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The Journal of Neuroscience, July 1, 2000, 20(13):4809-4820
Altered Stress-Induced Anxiety in Adenylyl Cyclase Type
VIII-Deficient Mice
Michele L.
Schaefer1,
Scott
T.
Wong2,
David F.
Wozniak3,
Lisa M.
Muglia1,
Jason A.
Liauw4,
Min
Zhuo4,
Anthony
Nardi3,
Richard E.
Hartman3,
Sherri K.
Vogt1,
Christina E.
Luedke6,
Daniel R.
Storm2, and
Louis J.
Muglia1, 5
Departments of 1 Pediatrics, 3 Psychiatry,
4 Anesthesiology and Anatomy and Neurobiology, and
5 Molecular Biology and Pharmacology and Obstetrics and
Gynecology, Washington University School of Medicine, St. Louis,
Missouri 63110, 2 Department of Pharmacology, University of
Washington, Seattle, Washington 98195, and 6 Division of
Endocrinology, Children's Hospital, Boston, Massachusetts 02115
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ABSTRACT |
Stress results in alterations in behavior and physiology that can
be either adaptive or maladaptive. To define the molecular pathways
involved in the response to stress further, we generated mice deficient
(KO) in the calcium-stimulated adenylyl cyclase type VIII (AC8) by
homologous recombination in embryonic stem cells. AC8 KO mice
demonstrate a compromise in calcium-stimulated AC activity in the
hippocampus, hypothalamus, thalamus, and brainstem. Hippocampal slices
derived from AC8 KO mice fail to demonstrate CA1-region long-term
depression after low-frequency stimulation, and AC8 KO mice also fail
to activate CRE-binding protein in the CA1 region after
restraint stress. To define the behavioral consequences of AC8
deficiency, we evaluated AC8 KO mice in the elevated plus-maze and open
field. Although naïve AC8 KO mice exhibit indices of anxiety
comparable with that of wild-type mice, AC8 KO mice do not show normal
increases in behavioral markers of anxiety when subjected to repeated
stress such as repetitive testing in the plus-maze or restraint
preceding plus-maze testing. These results demonstrate a novel role for
AC8 in the modulation of anxiety.
Key words:
adenylyl cyclase; cAMP response element-binding protein; hippocampus; knock-out mice; long-term depression; plus-maze
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INTRODUCTION |
Stress has been implicated in the
precipitation of common human diseases including major depression,
alcohol abuse, and cardiovascular disease (McEwen and Stellar, 1993 ;
McEwen, 1998b). Adaptations the body undergoes during stress to
maintain stability are critical for survival and incorporate neural,
neuroendocrine, and autonomic nervous system pathways (Chrousos and
Gold, 1992; Chrousos, 1995). Although acute activation of the adaptive
response during stress increases arousal and alters metabolism to
restore homeostasis, repeated or aberrant activation of this response
may instead worsen medical and psychiatric diseases (McEwen, 1998a).
Understanding the molecular mechanisms involved in the response to
stress is essential for the design of efficacious therapeutic agents.
Several mediators of the stress response have been characterized. These
include corticotropin-releasing hormone and arginine vasopressin
synthesized within the hypothalamic paraventricular nucleus that
augment adrenal glucocorticoid production and catecholamines from both
CNS and peripheral nervous system sources (Chrousos and Gold,
1992; Chrousos, 1995; Sawchenko et al., 1996). Corticotropin-releasing hormone (Potter et al., 1994; Smith et al., 1998), serotonin (Ramboz et
al., 1998; Grailhe et al., 1999), and glucocorticoid receptor-dependent (Tronche et al., 1999) signaling pathways have also been implicated in
the behavioral responses to acute stress. Additionally, an alteration
in synaptic transmission efficiency after stress, specifically long-term depression (LTD) in the CA1 region of the hippocampus, may
play a role in stress-dependent learning (Kim et al., 1996; Xu et al.,
1997; Manahan-Vaughan and Braunewell, 1999). Hippocampal LTD has been
associated with cAMP and protein kinase A (Brandon et al., 1995) and
glucocorticoid (Xu et al., 1998) signaling pathways, although the
importance of LTD in modifying stress-related behavior has not been established.
Production of cAMP by adenylyl cyclase (AC) provides an important
mechanism for the regulation of neuronal physiology. The calcium-stimulated AC isoforms have been strongly implicated in the
alteration of neuronal function based on previous stimulation (Cooper
et al., 1995; Sunahara et al., 1996). Targeted inactivation of the
calcium-stimulated AC type I (AC1) results in defective spatial
learning (Wu et al., 1995), motor coordination (Storm et al., 1998),
and hippocampal (Wu et al., 1995) and cerebellar long-term potentiation
(Storm et al., 1998). We initiated characterization of AC type VIII
(AC8) because of its possible role in the stress response. Like AC1,
AC8 undergoes robust stimulation by calcium and calmodulin and would be
anticipated to impart plasticity in neuronal responses in a
stimulus-dependent manner (Cali et al., 1994). Unlike AC1, AC8 is
expressed at high levels in the thalamus, habenula, and hypothalamic
paraventricular nucleus, sites involved in the neuroendocrine and
behavioral responses to stress, and in the olfactory bulb (Xia et al.,
1991; Matsuoka et al., 1994; Muglia et al., 1999). Within the
hippocampus, AC8 mRNA is expressed primarily in the CA1 region (Muglia
et al., 1999), whereas AC1 is robustly expressed in the dentate gyrus
and less so in the CA1 region (Xia et al., 1991).
We hypothesized that inactivation of AC8 would result in abnormal
behavioral and/or adrenal responses to psychological stressors. In the
current study, we test this hypothesis by generating AC8-deficient (KO)
mice and examining their responses to acute and chronic stress.
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MATERIALS AND METHODS |
Generation of AC8 KO mice
To generate a null AC8 allele, we ligated the 4.4 kb
BamHI-XbaI (excised from pBluescript SK II as
KpnI-XbaI) fragment from the 5'-flanking region
of the AC8 gene into KpnI- and XbaI-digested pPNT (Tybulewicz et al., 1991). This vector, designated
4.4AC8pPNT, was subsequently digested with NotI and
XhoI and ligated to a 5.5 kb HindIII (excised
from pBluescript SK II as NotI-XhoI) fragment distal to exon 1. Introduction of the linearized targeting vector into
R1 (129/Sv × 129/Sv-CP)F1 ES cells (Nagy et al., 1993)
resulted in 1 correctly targeted clone out of 150, as determined
by Southern blot analysis of ES DNA using a probe external to the
targeting vector, after selection with G418 and gancyclovir as
described previously (Muglia et al., 1994). Injection of the ES clone
3D6 into C57BL/6 blastocysts resulted in the production of several male
chimeras that transmitted the mutant allele through their germline.
