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The Journal of Neuroscience, May 1, 2003, 23(9):3837
Conditional Expression in Corticothalamic Efferents Reveals a
Developmental Role for Nicotinic Acetylcholine Receptors in Modulation
of Passive Avoidance Behavior
Sarah L.
King1,
Michael
J.
Marks2,
Sharon R.
Grady2,
Barbara J.
Caldarone1,
Andrei O.
Koren3,
Alexey G.
Mukhin3,
Allan C.
Collins2, and
Marina R.
Picciotto1
1 Department of Psychiatry, Yale University School of
Medicine, New Haven, Connecticut 06508, 2 Institute for
Behavioral Genetics, University of Colorado, Boulder, Colorado 80309, and 3 Brain Imaging Center, Intramural Research Program,
National Institute on Drug Abuse, Baltimore, Maryland 21224
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ABSTRACT |
Prenatal nicotine exposure has been linked to attention deficit
hyperactivity disorder and cognitive impairment, but the sites of action for these effects of nicotine are still under investigation. High-affinity nicotinic acetylcholine receptors (nAChRs) contain the
 2 subunit and modulate passive avoidance (PA) learning in mice.
Using an inducible, tetracycline-regulated transgenic system, we
generated lines of mice with expression of high-affinity nicotinic receptors restored in specific neuronal populations. One line of mice
shows functional 2 subunit-containing nAChRs localized exclusively
in corticothalamic efferents. Functional, presynaptic nAChRs are
present in the thalamus of these mice as detected by nicotine-elicited
rubidium efflux assays from synaptosomes. Knock-out mice lacking
high-affinity nAChRs show elevated baseline PA learning, whereas normal
baseline PA behavior is restored in mice with corticothalamic expression of these nAChRs. In contrast, nicotine can enhance PA
learning in adult wild-type animals but not in
corticothalamic-expressing transgenic mice. When these transgenic mice
are treated with doxycycline in adulthood to switch off nAChR
expression, baseline PA is maintained even after transgene expression
is abolished. These data suggest that high-affinity nAChRs expressed on
corticothalamic neurons during development are critical for baseline PA
performance and provide a potential neuroanatomical substrate for
changes induced by prenatal nicotine exposure leading to long-term
behavioral and cognitive deficits.
Key words:
nicotine; learning; transgenic mice; nicotinic
acetylcholine receptors; brain; prenatal
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Introduction |
The comorbidity of prenatal nicotine
exposure with psychiatric disorders and cognitive impairment suggests
that nicotinic acetylcholine receptors (nAChRs) may play a role in the
etiology of these disorders. In utero exposure to nicotine
can increase the risk of attention deficit hyperactivity disorder
(ADHD), lower IQ, and conduct disorder later in life (Ernst et al.,
2001 ; Wakschlag et al., 2002). Determining the nAChR subtypes and the
neuroanatomical loci responsible for these effects is difficult using
traditional pharmacological approaches because of the large
number of nAChR subtypes, their overlapping pharmacological profiles,
and their wide distribution in the brain. The thalamus has been
implicated in learning and memory, attention, arousal, and emotionality
(Masterman and Cummings, 1997; Kalivas et al., 1999). In addition, the
thalamus contains the highest density of high-affinity nAChRs in the
brain (composed predominantly of the  4 and 2 nAChR subunits)
(Zoli et al., 1998), but virtually nothing is known about the function of nAChRs in this region. There are two main neuronal types in thalamus: GABAergic interneurons and glutamatergic thalamocortical relay neurons (Guillery and Sherman, 2002). Glutamatergic sensory projections, glutamatergic projections from layer VI of the
cortex, and cholinergic pedunculopontine projections all synapse onto thalamic neurons (Hallanger et al., 1987). Functional nAChRs are present at several levels of this system on both cell bodies and terminals (Léna and Changeux, 1997; Lu et al., 1998; Corrigall et
al., 1999). The enrichment of high-affinity nAChRs in this region make
it a potential target for developmental changes caused by alterations
in nicotinic receptor function.
