Electron microscopy (EM) analysis of muskelin-specific immunoperoxidase
signals confirmed this view. Muskelin was identified at post-, but not presynaptic, sites of many but not all symmetric (inhibitory) synapses (Figures 1K and 1L), as well as at individual nonsynaptic intracellular vesicles (Figure 7C, arrow). To investigate the biological role of muskelin, we established a muskelin KO mouse. Exon 1 of the Mkln1 gene OSI-906 research buy (encoding muskelin) encodes only 32 amino acids. An OmniBank® ES cell clone ( Zambrowicz et al., 1998) with an insertion of a retroviral gene trapping vector in intron 1 (primary RNA transcript: position 6970 bp) of the Mkln1 locus ( Figure 2A) was used. Heterozygous animals were crossed to produce wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice for further analysis. PCR and Southern blotting confirmed the presence of one mutant allele in +/− and two mutant alleles in −/− animals, respectively ( Figures 2B and 2C). In addition, western blot analysis with a muskelin-specific antibody ( Ledee et al., 2005) confirmed that muskelin Raf targets protein levels were reduced by half in +/− and completely lost in −/− animals, as compared to +/+ genotypes ( Figure 2D). Accordingly, immunohistochemistry revealed a loss of muskelin signals in −/−, as compared to +/+
cerebellar and hippocampal tissue slices ( Figures 2E and 2F) and the use of a second and independent muskelin antibody ( Tagnaouti et al., 2007) failed to coprecipitate muskelin from −/−, but not from +/+ mice ( Figure 2G). We therefore conclude that muskelin expression is completely abolished in KO animals. Cresyl violet stainings revealed no gross histological abnormalities in KO brain tissue slices ( Figure 2H), suggesting that muskelin plays no major roles in brain development or anatomical changes might be subtle. Functional GABAergic synaptic transmission is essential for synchronizing the activity of neuronal networks giving rise to different sets of neuronal population rhythms in the hippocampus, i.e., theta, gamma, and
ripple oscillations (Buzsáki and Draguhn, 2004). All these hippocampal rhythms have been implicated in processes underlying the temporary storage and successive no consolidation of long-term memories (Buzsáki and Draguhn, 2004 and Diekelmann and Born, 2010). To assess the consequences of muskelin deficiency on the level of neuronal network synchronization, we analyzed sharp wave-associated ripples in acute hippocampal slices (Maier et al., 2003) from muskelin KO and control animals in area CA1 (Figures 2I and 2J). Spectral analysis of sharp wave ripples displayed a robustly enhanced power component in the ripple frequency range (Figure 2K). The distribution of cumulated ripple power also showed a systematic shift to higher values in slices from muskelin KO animals compared to controls (p = 1.