From the Department of Physiology and Biophysics, State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, New York 14214
Received for publication, March 19, 2003 , and in revised form, April 10, 2003.
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ABSTRACT |
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INTRODUCTION |
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Prefrontal cortex (PFC), one of the major target areas of basal forebrain cholinergic neurons, has long been associated with high-level, "executive" processes (12), particularly a form of short term information storage described as "working memory" (13). The cholinergic activity in frontal cortex is persistently increased in mice performing a working memory task (14). All the five subtypes of mAChRs, m1m5 (15), are expressed in cortical pyramidal neurons (1618). One of the important questions yet to be answered is the targets of muscarinic signaling that are involved in cognition and memory. Recent studies show that GABAergic inhibition in frontal cortex controls the timing of neuronal activities during cognitive processes, therefore, shaping the flow of information in cortical circuits (19). The critical involvement of cortical muscarinic signaling in cognition and AD, combined with the central role of GABAergic inhibition in working memory, prompts us to hypothesize that the GABA system might be a key cellular substrate for muscarinic signaling in cognition and memory, and its dysregulation by mAChRs in AD might contribute to the cognitive impairment.
Emerging evidence suggests that low concentrations of A peptides can
potently inhibit various cholinergic neurotransmitter functions independently
of concurrent neurotoxicity
(20). A
peptides are
produced by proteolytic cleavage of the
-amyloid precursor protein (APP)
(21). Most of the mutations in
the APP gene are clustered around the cleavage sites, which increases the rate
of cleavage, thereby generating more A
(22,
23). Transgenic mice
overexpressing mutant APP genes exhibit AD-like symptoms, including increased
A
deposits and deficits in spatial learning and memory
(2426).
In this study, we used this AD model to examine the muscarinic regulation of
GABAergic synaptic transmission in PFC pyramidal projection neurons.
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EXPERIMENTAL PROCEDURES |
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Electrophysiological Recordings in SlicesYoung adult rat or mouse slices containing PFC were prepared as described previously (27, 28). All experiments were carried out with the approval of State University of New York at Buffalo Animal Care Committee. In brief, animals were anesthetized by inhaling 2-bromo-2-chloro-1,1,1-trifluoroethane (1 ml/100 g, Sigma) and decapitated, and brains were quickly removed, iced, and then blocked for slicing. The blocked tissue was cut in 300400-µm slices with a Vibrotome while bathed in a low Ca2+ (100 µM), HEPES-buffered salt solution (in mM: 140 sodium isethionate, 2 KCl, 4 MgCl2, 0.1 CaCl2, 23 glucose, 15 HEPES, 1 kynurenic acid, pH = 7.4, 300305 mosM/liter). Slices were then incubated for 16 h at room temperature (2022 °C) in a NaHCO3-buffered saline bubbled with 95% O2, 5% CO2 (in mM): 126 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, 10 glucose, 1 pyruvic acid, 0.05 glutathione, 0.1 NG-nitro-L-arginine, 1 kynurenic acid, pH = 7.4, 300305 mosM/liter.
To evaluate the regulation of spontaneous IPSCs by muscarinic receptors in
PFC slices, the whole-cell patch technique was used for voltage-clamp
recordings using patch electrodes (59 M) filled with the
following internal solution (in mM): 100 CsCl, 30
N-methyl-D-glucamine, 10 HEPES, 1 MgCl2, 4
NaCl, 5 EGTA, 12 phosphocreatine, 2 MgATP, 0.2 Na3GTP, 0.1
leupeptin, pH = 7.27.3, 265270 mosM/liter. The slice
(300 µm) was placed in a perfusion chamber attached to the fixed-stage of
an upright microscope (Olympus) and submerged in continuously flowing
oxygenated artificial cerebrospinal fluid. For blocking glutamate
transmission, the
-amino-3-hydroxy-5-methyl-4-isoxazole proprionic
acid/kainate (AMPA/KA) receptor antagonist
6-cyano-7-nitroquinoxaline-2,3-dione (10 µM) and
N-methyl-D-aspartate receptor antagonist
D()-2-amino-5-phosphonopetanoic acid (25 µM)
were added to the recording solution. Cells were visualized with a 40x
water-immersion lens and illuminated with near infrared (IR) light, and the
image was detected with an IR-sensitive CCD camera. A Multiclamp 700A
amplifier (Axon instruments, Union City, CA) was used for these recordings.