Passive avoidance (PA) is a fear-associated learning test sensitive to
manipulations of the cholinergic system and a model for emotional
learning in humans. Cholinergic deafferentation of thalamus in rodents,
as in thiamine-deficient rats with reduced choline-acetyl-transferase
levels in thalamus (and other brain areas), is correlated with
impairments in PA learning (Nakagawasai et al., 2000). Senescence
accelerated mice also have lower levels of acetylcholine in thalamus
and hypothalamus and show impaired PA performance that is rectified by
acute, pretrial administration of nicotine (Meguro et al., 1994). 2
nAChR subunit knock-out (2 ko) mice lack all high-affinity binding
for nicotine and show abnormally high baseline PA and no improvement in
learning as a result of post-training nicotine administration when
compared with wild-type siblings (Picciotto et al., 1995). To identify where in the brain nicotinic receptors exert their actions on PA
learning, transgenic mice with temporally and spatially restricted expression of high-affinity nAChRs were generated and tested in PA.
These mice were used to identify the nAChR subtypes and neuronal populations that appear critical for the long-term behavioral consequences of genetic or pharmacological manipulations of nicotinic neurotransmission.
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Materials and Methods |
Animals. The cDNA encoding the 2 subunit was
generated by reverse transcription (RT)-PCR from mouse brain
mRNA (5', TTT AAG CTT- GCG CGG CTT CAG CAC CAC GGA CAG CGC; 3', TTT ACT
AGT- TCC ACC CAA TAC TAC TGA ACC) and subcloned into pTet-splice
(Invitrogen, Gaithersburg, MD). The plasmid was
sequenced, and the band containing the Tet-2 construct and SV40 3'
untranslated region was excised, purified by gel
electrophoresis, and microinjected into B6SJL oocytes (Yale University
Transgenic Facility). Founder mice carrying the transgene were crossed
with Line A NSE-tTA mice on the ICR background (Chen et al., 1998; Kelz
et al., 1999) and 2 ko mice on the C57BL/6J background (Picciotto et
al., 1995). C57BL/6J mice were obtained from The Jackson
Laboratory (Bar Harbor, ME). Genotyping was performed by PCR on
phenol-extracted tail DNA. Mice were crossed to generate 2 ko or
2 heterozygote (2 het) offspring with at least one copy of the
Tet-2 and NSE-tTA transgenes. Litters of mice on a mixed genetic
background of 2 wild-type (wt), 2 het, and 2 ko genotype with
copies of one or both of the transgenes were used for behavioral
experiments. 2 ko mice with both transgenes (2 tr) expressed the
2 subunit inducibly in different brain regions. 2 tr, 2 wt,
and het mice with or without the transgenes (wild type), and 2 ko
mice with no transgenes or only a single transgene (2 ko) were
compared. Mice were group housed with a maximum of five per cage in a
colony room maintained at 22°C on a 12 hr light/dark cycle, with
lights on at 7:00 A.M. Food and water were available ad
libitum. All animal procedures were in strict accordance with
NIH Care and Use of Laboratory Animals Guidelines and
were approved by the Yale Animal Care and Use Committee.
PA learning. Testing was performed in a mouse PA chamber
(Ugo Basile, Comerio, Italy). On day 1, the mouse was
placed in the light chamber and allowed to move freely between the two
compartments for 5 min. On day 2, the mouse was placed in the light
chamber, and latency to enter the dark chamber was measured.
After entry into the dark chamber, the door between compartments
closed, and a 2 sec electric shock (0.2 mA) was administered through
the grid floor. The animal was removed from the apparatus, given an
intraperitoneal injection (10 µl/gm) of 0.9% saline or 10 µg/kg
nicotine bitartrate (concentration calculated as free base), and
returned to a holding cage. On day 3, mice were again placed in the
light chamber, and time to enter the dark chamber was recorded. Mice
used in the initial passive avoidance experiment were aged from 2 to 9 months. No differences in PA behavior were seen with age. Another
cohort of mice (aged 4-12 months at testing) was divided into two
groups (balanced by genotype, sex, and age), and half were given
doxycycline (dox) in their drinking water (100 µg/ml) for 5 to 7 weeks before PA testing.
Shock reactivity test. Shock reactivity was measured as
described previously (Caldarone et al., 2000). Mice were given a series of 1 sec shocks starting at 0.05 mA and increasing to 1 mA in increments of 0.05 mA, with a 19 sec intershock interval. Mice were
scored by two observers for flinch (any observable reaction to the
shock), run, jump, or vocalization reactions. For each animal, the
experiment was stopped when the mouse had displayed all four reactions.
