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Neurotransmission selectively regulates synapse formation in parallel circuits in vivo.

Kerschensteiner D, Morgan JL, Parker ED, Lewis RM, Wong RO - Nature (2009)

Bottom Line: Time-lapse imaging revealed that this was caused by a reduced rate of synapse formation rather than an increase in synapse elimination.Similarly, TeNT-expressing ON bipolar cell axons generated fewer presynaptic active zones.Our results reveal an unexpected and remarkably selective role for activity in circuit development in vivo, regulating synapse formation but not elimination, affecting synapse number but not dendritic or axonal patterning, and mediating independently the refinement of connections from parallel (ON and OFF) processing streams even where they converge onto the same postsynaptic cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Washington University School of Medicine, St Louis, Missouri 63110, USA. KerschensteinerD@vision.wustl.edu

ABSTRACT
Activity is thought to guide the patterning of synaptic connections in the developing nervous system. Specifically, differences in the activity of converging inputs are thought to cause the elimination of synapses from less active inputs and increase connectivity with more active inputs. Here we present findings that challenge the generality of this notion and offer a new view of the role of activity in synapse development. To imbalance neurotransmission from different sets of inputs in vivo, we generated transgenic mice in which ON but not OFF types of bipolar cells in the retina express tetanus toxin (TeNT). During development, retinal ganglion cells (RGCs) select between ON and OFF bipolar cell inputs (ON or OFF RGCs) or establish a similar number of synapses with both on separate dendritic arborizations (ON-OFF RGCs). In TeNT retinas, ON RGCs correctly selected the silenced ON bipolar cell inputs over the transmitting OFF bipolar cells, but were connected with them through fewer synapses at maturity. Time-lapse imaging revealed that this was caused by a reduced rate of synapse formation rather than an increase in synapse elimination. Similarly, TeNT-expressing ON bipolar cell axons generated fewer presynaptic active zones. The remaining active zones often recruited multiple, instead of single, synaptic ribbons. ON-OFF RGCs in TeNT mice maintained convergence of ON and OFF bipolar cells inputs and had fewer synapses on their ON arbor without changes to OFF arbor synapses. Our results reveal an unexpected and remarkably selective role for activity in circuit development in vivo, regulating synapse formation but not elimination, affecting synapse number but not dendritic or axonal patterning, and mediating independently the refinement of connections from parallel (ON and OFF) processing streams even where they converge onto the same postsynaptic cell.

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Selective blockade of glutamate release from retinal ON BCsa, ON and OFF pathways. C, cone; B, bipolar cell; G, ganglion cell; O / IPL, outer / inner plexiform layer. ON (white), OFF (black) and ON-OFF responsiveness (gray) indicated by fill color. b, YFP expression in mGluR6-YFP/TeNT mouse at postnatal day 21 (P21) colocalizes with anti-CaBP5 labeling in ON but not OFF BCs 15. c - h, Overview (c - e) and enlargements of ON sublamina (f - h) from P21 mGluR6-YFP/TeNT (c, d, f, g) and wildtype retinas (e, h). YFP not shown in d and g. i - k, Representative traces (i, ON RGCs) and population data (mean ± s.e.m., j, k) for sEPSC recorded from RGCs at P21. l, m, Spatial profile (l, 100 ms before spike) and time course (m, average of ten pixels with highest SD) of STAs from representative RGCs. n, o, Summary data (mean ± s.e.m.) of receptive field extent (n, # pixels whose SD exceeded that of background pixels more than fourfold), and sensitivity (o, peak of average STA time course exemplified in m). * indicates p < 0.01.
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Figure 1: Selective blockade of glutamate release from retinal ON BCsa, ON and OFF pathways. C, cone; B, bipolar cell; G, ganglion cell; O / IPL, outer / inner plexiform layer. ON (white), OFF (black) and ON-OFF responsiveness (gray) indicated by fill color. b, YFP expression in mGluR6-YFP/TeNT mouse at postnatal day 21 (P21) colocalizes with anti-CaBP5 labeling in ON but not OFF BCs 15. c - h, Overview (c - e) and enlargements of ON sublamina (f - h) from P21 mGluR6-YFP/TeNT (c, d, f, g) and wildtype retinas (e, h). YFP not shown in d and g. i - k, Representative traces (i, ON RGCs) and population data (mean ± s.e.m., j, k) for sEPSC recorded from RGCs at P21. l, m, Spatial profile (l, 100 ms before spike) and time course (m, average of ten pixels with highest SD) of STAs from representative RGCs. n, o, Summary data (mean ± s.e.m.) of receptive field extent (n, # pixels whose SD exceeded that of background pixels more than fourfold), and sensitivity (o, peak of average STA time course exemplified in m). * indicates p < 0.01.

