, produces GFP under control of the CMV promoter/enhancer.
pCMV-GFP was cut with BglII/BamHI, and the 3.3 kb
fragment was isolated. This fragment was ligated into BamHI
cut pCI (Promega) to generate pCIGFP, containing
two transcriptional units in tandem, with the first possessing a
polylinker for introduction of a desired gene and the second expressing
GFP. The pCIGFP was cut with
EcoRI/SmaI, and the
HA-5-HT1B fragment used to produce pCI-HA1B was
ligated into it to produce pCIGFP-HA1B. All
plasmids were identified by multiple restriction cuts.
Amplicon construction and packaging
To produce an HSV amplicon producing both
HA-5-HT1B and GFP,
pCIGFP-HA1B was cut with XbaI and
BglII. This digest was then partially digested with
BamHI, and the 3.8 kb fragment was isolated. The resulting
fragment was ligated into XbaI/BamHI cut
pHSV-PrPUC (generously provided by Dr. Rachael Neve, McLean Hospital,
Boston, MA). This reconstructed amplicon contains two transcriptional units terminated by SV40 polyadenylation sites, the first producing HA-5-HT1B from an HSV promoter/enhancer and the
second producing GFP from a CMV promoter/enhancer. To make an amplicon
producing only GFP, the NheI/blunted XbaI
fragment of pCMV-GFP was ligated into
XbaI/(blunted)BamHI cut pHSV-PrPUC.
Each plasmid was identified by multiple restriction cuts. HSV amplicons
were then packaged either by Dr. Rachael Neve or in our laboratory as
described previously (Neve, 1999b).
Cell culture infections and transfections
PC12 cells were infected with packaged HSV amplicons as
described previously (Neve, 1999b). Briefly, PC12 cells were grown to
~80% confluence in DMEM containing 10% fetal bovine serum and penicillin/streptomycin/amphotericin B (100 U/ml, 100 µg/ml, and 0.25 µg/ml, respectively). Cells were harvested by a brief trypsin/EDTA treatment and passed through a 21 gauge syringe to dissociate aggregates. After counting on a hemocytometer, 3 × 105 cells were plated onto
poly-D-lysine-coated 24-well cell culture dishes, grown for
24 hr, and then treated with varying concentrations of packaged HSV
amplicon stocks (maximum of 1 µl of virus per milliliter of medium).
After 24 hr, cells were fixed in 4% paraformaldehyde/sodium phosphate
buffer for later fluoromicroscopy and immunocytochemistry. Viral titer
was determined from the number of GFP or hemagglutinin/GFP-positive cells.
HeLa, COS7, and CA77 cells were maintained as described
previously (Hamblin et al., 1992; Clark et al., 1995; Zhukovskaya and
Neumaier, 2000). HeLa cells were transfected essentially as described
previously (Tverberg and Russo, 1992). Briefly, cells were grown to
~80% confluence and harvested by a brief trypsin/EDTA treatment.
Cells (~1 × 106 cells per chamber)
were suspended in ice-cold
Ca2+/Mg2+-free
PBS, chilled 10 min on ice, and electroporated (0.300 kV, 1000 µF) in
an Electroporator 2 (Invitrogen, Carlsbad, CA) in the presence of 20 µg of transfection DNA. After electroporation, cells were mixed very
gently and placed on ice for another 10 min before plating. COS7 cells
were similarly transfected, except that the electroporation conditions
were 0.330 kV at 500 µF. CA77 cells were grown to 80% confluence and
infected with 3 ml of viral particles per 3 ml of medium per well in
six-well dishes. Twenty-four hours after transfection or infection,
cells were fixed in 4% paraformaldehyde or collected by centrifugation
at 500 × g for 10 min for subsequent RNA extraction.
Reverse transcribed-PCR
Total RNA from fresh CA77 cells infected with either
pHSV-HA1B/GFP or pHSV-GFP was purified using RNeasy columns (Qiagen, Valencia CA) and DNase I treatment. Total RNA from DRN was prepared from a 1 mm tissue punch containing DRN from a 2-mm-thick fresh brain
slice that contained the anterior DRN (approximately  6.5 to 8.5 mm
relative to bregma). The punched tissue was processed in RNAlater
(Ambion, Austin, TX), and total RNA was isolated as described for CA77
cells, using the manufacturer's recommended procedures followed by
DNase I treatment. RNA was quantified with RiboQuant (Molecular Probes,
Eugene, OR), and control DNA was quantified with PicoGreen assays
(Molecular Probes). Total RNA (1.5 µg for CA77 cells; 0.25 µg for
DRN) was reverse transcribed into first-strand cDNA using oligo-dT
primer and Moloney murine leukemia virus (Promega) in a final volume of
20 µl. HA-5-HT1B was selectively amplified by
35 cycles of PCR using a pair of primers that are specific for the
hemagglutinin tag (5'-ACCCATATGACGTCCCA-3') and the
5-HT1B sequence (5'-ACCGTGTACATGGTGCT-3'),
yielding a 350 nucleotide PCR product. Total
5-HT1B reverse transcribed (RT)-PCR was similarly
amplified using primers 5'-GGTCTTTTCACAGGTAGGTCAA-3' (upstream) and
5'-TTGACCTACCTGTGAAAAGACC-3' (downstream), yielding a 578 nucleotide
PCR product. PCR products were resolved using 1.3% Agarose gels and
stained with SYBR Gold (Molecular Probes) before photography.
Quantitative reverse transcribed-PCR
5-HT1B mRNA was quantified from
first-strand cDNA prepared from DRN as described above using real time
quantitative PCR with a LightCycler Instrument (Roche, Indianapolis,
IN) with SYBR Green detection of PCR product. A 61 nucleotide PCR
product was amplified using primers 5'-CCAAAAGGGCGGCCA-3' (upstream)
and 5'-TGGCAGCGAAATCGAGATG-3' (downstream) from 1 µl of template
containing either first-strand cDNA or known amounts of MG11B control
template (1 × 10 7 1 × 104 ng per reaction). The thermal
cycling procedures and quantitation procedures were based on the
manufacturer's recommendations. Briefly, a standard curve constructed
from the control template reactions was used to calculate the amount of
first-strand cDNA present in the samples. Each duplicate determination
was analyzed in three independent assays to calculate the relative
amount of first-strand cDNA from each tissue sample in a blinded
manner. Total 5-HT1B mRNA determinations from
each brain sample were standardized using glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) RT-PCR quantitation from the same
preparation, using the following primers: 5'-AACGACCCCTTCATTGAC-3' (upstream) and 5'-TCCACGACATACTCAGCAC-3' (downstream). After the code was broken, treatment group averages were calculated and are
expressed as percentage of control (pHSV-GFP). The efficiency of the RT
reaction was not calculated, but all samples were prepared in parallel
at each step.