Mean current thresholds to evoke each response were calculated and
averaged between observers.
In situ hybridization and equilibrium binding. Mice were
decapitated, and brains were removed, frozen on dry ice, and stored at
 80°C. Sections (12 µm) were cut at the cryostat, thaw mounted onto chrom-alum-coated slides [0.5% chromium (III) phosphate-0.5% gelatin] for radioligand binding or charged slides (Fisherbrand Superfrost/Plus; Fisher Scientific, Pittsburgh, PA) for
in situ hybridization. Sections were dried at room
temperature for 20 min and stored at 80°C. The large intracellular
loop of the 2 subunit (amino acids 338-456) was subcloned into
pBluescript. Antisense and sense cRNA probes were transcribed with T7
or SP6 RNA polymerase in the presence of
[35S]UTP (NEN, Boston, MA)
and purified through RNA mini-quick-spin columns (Roche,
Indianapolis, IN). Brain sections were postfixed in 4%
paraformaldehyde in 1× PBS (in mM: 1 KH2PO4, 10 Na2HPO4, 1.37 NaCl, and 2.7 KCl, pH 7.4), acetylated for 15 min in 0.1 M
tetraethylammonium and 0.25% acetic anhydride, and dehydrated through
an ethanol series. Slides were hybridized overnight at 60°C
(106 cpm per slide), washed in 2× SSC
(0.3 M sodium acetate and 0.03 M sodium citrate), treated with 20 µg/ml RNaseA
(Roche), washed in descending concentrations of SSC (to
0.1×) at 55°C, rinsed with water, dried, and exposed to
3H-Hyperfilm (Amersham
Biosciences, Arlington Heights, IL) for 15 d.
For radioligand binding, sections were thawed at room temperature and
incubated with 200 pM
[125I]A85380 for 30 min in 50 mM Tris-HCl, pH7.4, washed twice in the same buffer, dried,
and exposed to 3H-Hyperfilm for 2-7 d.
Tissue preparation and rubidium efflux. Mice were
killed, and brain regions were dissected on ice. Samples were
homogenized in cold isotonic sucrose (0.32 M
sucrose with 5 mM HEPES, pH 7.5) in a
tissue grinder with 16 strokes by hand. Homogenates were centrifuged at 12,000 × g for 20 min, and pellets were
resuspended in different buffers depending on the following assay.
Rubidium efflux experiments were performed as described previously
(Marks et al., 2002). Briefly, homogenized and centrifuged tissue was resuspended in buffer A (in mM: 140 NaCl, 1.5 KCl, 2 CaCl2, 1 MgSO4, 20 glucose, and 20 HEPES hemisodium, pH 7.5). Aliquots (25 µl) were
incubated with 4 µCi of
[86Rb+] for
30 min at 22°C in a final volume of 35 µl. Uptake was terminated by
filtration of each sample onto a Gelman A/E glass fiber filter under
gentle vacuum and washing once with 0.5 ml of buffer A. The washed
filters were transferred to a polypropylene platform and superfused
with buffer B (135 mM NaCl, 5 mM CsCl, 1.5 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 20 mM glucose, 50 nM
tetrodotoxin, 1 µM atropine, 20 mM HEPES hemisodium, and 0.1% bovine serum
albumin, pH 7.5). Buffer was applied at a rate of 2.5 ml/min and was
actively removed with a second peristaltic pump set for a flow rate of 3.2 ml/min. The buffer was pumped through a 200 µl Cherenkov cell in
-RAM Radioactivity HPLC detector (IN/US Systems, Tampa, FL) to allow
continuous monitoring of radioactivity. Samples were stimulated by
diverting the application buffer through a 200 µl loop containing the
test solution (30 µM nicotine). Stimulation time was 5 sec. Baseline efflux was calculated using points before and
after stimulation. Magnitude of
[86Rb+]
efflux was calculated as stimulation above basal and normalized to
basal efflux to allow comparison among regions and genotypes.
Epibatidine binding. Epibatidine binding was performed as
described previously (Marks et al., 2002). Samples were incubated in a
96-well polystyrene plate for 2 hr at 22°C. Final incubation was in
30 µl of binding buffer. Binding reactions (200 pM [125I]
epibatidine) were terminated by filtration onto Gelman A/E glass fiber
paper that had been treated with 0.5% polyethylenimine. Samples were
washed six times with cold load buffer. Bound ligand was quantified
using a Packard Cobra Gamma Counter. Counting efficiency was 85%.
