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. 2015 Jan 5;25(1):16-28.
doi: 10.1016/j.cub.2014.10.071. Epub 2014 Dec 4.

Neurobeachin Is Required Postsynaptically for Electrical and Chemical Synapse Formation

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Free PMC article

Neurobeachin Is Required Postsynaptically for Electrical and Chemical Synapse Formation

Adam C Miller et al. Curr Biol. .
Free PMC article

Abstract

Background: Neural networks and their function are defined by synapses, which are adhesions specialized for intercellular communication that can be either chemical or electrical. At chemical synapses, transmission between neurons is mediated by neurotransmitters, whereas at electrical synapses, direct ionic and metabolic coupling occur via gap junctions between neurons. The molecular pathways required for electrical synaptogenesis are not well understood, and whether they share mechanisms of formation with chemical synapses is not clear.

Results: Here, using a forward genetic screen in zebrafish, we find that the autism-associated gene neurobeachin (nbea), which encodes a BEACH-domain-containing protein implicated in endomembrane trafficking, is required for both electrical and chemical synapse formation. Additionally, we find that nbea is dispensable for axonal formation and early dendritic outgrowth but is required to maintain dendritic complexity. These synaptic and morphological defects correlate with deficiencies in behavioral performance. Using chimeric animals in which individually identifiable neurons are either mutant or wild-type, we find that Nbea is necessary and sufficient autonomously in the postsynaptic neuron for both synapse formation and dendritic arborization.

Conclusions: Our data identify a surprising link between electrical and chemical synapse formation and show that Nbea acts as a critical regulator in the postsynaptic neuron for the coordination of dendritic morphology with synaptogenesis.