Mentions: ON BCs, which depolarize in response to light, receive and invert photoreceptor signals through metabotropic glutamate receptors (mGluR6) on their dendrites. OFF BCs, instead, express ionotropic glutamate receptors and hyperpolarize upon illumination3,4. Thus, the parallel processing of light increment (ON) and decrement (OFF) signals that is central to visual system function is initiated at the first synapse in the retina. To create an imbalance of glutamate release from ON and OFF BCs in the inner plexiform layer (IPL) where their axons relay signals to RGCs, we generated transgenic mice in which a promoter fragment of mGluR65 drove expression of the light chain of TeNT and YFP (mGluR6-YFP/TeNT, Fig. 1a). TeNT, a bacterial protease, cleaves vesicle associated membrane protein 2 (VAMP2) and inhibits vesicle fusion 6. In mGluR6-YFP/TeNT mice, transgene expression was limited to ON BCs which ramify their axons in the inner half of the IPL (Fig. 1b), and was present throughout the period of BC - RGC synaptogenesis (Supplementary Fig. 1). We tested the proteolytic activity of TeNT using an antibody against VAMP27. Double-labeling for the vesicular glutamate transporter 1 (VGluT1), which is present in all BC axon terminals, verified that VAMP2 was selectively depleted from ON BCs (Fig. 1c - h). To examine glutamate release, we recorded spontaneous excitatory postsynaptic currents (sEPSCs) from large monostratified RGCs in retinal flat mount preparations (Fig. 1i). ON RGCs from mGluR6-YFP/TeNT mice showed very few and lower amplitude sEPSCs compared to wildtype, whereas OFF RGC sEPSCs were normal in frequency and amplitude (Fig. 1i - k). Focal application of the glutamate receptor agonist kainate (100 μM) near the soma of ON RGCs elicited excitatory currents of similar amplitude and without failure in wildtype and mGluR6-YFP/TeNT mice (Supplementary Fig. 2), arguing that the reduction in spontaneous events likely reflects a blockade of presynaptic transmitter release.


Neurotransmission selectively regulates synapse formation in parallel circuits in vivo.

Kerschensteiner D, Morgan JL, Parker ED, Lewis RM, Wong RO - Nature (2009)