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Animal husbandry |
To demonstrate germline transmission of the null AC8 allele,
chimeric males were mated with outbred Black Swiss females (Taconic, Germantown, NY). Agouti pups heterozygous for the mutated allele were
bred to generate homozygous-deficient mice and wild-type (WT) controls
of the same genetic background (mixed 129/Sv × Black Swiss) for
the following experiments, as well as heterozygotes for subsequent
strain propagation. The mice used in these studies (generations F2-F4)
were housed on a 12 hr:12 hr light/dark cycle with rodent chow
available ad libitum. All mouse protocols were in accordance
with National Institutes of Health guidelines and were approved by the
Animal Care and Use Committee of Washington University School of Medicine.
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Reverse transcription-PCR analysis |
Two micrograms of total hypothalamic or hippocampal RNA were
subjected to reverse transcription (RT)-PCR analysis, with
amplification conditions for all primer pairs as described previously
(Muglia et al., 1999). The sense primer (AD8-f2) of the 5' primer pair was encompassed by the region deleted in our targeting vector. We
therefore generated an intron-spanning primer pair that amplifies a 421 base region from the 3' end of the AC8 mRNA not deleted during
targeting (MSR, 5'-CTACGACCTTGTCTGCTCAG-3'; MAR,
5'-CTCTTCCACGTTATAGTCAC-3'). Intron-spanning primers from the
mouse vasopressin gene were included as a control for hypothalamic RNA integrity.
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Measurement of calcium-stimulated AC activity |
Excised brain regions from 12- to 14-week-old male AC8 KO and WT
mice were suspended in buffer A (20 mM Tris-HCl, pH 7.4, 2 mM MgCl2, 1 mM EDTA, 0.5 mM dithiothreitol, 5 µg/ml leupeptin, and 0.5 mM phenylmethanesulfonyl chloride) and broken at 4°C with a Dounce homogenizer. Cell homogenates were centrifuged at 600 × g for 2 min, and the resulting supernatants were centrifuged at 30,000 × g for 20 min. We assayed the resulting
membranes for AC activity. The enzyme assay was performed at 30°C for
25 min by adding membrane preparations (10-15 mg/ml of protein) to an assay cocktail containing 1 mM
[ -32P]ATP (500 cpm/pmol),
[3H]cAMP (15,000 cpm/mmol), 5 mM MgCl2, 0.2 mM EGTA, 1 mM EDTA, 2 mM cAMP, 5 mM theophylline,
5% bovine serum albumin, 20 mM creatine phosphate, and 100 U/ml creatine phosphokinase in 20 mM Tris-HCl, pH 7.4, in a final assay volume of
250 µl. The reaction was stopped by adding 750 µl of 1.5% SDS. The
reaction mixture was heated at 100°C for 2 min, and the
[32P]cAMP generated was recovered by
using Dowex AG-50 WX-4 and neutral alumina columns, as described
(Salomon et al., 1974). Free Ca2+
concentrations were calculated by using the Bound and Determined computer algorithm (Brooks and Storey, 1992). AC activity levels were
the means of triplicate determinations. Protein concentrations in the
cell membranes were determined by the method of Hill and Straka
(1988).
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In situ hybridization |
Paraformaldehyde-fixed brains from 8-week-old male AC8 KO and WT
mice were cut into 10 µm sections and hybridized with a probe specific for AC8 as described previously (Muglia et al., 1999). Adjacent sections were hybridized with an
[-33P]UTP-labeled RNA probe to AC1
that detected nucleotides 1557-1955 of the mouse AC1 mRNA (GenBank
accession number AF053980). After hybridization and washing, sections
were exposed to Hyperfilm  -max for 5-14 d.
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Hippocampal electrophysiology |
Male WT and AC8 KO mice at 6-8 weeks of age were anesthetized
with halothane. Transverse slices of hippocampus were rapidly prepared
and maintained in an interface chamber at 28°C, where they were
subfused with artificial CSF (ACSF) consisting of 124 mM NaCl, 4.4 mM KCl, 2.0 mM
CaCl2, 1.0 mM
MgSO4, 25 mM
NaHCO3, 1.0 mM
Na2HPO4, and 10 mM glucose, bubbled with 95% O2 and
5% CO2. Slices were kept in the recording
chamber for at least 2 hr before the experiments. A bipolar
tungsten-stimulating electrode was placed in the stratum radiatum in
the CA1 region, and extracellular field potentials were recorded using
a glass microelectrode (3-12 M ; filled with ACSF) also in the
stratum radiatum. Stimulus intensity was adjusted to produce a response
of ~1 mV amplitude. Test responses were elicited at 0.02 Hz.
Homosynaptic LTD was induced by prolonged low-frequency stimulation (1 Hz for 15 min) (Dudek and Bear, 1992). Data are presented as the
mean ± SEM. One-way ANOVA (with Duncan's multiple range
test for post hoc comparison) and Student's t
test were used for statistical analysis; p < 0.05 was
considered significant.
Behavioral analyses
General experimental design
Two sets of experiments (study 1 and study 2) were
conducted in separate groups of adult female mice. In study 1, AC8 KO
(n = 17) and WT controls (n = 14) that
were 3-4 months of age when behavioral testing began were evaluated
first for general locomotor activity during a 1 hr period and then on
the walking-initiation test from the sensorimotor battery (described
below). Next, we quantified anxiety-related behaviors during repeated
daily testing (over 3 consecutive days) in the elevated plus-maze. Two
different types of plus-maze experiments were conducted. In one
instance (study 1, experiment 1), plus-maze testing consisted of 5 min sessions conducted over 3 consecutive days with no other experimental manipulations included. The other plus-maze experiment (study 1, experiment 2), which was conducted 2 months after the first experiment,
included restraining the same mice for 30 min immediately before
testing them in the plus-maze on each of the 3 consecutive days.
Separate groups of 3-month-old naïve AC8 KO (n = 11) and WT (n = 10) females were evaluated on the
ledge, platform, and inclined-screen tests from the sensorimotor battery.
In study 2, a second set of experiments was conducted using different
groups of mice. In this second set of experiments, groups of
naïve KO (n = 18) and WT (n = 13) mice were subjected to plus-maze testing in which each of the
3 d test sessions was preceded by 30 min of restraint. One month
later the mice were retested on the plus-maze using the same
procedures. Two weeks after completing the second plus-maze experiment,
the mice were evaluated in terms of "open-field" behaviors
exhibited in the enclosure used in study 1 during the 1 hr locomotor
activity test.
One hour general locomotor activity
Locomotor activity of the mice was evaluated over a 1 hr period
as described previously (Brosnan-Watters et al., 1996). Testing was
performed in two transparent (47.6 × 25.4 × 20.6 cm high) polystyrene cages that contained a thin layer of wood chip bedding. Each cage was surrounded by a frame containing three pairs of photoelectric cells placed across the width of the cages, dividing the
cages into four equal quadrants along their length. Each pair of
photocells was connected to silent digital counters that recorded the
number of infrared beam breaks. Activity counts (beam breaks) were
summed across the three pairs of photocells at the end of the 1 hr
interval. Testing took place between 07:30 and 15:00 hr in a quiet room
continuously illuminated by fluorescent lights.
