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• W2012614147 abstract "It has long been recognized that muscarinic acetylcholine receptors (mAChRs) are crucial for the control of cognitive processes, and drugs that activate mAChRs are helpful in ameliorating cognitive deficits of Alzheimer's disease (AD). On the other hand, GABAergic transmission in prefrontal cortex (PFC) plays a key role in “working memory” via controlling the timing of neuronal activity during cognitive operations. To test whether the muscarinic and γ-aminobutyric acid (GABA) system are interconnected in normal cognition and dementia, we examined the muscarinic regulation of GABAergic transmission in PFC of an animal model of AD. Transgenic mice overexpressing a mutant gene for β-amyloid precursor protein (APP) show behavioral and histopathological abnormalities resembling AD and, therefore, were used as an AD model. Application of the mAChR agonist carbachol significantly increased the spontaneous inhibitory postsynaptic current (sIPSC) frequency and amplitude in PFC pyramidal neurons from wild-type animals. In contrast, carbachol failed to increase the sIPSC amplitude in APP transgenic mice, whereas the carbachol-induced increase of the sIPSC frequency was not significantly changed in these mutants. Similar results were obtained in rat PFC slices pretreated with the β-amyloid peptide (Aβ). Inhibiting protein kinase C (PKC) blocked the carbachol enhancement of sIPSC amplitudes, implicating the PKC dependence of this mAChR effect. In APP transgenic mice, carbachol failed to activate PKC despite the apparently normal expression of mAChRs. These results show that the muscarinic regulation of GABA transmission is impaired in the AD model, probably due to the Aβ-mediated interference of mAChR activation of PKC. It has long been recognized that muscarinic acetylcholine receptors (mAChRs) are crucial for the control of cognitive processes, and drugs that activate mAChRs are helpful in ameliorating cognitive deficits of Alzheimer's disease (AD). On the other hand, GABAergic transmission in prefrontal cortex (PFC) plays a key role in “working memory” via controlling the timing of neuronal activity during cognitive operations. To test whether the muscarinic and γ-aminobutyric acid (GABA) system are interconnected in normal cognition and dementia, we examined the muscarinic regulation of GABAergic transmission in PFC of an animal model of AD. Transgenic mice overexpressing a mutant gene for β-amyloid precursor protein (APP) show behavioral and histopathological abnormalities resembling AD and, therefore, were used as an AD model. Application of the mAChR agonist carbachol significantly increased the spontaneous inhibitory postsynaptic current (sIPSC) frequency and amplitude in PFC pyramidal neurons from wild-type animals. In contrast, carbachol failed to increase the sIPSC amplitude in APP transgenic mice, whereas the carbachol-induced increase of the sIPSC frequency was not significantly changed in these mutants. Similar results were obtained in rat PFC slices pretreated with the β-amyloid peptide (Aβ). Inhibiting protein kinase C (PKC) blocked the carbachol enhancement of sIPSC amplitudes, implicating the PKC dependence of this mAChR effect. In APP transgenic mice, carbachol failed to activate PKC despite the apparently normal expression of mAChRs. These results show that the muscarinic regulation of GABA transmission is impaired in the AD model, probably due to the Aβ-mediated interference of mAChR activation of PKC. Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; Aβ, β-amyloid peptide; mAChR, muscarinic acetylcholine receptor; PFC, prefrontal cortex; APP, β-amyloid precursor protein; IPSC, inhibitory postsynaptic current; sIPSC, spontaneous IPSC; GABA, γ-aminobutyric acid; GABAA, γ-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. is a devastating neurodegenerative disorder. Several prominent features consistently found in AD patients include: degeneration of basal forebrain cholinergic neurons and ensuing deficient cholinergic functions in cortex and hippocampus, extracellular protein aggregates containing β-amyloid peptides (Aβ) in these cholinergic target areas, and impairments in mental functions that are characterized by the loss of memory (1Whitehouse P.J. Price D.L. Struble R.G. Clark A.W. Coyle J.T. Delon M.R. Science. 1982; 215: 1237-1239Crossref PubMed Scopus (3013) Google Scholar, 2Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3668) Google Scholar, 3Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5168) Google Scholar). So far, the most effective therapeutic strategy in AD treatment is to enhance cholinergic transmission (4Sitaram N. Weingartner H. Gillin J.C. Science. 