cAMP determination
cAMP levels were assayed as described previously (Kohen et al.,
1996). Briefly, JEG-3 cells were grown in DMEM supplemented with
10% fetal bovine serum and 1% penicillin-streptomycin under 10%
CO2. Cells were seeded into 24-well plates and
grown to a density of ~50,000 cells per well. One to four hours
before transfection, the medium was replaced with 250 µl of DMEM
supplemented with 10% dialyzed fetal bovine serum and 1%
penicillin-streptomycin, after which the cells were switched to 5%
CO2. Cells were transiently transfected by a
calcium phosphate precipitation method as described previously
(Heidmann et al., 1998). Transfected DNA consisted of 1 ng of
5-HT1B (MG11B) or pHSV-HA1B/GFP plasmid (except
for controls in which no receptor was transfected), 50 ng of Rous sarcoma virus (RSV)- -galactosidase plasmid, 2.5 ng RSV-cAMP
responsive element (CRE)-luciferase plasmid (Mellon et al., 1989), and
plasmid Bluescript II KS() (Stratagene) as carrier DNA for a total of 250 ng of DNA in 25 µl per well. Twenty hours after transfection, cells were washed once with PBS, supplemented with 500 µl of serum- and serotonin-free medium (Complete Medium, Cellgro, Herndon, VA) with
1% penicillin-streptomycin, and switched back to 10% CO2. After another 24 hr, triplicate wells were
supplemented with 25 µl of forskolin (Calbiochem, San Diego, CA) for
a final concentration of 1 mM, and with 25 µl of 2 mM ascorbic acid alone or ascorbic acid containing 5-HT
(Sigma, St. Louis, MO) for a final concentration of 1 × 1011 M to 1 × 106 M. Five hours later,
cells were harvested in 100 µl of lysis buffer containing 100 nM KPO4, 6 mM
MgSO4, 1 mM dithiothreitol, and 0.1%
Triton X-100. To 350 µl of luciferase assay buffer (100 nM KPO4, 4 mM ATP,
6 mM MgSO4), 25 µl of cell extract
was added and incubated at room temperature for 30 min. Luciferase
activity was then assayed using an Autolumat LB 953 luminometer (EG and G Berthold, Bundoora, Australia) as described elsewhere (Migeon and Nathanson, 1994). Data were analyzed using the program Prism (GraphPad Software, San Diego, CA).
Stereotaxic injections and animal care
All animal procedures were approved by this institution's
animal care committee and handled in accordance with National
Institutes of Health guidelines. Male Sprague Dawley rats (180-250 gm)
were anesthetized with pentobarbital (0.9 mg/kg, i.p.) or isoflurane (2-3% in oxygen), the scalp fur was shaved, the animal was placed in
a Stoelting stereotaxic device, and the surgical site was cleaned with
betadine. After scalp incision, skull landmarks were visualized by
scraping of the periosteum. A small hole was bored at the site of
injection. To avoid penetration of the third ventricle, the DRN (7.7
from bregma, midline, 6.6 mm deep) was approached from an angle 20 or
25° off midline. The needle was slowly advanced over the course of 5 min, and 2 µl of viral particles (~200,000 infective units) was
injected from a Hamilton syringe (#30 needle) over 10 min using a
microprocessor-controlled pump (World Precision Instruments, Sarasota,
FL). The needle was left in place for 10 min after the injection and
then withdrawn slowly over 10 min. This injection volume and procedure
correspond to previous studies with pHSV-PrPUC-based amplicons
(Carlezon et al., 1997; Song et al., 1998). The skin was closed with
surgical methylacrylate glue, and in later injections the closure was
augmented with sterile 3-0 monofilament nylon sutures (Ethicon); the
rats were monitored until they recovered spontaneous movement.
Animals were allowed to recover for 48-96 hr before being killed. For
immunohistochemistry, rats were injected with heparin (1000 U, i.p.),
deeply anesthetized with pentobarbital, and intracardially perfused
with Tyrode's solution followed by 4% paraformaldehyde. The brains
were removed, post-fixed for 2 hr in 4% paraformaldehyde, and stored
in PBS at 4°C 1-2 d before being processed further. For immunoblot
analysis, fresh tissue was harvested, immediately frozen, and stored at 70°C. For RT-PCR, fresh tissue was processed as described below. For all elevated plus maze and pre-stress open-field testing, injection
location was confirmed in a blinded manner on perfused 40 µm tissue
slices prepared on a vibratome. Animals were excluded if >50% of
GFP-expressing neurons were outside the DRN or if there was any
evidence of trauma distorting any anatomic structures nearby.
To determine the number of infected neurons, 40 µm sequential
vibratome sections through the entire DRN were cut from perfused tissue
and mounted on slides. GFP-positive cell bodies within the DRN were
then counted manually. Although counting of the same GFP-positive cell
body in two sequential sections was theoretically possible, the
thickness of the slices relative to cell body size suggests that the
error introduced by counting a single cell twice is small relative to
the number of total neurons infected.
Immunocytochemistry, immunohistochemistry, and microscopy
For immunocytochemistry of cell cultures, the medium was
aspirated, and cultures were rinsed briefly in PBS and fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. Cells were
rinsed briefly in PBS blocked with 0.3% gelatin (bovine) in
PBS-0.025% Triton X-100 for 1 hr to overnight. Wells were incubated with mouse monoclonal anti-hemagglutinin antibody (1:1000 in 0.3% gelatin PBS-0.025% Triton X-100) (HA.11, Babco, Richmond, CA), rinsed
three times for 10 min with PBS-0.025% Triton X-100, and then
incubated with a goat anti-mouse Cy3-conjugated antibody (1:500 in
0.3% gelatin PBS-0.025% Triton X-100) (Jackson ImmunoResearch, West
Grove PA) for 1 hr at 37°. Wells were then rinsed three times for 10 min with PBS-0.025% Triton X-100 and rinsed briefly with deionized
water. Excess water was removed, and the bottoms of the wells were
coated with Gel/Mount (Biomeda, Foster City, CA). Immunofluorescence
was visualized with a Nikon inverted fluorescence microscope using an
FITC filter for detection of GFP fluorescence and a rhodamine filter
for detection of Cy3.
