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. 2006 Jul 26;26(30):7919-32.
doi: 10.1523/JNEUROSCI.1674-06.2006.

MuSK Expressed in the Brain Mediates Cholinergic Responses, Synaptic Plasticity, and Memory Formation

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Free PMC article

MuSK Expressed in the Brain Mediates Cholinergic Responses, Synaptic Plasticity, and Memory Formation

Ana Garcia-Osta et al. J Neurosci. .
Free PMC article

Abstract

Muscle-specific tyrosine kinase receptor (MuSK) has been believed to be mainly expressed and functional in muscle, in which it mediates the formation of neuromuscular junctions. Here we show that MuSK is expressed in the brain, particularly in neurons, as well as in non-neuronal tissues. We also provide evidence that MuSK expression in the hippocampus is required for memory consolidation, because temporally restricted knockdown after training impairs memory retention. Hippocampal disruption of MuSK also prevents the learning-dependent induction of both cAMP response element binding protein (CREB) phosphorylation and CCAAT enhancer binding protein beta (C/EBPbeta) expression, suggesting that the role of MuSK during memory consolidation critically involves the CREB-C/EBP pathway. Furthermore, we found that MuSK also plays an important role in mediating hippocampal oscillatory activity in the theta frequency as well as in the induction and maintenance of long-term potentiation, two synaptic responses that correlate with memory formation. We conclude that MuSK plays an important role in brain functions, including memory formation. Therefore, its expression and role are broader than what was believed previously.