Tight seals (210 G
) from visualized pyramidal neurons were
obtained by applying negative pressure. The membrane was disrupted with
additional suction and the whole-cell configuration was obtained. The access
resistances ranged from 13 to 18 M
and were compensated 5070%.
Cells were held at 70 mV for the recording of spontaneous IPSCs. To
examine the muscarinic regulation of intrinsic firing patterns, current-clamp
recordings were performed using patch electrodes filled with the internal
solution (in mM): 60 K2SO4, 60
N-methyl-D-glucamine, 40 HEPES, 4 MgCl2, 5
BAPTA, 12 phosphocreatine, 2 Na2ATP, 0.2 Na3GTP, 0.1
leupeptin, pH = 7.27.3, 265270 mosM/liter. The
resting membrane potential of the neurons was 61.4 ± 0.96 mV
(wild-type, n = 10) and 61.1 ± 1.02 mV (APP transgenic:
n = 9) in control Ringer's solution, and 59.3 ± 0.65 mV
(wild-type, n = 10) and 59.6 ± 0.73 mV (APP transgenic:
n = 9) in the presence of carbachol (10 µM).
Glutamatergic and GABAergic transmission was blocked to ensure that the
phenomena studied were independent of synaptic transmission. All experiments
were performed side by side with cells from wild-type versus APP
transgenic mice or non-treated versus A
-treated slices.
Mini Analysis Program (Synaptosoft, Leonia, NJ) was used to analyze synaptic activity. Individual synaptic events with fast onset and exponential decay kinetics were captured with threshold detectors in Mini Analysis software. All quantitative measurements were taken 46 min after drug application. IPSCs of 60 s (2001000 events) under each different treatment were used for obtaining the cumulative distribution plots. The detection parameters for analyzing synaptic events in each cell in the absence or presence of carbachol were the same. Statistical comparisons of the frequency and amplitude of synaptic currents were made using the Kolmogorov-Smirnov (K-S) test. Numerical values were expressed as mean ± S.E.
Muscarinic receptor ligand carbachol (CCh), atropine and pirenzepine
(Sigma), as well as second messenger reagents calphostin C,
bisindolylmaleimide I (i.e. GF109203X; Gö6850), and
myristoylated PKI-(1422) (Calbiochem) were made up as concentrated
stocks in water and stored at 20 °C. Stocks were thawed and diluted
immediately prior to use. The -amyloid peptide
A
2535 and the control peptide containing the reverse
sequence A
3525 were obtained from Sigma. These
peptides were resuspended in sterile distilled water at a concentration of 2
mM and incubated at 37 °C for 1 h to allow fibril
formation.
Western Blot AnalysisFor detecting activated PKC, a
phospho-PKC (pan) antibody that recognizes PKC,
I,
II,
,
, and
isoforms only when
phosphorylated at a carboxyl-terminal residue homologous to Ser660
of PKC
II was used in the Western blot analysis. After
incubation, slices were transferred to boiling 1% SDS and homogenized
immediately. Insoluble material was removed by centrifugation (13,000 x
g for 10 min), and protein concentration for each sample was
measured. Equal amounts of protein from slice homogenates were separated on
7.5% acrylamide gels and transferred to nitrocellulose membranes. The blots
were blocked with 5% nonfat dry milk for1hat room temperature. Then the blots
were incubated with the phospho-PKC (pan) antibody (Cell Signaling, 1:2000)
for1hat room temperature. After being rinsed, the blots were incubated with
horseradish peroxidase-conjugated anti-rabbit antibodies (Amersham
Biosciences, 1:2000) for 1 h at room temperature. Following three washes, the
blots were exposed to the enhanced chemiluminescence substrate. Then the blots
were stripped for 1hat50 °C followed by saturation in 5% nonfat dry milk
and incubated with a PKC antibody (Santa Cruz, 1:2000) recognizing the
,
, and
isoforms. Quantitation was obtained from
densitometric measurements of immunoreactive bands on autoradiograms. Data
correspond to the mean ± S.E. of 510 samples per condition and
were analyzed by ANOVA tests.