Blanks were determined with 100 µM nicotine,
and all results presented are specific binding.
GABA release. [3H]GABA
release from synaptosomes was performed as described previously (Lu et
al., 1998). Homogenates from cortex, thalamus, hippocampus, and
striatum were centrifuged at 12,000 × g for 20 min.
The resulting crude synaptosomal pellet was resuspended in uptake
buffer (in mM: 128 NaCl, 2.4 KCl, 1.2 KH2PO4, 1.2 MgSO4, 3.2 CaCl2, 25 HEPES,
and 10 glucose, pH 7.5) and incubated with 1 mM
aminooxyacetic acid at 37°C for 10 min. [3H]GABA (0.1 µM) and unlabeled GABA (0.25 µM) were added and incubated 10 min at 37°C.
Aliquots were collected onto glass fiber filters and washed with 0.5 ml
of perfusion buffer (119 mM NaCl, 3.6 mM KCl, 1.2 mM
MgSO4, 10 mM CsCl, 3.2 mM CaCl2, 25 mM HEPES, 10 mM glucose,
and 0.1% BSA, pH 7.5). Perfusion buffer was pumped over synaptosomes
at 1.8 ml/min (RT). Release was stimulated by a 12 sec exposure to 30 µM L-nicotine. Data (as
counts per minute released per fraction) were corrected for baseline
release from fractions collected before and after the agonist
stimulation. Data were then normalized to baseline, and fractions in
which release exceeded baseline by 10% or more were summed. Units of
release are as fraction of baseline.
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Results |
Inducible, region-specific expression of 2
subunit-containing nAChRs
Transgenic mice were generated that express the 2 subunit of
the nAChR under the control of a tetracycline-regulated promoter (TetOp-2). Founder mice were crossed with 2 ko mice carrying a
transgene encoding the neuron-specific enolase promoter driving expression of the tetracycline transactivator (NSE-tTA). In 2 tr
mice, all 2 subunit-containing nAChRs in the brain were expressed from the tetracyline-regulatable transgene. Equilibrium binding using
the nicotinic agonist [125I]epibatidine
in brain slices identified three TetOp-2 lines of mice with
increased densities of high-affinity binding sites (data not shown).
Region-specific patterns of binding were similar in all three lines,
and one line was picked for further characterization. This line was
crossed to three different NSE-tTA founder lines (Chen et al., 1998;
Kelz et al., 1999). Equilibrium binding studies using
[125I]A85380, a nicotinic ligand
specific to 2-containing nAChRs (Mukhin et al., 2000), showed
distinct patterns of expression in each of the NSE-tTA lines (Fig.
1). High-affinity nicotinic binding in
2 tr(CT) mice was restricted to cortex and thalamus. 2 tr(VN)
mice expressed high-affinity nAChRs in the optic tract, visual nuclei
of the thalamus, and superior colliculus. 2 tr(VTA) mice showed
high-affinity nicotinic binding predominantly in nucleus accumbens,
substantia nigra, ventral tegmental area, and striatum. In contrast,
2 het siblings of the 2 tr mice showed A85380 binding throughout
the brain, and 2 ko mice had no detectable binding. As a result of
the enrichment of nicotine binding in thalamus and cortex in the 2
tr(CT) transgenic mice, this line was selected for further
characterization in neurochemical and behavioral paradigms.
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Figure 1.
Transgenic mice with region-specific expression of
2 subunit-containing nAChRs. Mice from three transgenic lines
carrying the NSE promoter driving expression of the tetracycline
transactivator (NSE-tTA) (Chen et al., 1998; Kelz et al., 1999) were
crossed with mice carrying the TetOp promoter driving the 2 subunit
(TetOp-2) on a 2 ko background. [125I]A85380
binding is shown in 2 het, 2 ko, and three lines of 2 tr mice
(CT, VN, and VTA). Bregma coordinates are shown for the four levels
(Paxinos and Franklin, 1997).
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Inducible expression in 2 tr(CT) mice is restricted to
corticothalamic projection neurons
Nicotinic ligand binding was performed on membranes isolated from
several brain regions to provide a quantitative measure of
high-affinity nAChRs expressed in 2 tr(CT) mice (Fig.