One hour evaluation of open-field behaviors in previously
stressed mice
To assess exploratory behavior in mice that had been exposed
previously to stressors, we evaluated the open-field behaviors and
locomotor activity of the mice in study 2 during a 1 hr test. Testing
was performed in the same cages described above, although the data
collection and variables analyzed were different. A frame containing
four pairs of photocells along the shorter axis and eight pairs of
photocells along the longer axis surrounded the cages, and the output
of these photocell pairs was fed to an on-line computer
(Hamilton-Kinder, LLC, Poway, CA). The system software (Hamilton-Kinder, LLC) was used to define a 33 × 11 cm central zone and a peripheral or surrounding zone that was 5.5 cm wide with the
sides of the cage being the outermost boundary. This peripheral area
extended along the entire perimeter of the cage. Variables that were
analyzed included the total number of ambulations, as well as the
number of entries, the time spent and the distance traveled in the
center area, and the time spent and the distance traveled in the area
surrounding the center.
Sensorimotor battery
The general sensorimotor capabilities of the mice from study 1 were evaluated using the four tests described below that have been
found previously to be sensitive for detecting acute drug-induced sensorimotor disturbances in mice (Brosnan-Watters et al., 1996, 1999),
and a subset of the tests has been used to determine the onset of
chronic disturbances in certain transgenic mice (Chiesa et al., 1998).
Mice were evaluated over two test sessions for each of the four tests.
The first session commenced at 09:00 hr, and the second session
commenced 3 hr later.
Inclined screen. Each mouse was placed on an elevated (47 cm
above the floor) wire mesh grid (16 squares per 10 cm) that was inclined to 60°. The wire mesh grid was stretched across a wooden frame that was 15 × 52 cm, and tape was placed along the
perimeter to prevent the mice from climbing around to the back of the
apparatus. Each mouse was placed in the middle of the screen with its
head oriented downward and timed for either how long it remained on the
screen or how long it took to turn 180° and climb to the top of the
apparatus and rest its forepaws on top. A maximum score of 60 sec was
given if an animal did not fall.
Platform. Each mouse was timed for how long it remained on
an elevated (47 cm above the floor) circular platform (1.0 cm thick; 3.0 cm in diameter). A maximum score of 60 sec was assigned if the
mouse remained on the platform for the maximum amount of time or if it
could climb down on a very thin pole that supported the platform
without falling.
Ledge. Each mouse was timed for how long it could maintain
its balance on a narrow (0.75 cm thick) Plexiglas ledge without falling
(60 sec maximum). A score of 60 sec was also assigned if the mouse
traversed the entire length (51 cm) of the Plexiglas ledge and returned
to the starting place in <60 sec without falling.
Walking initiation. Each mouse was placed in the middle of a
square outlined by white cloth tape (21 × 21 cm) on a smooth black surface of a large table top. The time it took each mouse to
leave the square (place all four paws outside of the tape) was
recorded. The maximum time allowed was 60 sec.
Elevated plus-maze
Our apparatus is a modified version of that described by Lister
(1987) consisting of two opposing open arms [35 × 6.1 (outer width) × 0.3 cm] and two opposing enclosed arms (35 × 6.1 × 15 cm) that extend from a central platform (5.5 × 5.5 cm). The floor and walls of the maze were constructed of black
Plexiglas. The maze is equipped with pairs of photocells configured in
a 16 (x-axis) by 16 (y-axis) matrix, the
output of which is recorded by a computer and interface assembly
(Hamilton-Kinder, LLC). A software program (Hamilton-Kinder, LLC)
enables the beam break data to be recorded and analyzed to quantify
time spent, distance traveled, time spent at rest, and the numbers of
entries made into the open and closed arms and the center area. These
data were used to calculate the total arm entries, the percent of open
arm entries [e.g., (open arm entries/total arm entries) × 100],
the percent of closed arm entries, the percent of open arm time (e.g.,
[open arm time (sec)/300 sec] × 100), and the percent of closed arm
time. The numbers of fine movements and x- and
y-axes ambulations were also recorded throughout the maze.
The numbers of x- and y-axes ambulations predominantly reflect locomotor activity in the closed and open arms,
respectively, although a small percentage of x-axis
ambulations are measured in the open arms and the same is true for
y-axis ambulations in the closed arms. In study 2, various
behaviors were also quantified from a videotaped record of the test
sessions. The behaviors analyzed were stretch attend postures (SAPs) in the open and closed arms, open and closed arm head dips, and the time
taken to leave the center area to first enter an arm. The SAPs were
defined as exploratory postures in which the mouse stretches forward
and retracts to its original position without walking forward. Head
dips were characterized by exploratory movements of the head and
shoulders over the sides of the maze. In the closed arms, head dips
occur around the exiting corners of the arms nearest to the central
area. Mice were tested from 08:00 to 16:00 hr in a darkened room where
the only illumination came from a single incandescent (60 W) bulb. Test
sessions commenced by placing a mouse in an opaque plastic tube and
then removing the tube, allowing the mouse to explore the maze. Each
test session lasted 5 min, and mice were tested over 3 consecutive
days. Mice to be restrained were transported to a room separate from
the housing room and the behavioral-testing room. Each mouse was placed
in a ventilated 50 ml plastic tube. Mice were restrained for 30 min and
then taken into the testing room and evaluated on the plus-maze. The
mice were restrained before plus-maze testing on each of the 3 d
test sessions for each of the three experiments that involved restraint.
Olfactory testing
AC8 KO and WT male mice at 8-12 weeks of age (n = 4-5 per odorant concentration) were tested for the ability to detect
odorants and associate them with an aversive stimulus as described
previously (Griff and Reed, 1995). Briefly, after fluid restriction of
1 hr of access to fluid daily for 1 week, mice were provided solutions containing
10 3-108
M isovaleric acid or
103-104
dilutions of the pentadecalactone stock solution for 10 min followed by
intraperitoneal injection of 0.6 M lithium
chloride. Twenty-four hours later, two water bottles were placed in the
cages, one with odorant and one without. After 24 hr, the volume
ingested from each bottle was determined, and the positions of the
bottles were reversed. Volume was again measured after an additional 24 hr, and the preference ratio (volume of odorant solution ingested/total volume ingested) was determined for the 48 hr period. To ensure that an
underlying preference or aversion to the odorant-containing solution
was not present, we tested WT or KO mice (n = 4-5)
without the aversive stimulus.
Statistical analyses for the behavioral tests
The data from the behavioral tests were analyzed using ANOVA
models. Except for the sensorimotor and plus-maze data, one-way ANOVAs
were conducted for a given variable or test, e.g., the number of
entries into the central zone of the open field or locomotor activity
counts. Two-way ANOVAs containing one between-subjects variable, group
(AC8 KO vs WT), and one within-subjects (repeated measures) variable,
test sessions (one to two), were used to analyze the data from the
sensorimotor tests. Similar ANOVAs on repeated measures were used to
analyze each dependent variable generated in the plus-maze experiments
except that the within-subjects (repeated measures) variable was test
days (one to three). Simple main effects of group were calculated for
each test day after a significant main effect of group or a significant
group-by-test day interaction. levels were adjusted for the
within-subjects variables test days and the group-by-test days
interaction using the Greenhouse-Geisser correction to control for
violations of sphericity/compound symmetry.