For immunohistochemistry, free-floating sections (40 µm) were
prepared on a Leica VT1000S and rinsed in PBS. Sections were permeabilized in PBS-0.5% Triton X-100 for 30 min and then blocked with 0.3% gelatin (bovine) in PBS-0.025% Triton X-100 for 1 hr at
room temperature or overnight at 4°C. They were then incubated with
one or more primary antibodies concurrently: mouse monoclonal anti-hemagglutinin antibody (HA.11, Babco,), guinea pig
anti-5-HT1B (Chemicon, Temecula, CA), and guinea
pig anti-5-HT1A (Chemicon). All antibodies were
diluted 1:1000 in 0.3% gelatin in PBS-0.025% Triton X-100 and
incubated overnight at room temperature with gentle agitation. Sections
were then rinsed three times for 10 min with PBS-0.025% Triton X-100
and incubated with secondary antibodies [goat anti-mouse Alexa-633
conjugate and/or goat anti-guinea pig Alexa-568 conjugate (Molecular
Probes)], again concurrently, in 0.3% gelatin PBS-0.025% Triton
X-100 for 1 hr at room temperature. Secondary antibodies were diluted
as follows: Alexa-568 conjugate diluted to 5 µg/ml for
anti-5-HT1A and to 10 µg/ml for
anti-5-HT1B, and Alexa-633 conjugate diluted to
20 µg/ml. Sections were then rinsed three times for 10 min with
PBS-0.025% Triton X-100, rinsed briefly with deionized water, and
mounted on glass slides with Prolong Antifade mounting medium
(Molecular Probes). The sections were analyzed using a Bio-Rad Radiance
2000 confocal system (Bio-Rad, Hercules, CA) and an associated Nikon
fluorescence microscope using an argon/krypton laser and red laser
diode with appropriate Performance filters (Bio-Rad) for detection of
GFP, Alexa-568, and Alexa-633 fluorescence.
Immunoblot analysis
Frozen tissue was crushed on dry ice, placed into boiling 5%
SDS/50 mM Tris-HCl, pH 8.0, for 5 min, sonicated for 10 sec, and spun at 10,000 × g for 10 min. The
supernatant was retained and assayed for protein concentration by the
BCA Protein Assay (Pierce Biochemical, Rockford, IL). Protein samples
(50-100 µg) were separated on a 10% SDS-PAGE gel. Western blotting
using nitrocellulose was performed at 4°C in a Bio-Rad blotting
apparatus at 15 mV overnight or 35 mV for 2 hr. Blots were blocked in
5% instant milk in 50 mM Tris-0.9% saline
containing 0.025% Tween 20 (TBST) for 1-2 hr, incubated in
anti-hemagglutinin primary antibody (1:1000 in 5% non-fat instant
milk-TBST for 1 hr at room temperature) (HA.11, Babco), and rinsed
three times for 10 min in TBST. Blots were incubated in
anti-mouse-HRP-conjugated secondary antibody (1:1000 in 5% non-fat
instant milk-TBST for 1 hr at room temperature) and rinsed three times
for 10 min in TBST. Blots were developed with SuperSignal West Pico
chemiluminescent substrate (Pierce Biochemicals) for 5 min and exposed
to film. Biotin-conjugated molecular weight markers were visualized by
further incubating the blot in anti-biotin-HRP-conjugated antibody (New
England Biolabs, Beverly, MA) at 1:1000 for 1 hr, rinsing three times
for 10 min in TBST, and redeveloping in chemiluminescent substrate.
Behavioral testing procedures
Forced swim test. The FST was performed as described
previously (Porsolt et al., 1977; Detke et al., 1995). The FST
container was a 40-cm-tall Plexiglas cylinder with a 20-cm-diameter
base, mounted on a Plexiglas base. It was filled with tap water
(25°C) to 30 cm, a level deep enough to prevent the rat from resting on its extended tail. The forced swim stress session consisted of
placing the rat in the chamber for 15 min, followed by towel drying the
rat under warm lamps and returning it to the home cage. The following
day (between the hours of 9 and 11 A.M.), the test session was
performed by putting the rat into the water and videotaping its
behavior for 5 min. Each cylinder was cleaned between animals. FST
behaviors were scored in a blinded manner, by a different experimenter
(J.N.), using a time-sampling method (Detke et al., 1995). Every
5 sec the animal's behavior was scored as climbing, swimming, or
immobile; a total of 60 observations were made during the 5 min test
session. Statistical comparison of between group differences was
performed with the Mann-Whitney U test using GB-Stat software, with p  0.05 considered significant.
Increased immobility time was considered to represent behavioral
depression or behavioral despair but could also be operationally
defined as a behavioral pattern that is preventable by treating the
animal with antidepressants (Porsolt, 2000).
Water-restraint stress. Some animals were stressed for 15 min by water restraint on the third day after viral particle injection, as modified from a previously described procedure (Pare, 1994). Animals
were loosely restrained in an envelope constructed of plastic mesh so
that they could not make gross body movements, and they were suspended
to the level of their necks in 25°C water for 15 min (using an FST
chamber). The animals were then wiped briefly with a towel, dried under
a lamp, and returned to their home cage.
Open-field test. Animals were either tested 3 d after
viral particle injection (no stress exposure) or 24 hr later after
exposure to water-restraint stress. The open-field test (OFT) was
performed using a 45-cm-square black Plexiglas enclosure with
30-cm-tall walls set on a nonreflective black plastic base divided into
a grid of nine equal squares. The OFT arena was located in a small, quiet, light-proof room with video monitoring so that the researcher could leave the room immediately after placing the rat in the center of
the arena. Animals were tested between 4 and 6 P.M. under low
illumination red light, to which Sprague Dawley rats are blind, thereby
simulating darkness and increasing locomotor activity. Behavioral data
were collected by videotape for 10 min; the tape was scored by a
different experimenter (J.N.) in a blinded manner. The number of
entries into the central square over the first 3 min and the total
squares entered over 10 min were counted. The procedure and analysis
used were based on previous studies suggesting that centroid entering
on initial placement in the maze was most sensitive to stress-induced
anxiety states (Pare, 1994; Izumi et al., 1997; Durand et al., 1999).
Furthermore, factor analysis suggests that center entering assesses
approach/avoidance toward aversive stimuli, which is considered a
reliable index of fearfulness/anxiety and responds to anxiolytic agents
(Ramos et al., 1997). Statistical comparison of between group
differences was performed with the Mann-Whitney U test
using GB-Stat software, with p 0.05 considered significant.
Elevated plus maze. The EPM apparatus was constructed in
this lab from black Plexiglas with nonreflective painted surfaces. The
maze consisted of four runways (10 × 40 cm) joined by a central 10 × 10 cm square, 50 cm above the floor. Opposing arms
were either open (having only a 0.5 cm lip) or enclosed by 40-cm-high
walls. The maze was illuminated by a dim lamp above the apparatus (12 lux). Experimental methodology was based on previously published studies of the EPM (Handley and McBlane, 1993; Hogg, 1996). Percentage open arm entries was the key parameter assessed, because factor analysis had previously shown this index to be associated with fearfulness/anxiety (Ramos et al., 1997). Animals were
introduced into the center square facing an open arm, and behavior was
video recorded for 5 min and analyzed using the SMART computer analysis program (San Diego Instruments, San Diego, CA). The number of entries
into open or closed arms and total distance traveled were measured.
Statistical comparison of between group differences was performed with
the Mann-Whitney U test using GB-Stat software, with
p 0.05 considered significant. Open arm time, closed
arm time, open entries, and closed entries were also recorded. The center square was not considered to be part of either the open or
closed arms. To confirm our ability to detect anxiolytic and anxiogenic
effects in our EPM apparatus, we used the protocol of Grahn et al.