2A). There was a clear
effect of 2 subunit gene dosage on epibatidine binding in most brain
areas across 2 wt, het, and ko mice, as has been reported previously
(Marks et al., 1999); however, 2 tr(CT) mice showed significant
epibatidine binding in the cortex and thalamus only. In situ
hybridization was performed using the intracellular loop region of the
2 subunit as a probe in 2 tr(CT) mice. 2 subunit mRNA was
detected specifically in layer VI of the cerebral cortex, CA1, and
dentate gyrus of the hippocampal formation and throughout the dorsal
striatum, but no specific hybridization was seen in the thalamus (Fig.
2B). This pattern is similar to that seen for other
genes of interest driven by the same NSE-tTA line (Chen et al., 1998;
Kelz et al., 1999). Ectopic expression of the 2 subunit transgene in
cells not normally expressing the heteromeric nAChRs does not result in
the production of assembled nAChRs or high-affinity nicotinic
radioligand binding because the 2 subunit can only form a functional
channel in the presence of its partner subunits (Luetje and Patrick,
1991). The equilibrium binding data coupled with the in situ
hybridization studies suggest that 2 tr(CT) mice express functional
high-affinity nAChRs only in corticothalamic projection neurons.
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Figure 2.
2 subunit-containing nAChRs are expressed in
the cortex and thalamus of 2 tr(CT) mice. A,
Quantitiative epibatidine binding in microdissected brain regions of
2 wt, het, ko, and 2 tr(CT) transgenic mice
(n = 3 for each genotype). ANOVA showed a
significant effect of genotype on binding (p < 0.0001). Post hoc Newman-Keuls tests showed the 2
wt and 2 het mice to be different from each other and all other
genotypes for all brain areas (p < 0.05).
The 2 tr(CT) mice had greater binding than 2 ko mice in the
cortex and thalamus (p < 0.05). Binding in
the hippocampus (Hipp.) and striatum did not differ between 2 tr(CT)
and 2 ko mice (p > 0.05).
B, In situ hybridization with an
antisense riboprobe against the large intracellular loop region of the
2 subunit at the level of the thalamus (bregma +1.10 mm) and
prefrontal cortex (bregma 1.70 mm). Arrows point to the thalamus and
layer VI of the cortex.
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Functional characterization of 2 nAChRs in
corticothalamic terminals
To confirm that nicotinic binding in the thalamus represents
functional, terminal nAChRs and to identify the neurotransmitter released from these neurons, synaptosomal fractions were collected from
several brain areas and were assayed for rubidium
[86Rb] efflux (Fig.
3A) and
[3H]GABA release (Fig. 3B).
The [86Rb+]
efflux assay measures the opening of functional nicotinic receptor channels in synaptosomes in response to nicotine stimulation (Marks et
al., 1995). The expected gene dosage response in
[86Rb+]
efflux stimulated by nicotine was seen across 2 wt, het, and ko mice
(Fig. 3A) (Marks et al., 1999). A significant increase in
[86Rb+]
efflux was seen in the thalamus of 2 tr(CT) compared with 2 ko
mice. Efflux was similar to that seen in 2 het mice, suggesting that
2 tr(CT) mice express physiologically relevant levels of high-affinity nAChR (Fig. 3A). There was no increase in
[86Rb+]
efflux in cortical synaptosomes from 2 tr(CT) mice above levels seen
in 2 ko mice, demonstrating that nicotinic binding in this brain
region is on cell bodies rather than neuronal terminals. Furthermore,
no release was seen in striatum or hippocampus of 2 tr(CT) mice, as
expected from the lack of nicotinic binding in these brain areas.
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Figure 3.
2 tr(CT) mice have functional expression of
2 subunit-containing nAChRs on thalamic terminals of cortical
projection neurons. A, Effect of genotype on
nicotine-evoked rubidium efflux (30 µM nicotine) from
synaptosomal preparations of microdissected brain regions
(n = 4 for each genotype). ANOVA showed a
significant effect of genotype on [86Rb] efflux in
all brain areas (p < 0.0001). Post
hoc Newman-Keuls tests showed the 2 wt mice to be different
from all other genotypes in all brain areas
(p < 0.05). The 2 tr(CT) mice differed
from 2 ko mice only in the thalamus (p < 0.05). 2 tr(CT) did not differ from 2 het mice in
[86Rb]efflux from thalamic synaptosomes
(p > 0.05). B, Effect of
genotype on nicotine-evoked GABA release. [3H]GABA
release was measured in response to a 12 sec exposure to 30 µM nicotine in four brain regions (n = 4 for each genotype). ANOVAs of each brain area showed a significant
genotype effect on nicotine evoked [3H]GABA
release in all brain areas (p < 0.0001).