mRNA DetectionPFCs were dissected from wild-type and APP transgenic mouse brain slices (400 µm) and homogenized in 0.5 ml of TRIzol reagent (Invitrogen). Following 5 min of incubation at room temperature, 0.1 ml of chloroform was added and mixed with the homogenized samples. The tubes were incubated at 25 °C for 23 min and then centrifuged for 15 min at 4 °C. The upper aqueous phase containing RNA for each sample was transferred to a fresh tube. Then RNA was precipitated from the aqueous phase by mixing with 0.25 ml of isopropyl alcohol, incubating at room temperature for 10 min and then centrifuging for 10 min at 4 °C. The supernatant was removed, and the RNA pellet was washed with 75% ethanol. The RNA pellet was air-dried and then dissolved in diethyl pyrocarbonate-treated water.
Prior to reverse transcription-polymerase chain reaction (RT-PCR), the isolated RNA was treated with DNase I (Invitrogen) to eliminate genomic DNA. The reaction mixture (10 µl) contained 1 µg of RNA, 1 µl of 10x DNase I reaction buffer, 1 µl of DNase I (1 unit/µl), and 8 µl of diethyl pyrocarbonate-treated water. The tube was incubated at room temperature for 15 min. The reaction was terminated by adding 1 µl of 25 mM EDTA and heating for 10 min at 65 °C. RT-PCR analysis of muscarinic receptor cDNAs was performed as described previously (29, 30). PCR products were separated by electrophoresis in a 1.5% agarose gel stained with ethidium bromide. As a control for genomic contamination, samples were prepared as described above except that the RT was omitted in the reverse transcription procedure.
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RESULTS |
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We next examined whether the muscarinic modulation of GABAergic inhibitory
transmission is altered in the AD model. Compared with wild-type mice, APP
transgenic mice exhibited significantly higher (2030-fold) levels
of A
peptides at 2 months of age, even though no amyloid plaques,
neuronal death, or cognitive deficit were observed at the early stage
(25). Amyloid deposits were
found in frontal cortex, along with other brain regions, in aged APP
transgenic mice (25),
suggesting that the elevated A
expression is present in frontal cortical
neurons at the presymptomatic period. We first compared the basal properties
of sIPSCs in wild-type versus APP transgenic mice. No significant
difference was found between the two groups (mean amplitude: WT, 36.2 ±
3.7 pA, n = 18; APP transgenic, 34.4 ± 2.9 pA, n =
29, p > 0.05, ANOVA; mean frequency: WT, 4.2 ± 0.6 Hz,
n = 18; APP transgenic, 3.8 ± 0.4 Hz, n = 29,
p > 0.05, ANOVA). The lack of changes on the basal GABAergic
transmission in APP transgenic mice suggests that PFC GABAergic interneurons
are not lost or significantly impaired. We then examined the effect of
carbachol on sIPSCs in APP transgenic mice. As shown in
Fig. 1, DF,
bath application of carbachol (20 µM) failed to increase the
sIPSC amplitude in the mutant cell, but the carbachol-induced enhancement of
sIPSC frequency was intact. In a sample of PFC pyramidal neurons from APP
transgenic mice, carbachol caused little change in the mean amplitude of
sIPSCs (7.16 ± 3.2%, n = 29, p > 0.05, K-S test),
but still significantly increased the mean frequency of sIPSCs (198.6 ±
27.0%, n = 29, p < 0.001, K-S test). The effects of
carbachol on the sIPSC amplitude and frequency in PFC neurons from wild-type
versus APP transgenic mice are summarized in
Fig. 1G. It is evident
that muscarinic modulation of the sIPSC amplitude, but not the sIPSC
frequency, was significantly (p < 0.001, ANOVA) impaired in APP
transgenic mice.
Muscarinic Modulation of the sIPSC Amplitude Is Eliminated in Rat PFC
Slices Pretreated with the -Amyloid PeptideWe then
examined whether the altered muscarinic modulation of GABA transmission in APP
transgenic mice is attributable to the elevated
-amyloid protein
levels at an early age (25).