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Neuroendocrine response to restraint |
Three-month-old AC8 KO and WT males (n = 3-4
per group) that had been singly housed were subjected to retroorbital
phlebotomy 2-3 hr after lights-on either (1) in the basal state
without previous manipulation, (2) in the basal state but after 1 hr of
restraint stress daily for 1 week, (3) immediately after an initial 1 hr of restraint, or (4) after 1 hr of restraint preceded by 1 hr of
daily restraint for 1 week. Serum corticosterone was determined by RIA
as described previously (Muglia et al., 1995). Data are presented as
the mean ± SEM, with statistical significance determined by ANOVA.
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Immunohistochemistry |
Nonstressed AC8 KO and WT mice and KO and WT mice that had been
subjected to 1 hr of restraint stress and then returned to their home
cages for 2 hr (n = 3 males at 2-3 months of age per genotype and treatment group) were deeply anesthetized with 1 ml of
2.5% Avertin and transcardially perfused with Dulbecco's PBS
(D-PBS) followed by 4% paraformaldehyde in D-PBS. Brains were post-fixed by immersion in 4% paraformaldehyde for 24 hr at 4°C and
cryoprotected in 10% sucrose in D-PBS. Immunohistochemical analysis
was performed on free-floating sections cut at 35 58 m on a
cryostat. After blocking in 3% normal goat serum in PBS for 30 min,
sections were incubated with a 1:1000 dilution of a polyclonal rabbit
anti-phosphoCRE-binding protein (anti-phosphoCREB) antibody (New
England Biolabs, Beverly, MA) in D-PBS with 1% goat serum. Peroxidase
staining was visualized with a Vectastain Elite ABC kit (Vector
Laboratories, Burlingame, CA). Staining intensity was quantitated using
NIH Image software and analyzed for statistical significance between
genotypes by ANOVA.
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RESULTS |
Production of AC8 KO mice
To generate AC8 KO mice, we constructed a targeting vector
that replaces 2 kb of the AC8 5'-flanking region and all of exon 1 (encoding the translation initiation site and the first 320 amino acids
of the AC8 protein) with a phosphoglycerate kinase-neomycin resistance
cassette (Fig. 1A). One
of 150 G418- and gancyclovir-resistant ES clones underwent homologous
recombination as determined by Southern blot analysis using a
hybridization probe external to the region of homology contained within
the targeting vector. We generated several male chimeras from this ES
clone that transmitted the mutant allele through the germline.
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Figure 1.
Targeted inactivation of AC8.
A, Strategy for AC8 gene disruption. Homologous
recombination between the targeting vector and the WT AC8 locus results
in the deletion of exon 1 and 2 kb of the 5'-flanking region in the
targeted AC8 locus. The open box in exon
1 represents 5'-untranslated leader sequence, and the
stippled area shows the region coding for
the first 320 amino acids. Genes and restriction sites indicated are as
follows: B, BamHI; E,
EcoRI; H, HindIII;
pgk-neo, phosphoglycerate kinase promoter-neomycin
resistance gene; pgk-tk, phosphoglycerate kinase
promoter-herpes simplex virus thymidine kinase gene; S,
SacI; Xb, XbaI; and
Xh, XhoI. B, Absent AC8
mRNA in AC8 KO mice. Reverse-transcription PCR of total
hypothalamic RNA used gene-specific primers from the region
deleted after homologous recombination with the targeting vector
(5' AC8) and a region of the AC8 gene remaining after
targeting (3' AC8). Shown is an ethidium bromide-stained
agarose gel demonstrating the expected products for each primer pair in
WT (+/+) and heterozygous (+/ ) mice but not in the KO (/) mice.
The vasopressin (VP) mRNA amplification product is
detected in all samples, verifying hypothalamic RNA integrity.
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Matings of mice heterozygous for the disrupted AC8 allele resulted in
the generation of homozygous AC8-deficient mice in numbers not reduced
from the expected Mendelian frequency, with the homozygotes representing 17/79 (34%) of the initial progeny. To ensure that deletion of the promoter region and exon 1 had not allowed production of a truncated AC8 protein, we performed RT-PCR analysis of
hypothalamic RNA using amplification primers both from the region of
the AC8 gene that had been deleted and from a 3' region of the mRNA
that had not been deleted. No products from either primer pair were detected in AC8 KO hypothalamus (Fig. 1B) or
hippocampus (data not shown).
Pattern of calcium-stimulated AC activity in AC8 KO mice
To assess which regions of the brain would most likely manifest
altered function as a consequence of loss of AC8, we compared the
patterns of expression of AC8 and AC1, an abundant AC isoform in the
brain significantly stimulated by calcium and/or calmodulin (Xia et
al., 1991; Cooper et al., 1995), by in situ hybridization in
WT and AC8 KO mice. In general, despite expression of both AC isoforms
in many brain regions, the cell populations within these regions
expressing each AC isoform differed. Comparison of AC1 expression in WT
and AC8 KO mice revealed no qualitative alteration in the sites of AC1
mRNA expression as a consequence of loss of AC8 activity (Fig.
2). As we have reported previously (Muglia et al., 1999), AC8 mRNA is most abundantly expressed within the
olfactory bulb, thalamus, habenula, CA1 region of the hippocampus, and
hypothalamus in WT mice (Fig. 2).
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Figure 2.
Localization of AC1 and AC8 mRNA in WT and AC8 KO
brain. Representative in situ hybridization
autoradiograms of WT (top row) and AC8 KO
(bottom row) mice are shown. Adjacent
sagittal and coronal sections were hybridized to probes specific for
AC1 (left) or AC8 (right). AC1 mRNA
hybridization is strongest in the cerebellum (cb),
cerebral cortex (ctx), and dentate gyrus
(dg) of the hippocampus. AC8 mRNA is detected in the
olfactory bulb (ob), thalamus (th),
cortex, habenula (hb), CA1 region of the hippocampus,
and paraventricular nucleus of the hypothalamus
(pvn), as well as other brain regions in WT, but
not AC8 KO, mice.
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To assess alterations in calcium-stimulated AC activity in the AC8 KO
mice directly, we measured brain region-specific cAMP generation as a
function of calcium concentration (Fig.
3). Consistent with the in
situ hybridization findings, calcium-stimulated adenylyl cyclase
activity was almost entirely absent from the regions in which AC8
predominated such as the hypothalamus, olfactory bulb, thalamus, and
brainstem. Calcium-stimulated AC activity was reduced ~40-50% in
the hippocampus, in accord with the loss of AC8 activity within the CA1
region together with persistent hippocampal AC1 activity (Fig. 2).
Basal AC activity, as determined in the absence of calcium, is not
altered in the AC8 KO mice (Fig. 3).