Post hoc Newman-Keuls tests showed the 2 wt and 2
het mice to be different from all other genotypes in all brain areas
(p < 0.05). There was no increase in
[3H]GABA release in the 2 tr(CT) compared with
2 ko mice in any brain area (p > 0.05).
No significant differences were seen for baseline release for any
genotype in any brain area. Hipp., Hippocampus.
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Both GABAergic and glutamatergic terminals in the thalamus express
high-affinity nAChRs in wild-type mice. We therefore measured [3H]GABA release from synaptosomes in
response to treatment with 30 µM nicotine to identify the
neuronal subtypes expressing functional high-affinity nAChRs in 2
tr(CT) mice. As expected, 2 wt and het mice showed increased
[3H]GABA release in response to nicotine
treatment compared with 2 ko mice (Lu et al., 1998). In contrast,
2 tr(CT) mice show no increased GABA release over the level seen in
2 ko mice in any of the brain areas tested (Fig. 3B).
This implies that these transgenic mice do not express high-affinity
nAChRs in GABAergic neurons. Thus, nicotinic binding, in
situ hybridization, and release data together suggest that 2
tr(CT) mice express high-affinity nAChRs in glutamatergic, layer VI
cortical neurons projecting to the thalamus that are functional and can
regulate neurotransmitter release from synaptic terminals (Turner and
Salt, 1998).
Corticothalamic 2 nAChR expression restores baseline
PA behavior
We tested PA behavior in 2 tr(CT) mice and compared these mice
with sibling controls of different genotypes (Fig.
4). In contrast to 2 ko mice, which
showed hypersensitive PA learning compared with control siblings
(Picciotto et al., 1995), 2 tr(CT) mice had PA performance similar
to wild-type mice and had a significantly shorter latency to enter the
dark chamber than 2 ko mice (Fig. 4). Thus, restricted expression of
2 subunit-containing receptors to corticothalamic efferents is
sufficient for normal PA behavior.
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Figure 4.
Expression of 2 subunit-containing receptors in
corticothalamic terminals is sufficient for normal baseline PA
performance. PA performance was evaluated in wild-type (2 het and
2 wt), 2 ko, and 2 tr(CT) mice [wild type,
n = 17; 2 ko, n = 21; 2
tr(CT), n = 29]. A repeated-measures ANOVA of the
baseline PA performance with day (testing vs training) as the
within-subject variable and genotype [wild type, 2 ko, and 2
tr(CT)] as the between-subject variable revealed an interaction of day
and genotype (F(2,64) = 4.311;
p < 0.05). Post hoc Newman-Keuls
tests showed the 2 ko mice to have a longer latency to enter the
dark compartment on testing than the wild-type and 2 tr(CT) mice
(p < 0.05).
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We showed previously that nicotine is ineffective in improving PA
performance in 2 ko mice (Picciotto et al., 1995). In the current
study, although nicotine was able to enhance PA performance in
wild-type mice [saline, 23.7 ± 5.2 sec (n = 17);
nicotine, 56.4 ± 16.8 sec (n = 9); Newman-Keuls
test; p < 0.05], it had no effect on 2 tr(CT) mice
[saline, 31.8 ± 6.6 sec (n = 29); nicotine,
26.7 ± 1.6 sec (n = 11)]. Thus, expression of
high-affinity nAChRs on corticothalamic neurons rescues baseline
performance, although it is not sufficient to restore nicotine-mediated
enhancement of PA. These data suggest that nAChRs in other brain
regions, such as the amygdala, striatum, or hippocampus, may be
critical for the nicotine-mediated enhancement of PA learning.
The restored baseline PA learning in 2 tr(CT) mice was attributable
to 2 subunit expression rather than transgene insertion, because no
difference in baseline PA behavior was seen in mice carrying either the
NSE-tTA transgene or the TetOp-2 transgene alone (Fig.