To do so, we treated rat PFC slices with
-amyloid peptides
(A
) before examining carbachol effects on sIPSCs.
A
2535, which represents the biologically active region
of A
(32,
33), was aged to produce
aggregated A
2535. In non-treated rat slices, bath
application of carbachol caused a reversible increase in the amplitude and
frequency of sIPSCs (Fig. 2,
AC), similar to what was found in wild-type mice.
However, in A
2535-treated slices, carbachol failed to
increase the sIPSC amplitude, but still induced a potent enhancement of the
sIPSC frequency. A representative example from an
A
2535-treated neuron is shown in
Fig. 2, DF.
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To confirm the specificity of the action of A2535,
its control peptide containing the reverse sequence
A
3525 was used to pretreat PFC slices. Similar to
non-treated slices, in A
3525-treated slices, bath
application of carbachol induced a reversible increase in the sIPSC amplitude
(Fig. 2G). As
summarized in Fig. 2H,
in A
2535-treated pyramidal neurons, carbachol caused
little change in the mean amplitude of sIPSCs (4.27 ± 2.6%, n
= 17, p > 0.05, K-S test), which was significantly (p
< 0.001, ANOVA) different from the carbachol effect on the sIPSC amplitude
in non-treated neurons (78.6 ± 10.5%, n = 35, p <
0.001, K-S test) or neurons pretreated with the control peptide
A
3525 (77.4 ± 12.5%, n = 5, p
< 0.001, K-S test). However, the carbachol-induced increase in the mean
frequency of sIPSCs in A
2535-treated neurons (221.5
± 31.4%, n = 17, p < 0.001, K-S test) was similar
to the carbachol effect in non-treated neurons (245.4 ± 32.4%,
n = 35, p < 0.001, K-S test) or
A
3525-treated neurons (231.7 ± 38.3%,
n = 5, p < 0.001, K-S test). Despite the ability of
A
2535 to alter the muscarinic regulation of sIPSCs,
A
2535 itself had little direct effect on the sIPSC
amplitude (5.3 ± 2.1%, n = 10, p > 0.05, K-S test)
and frequency (8.9 ± 3.1%, n = 10, p > 0.05, K-S
test).
In prefrontal cortex, serotonin, by activating 5-HT2 receptors,
can also potentiate GABA transmission
(34). To test whether
-amyloid impairs the actions of 5-HT2 receptors, we examined
the serotonergic regulation of sIPSCs in A
2535-treated
PFC slices. Application of serotonin (20 µM) caused a potent
increase in the mean amplitude and frequency of sIPSCs in
A
2535-treated PFC pyramidal neurons (amplitude: 80.7
± 14.4% (Fig.
2I); frequency: 387.4 ± 75.0%, n = 6,
p < 0.001, K-S test), which was not significantly different from
the serotonin effect on sIPSCs in non-treated neurons (amplitude: 85.8
± 16.8% (Fig.
2I); frequency: 392.3 ± 65.6%, n = 10,
p < 0.001, K-S test), suggesting the lack of A
effect on
serotonin functions. Taken together, these results indicate that
-amyloid selectively alters the muscarinic regulation of GABA
transmission.
Muscarinic Modulation of GABA Transmission Is through a PKC-dependent MechanismTo find out the potential reason for the impairment of muscarinic modulation of GABA transmission in PFC from APP transgenic mice, we first examined the cellular mechanisms underlying the modulation of GABA transmission by mAChRs. It is known that activation of m1 receptors stimulates the hydrolysis of membrane phosphoinositol lipids, leading to PKC activation, while activation of m4 receptors inhibits adenylyl cyclase. To test whether the muscarinic modulation of GABA transmission is through the m1-activated PKC, we tested the effect of carbachol on IPSCs when PKC activation was blocked.