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Figure 3.
Brain-region specific AC activity in WT and AC8 KO
mice. Calcium-stimulated AC activity determinations from freshly
excised thalamus (A), hypothalamus
(B), olfactory bulb (C),
hippocampus (D), and brainstem
(E) from WT (closed
circles; n = 5) or AC8 KO
(open circles; n = 5)
mice are displayed. Nearly all AC8 KO brain regions, with the exception
of the hippocampus, demonstrated profound deficiency in the ability to
generate cAMP in response to Ca2+.
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Phenotype of AC8 KO mice
AC8 KO mice derived from heterozygous matings appeared grossly
indistinguishable from their WT and heterozygous littermates and were
fertile. Analysis of growth of the KO mice revealed a tendency for both
male and female AC8 KO mice to be somewhat smaller than the
corresponding WT controls (Fig.
4A,B). The AC8 KO
female mice gained weight in a manner similar to that of WT females
until day of life 30, at which point they began to diverge
significantly (p < 0.05). The AC8 KO females
then remained 10-15% smaller than WT females
(p < 0.0001). In contrast, KO male mice grew in
a manner identical to that of WT mice until day of life 45 and then
transiently grew more slowly between day of life 45 and 92 (p < 0.01), after which point differences in WT
and KO males were not significant. The observed differences in weight
gain were not caused by differences in appetite, because the absolute
amount of food ingested daily did not differ between the genotypes, and
food intake normalized to body weight was greater in the KO mice as
compared with WT (data not shown).
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Figure 4.
Growth and olfaction in AC8 KO mice.
A, Growth curves of WT (n = 23) and
AC8 KO (n = 27) male mice. Differences in weights
between males of each genotype achieved statistical significance
between day of life 45 and 92 (p < 0.01).
B, Growth curves of WT (n = 26) and
AC8 KO (n = 17) female mice. Differences in weights
between females of each genotype achieved statistical significance at
day of life 30 (p < 0.05). AC8 KO females
remained smaller than WT females as adults
(p < 0.0001). C, D,
Preference testing for isovaleric acid (C) and
pentadecalactone (D). Adult male mice
(n = 4-5 per odorant concentration and genotype)
were tested with and without (no cond) the conditioning
aversive stimulus. The preference ratio represents the total amount of
odorant-containing solution ingested per total amount of liquid
ingested.
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Because of the high level of expression of AC8 mRNA in the olfactory
bulb, we sought to determine whether olfactory learning or odorant
sensing had been impaired in the AC8 KO mice. We used a conditioned
avoidance assay (Griff and Reed, 1995) to determine whether the KO mice
could smell a given odorant and associate it with an aversive stimulus.
The preference ratio, defined as the volume of odorant solution
ingested per total amount of solution ingested as measured in a
two-bottle system, has been shown to reflect olfaction accurately. Both
WT and AC8 KO mice avoided the odorant (isovaleric acid or
pentadecalactone)-containing solutions when the odorants had been
paired with the aversive stimulus and showed similar thresholds for the
ability to detect the odorants in the drinking water (Fig.
4C,D).
Impaired long-term depression in AC8 KO mice
Although the loss of both AC1 and AC8 activity results in defects
in late-phase long-term potentiation (L-LTP) and long-term memory
(LTM), a single deficiency of each isoform causes impairment of neither
L-LTP nor LTM (Wong et al., 1999). Because behavioral stress-induced
learning and alteration in synaptic strength have been associated with
the induction of LTD in the CA1 region of the hippocampus (Kim et al.,
1996; Xu et al., 1997; Manahan-Vaughan and Braunewell, 1999), we
proceeded to evaluate the ability of hippocampal slices from AC8 KO
mice to demonstrate LTD after low-frequency (1 Hz; 15 min) CA1
stimulation. In contrast to WT mice, which demonstrated a significant
reduction in field EPSP after low-frequency stimulation, AC8 KO
mice did not elicit LTD (Fig. 5).
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Figure 5.
Impaired long-term depression in AC8 KO mice.
Low-frequency stimulation (1 Hz; 15 min; open
horizontal bar) induced LTD in the
CA1 region of the hippocampus from WT mice (open
circles; 61.8 ± 9.2%; n = 6 slices from 5 mice; p < 0.05 comparing the
potential 25-30 min after stimulation with the average of the
potential before stimulation). However, LTD was blocked in mice lacking
AC8 (closed circles; 101.4 ± 14.4%; n = 8 slices from 5 mice;
p < 0.05 vs WT after stimulation).
Inset, Representative recordings of the EPSP before and
30 min after low-frequency stimulation in WT (left) and
AC8 KO (right) mice are shown.
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Stress-related behavioral and neuroendocrine effects in AC8
KO mice
Previous behavioral evaluation of AC8 KO mice demonstrated normal
passive avoidance, contextual, and cued-learning responses (Wong et
al., 1999). Before analyzing the specific effects of stress on the
induction of anxiety in AC8 KO mice, they and WT mice were first
evaluated on a 1 hr test of general locomotor activity and a battery of
sensorimotor measures. These tests were done to ensure that there were
no inherent abnormalities in AC8 KO mice that might confound the
interpretation of behavioral performance on subsequent tests of anxiety.
Behavioral effects
No differences were found between the AC8 KO and WT mice in
general locomotor activity (see Fig. 9A). The two
groups also performed similarly on the ledge, platform, and
inclined-screen tests within the sensorimotor battery (Fig.
6A-C) demonstrating that they did not differ in balance, strength, and coordination. However, group performances significantly differed on the
walking-initiation portion of the battery (Fig. 6D).
The AC8 KO mice took less time to leave a defined perimeter outlined on
a large tabletop over two test sessions compared with WT mice
[F(1,29) = 6.71; p = 0.015], with the greatest difference between groups occurring during
the first test session (p = 0.008). Previous
drug studies in rats and mice (Wozniak et al., 1990; Brosnan-Watters et
al., 1996) suggest that these differences in the latency to initiate
movement in an open area reflect altered emotionality (e.g., anxiety)
rather than differing motoric capacity.
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Figure 6.
Sensorimotor analyses of AC8 KO and WT mice. Both
groups performed well on the ledge (A), platform
(B), and inclined-screen
(C) tests, with no differences found between
genotypes. On the walking-initiation test (D),
AC8 KO mice took significantly less time to leave a defined perimeter
over the two test sessions [F(1,29) = 6.71; p = 0.015]. The number of mice per group is
indicated in parentheses above the
corresponding bar. *, p = 0.008.
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To evaluate differences in anxiety further, we tested both groups of
mice on the elevated plus-maze. Spontaneous behaviors exhibited in the
maze reflect an innate aversion that rodents have to elevated, open
spaces. Anxiety is inferred from the analysis of dependent variables
that reflect an avoidance of the open arms. We conducted two groups of
studies to investigate the effects of two independent variables known
to alter plus-maze performance: repeated test exposure (Espejo, 1997)
and restraint stress immediately preceding plus-maze testing (Gorman
and Dunn, 1993).