Figures

Figure 1
Figure 1
Electrical synapses are disrupted in dis4 mutants. A. Model of the Mauthner (M) circuit. Neurons, synapses, and behavioral output are depicted. Hindbrain and two spinal segments are shown. Dotted arrows depict flow of circuit activity given the indicated stimulus. B–J. In this and all subsequent figures, except where noted, images are dorsal views of hindbrain and two spinal cord segments from M/CoLo:GFP larvae at 5 days post fertilization. The ′ figures are zooms of the region denoted by the dotted boxes. Hindbrain and spinal cord images are maximum intensity projections of ~30 and ~10uM, respectively. Anterior is to the left. Scale bar = 10 uM. Larvae are stained for GFP (magenta) and Connexin36 (Cx36, yellow) in all panels, neurofilaments (RMO44, blue) in B–E, and Neurobiotin (Nb, cyan) in G–J. Individual GFP, Cx36, and Nb channels are shown in neighboring panels. Graphs represent data as mean +/− SEM. Statistical significance compared to control is denoted as ** for P<0.01 and **** for P<0.0001. Associated experimental statistics are found in Table S2. B–E. The Cx36 staining found at M dendrites (B,B′) and M/CoLo synapses (C,C′) is reduced in dis4 mutant animals (D,E). F. Quantitation of Cx36 at M/CoLo synapses in wildtype and dis4 mutants. G–J. Electrical synapses are functionally defective in dis4 mutants. Retrograde labeling of M axons with the gap junction permeable dye Nb from a caudal transection. Spinal cord images are at the level of the CoLo cell bodies (arrowheads), which is dorsal to the synapses. G,H. Nb labels the M cell bodies and other caudally projecting neurons (G,G′) and passes through the Cx36 gap junctions to fill the CoLo cell bodies (H,H′, arrowheads). Other neurons are also labeled due to projections caudally into the lesion site. I,J. In dis4 mutants Nb labels M normally (I,I′) however the amount passing into CoLos is diminished (J,J′, arrowheads). K. Quantitation of ratio of Nb in CoLo to M cell bodies in wildtype and dis4 mutants.
Figure 2
Figure 2
The dis4 mutation disrupts neurobeachin. A. Genome wide RNA-seq-based mapping data. The average frequency of mutant markers (black marks) is plotted against genomic position. A single region on chromosome 10 (chr10) emerges with an allele frequency near 1 indicating linkage to the dis4 mutation (red arrow). Each chromosome is separated by vertical lines and labeled at the bottom. B. Detail of chr10, the average frequency of mutant markers (gray discs) is plotted against chromosomal position. A red box marks the region of tightest linkage. Each tick mark on the X-axis represents 10Mb. C. Mutant reads are shown aligned to the reference genome identifying a C to T transition in neurobeachin (nbea) creating a nonsense mutation at amino acid 906. Aligned reads are shown as grey boxes; differences from reference are highlighted by colored letters. D. nbea is downregulated presumably due to nonsense mediated decay (Cufflinks, log2 fold change = −0.82). E. Illustration of the primary structure of Nbea with protein domains depicted as colored boxes, the homology of zebrafish to human domains is labeled (% ident.), and the locations of the mutations are marked by dashed lines. F. Ratio of mutant to wildtype Connexin36 (Cx36) fluorescence at M/CoLo synapses for each allelic combination listed. Wildtype animals were siblings (homozygous and heterozygous) from a given cross. Data for fh364/fh364 is derived from that in Fig. 1F. G. Phylogenetic tree depicting relationships amongst Nbea gene family in common multicellular model organisms. Scale represents substitutions per site.
Figure 3
Figure 3
Glycinergic synapses are disrupted in nbea mutants. Larvae are stained for GFP (magenta), Connexin36 (Cx36, yellow), and glycine receptor (GlyR, cyan). Individual GFP and GlyR channels are shown in neighboring panels. Graphs represent data as mean +/− SEM. Statistical significance compared to control is denoted as **** for P<0.0001. Associated experimental statistics are found in Table S2. A–D. The GlyR staining found on M dendrites (A) and CoLo/CoLo synapses (B) is diminished in nbea mutant animals (C,D). E. Quantitation of the amount of GlyR in wildtype and mutant CoLo/CoLo synapses. See Fig. 1A for circuit diagram.
Figure 4
Figure 4
Electrical and chemical synaptic scaffolds are disrupted in nbea mutants. Larvae are stained for GFP (magenta) in all panels, Connexin36 (Cx36, yellow) and ZO-1 (green) in A–D, and Glycine receptor (GlyR, cyan) and Gephyrin (Geph, green) in F–I. Individual Cx36, ZO-1, GlyR, and Geph channels are shown in neighboring panels. Graphs represent data as mean +/− SEM. Statistical significance compared to control is denoted as ** for P<0.01. Associated experimental statistics are found in Table S2. Note that for each of spinal cord zoom there is only one M/CoLo and CoLo/CoLo synapse depicted – this is due to natural variation in the positions of CoLo neurons in the spinal cord. A–D. The electrical synapse scaffold ZO-1 is colocalized with Cx36 at M dendritic (A,A′) and spinal cord (B,B′) synapses. ZO-1 staining is diminished in mutants (C,D), but less severely than Cx36. E. Quantitation of ZO-1 at M/CoLo synapses in wildtype and nbea mutants. F–I. The inhibitory synapse scaffold Geph is colocalized with GlyR at M dendritic (F,F′) and CoLo spinal cord (G,G′) synapses. Geph staining is diminished in mutants (H,I), but less severely than GlyR. J. Quantitation of Geph at CoLo/CoLo synapses in wildtype and nbea mutants.
