Title: Redundant functions of RIM1α and RIM2α in Ca2+-triggered neurotransmitter release
Abstract: Article23 November 2006free access Redundant functions of RIM1α and RIM2α in Ca2+-triggered neurotransmitter release Susanne Schoch Corresponding Author Susanne Schoch Emmy Noether Research Group, Institute of Neuropathology, Department of Epileptology, University Bonn, Bonn, Germany Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Tobias Mittelstaedt Tobias Mittelstaedt Emmy Noether Research Group, Institute of Neuropathology, Department of Epileptology, University Bonn, Bonn, Germany Search for more papers by this author Pascal S Kaeser Pascal S Kaeser Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Daniel Padgett Daniel Padgett Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Nicole Feldmann Nicole Feldmann Emmy Noether Research Group, Institute of Neuropathology, Department of Epileptology, University Bonn, Bonn, GermanyPresent address: Department of Cell Physiology and Metabolism, University Medical Center, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland Search for more papers by this author Vivien Chevaleyre Vivien Chevaleyre Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Pablo E Castillo Pablo E Castillo Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Robert E Hammer Robert E Hammer Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Weiping Han Weiping Han Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USAPresent address: Singapore Bioimaging Consortium, Agency for Science, Technology and Research, Singapore 138667, Singapore Search for more papers by this author Frank Schmitz Frank Schmitz Institute of Anatomy and Cell Biology, Universität des Saarlandes, Homburg, Germany Search for more papers by this author Weichun Lin Weichun Lin Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Thomas C Südhof Thomas C Südhof Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Susanne Schoch Corresponding Author Susanne Schoch Emmy Noether Research Group, Institute of Neuropathology, Department of Epileptology, University Bonn, Bonn, Germany Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Tobias Mittelstaedt Tobias Mittelstaedt Emmy Noether Research Group, Institute of Neuropathology, Department of Epileptology, University Bonn, Bonn, Germany Search for more papers by this author Pascal S Kaeser Pascal S Kaeser Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Daniel Padgett Daniel Padgett Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Nicole Feldmann Nicole Feldmann Emmy Noether Research Group, Institute of Neuropathology, Department of Epileptology, University Bonn, Bonn, GermanyPresent address: Department of Cell Physiology and Metabolism, University Medical Center, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland Search for more papers by this author Vivien Chevaleyre Vivien Chevaleyre Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Pablo E Castillo Pablo E Castillo Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Robert E Hammer Robert E Hammer Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Weiping Han Weiping Han Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USAPresent address: Singapore Bioimaging Consortium, Agency for Science, Technology and Research, Singapore 138667, Singapore Search for more papers by this author Frank Schmitz Frank Schmitz Institute of Anatomy and Cell Biology, Universität des Saarlandes, Homburg, Germany Search for more papers by this author Weichun Lin Weichun Lin Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Thomas C Südhof Thomas C Südhof Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Author Information Susanne Schoch 1,2,3, Tobias Mittelstaedt1, Pascal S Kaeser2, Daniel Padgett2, Nicole Feldmann1, Vivien Chevaleyre4, Pablo E Castillo4, Robert E Hammer5, Weiping Han2, Frank Schmitz6, Weichun Lin2 and Thomas C Südhof2,3,7 1Emmy Noether Research Group, Institute of Neuropathology, Department of Epileptology, University Bonn, Bonn, Germany 2Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA 3Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA 4Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA 5Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA 6Institute of Anatomy and Cell Biology, Universität des Saarlandes, Homburg, Germany 7Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA *Corresponding author. Emmy Noether Research Group, Institute of Neuropathology, Department of Epileptology, University Bonn, Sigmund Freud Strasse 25, 53105 Bonn, Germany. Tel.: +49 228 287 19109; Fax: +49 228 287 