(1995) to assess our methodology. Compared with vehicle alone, we found
that diazepam (2 mg/kg, with 4 d pretreatment to allow
tolerance to motor effects of the drug) increases and methyl
6,7-dimethoxy-4-ethyl--carboline-3-carboxylate (a benzodiazepine inverse agonist, 0.4 mg/kg,) decreases percentage open arm entries while not altering total locomotion, as seen with our viral injections (data not shown). This finding indicates that we can detect both anxiolytic and anxiogenic effects in our EPM apparatus.
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RESULTS |
Coexpression of HA-5-HT1B and GFP in
vitro using multiple promoter/enhancer elements
Because 5-HT1B autoreceptors in DRN neurons
are translocated to axon terminals in forebrain (Hoyer and Middlemiss,
1989; Boschert et al., 1994; Ghavami et al., 1999), they may be
difficult to detect in the cell body. Accordingly, it would be useful
to express both the receptor and a marker protein that can be detected
in the cytosol of the transfected cell. GFP, which can be detected easily both in vivo and in vitro, can provide
such a cytosolic marker.
To accomplish our goal, we first introduced a hemagglutinin epitope
into the N terminus of the rat 5-HT1B cDNA to
facilitate detection of expressed transgenic receptor with commercially
available antibodies. The modified cDNA was then cloned into the
multiple cloning site of plasmid pCIGFP, in which
a complete expression unit containing the GFP gene driven by the CMV
immediate/early (IE) promoter/enhancer, and terminated with the
SV40 polyadenylation site, was inserted into the single
BamHI site of pCI (Fig. 1). The resulting plasmid expressed both GFP and
HA-5-HT1B, each from a separate transcriptional
unit. To determine whether this plasmid, pCIGFP-HA1B, did indeed express both genes, it
was transfected into COS7 cells by electroporation. After 72 hr, all
cells that displayed GFP fluorescence also displayed HA
immunoreactivity, which appeared to be concentrated at the cell
membrane (Fig.
2A,C). In contrast, cells transfected with pCIGFP
displayed only GFP fluorescence (Fig.
2B,D). Transfection of
pCIGFP-HA1B also led to expression of both gene
products in HeLa cells (data not shown).
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Figure 1.
Amplicon maps used for HA-5-HT1B and
GFP expression. The plasmids pHSV-HA1B/GFP and pHSV-GFP were
constructed as described in Materials and Methods and were confirmed by
sequence analysis. Note that either GFP alone or the
HA-5-HT1B and GFP sequences were inserted into the
pHSV-PrPUC backbone provided by Dr. Rachael Neve (Neve and Geller,
1995). In the latter case, the HA-5-HT1B and GFP gene
sequences were interrupted by an SV40 polyadenylation signal; the two
genes have different promoter/enhancers controlling expression (HSV IE
4/5 and CMV IE, respectively), to reduce competition effects.
ori S, HSV origin of replication; AmpR,
ampicillin resistance gene.
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Figure 2.
COS cells transfected with pCIGFP-HA1B
show dual expression. Cells were transfected with
pCIGFP-HA1B or pCIGFP by electroporation
as described. A and C show
pCIGFP-HA1B-transfected cells; B and
D show pCIGFP-transfected cells.
A and B show GFP fluorescence;
C and D show hemagglutinin
immunoreactivity. The HA-tagged 5-HT1B receptor could be
detected only in the pCIGFP-HA1B-transfected cells, and
there was no apparent interaction between GFP and HA-5-HT1B
expression in the same cells. Scale bar, 20 µm.
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HA-5-HT1B receptors coexpressed with GFP decrease cAMP
accumulation and forskolin-stimulated CRE activity
In mammalian cell culture, the rodent 5-HT1B
receptor is known to inhibit adenylate cyclase via activation of
Gi (Barnes and Sharp,
1999), thereby reducing the production of cAMP. To determine whether
the HA-tagged 5-HT1B receptor retains this activity when coexpressed with GFP, the ability of the receptor to
suppress forskolin-stimulated CRE-mediated expression of a reporter
gene (Mellon et al., 1989) was determined. As can be seen in Figure
3, transfection into JEG-3 cells with
either pCIGFP-HA1B or MG11B (wild-type receptor)
produced 5-HT-responsive reductions in luciferase expression with an
EC50 of 5.1 and 4.8 nM, respectively, agreeing with previously published values of ~6 nM
(Hamblin et al., 1992). Therefore, the HA epitope tag on the
5-HT1B amino terminus and coexpression of GFP do
not appear to change the apparent affinity or coupling efficiency of
the receptor.
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Figure 3.
HA-5-HT1B
receptors inhibit adenylate cyclase in JEG-3 cells. Adenylate
cyclase activity was assayed using a luciferase reporter gene assay as
described in Materials and Methods. Data points represent SD of
triplicate determinations; two replicate assays were performed. The
curve fits and EC50 determinations were calculated using
Prism 2.0. HA tagging of the 5-HT1B receptor did not appear
to reduce its ability to inhibit adenylate cyclase activity, and
coexpression of GFP from the same plasmid did not impair the level of
HA-5-HT1B expression or function. RLU,
Relative light units.
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HA-5-HT1B and GFP can be coexpressed from a single HSV
amplicon in vitro
To produce an HSV amplicon from pCIGFP-HA1B,
the 3.8 kb restriction fragment containing
5'-HA-5-HT1B-SV40 polyA-CMV I/E-GFP-SV40 polyA-3' was ligated into XbaI/BamHI cut
pHSV-PrPUC. In the resulting plasmid, pHSV-HA1B/GFP,
HA-5-HT1B cDNA is expressed from the HSV IE 4/5
promoter/enhancer, and GFP is expressed from the CMV promoter/enhancer transferred from pCIGFP-HA1B (Fig. 1). The
amplicon was then packaged using replication-deficient HSV as described
previously (Neve and Geller, 1995; Neve, 1999b). The recombinant viral
particles carrying pHSV-HA1B/GFP were titered using PC-12 cells by
assaying for HA immunocytochemistry and GFP fluorescence, and they
typically contained 1-2 × 108
infective units per milliliter. CA77 cells were infected with 3 µl of
viral particles per well, with 50-90% infection rates. Infected CA77
cells were examined for the presence of 5-HT1B
RNA. RT-PCR amplification of polyadenylated RNA from cells infected with pHSV-HA1B/GFP produced a robust RT-PCR product, whereas those infected with pHSV-GFP RNA had low levels of endogenous
5-HT1B mRNA (Clark et al., 1995), similar to that
in vehicle-treated cells (Fig.
4A). The HA-specific
RT-PCR product was evident only in pHSV-HA1B/GFP-infected cells. HA
specific antibodies labeled a single band of 65 kb only in
pHSV-HA1B/GFP infected cells (Fig. 4B). Taken
together, these in vitro studies showed that high levels of
functional HA-5-HT1B and GFP could be expressed
from separate promoter/enhancer elements using HSV amplicons.
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Figure 4.
