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BMC Neurosci
2010 Jan 19;11:6. doi: 10.1186/1471-2202-11-6.
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A glycine receptor is involved in the organization of swimming movements in an invertebrate chordate.
Nishino A
,
Okamura Y
,
Piscopo S
,
Brown ER
.
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BACKGROUND: Rhythmic motor patterns for locomotion in vertebrates are generated in spinal cord neural networks known as spinal Central Pattern Generators (CPGs). A key element in pattern generation is the role of glycinergic synaptic transmission by interneurons that cross the cord midline and inhibit contralaterally-located excitatory neurons. The glycinergic inhibitory drive permits alternating and precisely timed motor output during locomotion such as walking or swimming. To understand better the evolution of this system we examined the physiology of the neural network controlling swimming in an invertebrate chordate relative of vertebrates, the ascidian larva Ciona intestinalis.
RESULTS: A reduced preparation of the larva consisting of nerve cord and motor ganglion generates alternating swimming movements. Pharmacological and genetic manipulation of glycine receptors shows that they are implicated in the control of these locomotory movements. Morphological molecular techniques and heterologous expression experiments revealed that glycine receptors are inhibitory and are present on both motoneurones and locomotory muscle while putative glycinergic interneurons were identified in the nerve cord by labeling with an anti-glycine antibody.
CONCLUSIONS: In Ciona intestinalis, glycine receptors, glycinergic transmission and putative glycinergic interneurons, have a key role in coordinating swimming movements through a simple CPG that is present in the motor ganglion and nerve cord. Thus, the strong association between glycine receptors and vertebrate locomotory networks may now be extended to include the phylum chordata. The results suggest that the basic network for 'spinal-like' locomotion is likely to have existed in the common ancestor of extant chordates some 650 M years ago.
Figure 1. Simplified diagrams showing Ciona intestinalis larval body plan and 'central nervous system'. (A) Larval body plan showing the central nervous system divisions. Orange, brain vesicle (BV) containing photoreceptive ocellus and a gravity sensing otolith (black spots). Green, presumptive motor ganglion, known variously as the visceral, trunk or tail ganglion. Pink, nerve cord (NC). The upper dotted line shown the region of the section made when preparing 'headless' larvae. The lower section on the line a'-b' shows the region of the cross section shown diagrammatically in B. (B) Diagram showing the main features of the nerve cord (pink), muscle (green) at the cross-section at the line a'-b'. The blue arrow shows the angle of view in A. This diagram is used in the following figures to show the angle of view in the micrographs. Scale bar in A, 100 μm.
Figure 2. Swimming patterns and pharmacological effects in the larva of the ascidian Ciona intestinalis. (A) Montage of images from high speed video of swimming at 6 ms intervals showing alternating swimming movements and larval progression (in the last frame the final position overlays the first position (grey). Sequence runs from left to right and top to bottom. Scale bar in frame 13, 100 μm. (B) Traces showing strict left/right (L/R) alternation of tail movements during swimming strokes in a tethered decapitated larva in 1 mM L-glutamate. (C) Phase relation of the autocorrelation on the left side (blue) with the cross correlation (red) between left and right sides. (D) The same larvae showing loss of strict L/R alternation in the presence of strychnine. (E) Phase relation from the same larvae as in (C) showing a strong autocorrelation on the left side and no positive correlation with the right side (red) in the presence of strychnine.
Figure 3. Glycine immunocytochemistry at the junction of the 'tail' and 'head' region of Ciona larvae. (A) Fluorescent image of the junction of tail and 'head' showing the location of glycine positive elements (red). Note the two clearly discernable glycine-positive cells (small arrows) in the nerve cord. (B) Brightfield image of the same area. (C) Fluorescent image of a similar area as in A showing dual staining of glycine positive material (red) and nuclei (green). In this case, at least four glycine positive elements may be identified as being cellular (yellow co-label). Large arrows indicate the junction between the 'head' and tail of the larva. Scale bars = 10 μm.
Figure 4. Expression pattern of Ci-GlyR. (A-E) Whole-mount in situ hybridization of the Ci-GlyR antisense probe during larval development. The signal in the nervous system is marked with red arrowheads, and that in muscle cells is with open black arrowheads. eTB, early tailbud stage; mTB, mid-tailbud stage; lTB, late tailbud stage; hL, hatched larva stage. Scale bar on E = 100 μm for A-E. (F-G). Pseudo-color images of fluorescently detected Ci-GlyR and Ci-ChAT expression. (F) In early tailbud (eTB), Ci-GlyR (green, yellow arrowheads) and Ci-ChAT (magenta) expression can be detected in the cytoplasm of single neuronal cells (determined by labeling the cell nuclei (blue) with SYTOX Green), although it should be noted that the expression patterns do not overlap until the mid-tailbud stage (though nuclear staining show that the signals are in the same cell). Scale bar = 10 μm. Beside the scale bar, the location of the depicted region is outlined with an orange rectangle. (G) Ci-GlyR and Ci-ChAT transcripts are co-localized at the mid tailbud stage (mTB). The dashed line indicates the midline of the nerve cord. Scale bar = 10 μm.
Figure 5. (A-C) Expression of enhanced GFP under the control of a 2.5 kb 5'-flanking sequence of the Ci-GlyR gene containing a putative Ci-GlyR promoter. (A) A brightfield image partially merged with the GFP expression pattern (red rectangle that corresponds with the view in C). (C, D) The two image planes on right (C) and left (D) muscle bands from a typical sample that shows GFP expression in muscle cells and motoneurons (large yellow arrows). Some nerve terminals are branched (red asterisks), while others have long axons that enter the tail nerve cord (small yellow arrowheads not resolvable easily in I). Ciona muscle bands are composed of three rows of cells, dorsal (D), middle (M), and ventral (V), and are numbered from anterior to posterior. Muscle cells with the highest expression in this specimen are underlined. The background signal in the tunic cells on the surface of the larva is due to autofluorescence that is also present in the controls. Scale bar = 100 μm.
Figure 6. Function and evolution of Ci-GlyR. (A, B) Effect of gene suppression on larval swimming by morpholino injection into developing eggs. (A) Antisense morpholino disrupts alternating swimming in a similar manner to strychnine. (B) 5-base mismatched control morpholino is without effect on swimming, larvae are capable of normal alternating swimming (Supp. Video 2). (C) The phylogeny of Ci-GlyR compared to other members of the Cys-loop superfamily of ligand gated chloride channels (Ci-GlyR is indicated in the box). Hs: H. sapiens, Zf: D. rerio (zebrafish), Ci: C intestinalis, Ce: C. elegans, Dm: D. melanogaster, Sp: S. purpuratus (sea urchin).
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