We first preincubated rat PFC slices with the cell-permeable and specific PKC inhibitor calphostin C (1 µM) for 1 h, followed by the examination of carbachol effects on sIPSCs. As shown in Fig. 3, A and B, carbachol failed to enhance sIPSC amplitudes in the calphostin C-treated slice. The carbachol enhancement of sIPSC frequencies was not significantly affected by calphostin C (Fig. 3C). Another potent and selective PKC inhibitor, bisindolylmaleimide (1 µM), gave similar results as calphostin C, eliminating the carbachol enhancement of sIPSC amplitudes (data not shown). To confirm the specific involvement of m1/PKC in the muscarinic regulation, PFC slices were preincubated with the cell-permeable myristoylated PKA inhibitor PKI-(1422) (1 µM) to test the potential role of m4/PKA in this process. As shown in Fig. 3, DF, carbachol still induced a potent increase in sIPSC amplitudes and frequencies in the PKI-(1422)-treated slice, similar to what was obtained in the non-treated slice (Fig. 2, AC), indicating that PKA inhibition did not affect the muscarinic regulation of GABA transmission. As summarized in Fig. 3G, the carbachol effect on sIPSC amplitudes in the presence of PKC inhibitor calphostin C (21.6 ± 5.8%, n = 6, p > 0.05, K-S test) or bisindolylmaleimide (19.6 ± 4.3%, n = 7, p > 0.05, K-S test) was significantly (p < 0.001, ANOVA) different from that in the presence of PKA inhibitor PKI-(1422) (76.7 ± 11.3%, n = 5, p < 0.01, K-S test). The carbachol effect on sIPSC frequencies was similar with these treatments (Fig. 3H, calphostin C: 180.4 ± 32.7%, n = 6; bisindolylmaleimide: 200.8 ± 41.7%, n = 7; PKI-(1422): 210.8 ± 38.8%, n = 5). These data suggest that the muscarinic modulation of sIPSC amplitudes (but not frequencies) depends on the activation of PKC.
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Muscarinic Activation of PKC Is Lost in APP Transgenic
MiceGiven the PKC dependence, we speculated that the underlying
mechanism for the loss of muscarinic modulation of GABA transmission in APP
transgenic mice is the impaired muscarinic activation of PKC in these mutants.
To test this, we compared the muscarinic activation of PKC in PFC slices from
wild-type and APP transgenic mice. Because the catalytic competence of many
PKC isozymes depends on autophosphorylation at the carboxyl terminus on a
conserved residue (35), a
phosphospecific pan PKC antibody that detects PKC isoforms only when
phosphorylated at this residue was used for measuring activated PKC. As shown
in Fig. 4, A and
B, carbachol potently increased the activated PKC in
wild-type slices (2.51 ± 0.17-fold, n = 12, p <
0.001, ANOVA), but this effect was almost completely abolished in slices from
APP transgenic mice (1.10 ± 0.06-fold, n = 12, p >
0.05, ANOVA). These results suggest that the mAChR-mediated second messenger
cascade is altered by A, which apparently leads to the loss of
muscarinic modulation of GABAergic signaling.
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To examine whether the impaired PKC activation by carbachol in APP transgenic mice is due to the loss of mAChR expression, we compared the m1m5 mRNA levels in PFC from wild-type versus APP transgenic mice. As shown in Fig. 4C, the expression levels of m1m5 mRNAs detected with RT-PCR were almost identical in PFC tissues from wild-type versus APP transgenic mice. To provide a semiquantative analysis of m1 mRNA, serial dilution experiments were performed (30). As shown in Fig. 4D, using increasing dilutions (1:10 to 1:105) of the cDNAs from wild-type versus APP transgenic mice produced almost the same amount of PCR products of m1, implicating similar abundance of the m1 mRNA in wild-type and APP transgenic mice. These results suggest that the expression of mAChRs is not significantly altered in APP transgenic mice.
Muscarinic Modulation of Intrinsic Firing Is Not Altered in APP
Transgenic MiceSince muscarinic modulation of GABAergic
transmission is impaired in PFC neurons from APP transgenic mice or
A2535-treated rat slices, we would like to know
whether the impairment is due to a selective change in the signaling
mechanisms underlying muscarinic regulation of the GABA system or a general
dysfunction of muscarinic receptors caused by elevated
-amyloid
peptides. To test this, we examined the effects of mAChRs on the intrinsic
firing pattern of pyramidal neurons in PFC slices when glutamatergic and
GABAergic synaptic transmission was blocked.