In the first experiment (study 1, experiment 1), we tested mice over 3 successive days without restraint before any test. ANOVAs conducted on
the plus-maze variables indicated that there were no significant
overall main effects of group for any variable (Fig.
7). However, because a single previous
undrugged exposure to plus-maze testing reduces or abolishes the
subsequent efficacy of anxiolytics (Rodgers et al., 1992; Rodgers and
Shepherd, 1993), we postulated that daily exposure to testing may have
altered innate differences in anxiety-related behaviors between groups on specific days. We therefore proceeded to conduct pairwise
comparisons for each dependent variable as a function of test day. On
the first test day, significant differences between the naïve
AC8 KO and WT mice were found only for y-axis ambulations,
which reflect open arm locomotor activity
[F(1,28) = 6.12; p = 0.02; Fig. 7D]. However, on the second test day differences
emerged on three variables related to open arm behaviors. The KO mice
spent a significantly greater percentage of time
[F(1,28) = 6.16; p = 0.019; Fig. 7A] and traveled a significantly greater
distance [F(1,28) = 4.57; p = 0.041; Fig. 7B] in the open arms than
did the WT mice, and the percent of open arm entries was significantly
greater for the KO compared with the WT mice (data not shown). The
trends for the variables continued, although they did not achieve
statistical significance, when repeated on day 3 (Fig.
7A,B). The significant pairwise comparisons found on
specific test days in study 1, experiment 1, suggested that AC8 KO mice
displayed less anxiety than did WT mice in the plus-maze, particularly
in the degree to which anxiety increased across test days.
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Figure 7.
Restraint stress and repeated testing alters
anxiety-related behaviors in the elevated plus-maze. Effects on four
different variables are shown: the percent of time in the open arms
(A), the distance traveled in the open arms
(B), the number of x-axis (closed
arm) ambulations (C), and the number of
y-axis (open arm) ambulations (D).
Mice were tested on 3 consecutive test days under each of the following
conditions: study 1, experiment 1, mice naïve to the plus-maze
on test day 1 and no restraint before testing; study 1, experiment 2, same mice used in study 1, experiment 1, except it is 2 months later
with restraint for 30 min each day before testing; study 2, experiment
1, different naïve groups of mice restrained for 30 min each
day before testing; study 2, experiment 2, same mice used in study 2, experiment 1, retested with the same restraint protocol 1 month
later.  , Significant main effect of group; *, significant simple main
effects of group; +, borderline nonsignificance of simple main effects.
EXP., Experiment.
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We next subjected the same AC8 KO and WT mice to 30 min of restraint
stress before testing them on the plus-maze to potentiate innate
differences in anxiety between the two groups. Restraining the mice
before testing them on the plus-maze (study 1, experiment 2) resulted
in significant main effects of group for several dependent variables.
The AC8 KO mice spent a significantly greater amount of time, and
traveled a significantly greater distance, in the open arms than did
the WT mice [F(1,28) = 5.68 (p = 0.024) and F(1,28) = 5.79 (p = 0.023), respectively; Fig.
7A,B]. Evaluation of the numbers of ambulations along the
x- and y-axes also yielded significant main
effects of group, with the KO mice exhibiting greater levels of
activity in both the open and closed arms
[F(1,28) = 8.40 (p = 0.007) and
F(1,28) = 4.67 (p = 0.039), respectively; Fig.
7C,D]. Thus, the restraint stress and/or second exposure to
repeated daily testing in the plus-maze increased differences in
anxiety-related behaviors, with the KO mice consistently exhibiting less evidence of anxiety.
To confirm the findings in study 1, experiment 2, we subjected
additional groups of naïve AC8 KO and WT mice to the same 3 d restraint stress and plus-maze protocol (study 2, experiment 1). These results were similar to those observed in study 1, experiment 2, again indicating that the repeated restraint and testing resulted in
more anxiety in WT than in AC8 KO mice (Fig. 7). For example, significant main effects of group were found for ambulations along the
x- and y-axes indicating that the AC8 KO mice
were more active within both arms compared with the WT mice when
restraint preceded plus-maze testing
[F(1,29) = 8.97 (p = 0.006) and
F(1,29) = 4.24 (p = 0.049), respectively; Fig.
7C,D]. In the second experiment conducted in study 2, we
retested mice on the same restraint and plus-maze protocol used in
experiment 1 after an intervening period of 1 month, during which time
no behavioral testing was conducted. This second exposure revealed even
larger differences in open arm behaviors between the groups (Fig.
7).
Ethologically based indices of anxiety (Rodgers and Cole, 1993; Espejo,
1997) such as head dips and SAPs were also analyzed in the plus-maze.
In study 2, experiment 1, WT mice emitted more closed arm SAPs
(p < 0.0005; Fig.
8A) and head dips
(p = 0.001; Fig. 8C) than did AC8 KO
mice on the first test day, suggesting greater risk assessment by the
WT mice. In agreement with greater risk assessment, the WT mice also
consistently took longer to leave the center area to first enter an arm
of the plus-maze (p = 0.023; Fig.
8B). In contrast, the AC8 KO mice emitted more head dips over the side of the open arms (a behavior anticipated to induce
anxiety) than did the WT mice in both studies, achieving a significant
main effect of group during study 2, experiment 2 (p = 0.036; Fig. 8D).
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Figure 8.
Effects of restraint stress and repeated plus-maze
testing on ethological variables. A, C, When first
exposed to the restraint/plus-maze protocol (study 2, test day 1, experiment 1), the WT mice (n = 13) exhibited
significantly (*) greater numbers of closed arm SAPs
(A) and closed arm head dips
(C) compared with that of the AC8 KO mice
(n = 18). These effects were large enough to
produce significant main effects of group () for both variables in
experiment 1. B, The WT mice compared with the AC8 KO
mice took consistently longer to exit the center area with a
significant main effect of group () found in study 2, experiment 1. Substantial differences (+) exhibited on test day 1 (p = 0.051) contributed greatly to this
effect. D, In contrast, the AC8 KO mice consistently
exhibited more head dips in the open arms. A significant main effect of
group () was found for study 2, experiment 2, and analysis of the
simple main effects of group indicated that the KO mice compared with
the WT mice exhibited significantly (*) more open arm head dips on test
day 2 (experiment 2).
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Two weeks after completion of the second restraint and plus-maze
experiment in study 2, we evaluated the same mice on several measures
in the open field. In agreement with findings from the restraint and
plus-maze testing, the AC8 KO mice exhibited a greater number of
ambulations in the open field compared with the WT mice (Fig.
9A). In addition, the KO mice
also made more entries into and traveled a greater distance within the
central area relative to the WT mice (Fig. 9B,C).
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Figure 9.
Analysis of open-field behavior. A,
In study 1 before being restrained or tested on the plus-maze, the AC8
KO (n = 17) and WT (n = 14)
mice were screened on a 1 hr general locomotor activity test using an
open field containing relatively low photobeam resolution capabilities.