1978; 201: 274-276Crossref PubMed Scopus (394) Google Scholar, 5Weinstock M. Neurodegeneration. 1995; 4: 349-356Crossref PubMed Scopus (98) Google Scholar). It has long been recognized that muscarinic acetylcholine receptors (mAChRs) are crucial for the control of high level cognitive processes (6Nathanson N.M. Annu. Rev. Neurosci. 1987; 10: 195-236Crossref PubMed Google Scholar, 7Hasselmo M.E. Trends Cogn. Sci. 1999; 3: 351-359Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). Drugs that antagonize mAChRs worsen the performance of human subjects and animals in learning and memory tasks (8Coyle J.T. Price D.L. DeLong M.R. Science. 1983; 219: 1184-1190Crossref PubMed Scopus (2614) Google Scholar, 9Whishaw I.Q. Tomie J.A. Behav. Neurosci. 1987; 101: 603-616Crossref PubMed Scopus (111) Google Scholar), while drugs that activate mAChRs are helpful in ameliorating cognitive deficits of AD (10Whitehouse P.J. Acta Neurol. Scand. Suppl. 1993; 149: 42-45PubMed Google Scholar, 11Brown J.H. Taylor P. Hardman J.G. Limbird L.E. The Pharmacological Basis of Therapeutics. 9th Ed. McGraw-Hill, New York1996: 141-160Google Scholar). Despite the discovery of correlation between cholinergic hypofunction and AD, the cellular and molecular mechanisms underlying the function and dysfunction of mAChRs in normal cognition and dementia remain elusive. Prefrontal cortex (PFC), one of the major target areas of basal forebrain cholinergic neurons, has long been associated with high-level, “executive” processes (12Miller E.K. Neuron. 1999; 22: 15-17Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), particularly a form of short term information storage described as “working memory” (13Goldman-Rakic P.S. Neuron. 1995; 14: 477-485Abstract Full Text PDF PubMed Scopus (1870) Google Scholar). The cholinergic activity in frontal cortex is persistently increased in mice performing a working memory task (14Durkin T.P. Neuroscience. 1994; 62: 681-693Crossref PubMed Scopus (46) Google Scholar). All the five subtypes of mAChRs, m1–m5 (15Bonner T.I. Buckley N.J. Young A.C. Brann M.R. Science. 1987; 237: 527-532Crossref PubMed Scopus (1220) Google Scholar), are expressed in cortical pyramidal neurons (16Levey A.I. Kitt C.A. Simonds W.F. Price D.L. Brann M.R. J. Neurosci. 1991; 11: 3218-3226Crossref PubMed Google Scholar, 17Stewart A.E. Yan Z. Surmeier D.J. Foehring R.C. J. Neurophysiol. 1999; 81: 72-84Crossref PubMed Scopus (43) Google Scholar, 18Ma X.H. Zhong P. Gu Z. Feng J. Yan Z. J. Neurosci. 2003; 23: 1159-1168Crossref PubMed Google Scholar). 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 (19Constantinidis C. Williams G.V. Goldman-Rakic P.S. Nat. Neurosci. 2002; 5: 175-180Crossref PubMed Scopus (271) Google Scholar). 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 Aβ peptides can potently inhibit various cholinergic neurotransmitter functions independently of concurrent neurotoxicity (20Auld D.S. Kar S. Quirion R. Trends Neurosci. 1998; 21: 43-49Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Aβ peptides are produced by proteolytic cleavage of the β-amyloid precursor protein (APP) (21Selkoe D.J. Trends Cell Biol. 1998; 8: 447-453Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar). Most of the mutations in the APP gene are clustered around the cleavage sites, which increases the rate of cleavage, thereby generating more Aβ (22Selkoe D.J. J. Biol. Chem. 1996; 271: 18295-18298Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar, 23Tanzi R.E. Bertram L. Neuron. 2001; 32: 181-184Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). Transgenic mice overexpressing mutant APP genes exhibit AD-like symptoms, including increased Aβ deposits and deficits in spatial learning and memory (24Games D. Adams D. Alessandrini R. Barbour R. Berthelette P. Blackwell C. Carr T. Clemens J. Donaldson T. Gillespie F. Nature. 1995; 373: 523-527Crossref PubMed Scopus (2247) Google Scholar, 25Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-103Crossref PubMed Scopus (3700) Google Scholar, 26Chapman P.F. White G.L. Jones M.W. Cooper-Blacketer D. Marshall V.J. Irizarry M. Younkin L. Good M.A. Bliss T.V. Hyman B.T. Younkin S.G. Hsiao K.K. Nat. Neurosci. 1999; 2: 271-276Crossref PubMed Scopus (816) Google Scholar). In this study, we used this AD model to examine the muscarinic regulation of GABAergic synaptic transmission in PFC pyramidal projection neurons. An AD Model—Transgenic mice carrying the human APP 695 with the double mutation K670N and M671L (Swedish mutation) were created as described previously (25Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-103Crossref PubMed Scopus (3700) Google Scholar). Eight-week-old transgenic males (on B6SJLF1 hybrid background) were bred with mature B6SJLF1 females. Genetic background of these mice is the same with this breeding scheme. Genotyping were performed by PCR. Male transgenic (1–2 months old) and age-matched wild-type littermates were used in the experiments. Electrophysiological Recordings in Slices—Young adult rat or mouse slices containing PFC were prepared as described previously (27Feng J. Cai X. Zhao J. Yan Z. J. Neurosci. 