In vitro expression of
pHSV-HA1B/GFP in CA77 cells. A, Twenty-four hours after
infection with pHSV-GFP (lanes 1, 2) or
pHSV-HA1B/GFP (lanes 3, 4) (3 µl
per well in six-well tissue culture plates; 1-2 × 108 infective units per milliliter), CA77 cells were
harvested and processed for either immunoblot analysis or poly-A RNA
extraction. GFP expression and HA immunoreactivity were robustly
detected in ~50-90% of cells (data not shown). RT-PCR of poly-A RNA
from these cells showed the presence of 5-HT1B mRNA using
total 5-HT1B primers for amplification (lanes
1, 3) and HA-specific primers (lanes
2, 4). Vehicle and pHSV-GFP-treated CA77
cells express low levels of 5-HT1B RNA (lane
1) and no HA epitope (lane 2).
pHSV-HA1B/GFP-treated cells showed dramatically more total
5-HT1B message (lane 3) and a strong
HA-5-HT1B-specific PCR product (lane
4). Vehicle-treated CA77 cells showed low levels of
5-HT1B mRNA, similar to pHSV-GFP (data not shown).
B, Protein samples (5 µm) from pHSV-GFP (lane
1)- or pHSV-HA1B/GFP (lane 2)-infected CA77
cells were separated by PAGE and immobilized on membranes by Western
blot. HA-specific immunoreactive protein was detected only in
pHSV-HA1B/GFP-infected cells.
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HSV amplicons are capable of expressing HA-5-HT1B and
GFP gene products in vivo
To determine whether pHSV-HA1B/GFP is capable of infecting
mammalian brain and inducing gene expression of both
HA-5-HT1B and GFP, we injected viral particles
into the rat DRN by stereotaxic surgery. After microinjection into the
DRN, animals were returned to home cages for 4 d and then
euthanized. The brains were either prepared for immunocytochemistry or
rapidly frozen for subsequent Western blot. Tissue sections were
examined for GFP fluorescence and immunostained for the presence of the
HA epitope. Large numbers of GFP-positive neurons were detected at the
sites of injection (Fig. 5A).
In six consecutive brains examined 4 d after gene transfer, 930 ± 160 GFP-positive cells were counted (mean ± SEM).
These counts may be an underestimate of total transgene expression
because gene expression peaks on day 3 and declines gradually
thereafter (Carlezon et al., 2000a; Pliakas et al., 2001), but these
numbers are comparable to previous studies that used gene transfer with this vector or similar strategies (Carlezon et al., 1997; Fabre et al.,
2000; Pliakas et al., 2001). There are ~11,000-15,000 serotonergic
neurons in the full rostral-caudal extent of the nucleus (Wiklund and
Bjorklund, 1980; Vertes and Crane, 1997). Therefore, we estimate that
~10% of serotonergic neurons in the anterior dorsal raphe nucleus,
the area targeted in the injections, expressed
HA-5-HT1B. As shown in confocal micrographs,
individual DRN neurons infected with pHSV-HA1B/GFP coexpressed GFP and
HA-5-HT1B signals (Fig.
5B,C). Furthermore, most of the
transgene-expressing DRN neurons were serotonergic, as indicated by
5-HT1A immunostaining (Fig.
5D,E). In some cases, either GFP or
anti-HA label was apparent in a particular neuron or a specific
confocal plane, but in most cases both signals colocalized in the same
neurons (Fig. 5E). Specifically, immunolabeling was most
intense near the surfaces of the tissue section, likely because of
antibody penetration, whereas GFP expression was equally intense at all
depths within a tissue section.
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Figure 5.
Coexpression of
HA-5-HT1B and GFP in
vivo. pHSV-HA1B/GFP viral particles were injected
stereotaxically into DRN, and animals were killed 4 d
later for evaluation of transgene expression by immunostaining and
confocal microscopy. A, A composite image of several
10× fields shows clear GFP localization within the anatomic region of
the DRN. The fourth ventricle (4V) has been
colored light green for clarity. Scale bar, 100 µm. B,
A 20× image of DRN shows GFP fluorescence in cells and beaded fibers.
C, HA-5-HT1B immunostaining of the same
region shown in B. D, 5-HT1A
immunostaining of the same region shown in B and
C. Because of the lower laser strength available for the
secondary dye used (Alexa-633), the intensity of positive staining,
typically visible as rings around darker nuclei, is relatively low.
E, A composite image of all three signals displays cells
positive for GFP, HA-5-HT1B, and
5-HT1A, demonstrating that serotonergic neurons have
been infected and that these neurons produce both viral transgene
products in vivo. A count of cells positive for both GFP
and 5-HT1A in images obtained for this study showed that
80% of GFP-positive neurons were also positive for 5-HT1A.
Scale bar, 100 µm.
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5-HT1B immunostaining was much more intense in
GFP-expressing cells than in other serotonergic cells expressing only
endogenous 5-HT1B receptor within the DRN (Fig.
6A-D). In a
subset of injected brains, the DRN was removed by punch, total RNA was
extracted, and total 5-HT1B mRNA was quantified
by real-time RT-PCR. This was normalized for GAPDH mRNA. DRNs that were
injected with pHSV-HA1B/GFP had approximately threefold more
5-HT1B mRNA than pHSV-GFP-injected brains (Fig.
6E). This reflects total expression in the DRN;
therefore, the infected neurons probably had significantly higher
levels of expression than suggested above.
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Figure 6.
Injection of pHSV-HA1B/GFP increases
5-HT1B expression in vivo. Sections through
the DRN were taken from animals injected at that site with
pHSV-HA1B/GFP viral particles, immunostained for
5-HT1B, and examined by confocal microscopy.
A, GFP-containing cells and associated fibers may be
clearly seen in the DRN, as shown previously. B,
5-HT1B immunoreactivity is detected within both GFP- and
non-GFP-containing cells. C, When GFP fluorescence and
5-HT1B immunoreactivity are overlapped, GFP-positive cells
are shown to typically display intense 5-HT1B
immunoreactivity, as indicated by the yellow-green
coloration of these cells. A-C, Magnification:
20×. Scale bar, 100 µm. D, In another focal plane
from the same section shown in A-C,
5-HT1B immunoreactivity is much more intense in a
GFP-positive cell (indicated by a large white arrow)
compared with endogenously expressed 5-HT1B protein in
non-GFP-positive cells (small arrows). The GFP-positive
cell displays much greater 5-HT1B immunoreactivity,
although both types of cells display what appears to be membrane-bound
localization of 5-HT1B signal. Magnification: 40×. Scale
bar, 100 µm. E, DRN punches from animals injected with
either pHSV-GFP or pHSV-HA1B/GFP viral particles were analyzed by
quantitative RT-PCR for 5-HT1B message content
(n = 9 for each group). 5-HT1B mRNA
levels, normalized for GAPDH mRNA expression, were 3.16-fold times
higher in animals injected with pHSV-HA1B/GFP than in animals injected
with pHSV-GFP (***p = 0.001; Student's
t test).