Under these conditions, no differences were found between the groups
for locomotor activity. In study 2, the mice were evaluated on a 1 hr
open-field test using a high-resolution photobeam system after they had
been subjected to the restraint/plus-maze test sessions. Under these
conditions, the AC8 KO mice (n = 18) were found to
be significantly (*) more active in the numbers of ambulations than
were the WT mice (n = 13)
[F(1,29) = 5.67; p = 0.024]. B, C, The KO mice also made significantly
more entries into the center area
[F(1,29) = 5.69; p = 0.024] of the open field (B) and traveled a
significantly greater distance
[F(1,29) = 4.32; p = 0.047] in the center area (C) compared with
the WT mice.
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Neuroendocrine effects
Because AC8 is the only calcium-stimulated AC expressed in the
hypothalamus, we evaluated the response of the
hypothalamic pituitary-adrenal axis to acute and chronic stress to
determine whether glucocorticoid production had been impaired. We
subjected AC8 KO and WT mice to either acute (1 hr) or chronic
restraint (1 hr daily for 7 d) stress followed by determination of
plasma corticosterone concentration. In naïve KO and WT mice
before their initial restraint, no difference in plasma corticosterone
concentration was found (Fig. 10).
After the initial 1 hr of restraint, corticosterone levels increased equivalently in both genotypes. Adaptation to chronic restraint stress,
however, differed between the two genotypes. Evaluation of serum
corticosterone in WT mice immediately before their seventh day of 1 hr
restraint stress showed no difference from the basal corticosterone in
naïve WT mice. The chronically restrained WT mice, however,
exhibited a significantly augmented production of corticosterone after
the seventh restraint compared with that of WT mice after the initial
restraint (Fig. 10). In contrast, AC8 KO mice exhibited an elevated
basal corticosterone level before their final restraint in comparison
with naïve KO and WT mice. No significant augmentation of serum
corticosterone after the final restraint in comparison with that after
the first restraint occurred in the KO mice, but the corticosterone
measured after the final restraint did not significantly differ from
that achieved by WT mice (Fig. 10).
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Figure 10.
Adrenal response to restraint stress in AC8 KO
mice. AC8 KO and WT adult male mice (n = 3-4 per
group) underwent retroorbital phlebotomy for plasma corticosterone
measurement before any restraint (acute basal),
after one episode of 1 hr of restraint (acute
restraint), before a seventh episode of 1 hr per day of
restraint (chronic basal), or immediately after a
seventh episode of 1 hr per day of restraint (chronic
restraint). Data are presented as the mean ± SEM. *,
p < 0.05 versus WT acute
restraint; **, p < 0.01 versus KO
acute basal, or p < 0.05 versus WT acute and chronic
basal.
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Cellular basis of the altered stress response in AC8 KO mice
AC8 is expressed in several brain regions associated with the
stress response and is completely deficient in all of these areas in
the AC8 KO mice. Reduced AC activity should result in diminished
downstream signaling including protein kinase A-mediated CREB
activation. To implicate specific brain regions in the alteration in
stress-induced anxiety in the KO mice, we analyzed the pattern of
expression of the active phosphorylated form of CREB (pCREB) in WT and
KO mice (Fig. 11). Basal (unstressed)
pCREB immunoreactivity revealed no significant differences between AC8
KO and WT mice in any brain region (Fig. 11B). To
evaluate sustained pCREB induction, we analyzed KO and WT mice 2 hr
after restraint stress. We found pCREB immunoreactivity consistently
and significantly decreased in the KO mice in the CA1 region of the
hippocampus after restraint in comparison with WT mice (Fig. 11). No
consistent differences in pCREB expression were observed in the dentate
gyrus and cerebral cortex (Fig. 11) or other brain regions including
the hypothalamus, pyriform cortex, amygdala, and thalamus (data not
shown). Interestingly, analysis of pCREB expression immediately after 1 hr of restraint revealed significant immunoreactivity in the dentate
gyrus of both genotypes but no CA1 staining in either WT or KO mice
(n = 3 per genotype; data not shown). Thus, CA1 CREB
phosphorylation is a sustained, time-dependent process, which is
disrupted in the AC8 KO mice.
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Figure 11.
pCREB immunohistochemistry in AC8 KO and
WT mice. A, Representative sections through the
hippocampus of AC8 KO (right) and WT
(left) mice 2 hr after restraint stress stained with a
pCREB antibody. Positive cells are demonstrated by deposition of a dark
nuclear precipitate after peroxidase staining. A specific deficit in
pCREB immunoreactivity is seen in the CA1 region of the hippocampus of
the AC8 KO mice, whereas cortex and dentate gyrus show no difference
from WT. B, Quantitation of pCREB immunohistochemistry.
Densitometric analysis of sections from n = 3 WT
and KO mice per treatment (basal or 2 hr after restraint) is shown. The
average density of the WT postrestraint group is normalized to 1 for
each brain region. *, p < 0.05 versus WT CA1.
Ctx, Cortex.
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DISCUSSION |
To determine the role of the calcium-stimulated ACs in the
behavioral and neuroendocrine response to stress, we report the generation and characterization of AC8-deficient mice. Our targeting strategy, which deleted the AC8 gene promoter region and exon 1, resulted in complete AC8 deficiency as demonstrated by the absence of
AC8 mRNA and the loss of calcium-stimulated AC activity in brain
regions where AC8 predominates. In situ hybridization studies in AC8 KO mice, together with the direct analysis of AC activity, further demonstrated that significant induction of AC1 in
cell populations primarily expressing AC8 in WT mice does not occur.
Surprisingly, despite normal hippocampal L-LTP and LTM (Wong et al.,
1999), AC8 KO mice manifest significant impairment in hippocampal CA1
LTD. Calcium-stimulated AC activity is reduced, but not absent, in the
hippocampi of AC8 KO mice, as would be expected on the basis of
abundant AC1 expression in the dentate gyrus, lower AC1 expression in
the CA1 region, and loss of AC8 in the CA1 region. Despite evidence of
AC1 mRNA in the CA1 region, we find that loss of AC8 cannot be
compensated for by the residual AC1 activity. The inability of AC1 to
maintain LTD in AC8 KO mice could result from differences between the
isoforms including sensitivity to calcium for activation (Villacres et
al., 1995), subcellular localization, or subpopulations of expressing
cells. These possibilities will be evaluated in our ongoing studies.
Hippocampal CA1 LTD has been found previously to be dependent on NMDA
receptor (Dudek and Bear, 1992; Kim et al., 1996; Zhuo et al., 1999)
and protein kinase A RI and C1 subunit
(Brandon et al., 1995; Qi et al., 1996) function. Our demonstration of
a critical role for AC8 in CA1 LTD further elucidates the molecular
basis of this response by defining the AC isoform linking the
intracellular influx of calcium resulting from NMDA receptor activation
to the generation of cAMP for activation of protein kinase A. The
dependence of CA1 LTD on AC8 activity suggests region-specific
differences in the mechanisms for LTD within the hippocampus. For
example, mossy fiber synapses on CA3 pyramidal cells require
metabotropic glutamate receptor-mediated decreases in adenylyl cyclase
and protein kinase A activity for the induction of LTD (Tzounopoulos et
al., 1998), whereas LTD in the medial perforant path of the dentate
gyrus is contributed to by activation of both protein kinase A and C (Huang et al., 1999).