2001; 21: 6502-6511Crossref PubMed Google Scholar, 28Cai X. Flores-Hernandez J. Feng J. Yan Z. J. Phyisol. 2002; 540: 743-759Crossref PubMed Scopus (83) Google Scholar). 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 300–400-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, 300–305 mosm/liter). Slices were then incubated for 1–6 h at room temperature (20–22 °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 N G-nitro-l-arginine, 1 kynurenic acid, pH = 7.4, 300–305 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 (5–9 MΩ) 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 = 7.2–7.3, 265–270 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 α-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 40× 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 (2–10 GΩ) 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 MΩ and were compensated 50–70%. 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 = 7.2–7.3, 265–270 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 Aβ-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 4–6 min after drug application. IPSCs of 60 s (200–1000 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-(14–22) (Calbiochem) were made up as concentrated stocks in water and stored at –20 °C. Stocks were thawed and diluted immediately prior to use. The β-amyloid peptide Aβ25–35 and the control peptide containing the reverse sequence Aβ35–25 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 Analysis—For detecting activated PKC, a phospho-PKC (pan) antibody that recognizes PKCα, βI, βII, ∈, η, and δ isoforms only when phosphorylated at a carboxyl-terminal residue homologous to Ser660 of PKCβII 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 × 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 α, β, and γ isoforms. Quantitation was obtained from densitometric measurements of immunoreactive bands on autoradiograms. Data correspond to the mean ± S.E. of 5–10 samples per condition and were analyzed by ANOVA tests. mRNA Detection—PFCs 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 2–3 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 10× 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 (29Yan Z. Surmeier D.J. J. Neurosci. 1996; 16: 2592-2604Crossref PubMed Google Scholar, 30Yan Z. Flores-Hernandez J. Surmeier D.J. Neuroscience. 2001; 103: 1017-1024Crossref PubMed Scopus (141) Google Scholar). 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. Muscarinic Modulation of the Spontaneous IPSC Amplitude Is Abolished in APP Transgenic Mice—To test the impact of muscarinic receptors (mAChRs) on GABAergic inhibitory transmission in PFC, we first examined the effect of mAChR agonist carbachol on spontaneous inhibitory postsynaptic currents (sIPSCs) in mouse pyramidal neurons located in deep layers of PFC. Application of the GABAA receptor antagonist bicuculline (10 μm) completely blocked the sIPSCs (n = 5, data not shown), indicating that these synaptic currents are mediated by GABAA receptors. Bath application of carbachol (20 μm), a broad-spectrum cholinergic agonist, caused a reversible increase in the amplitude and frequency of sIPSCs (Fig. 1, A–C). The increase developed gradually and reached a plateau 4–6 min after the application of carbachol. A tonic increase of sIPSCs, rather than periodical bursts of sIPSCs (31Kondo S. Kawaguchi Y. Neurosience. 2001; 107: 551-560Crossref PubMed Scopus (20) Google Scholar), was observed in response to carbachol in most of our cells. In a sample of PFC pyramidal neurons examined, carbachol increased the mean amplitude of sIPSCs by 62.4 ± 9.7% (n = 18, p < 0.001, K-S test) and the mean frequency of sIPSCs by 220 ± 35.1% (n = 18, p < 0.001, K-S test). In the presence of pirenzepine (1 μm), an antagonist for m1/m4 receptors, carbachol failed to cause a significant change in sIPSCs (mean amplitude: 5.5 ± 3.0%; mean frequency: 5.3 ± 7.0%; n = 5, p > 0.05, K-S test), suggesting the mediation by m1 or m4 mAChRs. Since m1 is the most prominent subtype abundantly expressed in the majority of cortical neurons (16Levey A.I. Kitt C.A. Simonds W.F. Price D.L. Brann M.R. J. Neurosci. 1991; 11: 3218-3226Crossref PubMed Google Scholar, 18Ma X.H. Zhong P. Gu Z. Feng J. Yan Z. J. Neurosci. 