19110; E-mail: [email protected] The EMBO Journal (2006)25:5852-5863https://doi.org/10.1038/sj.emboj.7601425 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info α-RIMs (RIM1α and RIM2α) are multidomain active zone proteins of presynaptic terminals. α-RIMs bind to Rab3 on synaptic vesicles and to Munc13 on the active zone via their N-terminal region, and interact with other synaptic proteins via their central and C-terminal regions. Although RIM1α has been well characterized, nothing is known about the function of RIM2α. We now show that RIM1α and RIM2α are expressed in overlapping but distinct patterns throughout the brain. To examine and compare their functions, we generated knockout mice lacking RIM2α, and crossed them with previously produced RIM1α knockout mice. We found that deletion of either RIM1α or RIM2α is not lethal, but ablation of both α-RIMs causes postnatal death. This lethality is not due to a loss of synapse structure or a developmental change, but to a defect in neurotransmitter release. Synapses without α-RIMs still contain active zones and release neurotransmitters, but are unable to mediate normal Ca2+-triggered release. Our data thus demonstrate that α-RIMs are not essential for synapse formation or synaptic exocytosis, but are required for normal Ca2+-triggering of exocytosis. Introduction In presynaptic nerve terminals, exocytosis of transmitter-filled synaptic vesicles at the plasma membrane is under tight spatial and temporal control. Synaptic vesicle exocytosis is restricted to a specialized area of the plasma membrane, the active zone. The protein network that constitutes the active zone organizes the docking and priming of synaptic vesicles. In addition, the active zone mediates use-dependent changes in release during short- and long-term forms of presynaptic plasticity (reviewed in Dresbach et al, 2001; Rosenmund et al, 2003; Kaeser and Südhof, 2005). The active zone is composed of a network of proteins that includes RIMs (for Rab3-interacting molecule; see Wang et al, 1997, 2000) as central components (Koushika et al, 2001; Castillo et al, 2002; Schoch et al, 2002). RIMs are multidomain proteins that are expressed in variable forms: two α-RIMs (RIM1α and 2α) that include a full complement of RIM domains (an N-terminal region that comprises a Rab3-binding sequence and a Munc13-binding zinc-finger; a central PDZ-domain; and two C-terminal C2-domains that however do not bind Ca2+), a single β-RIM (RIM2β) that contains all of the domains of α-RIMs except for the N-terminal Rab3- and Munc13-binding sequences;, and three γ-RIMs (RIM2γ, 3γ, and 4γ) that are composed of only the C-terminal C2-domain preceded by a short N-terminal flanking sequence (Wang et al, 2000). RIMs are encoded by four genes, of which the RIM1, RIM3, and RIM4 genes express only a single isoform (RIM1α, 3γ, and 4γ, respectively), whereas the RIM2 gene expresses three isoforms (RIM2α, 2β, and 2γ; Wang and Südhof, 2003). Further variation is introduced into α- and β-RIMs (but not γ-RIMs) by extensive alternative splicing (Wang et al, 2000; Johnson et al, 2003; Wang and Südhof, 2003). In addition to interacting with Munc13 and Rab3, α-RIMs bind to multiple other synaptic proteins: ELKS via the central PDZ-domain (Ohtsuka et al, 2002; Wang et al, 2002), RIM-BPs via an SH3-domain-binding sequence between the two C2-domains (Wang et al, 2000), and α-liprins and synaptotagmin 1 via the C-terminal C2-domain (Coppola et al, 2001; Schoch et al, 2002). Furthermore, in vitro interactions with several proteins have been described, including cAMP–GEFII (guanine nucleotide-exchange factor II) (Ozaki et al, 2000), SNAP-25 (Coppola et al, 2001), N-type Ca2+ channels (Coppola et al, 2001), and 14-3-3 adaptor proteins (Sun et al, 2003; Simsek-Duran et al, 2004). RIMs are connected indirectly with the active zone proteins Piccolo and Bassoon via ELKS (Takao-Rikitsu et al, 2004) and with receptor tyrosine phosphatases via liprins (Serra-Pages et al, 1998). Of these interactions, only RIM1α and 2α bind to Munc13 and Rab3, whereas γ-RIMs bind only to α-liprins and synaptotagmin 1. The binding of the N-terminal region of α-RIMs to Rab3 on synaptic vesicles and Munc13s is particularly interesting because a relatively short sequence (<150 residues) contains two nested subdomains, an α-helical region that binds to Rab3 (Wang et al, 2001) and a zinc-finger that binds to Munc13 (Betz et al, 2001; Dulubova et al, 2005). This binding is mutually compatible with each other, resulting in a trimeric complex in which the α-RIM/Munc13 dimer on the active