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Because the 5-HT1B receptor is translocated to
axon terminals, only a small proportion of 5-HT1B
protein would be expected in the soma, whereas the majority would be
expected to be present diffusely among the termination zones of DRN
axonal projections such as frontal cortex and striatum (Molliver,
1987). To determine whether midbrain DRN infection with pHSV-HA1B/GFP
and pHSV-GFP induces gene expression and protein translocation to
forebrain, immunocytochemistry was performed on striatal sections for
HA epitope. HA immunoreactive fibers with morphology characteristic of
DRN projections (Kosofsky and Molliver, 1987) are seen in the striatum
of pHSV-HA1B/GFP-injected animals (Fig.
7A) but not pHSV-GFP-injected animals (Fig. 7B). To confirm this data, protein samples
from frontal cortex and striatum were subjected to PAGE and immunoblot analysis. As can be seen in Figure 7C, rat forebrain
contains an HA-immunoreactive band at 65 kDa, approximately the
same size as photoaffinity-labeled CNS 5-HT1B
receptor (Hamblin et al., 1988) and slightly larger than that seen
in vitro (Fig. 4B). The HA
immunoreactivity was more intense in striatum than frontal cortex,
perhaps reflecting the greater density of 5-HT terminals in striatum.
These findings strongly suggest that HA-5-HT1B
receptors introduced by viral-mediated gene transfer are translocated
from DRN to serotonergic axon terminals in forebrain.
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Figure 7.
HA-5-HT1B
is translocated to the forebrain. Striatal sections of animals
injected in the DRN with either pHSV-HA1B/GFP or pHSV-GFP viral
particles were immunostained to detect the presence of
HA-5-HT1B immunoreactivity and examined by confocal
microscopy. A, In animals injected with pHSV-HA1B/GFP,
beaded fibers with anti-HA immunoreactivity may be clearly seen. The
fibers demonstrate typical pleiomorphic varicosities, suggesting
multiple sites of neurotransmitter release that are characteristic of
DRN axons (Kosofsky and Molliver, 1987). B, In animals
injected with pHSV-GFP, only background is present. In neither case was
GFP detected (data not shown). Images shown are flattened, 60×
confocal stacks. Scale bar, 50 µm. C, Western blot of
HA-immunostained protein from terminals field of DRN axonal
projections to forebrain. Protein samples from frontal cortex
(lanes 1, 2) or striatum (lanes 3,
4) after DRN injection of pHSV-GFP () or
pHSV-HA1B/GFP (+) viral particles. The single immunoreactive
HA-5-HT1B band migrated at an apparent size of 65 kDa, perhaps reflecting glycosylation and/or other
posttranslational modifications of the 49 kDa predicted
protein.
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HA-5-HT1B overexpression in rat DRN alters
anxiety-related behavior after an inescapable stressor
5-HT1B terminal autoreceptors are
downregulated by antidepressants (Artigas et al., 1996; Sayer et al.,
1999) and may be upregulated in learned helpless rats (Edwards et al.,
1991; Neumaier et al., 1997) but are difficult to manipulate in
behavioral models without also impinging on
5-HT1B heteroreceptors. Therefore we sought to
determine whether increasing 5-HT1B mRNA in DRN
would induce behaviors relevant to the symptoms of depression and
anxiety. We first hypothesized that because antidepressants reduce
immobility in the Porsolt FST (Porsolt et al., 1977), overexpression of
5-HT1B autoreceptors would increase immobility in
the same test. Animals received stereotaxic injections of viral
particles containing either pHSV-HA1B/GFP or pHSV-GFP into DRN and were
subjected to forced swim 3 d later and tested the following
morning. We used the pHSV-GFP amplicon as a control treatment because
it controlled for the surgical procedure, infection with viral
particles, the presence of viral particle constituents, transgenic RNA
expression, and expression of GFP. We believe that this represents a
good control strategy for viral-mediated gene transfer studies in rat brain. The animals' behavior was coded as climbing, swimming, or
immobile, as described previously (Detke et al., 1995). There were no
statistically significant changes in any of these behaviors in control
or experimental animals (Fig. 8).
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Figure 8.
HA-5-HT1B
expression in DRN neurons did not alter immobility in the
forced swim test. Animals received injections of pHSV-HA1B/GFP
(n = 11) or pHSV-GFP (n = 8) in
DRN and were subjected to the standard FST procedure on days 3 and 4 as
described in Materials and Methods. Behaviors were counted as swimming,
climbing, or immobile as described in Materials and Methods. Numerical
data are as follows and are presented as mean ± SEM
(climb: GFP 14 ± 2.0, HA1B/GFP 17 ± 2.5;
swim: GFP 14 ± 2.9, HA1B/GFP 12 ± 2.1;
tread: GFP 31 ± 4.5, HA1B 31 ± 3.4). There
were no significant differences in these behaviors between treatment
groups.
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The OFT has been used to model "emotionality" or behavioral anxiety
in rodents, whose open-field behavior is altered by both stressors and
antidepressants (Stockert et al., 1988; Kelly and Leonard, 1994; Pare,
1994; Meerlo et al., 1996; Izumi et al., 1997; Ramos et al., 1997;
Durand et al., 1999). Factor analysis suggests that center entries are
most related to approach/avoidance toward potentially aversive stimuli,
which responds more strongly to anxiolytic drugs such as diazepam and
is considered an index of anxiety/fearfulness (Ramos et al., 1997).
Thus, we hypothesized that open entries would be altered in animals
overexpressing 5-HT1B in the DRN. Animals were
injected in DRN with viral particles carrying either pHSV-HA1B/GFP or
pHSV-GFP, housed in routine conditions for 3 d without specific
stress exposure, and then tested with the OFT. In the absence of
specific stress exposure, pHSV-HA1B/GFP-treated animals showed greater
exploration of the center of the open field, with 35% more entries
into the center square as compared with pHSV-GFP-treated animals (Fig.
9A) (p = 0.05). There was no difference in total locomotor activity between
groups as shown by total square entries (Fig. 9B),
indicating that overexpression of 5-HT1B
receptors in DRN did not alter general locomotor activity. In a
separate experiment, animals received viral injections followed by
water-restraint stress 3 d later and were then tested in the OFT
24 hr later. This stress paradigm differs from the FST in that it
prevents gross body movements (Pare, 1994). The pHSV-HA1B/GFP-injected animals entered the central region of the arena 30% less frequently than the pHSV-GFP-treated animals (Fig. 9C)
(p = 0.044). Total locomotor activity, as
determined by total square entries, was not affected by
HA-5-HT1B expression (Fig. 9D). This
suggests that the animals that received pHSV-HA1B/GFP were more
sensitive to water-restraint stress than GFP controls.
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Figure 9.