2003; 23: 1159-1168Crossref PubMed Google Scholar), the potent effect of carbachol on GABA transmission found in about 90% of the PFC pyramidal neurons we examined is likely to be mediated by the m1 receptor. 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 (∼20–30-fold) levels of Aβ peptides at 2 months of age, even though no amyloid plaques, neuronal death, or cognitive deficit were observed at the early stage (25Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-103Crossref PubMed Scopus (3700) Google Scholar). Amyloid deposits were found in frontal cortex, along with other brain regions, in aged APP transgenic mice (25Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-103Crossref PubMed Scopus (3700) Google Scholar), suggesting that the elevated Aβ 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, D–F, 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 β-Amyloid Peptide—We then examined whether the altered muscarinic modulation of GABA transmission in APP transgenic mice is attributable to the elevated β-amyloid protein levels at an early age (25Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-103Crossref PubMed Scopus (3700) Google Scholar). To do so, we treated rat PFC slices with β-amyloid peptides (Aβ) before examining carbachol effects on sIPSCs. Aβ25–35, which represents the biologically active region of Aβ (32Yankner B.A. Duffy L.K. Kirschner D.A. Science. 1990; 250: 279-282Crossref PubMed Scopus (1910) Google Scholar, 33McDonald D. Bamberger M. Combs C. Landreth G. J. Neurosci. 1998; 18: 4451-4460Crossref PubMed Google Scholar), was aged to produce aggregated Aβ25–35. In non-treated rat slices, bath application of carbachol caused a reversible increase in the amplitude and frequency of sIPSCs (Fig. 2, A–C), similar to what was found in wild-type mice. However, in Aβ25–35-treated slices, carbachol failed to increase the sIPSC amplitude, but still induced a potent enhancement of the sIPSC frequency. A representative example from an Aβ25–35-treated neuron is shown in Fig. 2, D–F. To confirm the specificity of the action of Aβ25–35, its control peptide containing the reverse sequence Aβ35–25 was used to pretreat PFC slices. Similar to non-treated slices, in Aβ35–25-treated slices, bath application of carbachol induced a reversible increase in the sIPSC amplitude (Fig. 2G). As summarized in Fig. 2H, in Aβ25–35-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 Aβ35–25 (77.4 ± 12.5%, n = 5, p < 0.001, K-S test). However, the carbachol-induced increase in the mean frequency of sIPSCs in Aβ25–35-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 Aβ35–25-treated neurons (231.7 ± 38.3%, n = 5, p < 0.001, K-S test). Despite the ability of Aβ25–35 to alter the muscarinic regulation of sIPSCs, Aβ25–35 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 (34Zhou F.M. Hablitz J.J. J. Neurophysiol. 1999; 82: 2989-2999Crossref PubMed Scopus (202) Google Scholar). To test whether β-amyloid impairs the actions of 5-HT2 receptors, we examined the serotonergic regulation of sIPSCs in Aβ25–35-treated PFC slices. Application of serotonin (20 μm) caused a potent increase in the mean amplitude and frequency of sIPSCs in Aβ25–35-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 Aβ effect on serotonin functions. Taken together, these results indicate that β-amyloid selectively alters the muscarinic regulation of GABA transmission. Muscarinic Modulation of GABA Transmission Is through a PKC-dependent Mechanism—To 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-(14–22) (1 μm) to test the potential role of m4/PKA in this process. As shown in Fig. 3, D–F, carbachol still induced a potent increase in sIPSC amplitudes and frequencies in the PKI-(14–22)-treated slice, similar to what was obtained in the non-treated slice (Fig. 2, A–C), 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-(14–22) (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-(14–22): 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. Muscarinic Activation of PKC Is Lost in APP Transgenic Mice—Given 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 (35Behn-Krappa A. Newton A.C. Curr. Biol. 1999; 9: 728-737Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), a phosphospecific pan PKC antibody that de" @default.
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• W2012614147 date "2003-07-01" @default.
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• W2012614147 title "Impaired Modulation of GABAergic Transmission by Muscarinic Receptors in a Mouse Transgenic Model of Alzheimer's Disease" @default.
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• W2012614147 doi "https://doi.org/10.1074/jbc.m302789200" @default.
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