5A). The ability of transgenic
expression from a different locus to restore a phenotype altered in the
2 ko mice also confirms that the original phenotype was not
attributable to effects of flanking genes surrounding the mutant allele
(Banbury Conference on Genetic Background in Mice, 1997; Bolivar et
al., 2000). Furthermore, the difference in PA performance in 2
tr(CT) mice cannot be explained by altered pain sensitivity because no
differences were seen in shock reactivity at intensities similar to
those used in PA training (Fig. 5B).
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Figure 5.
The PA phenotype is not attributable to transgene
insertion or alteration of shock sensitivity. A, The
insertion of the individual transgenes on the 2 het or 2 ko
backgrounds does not affect PA performance. 2 het and 2 ko mice
with either the TetOp-2 or NSE-tTA transgene alone were tested for
PA behavior (2 het/TetOp-2, n = 8; 2
het/NSE-tTA, n = 9; 2 ko/TetOp-2,
n = 15; 2 ko/NSE-tTA, n = 12). Repeated-measures ANOVA with day (testing vs training) as the
within-subject variable and genotype (2 het vs 2 ko) and
transgene (TetOp-2 vs NSE-tTA) as between-subject variables showed
an interaction of day and genotype
(F(1,40) = 9.015; p < 0.01) but no main effect or interaction with single transgenes.
B, The altered PA phenotype is not a result of
differential shock reactivity. Mice of different genotypes [2 wt
and 2 het, 2 ko and 2 tr(CT)] were tested for response to
increasing levels of shock. (2 wt and 2 het were combined as
"wild types" for subsequent analysis). Mean lowest shock that
evoked the responses flinch, run, and vocalize for each of the three
genotypes (n = 7 per genotype) is plotted. ANOVA
showed no effect of genotype (p > 0.05 in
all cases).
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Developmental expression of 2 nAChRs is critical for
baseline PA
One possible role for high-affinity nAChRs in thalamus could be to
modulate sensory processing through thalamus to cortex by altering
synaptic strength in glutamatergic neurons during development. To
determine whether high-affinity nAChRs act acutely to regulate PA
behavior in the adult or whether the receptors are critical earlier in
development, adult mice were administered 100 µg/ml dox in their
drinking water for 30 d to abolish expression of
high-affinity nAChRs in 2 tr(CT) mice (Fig.
6A). dox administration had no effect on PA performance of mice of any genotype (Fig. 6B). These data suggest that expression of 2
subunit-containing nAChRs in corticothalamic efferents during
development results in a long-lasting alteration of this pathway that
facilitates PA learning in the adult.
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Figure 6.
Expression of the 2 subunit during development
is necessary for normal PA learning. A, 2 het, 2
ko, and 2 tr(CT) mice were treated with dox (100 µg/ml) in their
drinking water for 4 weeks. No change was seen in
[125I]A85380 binding in 2 wt (data not shown)
or 2 het mice after dox treatment, but no binding was detectable in
2 tr(CT) mice administered dox. B, Four weeks of dox
treatment in adult mice does not alter PA performance in 2 tr(CT)
mice [2 het, no dox, n = 4; 2 het
with dox, n = 6; 2 ko, no dox,
n = 4, 2 ko with dox, n = 9;
2 tr(CT), no dox, n = 10; 2 tr(CT) with dox,
n = 11]. A repeated-measures ANOVA with day
(training vs testing) as the within-subject variable and dox treatment
(on or off) and genotype [2 het, 2 ko, and 2 tr(CT)] as the
between-subject variables showed an interaction of day and genotype
(F(2,39) = 13.382;
p < 0.01) but no main effect or interaction with
dox.
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Discussion |
We generated transgenic mice with inducible, region-specific
expression of 2 subunit-containing nAChRs in corticothalamic projection neurons. Using these mice, we showed that alterations in
neurotransmission through nAChRs during development can affect PA
performance in the adult. Nicotine treatment during development is
known to have neurodevelopmental and behavioral consequences. In
humans, maternal smoking has been associated with babies of lower birth
weight, increased risk of sudden infant death syndrome, and increased
risk of psychiatric disorders in childhood and adolescence (Ernst et
al., 2001), including ADHD (Milberger et al., 1996; Milberger et al.,
1998). Cognitive impairments and hyperactivity have also been reported
in animals exposed to nicotine during development (Genedani et al.,
1983; Sorenson et al., 1991; Pennington et al., 1994; Shacka et al.,
1997). A blunting of both cholinergic and catecholaminergic function is
also seen in rats prenatally exposed to nicotine (Navarro et al., 1988,
1989; Oliff and Gallardo, 1999). Thus, expression of nAChRs during
prenatal and postnatal development is likely to be critical for normal
cognitive behavior in the adult.