HA-5-HT1B expression in DRN neurons
increased avoidance of the center of an open field only after
water-restraint stress. Animals received injections of pHSV-HA1B/GFP or
pHSV-GFP and either were tested in the OFT 3 d later
(A, B) or subjected to water-restraint
stress on day 3 and tested in the OFT 24 hr later (C,
D). The numbers of entries into the central square
during the first 3 min were counted and are shown as mean ± SEM
(A, GFP 4.5 ± 0.56, HA1B/GFP 6.5 ± 0.64;
C, GFP 8.4 ± 0.86, HA1B/GFP 5.9 ± 0.58).
HA-5-HT1B expression increased central square entries in
the absence of a specific stress exposure (#p = 0.05) but reduced entries into the central region after stress by 30%
(*p = 0.044). The total number of zone crossings,
shown as mean ± SEM (B, GFP 102 ± 9, HA1B/GFP 123 ± 10; D, GFP 160 ± 13, HA1B/GFP
162 ± 20), was not different between pHSV-HA1B/GFP and pHSV-GFP.
n = 8-14 animals in each treatment
condition.
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To further examine the effects of 5-HT1B
overexpression in DRN on stress-induced behaviors, we used the EPM,
another commonly used test for anxiety-like behaviors (Handley and
McBlane, 1993; Hogg, 1996). As in the OFT, we assessed behavior most
associated with indices of anxiety. On the basis of factor analysis
(Ramos et al., 1997) and response to anxiolytics (Handley and
McBlane, 1993; Hogg, 1996), we chose to examine percentage open arm
entries as our primary measure. Indeed, our preliminary studies with
diazepam and the benzodiazepine inverse agonist methyl
6,7-dimethoxy-4-ethyl--carboline-3-carboxylate (Grahn et al., 1995)
demonstrated that this parameter was the most robust and least variable
indicator of anxiety in our protocol (data not shown). Animals were
exposed to water-restraint stress 3 d after viral vector injection
into DRN and were tested in the EPM 24 hr later. Animals treated with
pHSV-HA1B/GFP demonstrated greater anxiety-like behavior than pHSV-GFP
controls, as indicated by a 20% reduction in percentage entries into
the open arms of the maze (Fig.
10A)
(p = 0.047). 5-HT1B
overexpression did not significantly affect total distance traveled in
the EPM (Fig. 10B).
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Figure 10.
HA-5-HT1B
expression in DRN reduced open arm entries in the EPM 24 hr
after water-restraint stress. Animals received injections of
pHSV-HA1B/GFP (n = 13) or pHSV-GFP
(n = 8), were subjected to water-restraint stress
3 d later, and were tested in the EPM 24 hr later. The rat's
behavior was recorded and analyzed by computer-assisted video
monitoring. A shows the percentage of open arm/total arm
entries; HA-5-HT1B-expressing animals had significantly
reduced percentage of entries into open arms (GFP 55 ± 4%;
HA1B/GFP 44 ± 3%; mean ± SEM shown);
*p = 0.047. B, There was no
significant difference in total distance traveled between treatment
groups (GFP 415 ± 38 cm; HA1B/GFP 450 ± 40 cm; mean ± SEM shown). Other parameters measured include percentage open time (GFP
29.6 ± 8.9; HA1B/GFP 22.3 ± 4.0), percentage closed time
(GFP 51.5 ± 8.1, HA1B/GFP 54.2 ± 5.2), open entries (GFP
8.2 ± 1.0, HA1B/GFP 7.2 ± 1.1), and closed entries (GFP
7.4 ± 1.5, HA1B/GFP 9.1 ± 1.2); mean ± SEM shown.
Although these parameters did not reach statistical significance
(p > 0.05), all trends are consistent with
anxiogenic effects in HA-5-HT1B-expressing animals.
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DISCUSSION |
Validation of HA-5-HT1B and GFP
dual expression
Several recombinant viral vectors have been developed for use in
the mammalian CNS, including gene transfer systems derived from
adenovirus, adeno-associated virus, HSV, and others (Zlokovic and
Apuzzo, 1997). The HSV system used here possesses many advantages for
delivery of genes into postmitotic neurons, including neurotropic specificity, high infectivity, efficient extrachromosomal gene expression, and low toxicity (Neve and Geller, 1995). HSV amplicons have been used previously to introduce transgenes for the purpose of
overexpressing an endogenous protein of interest in discrete structures
of the rat brain (Chiocca et al., 1990; Geller et al., 1991; Wood et
al., 1994; Carlezon et al., 1997, 2000b; Song et al., 1998; Neve,
1999a). Double infection with two HSV vectors has been used to
introduce two genes of interest into primary cortical neurons in
culture (Coopersmith and Neve, 1999). Although using two viral
amplicons simultaneously can lead to dual infection of some neurons,
essentially all neurons that are infected with pHSV-HA1B/GFP should
express both genes. This report is the first to use a single amplicon
to introduce and express two genes of interest in rat brain. When viral
particles were injected into DRN in rat midbrain, expression of
HA-5-HT1B and GFP in individual neurons could be
detected, and HA immunoreactivity was found in the striatum and frontal
cortex only when HA-5-HT1B/GFP was injected into
DRN. We have validated that both genes are expressed in
vitro and in vivo, preserving protein activity and
receptor localization.
This approach made it possible to manipulate
5-HT1B presynaptic autoreceptors separately from
5-HT1B postsynaptic heteroreceptors in forebrain
tissue. Such discrimination is critical in examining the role of DRN
5-HT1B autoreceptors in depression and anxiety, because most of the 5-HT1B receptors in the brain
are postsynaptic heteroreceptors, located on nonserotonergic neurons,
that are intermingled with 5-HT1B
autoreceptor-containing serotonergic fibers (Verge et al., 1986; Offord
et al., 1988; Jacobs and Azmitia, 1992; Sexton et al., 1999).
Manipulating expression of the autoreceptor population selectively
offers the opportunity to examine the behavioral role of a small but
very important subpopulation of 5-HT1B receptors that has not been possible using other genetic or pharmacologic techniques. Fabre et al. (2000) recently used nonviral gene transfer to
alter serotonin transporter expression in the rat DRN, observing alterations of circadian rhythms in animals transfected with an antisense-expressing plasmid. The nonviral approach transfects both
glia and neurons. However, both viral and nonviral methods are well
suited to altering gene expression in DRN because of its small size and
mostly homogenous neuron type (i.e., 70% serotonergic). Although our
infection rate of 10% of DRN neurons appears fairly low, previous
studies using viral-mediated gene transfer demonstrating robust
biological effects have often targeted larger brain regions than the
DRN while infecting approximately as many neurons (Chiocca et al.,
1990; Geller et al., 1991; Wood et al., 1994; Carlezon et al., 1997,
2000b; Song et al., 1998; Neve, 1999a). Because DRN has only
11,000-15,000 serotonergic neurons and we targeted the anterior
section of the nucleus, we achieved an equal or higher proportion of
transgene-expressing neurons within the region of interest compared
with previous studies. However, our findings do not rule out the
possibility of differential involvement of 5-HT1B
autoreceptors in anxiety behaviors between subregions of the DRN or in
other raphe nuclei such as the median raphe nucleus. Further
investigation will likely prove useful in addressing
these issues.