Figure 5
Figure 5
nbea mutants have defects in eliciting normal behavior. A–D. Individual frames from a 500-frame/second movie. Arrowheads point to eyes. Auditory startle stimulus was applied at time = 0. msec = millisecond. Graphs represent data as mean +/− SEM from three separate trials. Statistical significance compared to control is denoted as * for P<0.05 and ns for not significant. Associated experimental statistics are found in Table S3. A. M is responsible for a fast-escape response to threatening stimuli and initiates a turn away from stimulus that is completed in 10 msec. B–D. nbea mutant animals often fail to initiate turns (B), and are frequently found lying on their side (C). However, when they do respond the behavior produced is indistinguishable from wildtype (D). E,F. Quantitation of escape response and balance defects in a separate wildtype line (wt) and nbea mutant animals and their wt siblings (wt sibs). Each animal was tested in three separate trials for its response to stimulus. The average number of trials with response (E) or with animals on their sides (F) was recorded.
Figure 6
Figure 6
Neurobeachin is necessary and sufficient in the postsynaptic neuron for electrical and chemical synapse formation. Dorsal views of chimeric larvae at 6 day post fertilization containing GFP-marked cells transplanted from an M/CoLo:GFP embryo into an unmarked host. Larvae are stained for GFP (magenta), Connexin36 (Cx36, yellow), and glycine receptor (GlyR, cyan). Individual Cx36 and GlyR channels are shown in neighboring panels. The arrows point M/CoLo electrical synapses. The arrows also point to the adjacent postsynaptic side of the CoLo/CoLo synapse associated with the upper left CoLo neuron. The arrowheads point to the presynaptic side of the glycinergic synapse associated with the upper left CoLo neuron. Graphs represent data as mean +/− SEM. Statistical significance compared to control is denoted as ** for P<0.01, *** for P<0.001, **** for P<0.0001, and ns for not significant. Associated experimental statistics are found in Table S4. A–C. Individual examples of control chimeric larvae with cells transplanted from a M/CoLo:GFP embryo into an unmarked wildtype host (wt > wt). D–F. Examples of chimeric larvae testing where Nbea is necessary, with cells transplanted from a nbea mutant M/CoLo:GFP embryo into an unmarked wildtype host (nbea > wt). Note the greatly diminished synapses when the nbea mutant cell is the postsynaptic neuron (D,F). G–I. Examples of chimeric larvae testing where Nbea is sufficient, with cells transplanted from an M/CoLo:GFP embryo into an unmarked nbea mutant host (wt > nbea). Note that synapses are rescued when the postsynaptic neuron is wildtype for Nbea (G,I). J–M. Quantitation of the ratio of Cx36 fluorescence at M/CoLo or GlyR fluorescence at CoLo/CoLo synapses at transplant-associated neurons compared to unassociated neurons within the same animal in wt > wt and nbea > wt chimeras (J,L) and wt > nbea chimeras (K,M).
Figure 7
Figure 7
Neurobeachin is required autonomously to maintain dendritic complexity. A–C. Cross section views of individual chimeric larvae containing GFP-marked cells from M/CoLo:GFP embryos in unmarked hosts at 6 days post fertilization. Images are maximum intensity projections of ~20uM from digitally rendered cross sections. For clarity, fluorescent signal outside of the GFP-labeled neurons was digitally removed. Ventral is down, lateral is to the left. Scale bar = 10 uM. Larvae are stained for GFP (magenta), Connexin36 (Cx36, yellow), and glycine receptor (GlyR, cyan). The GFP channel is shown in neighboring panels. Graphs represent data as mean +/− SEM. Statistical significance compared to control is denoted as *** for P<0.001 and ns for not significant. Associated experimental statistics are found in Table S5. A. In wildtype (wt > wt) Mauthner elaborates complex dendrites with three main compartments – ventral (arrow), lateral (arrowhead), and somatic (double arrowhead). B. When M is nbea mutant in an otherwise wildtype host (nbea > wt) the M dendrites lose the fine terminal branches of the dendritic arbor. C. When M is wildtype in a nbea mutant host (wt > nbea) the dendritic complexity is similar to wildtype. D–G. Quantitation of M ventral dendrite parameters in chimeric embryos. H–K. Quantitation of changes in the ventral dendrite from 1 to 5 days post fertilization (dpf) in wildtype and nbea mutant embryos. “Longest path” is the longest continuous main path from cell body to dendrite tip. “Total length” is the sum of the lengths of all the dendritic branches. “Branches” is the sum of the number of branches made off the main, longest branch. “Branch depth” is the maximum depth of branching, with the main branch being primary, and all subsequent branches being labeled sequentially.

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