zone is coupled to the synaptic vesicle protein Rab3 (Dulubova et al, 2005). Finally, RIMs are substrates for cAMP-dependent protein kinase (PKA) that phosphorylates RIM1α and RIM2α/β at two sites (Lonart et al, 2003). Analysis of RIM1α knockout (KO) mice showed that RIM1α plays a key regulatory role in synaptic vesicle exocytosis at the active zone, from vesicle priming to short- and long-term synaptic plasticity (Castillo et al, 2002; Schoch et al, 2002; Calakos et al, 2004). RIM1α-deficient synapses did not exhibit major changes in ultrastructure, suggesting that it is essential only for regulating exocytosis, and not for building an active zone architecture (Schoch et al, 2002). Although important, loss of this function does not impair mouse survival, as RIM1α KO mice have a normal apparent life expectancy (Schoch et al, 2002). The importance of RIM1α function nevertheless is apparent from the severe behavioral abnormalities observed in these mice, which include impairments in spatial learning and in fear conditioning as well as an increase in locomotor responses to novelty (Powell et al, 2004). The currently available data confirm that RIM1α is an active zone protein with a central role in regulating neurotransmitter release, and suggest that the other RIM isoforms may also be involved in the regulation of synaptic vesicle exocytosis. However, so far, only RIM1α has been analyzed. Although the various RIM isoforms are coexpressed in brain, their relative expression patterns are unknown, and it is unclear how much potential redundancy may exist among RIM isoforms. Such redundancy could exist, for example, between RIM1α and RIM2α because both of these RIM isoforms bind to Munc13 and to Rab3 (Dulubova et al, 2005), although they are the only isoforms that do so. Therefore, major questions remain unanswered: (1) in which cell types are the various RIM isoforms expressed? (2) Are RIM1α and RIM2α functionally redundant? (3) How do the two α-RIMs relate to each other? (4) Does the deletion of both α-RIMs lead to ultrastructural changes? To examine the role of the α-RIMs in synaptic transmission, we generated single and double KO mice (DKO) lacking either or both α-RIMs. Our data demonstrate that the RIM-α-isoforms are essential for survival and exhibit partially overlapping functions in the regulation of synaptic transmission, but are not required for building a normal synapse. Results Differential expression of RIM1 and RIM2 isoforms To examine whether RIM1α, RIM2α, RIM2β, and RIM2γ are differentially expressed in brain, we performed in situ hybridizations on brain sections from adult rats (Figure 1A, left panels). Two oligonucleotides were used for each RIM isoform to ensure that the same labeling patterns were obtained (data not shown). This labeling was abolished when excess unlabeled oligonucleotides were added to the hybridization mix (Figure 1A, right panels). Figure 1.In situ hybridization of RIM mRNAs in the rat brain. (A) Film images showing the distribution of RIM mRNAs in the adult rat brain (CB, cerebellum; CX, cerebral cortex; DG, dentate gyrus; HC, hippocampus; MB, midbrain; OB, olfactory bulb; Pn, pontine nucleus; sTn, subthalamic nucleus; ST, striatum; TH, thalamus; MO medulla oblongata). (B) Dark-field images of emulsion-dipped sections from rat hippocampus, olfactory bulb, and cerebellum (AON, anterior olfactory nucleus; DG, dentate gyrus; EPL, external plexiform layer; GL, glomerular layer; GRL, granule cell layer; MCL, mitral cell layer; ML, molecular layer; PCL, Purkinje cell layer; scale bar B, a–d=100 μm, B, e–f 20 μm). Download figure Download PowerPoint Autoradiographs of hybridized rat brain sections revealed differential but overlapping expression patterns of RIM mRNAs. In each case, regions rich in glial cells (e.g., white matter of cerebral cortex and cerebellum) were unlabeled, indicating a neuron-specific expression of RIM isoforms (Figure 1A, left panel). RIM1α mRNA is present throughout the brain, with the highest levels in the cortex, cerebellum, hippocampus and thalamus. RIM2α mRNA is concentrated in the cerebellum, the olfactory bulb and the dentate gyrus of the hippocampus. However, comparison of the signals obtained with the negative control indicates that RIM2α is also ubiquitously expressed in brain, albeit at low levels. RIM2β and RIM2γ mRNAs are present in a pattern similar to RIM1α, but both are probably expressed at lower levels based on the hybridization signal and exhibit regional differences (e.g., RIM2β is expressed more in the thalamus, and RIM2γ more in the