Analyses of behavior after HA-5-HT1B/GFP
infection of dorsal raphe nucleus
The second purpose of this study was to characterize the
behavioral effects of HA-5-HT1B/GFP expression in
DRN neurons. We examined several behavioral paradigms to
elucidate the role of 5-HT1B autoreceptors in
depression and anxiety. The FST has been used to predict the
antidepressant activity of drugs, whereas the OFT and EPM have been
used to detect changes in anxiety-like behavior (Porsolt, 2000). We did
not detect an effect of HA-5-HT1B/GFP expression
in DRN on immobility or struggling behaviors using the FST. This
negative result could be explained by several interpretations. First,
the FST may be more sensitive in detecting antidepressant activity than prodepressant activity. It is also possible that forced
swim stress does not activate DRN mechanisms to the same extent as
other stress procedures. Forced swim does cause region-specific changes
in 5-HT release and metabolism immediately after forced swim, but no
changes were detected 24 hr later (Kirby and Lucki, 1998), suggesting
that forced swim either does not alter 5-HT1B receptor activity or does so at a different time point than we tested
using the standard FST procedure. Previously we found
5-HT1B mRNA to be elevated in rats displaying
learned helplessness in shuttle box testing after inescapable restraint
and tail shock (Neumaier et al., 1997). Tail shock stress alters DRN
function for at least 24 hr (Maier et al., 1995; Sutton et al., 1997;
Grahn et al., 1999). Different stress paradigms have variable effects on monoamine activity in general and have different impacts on dorsal
versus median raphe activity in particular (Adell et al., 1997; Durand
et al., 1999). Thus, it is important to consider a range of behavioral
measures in assessing the role of 5-HT1B autoreceptors in depression- and anxiety-related behavior.
Toward this end we also analyzed the effect of water-restraint stress
on OFT and EPM behavior after HA-5-HT1B/GFP
infection of DRN. We used water restraint because it has previously
been found to be useful in a rodent model of stress-induced depression, Wistar Kyoto rats (Pare, 1994), and was similar to our FST procedure. Using this assay, we found a significant reduction in central square
entries after an inescapable stressor in the pHSV-HA1B/GFP group as
compared with the pHSV-GFP control group. OFT procedures differ widely
between different research groups, particularly in the size, shape, and
lighting of the testing arena and in the behavioral outcomes measured.
In this study, rats were tested in the afternoon to maximize the
potential contribution of 5-HT1B autoreceptors
(Sayer et al., 1999) and under low intensity red illumination because
this leads to greater overall locomotor activity. A number of behaviors
have been measured in previous OFT studies, including total locomotor
activity, central entries, rearing, defecation, and others (Plaznik et
al., 1988; Stockert et al., 1988; Kelly and Leonard, 1994; Pare, 1994;
Meerlo et al., 1996; Izumi et al., 1997; Durand et al., 1999). We chose
to measured central square entries, which reflects an
approach/avoidance conflict, because this was recently shown to be
particularly sensitive to stress-induced anxiety states (Ramos et al.,
1997; Durand et al., 1999). There was no change in overall animal
locomotor activity in the OFT, ruling out a nonspecific change in
locomotor activity. Similarly, rats with 5-HT1B
overexpression in DRN who were stressed by water restraint avoided the
open arms of the EPM, consistent with increased anxiety-like behavior,
but had no greater total locomotor activity than GFP controls.
Therefore, 5-HT1B overexpression in DRN combined
with exposure to a stressor increased anxiety-like behavior 24 hr
later, suggesting that the 5-HT1B overexpression induced an enduring change in behavior after stress.
There are at least two possible interpretations of this data.
Increasing 5-HT1B autoreceptor expression in
serotonergic DRN neurons either makes the animals more anxious directly
or increases the impact of stress on anxiety behaviors. When we
examined open-field behavior in the absence of stress exposure,
pHSV-HA1B/GFP did not decrease entries into the center of the open
field. Indeed, 5-HT1B overexpression in DRN in
the absence of stress increased exploration of the central square.
Although it is not clear why increased 5-HT1B
autoreceptor expression might lower anxiety behavior in an unstressed
animal, we have recently observed that 5-HT1B mRNA is elevated in the stress-resistant group from two models of
differential stress susceptibility when the animals had not been
stressed (Neumaier et al., 2002). The findings suggest a complex
role for the 5-HT1B autoreceptor in modulating
anxiety. Our working hypothesis is that the impact of
5-HT1B overexpression in DRN is dependent on
context (in this case, exposure to stress). Thus in the absence of
stress, 5-HT1B autoreceptor overexpression may
increase an approach toward potentially aversive stimuli, whereas in
the presence of stress, these stimuli may provoke increased anxious
behavior. Because serotonin release is increased or decreased by stress
in different brain regions at different time points (Adell et al.,
1997; Amat et al., 1998; Kirby and Lucki, 1998), there are likely to be
discrete regulatory mechanisms that have yet to be elucidated.
Although OFT, EPM, and FST behaviors may involve different behavioral
circuits and do not necessarily change in a unified manner (Plaznik et
al., 1988; West and Weiss, 1998; Durand et al., 1999; Page et al.,
1999), forced swim appears to have less impact on
5-HT1B autoreceptor mechanisms than inescapable
water-restraint (this study) or inescapable restraint with tail shock
(Neumaier et al., 1997). Controllability of stress is especially
important in activating the DRN (Grahn et al., 1999). This may explain
why we detected larger effects of water restraint than forced swim in
this study: the combination of water stress and restraint may have
potently impacted the dimension of controllability. The behavioral measures used in this study may be less dependent on the amygdala, which receives serotonergic innervation predominantly from DRN, than
other models such as learned helplessness, fear conditioning, and
social interaction indices of stress (Maier et al., 1993; Gonzalez et
al., 1996; Amat et al., 1998). It will be interesting to examine the
effect of HA-5-HT1B/GFP expression using other stress paradigms such as inescapable tail shock, and in other testing
paradigms, to understand the role of 5-HT1B
autoreceptors more fully.
In summary, we have used a modification of HSV-based gene delivery to
overexpress 5-HT1B mRNA in DRN and to express GFP
as a vital marker of neuronal infection. The dual expressing amplicon led to functional expression of membrane-bound, epitope-tagged 5-HT1B receptors in vitro and in
vivo. Overexpression of HA-5-HT1B/GFP in DRN
had marked effects on stress-sensitive behaviors in the open-field
paradigm. This manipulation is particularly well suited to studying the
5-HT1B autoreceptor in DRN because only the
region of interest is directly altered, and exogenous agonist treatment is not necessary. Because 5-HT1B autoreceptors
are thought to be predominantly active in the axon terminals and not in
the vicinity of DRN (Pineyro et al., 1995),
HA-5-HT1B/GFP effects in animal models of
depression or anxiety can be attributed mainly to these axonal
projection |
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ABSTRACT
INTRODUCTION