As found in several neuronal populations
(36,
37), bath application of
carbachol (5 or 10 µM) to PFC slices blocked the slow
afterhyperpolarization that followed a train of action potentials elicited by
current steps, induced a slow after depolarization that gives rise to a
plateau potential accompanied by spiking (n = 10). A representative
example from a wild-type mouse is shown in
Fig. 5A. This
carbachol-induced generation of persistent activity has been proposed to
provide a cellular mechanism for the delayed activity observed during working
memory tasks (38). It is
mediated by m1 activation of a Ca2+-sensitive,
voltage-dependent nonspecific cationic current
(39,
40,
37). In PFC pyramidal neurons
from APP transgenic mice, bath application of carbachol also elicited a
persistent firing (n = 9; a representative example shown in
Fig. 5B), which was
similar to what was found in wild-type mice. Moreover, the ability of
carbachol to trigger sustained spiking activity was intact in PFC neurons from
A2535-treated slices (n = 4, data not
shown).
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We next examined the potential reason for the intact muscarinic regulation
of intrinsic firing in APP transgenic mice. Since the A-caused loss of
muscarinic activation of PKC apparently leads to the impaired muscarinic
regulation of GABA transmission, we tested whether PKC activation is required
for the muscarinic regulation of firing. To do so, we preincubated rat PFC
slices with the cell-permeable and specific PKC inhibitor bisindolylmaleimide
(1 µM), followed by the examination of carbachol effect on the
intrinsic firing pattern. Application of carbachol (10 µM)
triggered sustained spiking activity in bisindolylmaleimide-treated neurons
(n = 7, a representative example shown in
Fig. 5C), which was
almost identical to the carbachol effect in non-treated cells (n = 3,
data not shown). These results show that the muscarinic induction of
persistent firing is through a PKC-independent mechanism, which is not
disrupted in APP transgenic mice. Taken together, these functional studies
suggest that elevated levels of A
are unable to affect muscarinic
receptors per se, at least at the early stage, but are more likely to
impair selective signaling events mediated by muscarinic receptors.
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DISCUSSION |
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Muscarinic receptors could enhance GABAergic synaptic transmission through a presynaptic and a postsynaptic mechanism. In this study, activation of mAChRs with carbachol induced an increase in the frequency of sIPSCs and shifted their amplitude distribution toward larger sizes. Several mechanisms could account for the carbachol effects on GABA transmission. First, mAChRs increase the excitability of GABAergic interneurons, therefore leading to the elevated probability of GABA release. Direct excitation of GABAergic interneurons by activation of cholinergic receptors has been reported in the cortex in vitro (5052, 34). The ionic basis for the excitatory actions of muscarinic receptors on these interneurons is not clear yet. Muscarinic suppression of potassium conductances and potentiation of cation currents (53, 39) could be potential mechanisms. Second, mAChRs enhance the probability of action potential-dependent GABA release from axon terminals, therefore leading to the increase of the contribution of large size (multiquantal) sIPSCs to the overall population of synaptic events. The underlying mechanisms for this muscarinic action await to be elucidated. One possibility is that the m1/phospholipid/PKC signaling regulates the presynaptic protein synaptotagmine that functions as a calcium sensor to trigger synchronous vesicle fusion events (5456), thus facilitating the Ca2+ cooperativity of transmitter release. Third, mAChRs potentiate the postsynaptic GABAA receptor functions, therefore resulting in the bigger response to GABA. The muscarinic modulation of postsynaptic GABAA receptor properties could be attributed to changes in the phosphorylation state of GABAA receptor subunits by PKC-activated Src kinase in response to m1 receptor stimulation (57, 58, 18).