brainstem; Figure 1A). These results show that the various RIM1- and RIM2-isoforms exhibit a differential, but highly overlapping expression pattern in rat brain. To determine the cellular distribution of RIM mRNA species, we analyzed emulsion-dipped sections. In the hippocampus, RIM1α can be detected at high levels in the dentate gyrus and the CA3 region, and at lower levels in the CA1 region (Figure 1B). In contrast, strong labeling for RIM2α was largely restricted to the dentate gyrus, with lower levels observed in the CA3 region. In the olfactory bulb, significant RIM1α expression was detected in the mitral cell layer (MCL) and in some cells of the external plexiform layer (EPL), whereas the cells of the granule cell layer (GRL) and the glomerular layer (GL) were devoid of a RIM1α signal (Figure 1B). In contrast, RIM2α showed strong expression in the GL, the MCL, and the GRL. Within the cerebellum, RIM1α and RIM2α expression is highly concentrated in the GRL (Figure 1B). Clearly, most neurons express multiple isoforms of RIMs with distinct relative expression levels. Characterization of RIM2α KO mice We generated mutant mice in which the fifth exon of the RIM2α gene (which encodes part of the zinc-finger; Wang and Südhof, 2003) is flanked by loxP sites (Figure 2A). Mice containing the targeted floxed RIM2α gene synthesized RIM2α at approximately wild-type levels (data not shown). We then crossed the floxed mice to transgenic mice that express cre recombinase in the male germ line to produce KO mice in which the floxed exon was deleted (Figure 2A) (O'Gorman et al, 1997). The conditional KO mice may prove to be useful in the analysis of the function of the α-RIMs in specific brain regions. However, in the present study, we focused on the constitutive RIM2α KO mice in order to establish the baseline function of the α-RIM isoforms. Figure 2.Generation of RIM2α KO mice. (A) Structures of the RIM2 wild-type gene (wild-type allele), of the targeting vector used for homologous recombination (targeting construct), and of the mutant RIM2 alleles after homologous recombination before and after further recombination of flp and cre recombinases. In the targeting vector, exon 5 is flanked by loxP sites (black triangles) and the neomycin resistance gene cassette (neo) that is flanked by flp recombination sites (black circles). A diphtheria toxin gene (DT) is included for negative selection. (B) Weights of male RIM2α KO and littermate control mice (N=36, *P<0.001). (C) Immunoblots of E18.5 embryonic and adult wild-type, RIM1α KO, RIM2α KO and embryonic DKO whole brain homogenates. (D) α-RIM immunoblots of proteins from different brain regions from adult RIM1α KO, RIM2α KO, and wild-type control mice; blots were probed with an antibody that recognizes both RIM1α and RIM2α (abbreviations of brain areas: OB, olfactory bulb; STR, striatum; CTX, cortex; HC, hippocampus; TH, thalamus; MB, midbrain, BS, brain stem; CB, cerebellum; *, bands of alternatively spliced RIM2α isoforms of higher molecular weight). Download figure Download PowerPoint Homozygous RIM2α KO mice were viable and fertile. However, systematic analysis of the offspring from heterozygous matings revealed that homozygous KO mice were slightly smaller than littermate wild-type controls (males, N=36) (Figure 2B), and less frequent than would be expected based on Mendelian inheritance (24.1% wild type, 56.3% heterozygous, and 19.6% homozygous mutant mice in offspring at >1 month of age; N=591; P<0.01). Immunoblotting confirmed that the KO mice lacked RIM2α (Figure 2C and D and Supplementary Figure 2). Similar to the RIM1α KO mice (Schoch et al, 2002), RIM2α mutant mice exhibited a deficit in maternal behavior, as they did not take care of their litters even after multiple pregnancies. Brains of single RIM2α KO mice showed a normal cell density, cytoarchitecture, connectivity, distribution/density of synapses, and ultrastructural morphology as assessed by H&E staining, NeuN immunohistochemistry, and electron microscopy (Supplementary Figure 1). Taken together, these data demonstrate that although RIM2α KO mice are viable and exhibit no apparent developmental abnormalities, the behavior and survival of these mice are slightly impaired. To search for compensatory changes in the expression of RIM1α as the only other α-RIM isoform in RIM2α KO mice, we compared the relative expression of RIM1α and RIM2α in various brain regions in wild-type, RIM1α KO, and RIM2α KO mice (Figure 2D and Supplementary Figure 2). Antibodies to the zinc-finger region of RIM1α and RIM2α often crossreact because