Figures

Figure 1.
Figure 1.
MuSK is expressed in adult rat brain. A–C, Cloning of MuSK in the brain. A, Schematic representation of MuSK isoforms expressed in the brain. SS, Signal sequence; IgI–IgIV, Ig-like domains; C6, cysteine rich domain; KD, kinase domain; E, glutamic acid; W, tryptophan; S, serine; V, valine; K, lysine. Arrows in different colors indicate the five sets of primers used for PCRs. B, Electrophoretic analysis of PCR amplifications generated with the second set of primers (shown in green in Fig. 1A). M, Molecular weight marker. C, Electrophoretic analysis of PCR amplifications with a set of primers that flanked the entire MuSK ORF. D, Musk mRNA distribution in adult rat brain and HNCs revealed by in situ hybridization. Representative examples of rat adult brain coronal sections and HNCs hybridized with antisense (AS) or sense (S) probes. DG, Dentate gyrus. CA1 regions of the hippocampus, somatosensory cortex, cerebellum, and HNCs are shown. Magnifications: 2.5 and 40×. Data are representative of results obtained from eight experiments.
Figure 2.
Figure 2.
MuSK expression is detected in several tissues by Western blot analysis. A, B, Validation of anti-MuSK antiserum specificity. A, Western blot analysis of recombinant N-terminal portion of rat MuSK (rrMuSK). B, Western blot analysis of protein extracts obtained from nontransfected HEK293 cell line (293-), HEK293 transfected with rat MuSK tagged with the Myc sequence (293M), 293M treated with siRNA specific for MuSK (siRNA-MuSK) or with control, sense sequence (siRNA-control). The membrane was stained with anti-MuSK antiserum, stripped, and restained with anti-Myc antiserum. C, MuSK is expressed in several tissues. Western blot analysis was performed on extracts obtained from the indicated tissues. C2C12 and 293M were used as positive controls. Arrows indicate nonspecific bands that are not competed by the recombinant MuSK. D, At least two specific bands are detected with anti-MuSK antiserum in different tissues tested. Hippocampus and C2C12 are shown as representative examples. E, 2D gel analysis of extracts obtained from E18 brain and E18 muscle stained with anti-MuSK antibody. Muscle and brain MuSK isoforms show different isoelectric point (pI) migration.
Figure 3.
Figure 3.
Knockdown of hippocampal MuSK blocks IA memory consolidation. A, Biotinylated M-ODN diffusion and relative concentrations after hippocampal injection. Representative sections of a rat brain at different bregma (left to right) and representative sections injected with biotinylated M-ODN and killed either 2 or 8 h after injection (−2.8 μm from bregma) are shown. Representative examples of three independent experiments. B, Injection of M-ODN or S-ODN, training and testing time points. C, Quantitative Western blot analyses of hippocampal extracts from trained rats that underwent the protocols shown in B. Blots were stained with anti-MuSK antibody stripped and restained with anti-GluR1 antibody. Staining for actin was used for normalization of both MuSK and GluR1 values. A representative blot per condition is shown. Graphs represent the densitometric analysis of all data. Data are expressed as a mean percentage ± SEM of the S-ODN/PBS (S/P) control group (100%). D, Memory retention expressed as mean latency ± SEM (in seconds) of trained rats that received the treatments shown in B. M, M-ODN; S, S-ODN.
Figure 4.
Figure 4.
Hippocampal disruption of MuSK prevents the activation of the CREB–C/EBPβ pathway after training. A, Schematic representation of injection of M-ODN or S-ODN, training, and killing time points. Quantitative Western blot analysis of hippocampal extracts from untrained and trained rats that received the above treatments. Blots were stained with anti-pCREB (Ser133) antibody, stripped, and restained with anti-CREB, anti-NP62 antibodies and finally with anti-actin, which was used for normalization. A representative blot per condition is shown. Graphs represent the statistical densitometric analysis of all data. Data are expressed as a mean percentage ± SEM of the S-ODN (S) unpaired (S-unp) control group (100%). M, M-ODN; M-unp, M-ODN unpaired. B, Injection of M-ODN or S-ODN, training, and killing time points. Quantitative Western blot analysis of hippocampal extracts from unpaired and trained rats that received the above treatments. Blots were stained with anti-C/EBPβ antibody, stripped, and restained with anti-NP62 antibody and finally with anti-actin, which was used for normalization. A representative blot per condition is shown. Graphs represent the statistical densitometric analysis of all data. Data are expressed as a mean percentage ± SEM of the S-ODN unpaired control group (100%). C, Injection of M-ODN or S-ODN training and testing time points. Quantitative Western blot analysis of hippocampal extracts obtained from trained rats that received the above treatments (experiment described in Fig. 3). Blots were stained with anti-C/EBPβ antibody, stripped, and restained with anti-NP62 antibody and finally with anti-actin, which was used for normalization. A representative blot per condition is shown. Graphs represent the statistical densitometric analysis of all data. Data are expressed as a mean percentage ± SEM of the S-ODN trained group (100%).
Figure 5.
Figure 5.
MuSK plays a critical role in cholinergic oscillatory activity and long-term potentiation in the CA3–CA1 synapse. A, Left, Carbachol perfusion (50 μm) induced periodic bursts (top) recorded extracellularly in area CA1 of slices from S-ODN-injected hippocampi. Representative experiment of six. Expanded traces (bottom) show that each burst consisted of a series of field events, occurring at ∼10 Hz. Right, Hippocampal M-ODN injection disrupted cholinergic field oscillatory responses in the CA3–CA1 synapse, resulting in regularly spaced unitary events (top). Representative experiment of six; expanded trace shown below. Calibration: top, 0.2 mV, 60 s; bottom, 0.2 mV, 1 s. B, Summary data from all carbachol experiments. The majority (66.7%) of S-ODN slices displayed periodic bursting activity (left), whereas only 8.3% of the M-ODN slices exhibited such activity. The difference is statistically significant (*p < 0.01). Of those M-ODN slices showing nonbursting periodic responses (right), 54.5% displayed pacemaking (PACE) activity, with the remainder showing little or no such activity. Carbachol elicited pacemaking activity in only 11.1% of the S-ODN slices (right). INACT, Inactivity. C, D, Hippocampal M-ODN injection did not affect basal synaptic function. C, Input/output curve of M-ODN-treated slices was indistinguishable from that obtained from S-ODN-treated slices. D, There was no statistical difference between the degree of paired-pulse facilitation (PPF) produced in S-ODN and M-ODN slices. Data are expressed as mean percentage ± SEM. E, Hippocampal M-ODN injection impaired long-term potentiation induced by two trains of HFS (delivered at t = 0) in area CA1. In slices from hippocampi injected with S-ODN, HFS induced stable LTP that persisted for at least 2 h (filled circles). However, injection with M-ODN impaired LTP as early as 5–15 min post-HFS (for fEPSP slope measured over the 5–15 post-HFS period and normalized to baseline, S-ODN vs M-ODN, p < 0.01). After this period, LTP continued to decay in the M-ODN-treated slices relative to controls. The dashed line represents the normalized baseline value of 100%. Inset traces show superimposed sample fEPSPs recorded during the baseline period and 2 h after HFS in S-ODN-treated slices (left traces) or M-ODN-treated slices (right traces). Error bars represent SE. Calibration: 0.5 mV, 10 ms. F, Treatment with M-ODN did not affect the field potential evoked by HFS. Left, Slices were stimulated with two trains of HFS in the presence of 50 μm d-APV, and a second pair of trains was delivered 30 min after APV washout. The field potentials recorded during the second train from each set are shown here. Note the additional field negativity that was observed in the absence of APV, representing the NMDAR-mediated component of the field potential. Right, The AUCs for the potentials obtained in the presence and absence of APV were determined, and the NMDAR-mediated component was calculated by subtracting the +APV AUC from the −APV AUC (total AUC). Treatment with M-ODN affected neither the total AUC nor the NMDAR-mediated component. Calibration: 1 mV, 200 ms.
Figure 6.
Figure 6.
MuSK and agrin expression is increased in rat hippocampus after IA training. A, TaqMan-PCR analysis of MuSK on hippocampal extracts taken from rats trained in IA at 0 h−, 20 h−, and 20 h+. The concentration of mRNA was normalized against that of GAPDH. Data are expressed as mean fold change ± SEM of the ratio 20 h+:0 h− versus 20 h−:0 h− (∗p < 0.05). B, Quantitative Western blot analysis of hippocampal extracts taken from 0 h−, unpaired (unp), and IA trained (20 h+) individual rats. A representative blot per condition is shown. Graphs represent the statistical densitometric analysis of all data. Data were normalized against actin and are expressed as mean percent ± SEM of the 0 h− control mean values (100%). C, Quantitative Northern blot analysis of agrin expressed in hippocampal RNA extracts taken from 0 h−, unpaired, and 20 h+ rats. A representative blot per condition is shown. Graphs represent the statistical densitometric analysis of all data. The same membranes were also hybridized with a cyclophilin probe, which was used for normalization. Data are expressed as mean percentage ± SEM of the 0 h− control mean values (100%). ∗∗p < 0.01; ∗p < 0.05. D, Quantitative Western blot analysis of hippocampal extracts taken from 0 h−, unpaired, and 20 h+ rats. A representative blot per condition is shown. Graphs represent the statistical densitometric analysis of all data. Data were normalized against GAPDH and are expressed as mean percentage ± SEM of the 0 h− control mean values (100%), ∗p < 0.05.

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