The first 2 weeks of life are a critical period for maturation of
glutamatergic synapses on corticothalamic neurons, with targeting of
glutamate receptor subunits such as mGluR1a to the distal dendrites of
these synapses during the second postnatal week of rat development (Liu
et al., 1998). Chronic nicotine treatment at this time has been shown
to enhance glutamatergic (NMDA receptor-mediated) transmission in the
neocortex of rats (Aramakis and Metherate, 1998), resulting in a
subsequent increase in NMDA receptor subunit NR2A mRNA expression in
layer VI of the auditory cortex that lasts for 2 weeks (Hsieh et al.,
2002). These data suggest that expression of high-affinity nAChRs
during this period of development can affect maturation of
glutamatergic synapses. In rat, the NSE promoter drives expression
early in the development of layer VI neurons and remains on throughout
development, with a dip on postnatal day 10, before rising again to
adult levels by day 20 (Hamre et al., 1989). Our data suggest that the
presence of 2 subunit-containing nAChRs during these critical
periods of synaptogenesis is essential for the long-term modulation of
PA performance.
The nicotine-induced enhancement of PA learning in adult mice is not
mediated by nAChRs on corticothalamic efferents, however. We
hypothesize that nAChRs in other brain regions are critical for the
effect of nicotine on PA learning in adulthood, and potential brain
regions that could be explored include the amygdala (Riekkinen et al.,
1993), striatum (Sandberg et al., 1984; Giordano et al., 1998), or
hippocampus (Pope et al., 1985; Bailey et al., 1986), all of which have
been identified as critical sites for plastic changes associated with
consolidation of PA learning (McGaugh et al., 1996).
High-affinity nAChRs are normally expressed throughout the brain and
are likely to affect the development and function of synaptic activity
in many brain areas. Mutations in the 4 and 2 subunits of the
nAChR in several human families result in autosomal dominant nocturnal
frontal lobe epilepsies (Sutor and Zolles, 2001), which have been
associated with an increased probability of learning and developmental
disorders (Gunduz et al., 1999; Verrotti et al., 2000). It should be
noted, however, that it is unclear whether the cognitive impairment in
these patients results directly from altered nAChR activity during
development or indirectly from repeated seizures, which are known to
cause cognitive impairment. We showed that mutation of the 2 subunit
in mice results in abnormal sensitivity to PA learning and have
identified corticothalamic efferents as the anatomical locus for this
effect. Abnormalities in nAChRs in human subjects may also result in
developmental changes in corticothalamic signaling that could affect
emotional learning. Prenatal exposure to nicotine in humans may
desensitize and functionally downregulate high-affinity nAChRs, as is
seen with chronic nicotine exposure in mice (Marks et al., 1993;
Zachariou et al., 2001). Thus, chronic exposure to nicotine during
development could alter nicotinic neurotransmission in corticothalamic
circuits and lead to inappropriate emotional learning during adulthood.
| |
FOOTNOTES |
Received Dec. 9, 2002; revised Jan. 29, 2003; accepted Feb. 12, 2003.
This work was supported by National Institute on Drug Abuse Grants
DA00436, DA14241, DA10455, and DA84733 (M.R.P.) and DA03194 and DA00197
(A.C.C). We thank Dr. Eric Nestler for the generous gift of NSE-tTA
mice and valuable input on many aspects of this study. We thank Drs.
Ronald Duman and Angus Nairn for reading this manuscript and for
helpful conversations about this work. We thank Drs. Max Kelz and
Jingshan Chen for providing information and primers for characterizing
NSE-tTA transgenic mice.
Correspondence should be addressed to Marina R. Picciotto, Department
of Psychiatry, Yale University School of Medicine, 34 Park Street,
Third Floor Research, New Haven, CT 06508. E-mail: marina.picciotto{at}yale.edu.
| |
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TOP
ABSTRACT
Introduction
Gene