of their structural similarities (Schoch et al, 2002). Moreover, these antibodies detect multiple bands even when one of the two α-RIM isoforms is deleted because of the extensive alternative splicing of RIMs (Wang and Südhof, 2003). Consistent with the in situ hybridization data, immunoblotting of different brain regions in single RIM1α and RIM2α KO mice showed that RIM1α is the more abundant isoform, whereas RIM2α is found at low levels in most rostral brain regions examined, but at high levels in the cerebellum and olfactory bulb (Figure 2D). However, we detected no region-specific compensatory changes in RIM1α protein levels in RIM2α KO mice, or conversely in RIM2α protein levels in RIM1α KO mice (Figure 2D and Supplementary Figure 2). Interestingly, alternative splicing of RIM2α seems to be regulated in a region-specific manner. Although in most brain regions, a smaller isoform is detected, in cerebellum and olfactory bulb an additional larger protein could be observed (asterisks in Figure 2D and Supplementary Figure 2). In RIM1α KO mice, synaptic transmission in the hippocampus is severely impaired (Castillo et al, 2002; Schoch et al, 2002; Calakos et al, 2004). To test whether RIM2α performs a similar fundamental role in synaptic transmission in the hippocampus, we recorded excitatory and inhibitory synaptic responses in acute hippocampal slices (Supplementary Figure 3). We first probed excitatory synaptic transmission in the CA1 region of the hippocampus, and compared synaptic responses of wild-type and KO mice in four paradigms that measure different types of short-term synaptic plasticity: paired-pulse facilitation (Supplementary Figure 3A), post-tetanic potentiation (Supplementary Figure 3B), and use-dependent depression (Supplementary Figure 3C). In addition, we examined the synaptic release probability by analyzing the progressive block of NMDA-dependent synaptic responses by the irreversible NMDA receptor antagonist MK801 (Supplementary Figure 3D). In all of these measurements, we observed no significant difference between wild-type and RIM2α KO mice. Next, we tested whether RIM2α KO mice exhibited a change in mossy fiber LTP in excitatory synapses of the CA3 region, which is abolished in RIM1α KO mice (Castillo et al, 2002). Again, we found no change in RIM2α KO mice (Supplementary Figure 3E). Finally, we examined inhibitory synaptic transmission at CA1 pyramidal cells, but failed to detect significant changes in paired-pulse-ratio (Supplementary Figure 3F). Thus, RIM2α-deficient synapses, different from RIM1α-deficient synapses, do not exhibit a major impairment of synaptic transmission in the hippocampus. Impaired survival of RIM1α/2α DKO mice To assess the redundant or divergent functions of the two α-RIMs, we generated RIM1α/2α DKO mice. Analysis of the offspring from systematic breedings of double heterozygous RIM1α/2α KO mice revealed that the survival of RIM1α or RIM2α homozygous mutant mice exhibited no significant decrease as long as two RIM1α or RIM2α wild-type alleles were present (Figure 3A). However, in >100 crossings, no surviving mouse that was homozygous mutant for both RIM1α and RIM2α was observed, demonstrating that RIM1α and RIM2α are redundant in terms of survival. Moreover, even single homozygous RIM1α or RIM2α KO mice exhibited significantly impaired survival when one of the other two α-RIM alleles was also deleted (i.e., in the RIM1α−/−2α+/− or RIM1α+/−2α−/− genotypes, P<0.001; see Figure 3A). Figure 3.(A) Survival analysis of the offspring from matings of double heterozygous RIM1α/2α mutant mice. The black bars plot the observed frequency of the indicated genotypes as percentage of the total, whereas the gray background indicates the expected frequency based on Mendelian inheritance (N=323). (B, C) Images of E18.5 RIM1α/2α DKO mutant and control littermate mice overall morphology (B) and skeleton (C, bones are stained in blue and cartilage in pink; black arrows point to ribcage and cervical vertebrae). Download figure Download PowerPoint RIM1α/2α DKO mice died immediately after birth because they could not breathe. The mutant mice did not respond to tactile stimuli, and exhibited an abnormal body posture similar to synaptobrevin 2 KO mice (Figure 3; Schoch et al, 2001), suggesting that they were paralyzed. Double KO mice could be recovered alive, however, at E18.5 as embryos by hysterectomy; at this age, DKO mice were present in a normal Mendelian ratio, suggesting that the deletion of both α-RIMs does not impair survival in utero (data not shown). Thus, for all subsequent