In addition to cholinergic hypofunction, genetic studies have implicated
the importance of A deposition in the pathogenesis of AD
(3). A
is synthesized and
secreted by brain cells (59,
60) and deposited proximal to
nerve terminals by axonally transported APP
(61). It is generally accepted
that accumulation of diffuse deposits of A
in the brain is an early
event in the development of AD, which emphasizes the importance of elucidating
the neuronal response to A
fibrils before clinical symptoms arise. We
propose that the early pathophysiological changes could contribute
significantly to later cognitive impairments, which are not accompanied by
massive neurodegeneration
(26). Several lines of
evidence have suggested that A
has pleiotropic actions on the
cholinergic system (20),
including the suppression of acetylcholine synthesis in primary cultures of
basal forebrain neurons (62),
the inhibition of acetylcholine release from hippocampal slices
(63), and the disruption of
muscarinic receptor-G-proteins coupling in cortical cultures
(64). In the present study, we
found that in neurons from APP transgenic mice or neurons exposed to fibrillar
A
2535, the carbachol enhancement of sIPSC amplitudes
was impaired, whereas the carbachol-induced increase in sIPSC frequencies was
intact. It suggests that A
partially affects the muscarinic modulation
of GABAergic interneuron excitability, leading to the impairment in muscarinic
regulation of multiquantal release of GABA. Since young APP transgenic mice
demonstrate little or no neuronal loss
(25,
65) and apparently normal
expression of mAChRs (Fig. 4, C
and D), the altered regulation of GABA transmission by
muscarinic receptors in these mutants are most likely due to the specific
impairment of muscarinic signaling. Our result is consistent with the notion
that A
can directly induce cholinergic hypofunction without apparent
neurotoxicity (20). The
specific impairment in muscarinic regulation of GABA transmission, but not of
intrinsic firing, in APP transgenic mice suggests that not all of the
cholinergic functions are altered by A
. Given the significance of PFC
GABAergic inhibition in working memory
(19), the alteration of
muscarinic regulation of GABAergic signaling in APP transgenic mice could
contribute to the deficit in cognition and memory associated with AD.
Earlier observations reported that exposure cortical cultures to A
peptides reduces carbachol-stimulated GTPase activity, inositol phosphate
accumulation, and intracellular Ca2+ increase
(64). It prompted us to
hypothesize that one potential mechanism for the loss of muscarinic modulation
of GABA transmission in the mutant APP-overexpressing mice is the impairment
of m1/G-protein-mediated signal transduction by elevated levels of A
. In
agreement with this, we found that mAChRs regulate GABA transmission partly
through a PKC-dependent mechanism, and mAChRs fail to activate PKC in APP
transgenic mice. It suggests that the A
-induced disruption of muscarinic
activation of PKC contributes to the impaired muscarinic regulation of GABA
signaling. Interestingly, previous studies have shown that activation of PKC
by stimulation of m1 receptors markedly inhibits the production and release of
A
in cell lines
(6668).
The significant attenuation of muscarinic activation of PKC in APP transgenic
mice provides a mechanism for preventing the m1/PKC-mediated down-regulation
of amyloidogenic A
generation. The loop between A
and ACh could
lead to enhanced A
production and exacerbated cholinergic deficiencies
in AD.
Taken together, the central finding of this study is that activation of mAChRs enhances GABA transmission in PFC pyramidal neurons, and the m1-mediated, PKC-dependent modulation of GABA signaling is impaired in neurons from APP transgenic mice, which is attributable to the loss of muscarinic activation of PKC in these mutants. This finding provides a possible connection between amyloid burden and cholinergic dysfunction in AD. Elucidation of the functional abnormalities in the transgenic mouse models of AD may offer valuable insights into the pathogenesis of the disease and potential therapeutic interventions.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Physiology and
Biophysics, State University of New York at Buffalo, 124 Sherman Hall,
Buffalo, NY 14214. E-mail:
zhenyan{at}buffalo.edu.
1 The abbreviations used are: AD, Alzheimer's disease; A,
-amyloid peptide; mAChR, muscarinic acetylcholine receptor; PFC,
prefrontal cortex; APP,
-amyloid precursor protein; IPSC, inhibitory
postsynaptic current; sIPSC, spontaneous IPSC; GABA,
-aminobutyric
acid; GABAA,
-aminobutyric acid, type A; PKC, protein kinase
C; PKA, cAMP-dependent protein kinase; BAPTA,
1,2-bis(2-aminophenoxy)ethane-N,N,N',
N'-tetraacetic acid; K-S, Kolmogorov-Smirnov; CCh, carbachol;
PKI, PKA inhibitor; ANOVA, analysis of variance; RT, reverse
transcription.
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ACKNOWLEDGMENTS |
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REFERENCES |
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