Enhancement of LTD occurs after behavioral stress in normal rodents
(Kim et al., 1996; Xu et al., 1997; Manahan-Vaughan and Braunewell,
1999) and has been proposed as an important component of stress-induced
learning. The behaviors exhibited by AC8 KO mice in the elevated
plus-maze together with their impaired LTD are in accord with this
hypothesis. Indeed, although naïve AC8 KO mice did not differ
from WT mice in the exploration of the open arms of the elevated
plus-maze, when repetitively tested or subjected to a defined stressor
such as restraint or previous exposure to the plus-maze, clear
genotypic differences emerged. WT mice exhibited evidence of heightened
anxiety after previous testing or restraint in the elevated plus-maze,
whereas the KO mice had little change in their responses. Videotape
analysis of ethological variables showed further differences between
AC8 KO and WT mice. The greater number of SAPs and closed arm head dips
exhibited by WT mice suggest that the WT mice engage more frequently in
risk assessment behaviors related to their defense repertoires when
first exposed to the relatively protected closed arms of the plus-maze.
The number of times these behaviors were exhibited dropped
precipitously in the WT mice on subsequent exposures to the plus-maze
or with restraint, which may have been caused by fear sensitization
from the repeated testing and/or additional stressor (Rodgers et al.,
1997). On the other hand, the AC8 KO mice demonstrated a greater number
of open arm head dips, a behavior that would be expected to produce
substantial anxiety in WT mice (Rodgers and Shepherd, 1993; Espejo,
1997).
Consistent with the findings in the elevated plus-maze, AC8 KO mice
exhibited increased locomotor activity in the open field after previous
exposure to the restraint and plus-maze protocol but indistinguishable
general locomotor activity in comparison with WT mice when both
genotypes were naïve. Thus, the differences in anxiety observed
between the AC8 KO and WT mice in the plus-maze were not specific to
only this testing technique as demonstrated by the open-field results,
which closely paralleled the plus-maze findings. Furthermore, in accord
with the heightened anxiety observed in WT mice, WT mice significantly
augmented peak adrenal glucocorticoid synthesis after repeated
restraint stress. Although AC8 KO mice did not augment peak plasma
glucocorticoid levels immediately after the final restraint, suggesting
less sensitization as a result of the previous stress, we cannot
exclude an alteration in the temporal pattern of the KO corticosterone
response. Differences in the timing of the corticosterone response
between genotypes may be reflected by the elevated basal corticosterone
level found in the AC8 KO mice preceding the final restraint.
Alternatively, the elevated basal corticosterone level in the AC8 KO
mice, a finding also reported after surgical lesions of the hippocampus (Jacobson and Sapolsky, 1991), may be another manifestation of impaired
hippocampal function along with attenuated LTD and CREB phosphorylation.
The mechanism by which AC8 deficiency results in impaired
stress-induced anxiety may be quite complex, because AC8 is expressed in several regions associated with the stress response. Although impaired LTD implicates the CA1 region of the hippocampus as a potential contributor to the alteration in their behavioral stress response, the altered response to chronic stress we describe in the AC8
KO mice could have resulted from the abnormal integration of stimuli in
more than one brain region. To assess biochemical changes in specific
regions throughout the CNS associated with the response to restraint
stress, we analyzed pCREB immunoreactivity. Despite substantial
reductions in calcium-stimulated AC activity in many brain regions in
AC8 KO mice, no consistent differences in pCREB between WT and KO mice
were found outside the CA1 region after stress. Although this finding
does not exclude a role for AC8 in modulating stress-induced neuronal
function in sites such as the thalamus, amygdala, and hypothalamus,
intact CREB activation in these other sites indicates that other ACs or
AC-independent mechanisms of CREB activation (e.g., calcium- and/or
calmodulin-stimulated kinases) can compensate for AC8 loss in these regions.
AC8 is most highly expressed in the olfactory bulb, yet AC8 KO mice
exhibit no alteration in odorant sensing or the ability to associate an
odorant with an aversive stimulus. Why does global AC8 loss manifest a
phenotype most reflective of abnormal function in the CA1 region, and
what can this teach us about the stress response? The hippocampus and
notably its CA1 region possess the highest concentration of type II
glucocorticoid receptors in the brain (McEwen et al., 1986; Van Eekelen
et al., 1988; Stumpf et al., 1989). It is these lower affinity
glucocorticoid receptors that undergo occupation specifically during
stress-associated glucocorticoid increases, as opposed to at basal
glucocorticoid concentrations (Jacobson and Sapolsky, 1991).
Furthermore, exogenous glucocorticoid administration, as well as
changes in endogenous glucocorticoid after stress, increase the
induction of LTD in the CA1 region (Xu et al., 1998) by action on type
II glucocorticoid receptors. This action of glucocorticoid on
hippocampal neurons for induction of LTD requires new protein synthesis
(Xu et al., 1998), with important physiological sequelae including
increased Ca2+ action potentials and
voltage-activated Ca2+ currents (Kerr et
al., 1992). We postulate that the CA1 region provides a unique site for
integrating changes in serum and CNS glucocorticoids arising during the
response to stress with calcium-mediated changes in CREB activation.
Our data in AC8 KO mice implicate AC8 as a critical molecule for the
translation of increased intracellular calcium to altered protein
kinase A and/or CREB-mediated changes in gene expression that impact
upon induction of LTD and the response to stress. The altered response
to chronic stress observed in the AC8 KO mice, together with an absence
of other significant consequences of loss of AC8 activity, implicates
pharmacological modulation of AC8 action as a possible therapeutic
intervention for stress-related disorders.
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FOOTNOTES |
Received Feb. 23, 2000; revised April 11, 2000; accepted April 12, 2000.
This work was supported by grants from the National Institutes of
Health, Howard Hughes Medical Institutes, and Monsanto Company and a
Burroughs Wellcome Fund Career Development Award in the Biomedical
Sciences to L.J.M, a grant from the Alzheimer's Disease Research
Center at Washington University School of Medicine to M.Z., and
National Institutes of Health Grant NS 20498 to D.R.S. We thank Drs. A. Imamura and G. Gurtner for assistance with experiments and Drs. J. Gitlin and D. Pfaff for helpful discussions.
Correspondence should be addressed to Dr. Louis J. Muglia, Washington
University School of Medicine, One Children's Place, Box 8116, St.
Louis, MO 63110. E-mail: Muglia L{at}kids.wustl.edu.
M.L. Schaefer's present address: Neurosciences Program, University of
Colorado Health Sciences Center, Denver, CO 80262.
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ABSTRACT
INTRODUCTION