studies on DKO mice, we analyzed embryos at E18.5. Although whole-body staining for bone and cartilage revealed no major developmental defects in the DKO mice, their vertebrae in the KO skeleton seemed more compact at the cervical level, and the ribcage appeared enlarged (Figure 3C), possibly because of permanent paralysis of the embryo during development. Brain composition and synapse structure of RIM1α/2α double KO mice Histochemistry and immunocytochemistry of brain sections revealed that RIM1α/2α DKO mice displayed no obvious impairments in central nervous system development (Figure 4A and data not shown). However, H&E- and NeuN-stained vibratome sections of paraffin-embedded spinal cords showed an increased number of ventral horn motoneurons at all cervical levels in RIM1α/2α DKO mice as compared with control littermates (Figure 4B). A morphometric analysis of NeuN-immunopositive motoneurons revealed a significant increase (**P<0.005) in motoneuron density in E18.5 DKO spinal chords (Figure 4C). No sign of neurodegeneration was detectable in the spinal cord by TUNEL or Fluoro JadeB staining (data not shown). The increase in motoneurons may be due to decreased synaptic transmission at the neuromuscular junction (NMJ) (see below) (Harris and McCaig, 1984; Oppenheim, 1991). Figure 4.Morphology of RIM1α/2α DKO brains and spinal cord. (A) H&E- and NeuN-stained sagittal section of brains from E18.5 mice of the indicated genotype (scale bar=2 mm). (B) NeuN-stained coronal sections of spinal cord from E18.5 mice of the indicated genotype (scale bar=100 μm). (C) Morphometric analysis of NeuN-immunopositive spinal motoneurons (**P<0.005). (D) Electron micrographs of synapses in the spinal cord. The arrows in (c, f) point to presynaptic dense projections in active zones. Abbreviations: pr, presynaptic; po, postsynaptic; sv, synaptic vesicles; pm, presynaptic plasma membrane. Scale bars, a=300 nm, d=370 nm, b=300 nm, e=240 nm, c=100 nm, f=130 nm. Download figure Download PowerPoint To test whether deletion of α-RIMs causes a major change in the protein composition of the brain, we quantified the levels of neuronal proteins in adult littermate wild-type, heterozygous, and homozygous mutant RIM2α KO mice (Figure 5A and C and Supplementary Table I) and in E18.5 embryos that were homozygous for the RIM1α KO allele and wild-type, heterozygous, or homozygous mutant for the RIM2α KO allele (Figure 5B and D and Supplementary Table II). We detected no significant changes in any protein in adult RIM2α KO mice compared with controls; in particular, no significant decrease in Munc13-1 in RIM1α/2α DKO embryos compared with RIM1α KO embryos was observed. To rule out region-specific changes in Munc13-1 level, we investigated different brain areas, but detected no obvious alterations (Figure 5E). These results were unexpected because previous studies showed that Munc13-1 is decreased ∼50% in RIM1α KO mice (Schoch et al, 2002), which prompted the hypothesis that the remaining Munc13-1 is stabilized by RIM2α. As shown here, however, additional deletion of RIM2α does not cause a major further decrease in Munc13-1 levels beyond that observed in RIM1α KO mice. A recent report described a role for RIM1α in the regulation of presynaptic recruitment of Munc13-1 and ubMunc13-2 (Andrews-Zwilling et al, 2006). Therefore, we examined if deletion of both α-RIMs results in a redistribution of Munc13-1 from the membrane-bound to the soluble fraction (Supplementary Figure 4B and data not shown). Soluble and membrane-bound protein fractions from wild-type and RIM1α/2α DKO embryonic brains were separated by ultrathurrax homogenization in buffer or in the presence of detergents of different strength and high-speed centrifugation. In agreement with Andrews-Zwilling et al (2006) we detected an increase of Munc13-1 in the soluble fraction in the brains of DKO mice. However, the majority of Munc13 was still found in the insoluble fraction with buffer or nondenaturing detergents but was solubilized with SDS. Thus, consistent with the fact that only some of the Munc13 isoforms contain the N-terminal C2-domain that binds to α-RIMs (Brose et al, 1995; Betz et al, 2001; Dulubova et al, 2005), only approximately half of the synaptic Munc13-1 depends on α-RIMs, and α-RIMs are not absolutely essential for Munc13 function. Figure 5.Analysis of synaptic protein levels in brains from RIM2α KO mice and RIM1α/2α DKO embryos. Brain homogenates of the indicated genotypes were analyzed by immunoblotting using antibodies to the indicated proteins (A, B), the blots were quantified