Selective blockade of glutamate release from retinal ON BCsa, ON and OFF pathways. C, cone; B, bipolar cell; G, ganglion cell; O / IPL, outer / inner plexiform layer. ON (white), OFF (black) and ON-OFF responsiveness (gray) indicated by fill color. b, YFP expression in mGluR6-YFP/TeNT mouse at postnatal day 21 (P21) colocalizes with anti-CaBP5 labeling in ON but not OFF BCs 15. c - h, Overview (c - e) and enlargements of ON sublamina (f - h) from P21 mGluR6-YFP/TeNT (c, d, f, g) and wildtype retinas (e, h). YFP not shown in d and g. i - k, Representative traces (i, ON RGCs) and population data (mean ± s.e.m., j, k) for sEPSC recorded from RGCs at P21. l, m, Spatial profile (l, 100 ms before spike) and time course (m, average of ten pixels with highest SD) of STAs from representative RGCs. n, o, Summary data (mean ± s.e.m.) of receptive field extent (n, # pixels whose SD exceeded that of background pixels more than fourfold), and sensitivity (o, peak of average STA time course exemplified in m). * indicates p < 0.01.
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Figure 1: Selective blockade of glutamate release from retinal ON BCsa, ON and OFF pathways. C, cone; B, bipolar cell; G, ganglion cell; O / IPL, outer / inner plexiform layer. ON (white), OFF (black) and ON-OFF responsiveness (gray) indicated by fill color. b, YFP expression in mGluR6-YFP/TeNT mouse at postnatal day 21 (P21) colocalizes with anti-CaBP5 labeling in ON but not OFF BCs 15. c - h, Overview (c - e) and enlargements of ON sublamina (f - h) from P21 mGluR6-YFP/TeNT (c, d, f, g) and wildtype retinas (e, h). YFP not shown in d and g. i - k, Representative traces (i, ON RGCs) and population data (mean ± s.e.m., j, k) for sEPSC recorded from RGCs at P21. l, m, Spatial profile (l, 100 ms before spike) and time course (m, average of ten pixels with highest SD) of STAs from representative RGCs. n, o, Summary data (mean ± s.e.m.) of receptive field extent (n, # pixels whose SD exceeded that of background pixels more than fourfold), and sensitivity (o, peak of average STA time course exemplified in m). * indicates p < 0.01.
Mentions: ON BCs, which depolarize in response to light, receive and invert photoreceptor signals through metabotropic glutamate receptors (mGluR6) on their dendrites. OFF BCs, instead, express ionotropic glutamate receptors and hyperpolarize upon illumination3,4. Thus, the parallel processing of light increment (ON) and decrement (OFF) signals that is central to visual system function is initiated at the first synapse in the retina. To create an imbalance of glutamate release from ON and OFF BCs in the inner plexiform layer (IPL) where their axons relay signals to RGCs, we generated transgenic mice in which a promoter fragment of mGluR65 drove expression of the light chain of TeNT and YFP (mGluR6-YFP/TeNT, Fig. 1a). TeNT, a bacterial protease, cleaves vesicle associated membrane protein 2 (VAMP2) and inhibits vesicle fusion 6. In mGluR6-YFP/TeNT mice, transgene expression was limited to ON BCs which ramify their axons in the inner half of the IPL (Fig. 1b), and was present throughout the period of BC - RGC synaptogenesis (Supplementary Fig. 1). We tested the proteolytic activity of TeNT using an antibody against VAMP27. Double-labeling for the vesicular glutamate transporter 1 (VGluT1), which is present in all BC axon terminals, verified that VAMP2 was selectively depleted from ON BCs (Fig. 1c - h). To examine glutamate release, we recorded spontaneous excitatory postsynaptic currents (sEPSCs) from large monostratified RGCs in retinal flat mount preparations (Fig. 1i). ON RGCs from mGluR6-YFP/TeNT mice showed very few and lower amplitude sEPSCs compared to wildtype, whereas OFF RGC sEPSCs were normal in frequency and amplitude (Fig. 1i - k). Focal application of the glutamate receptor agonist kainate (100 μM) near the soma of ON RGCs elicited excitatory currents of similar amplitude and without failure in wildtype and mGluR6-YFP/TeNT mice (Supplementary Fig. 2), arguing that the reduction in spontaneous events likely reflects a blockade of presynaptic transmitter release.

Bottom Line: Time-lapse imaging revealed that this was caused by a reduced rate of synapse formation rather than an increase in synapse elimination.Similarly, TeNT-expressing ON bipolar cell axons generated fewer presynaptic active zones.Our results reveal an unexpected and remarkably selective role for activity in circuit development in vivo, regulating synapse formation but not elimination, affecting synapse number but not dendritic or axonal patterning, and mediating independently the refinement of connections from parallel (ON and OFF) processing streams even where they converge onto the same postsynaptic cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Washington University School of Medicine, St Louis, Missouri 63110, USA. KerschensteinerD@vision.wustl.edu

ABSTRACT
Activity is thought to guide the patterning of synaptic connections in the developing nervous system. Specifically, differences in the activity of converging inputs are thought to cause the elimination of synapses from less active inputs and increase connectivity with more active inputs. Here we present findings that challenge the generality of this notion and offer a new view of the role of activity in synapse development. To imbalance neurotransmission from different sets of inputs in vivo, we generated transgenic mice in which ON but not OFF types of bipolar cells in the retina express tetanus toxin (TeNT). During development, retinal ganglion cells (RGCs) select between ON and OFF bipolar cell inputs (ON or OFF RGCs) or establish a similar number of synapses with both on separate dendritic arborizations (ON-OFF RGCs). In TeNT retinas, ON RGCs correctly selected the silenced ON bipolar cell inputs over the transmitting OFF bipolar cells, but were connected with them through fewer synapses at maturity. Time-lapse imaging revealed that this was caused by a reduced rate of synapse formation rather than an increase in synapse elimination. Similarly, TeNT-expressing ON bipolar cell axons generated fewer presynaptic active zones. The remaining active zones often recruited multiple, instead of single, synaptic ribbons. ON-OFF RGCs in TeNT mice maintained convergence of ON and OFF bipolar cells inputs and had fewer synapses on their ON arbor without changes to OFF arbor synapses. Our results reveal an unexpected and remarkably selective role for activity in circuit development in vivo, regulating synapse formation but not elimination, affecting synapse number but not dendritic or axonal patterning, and mediating independently the refinement of connections from parallel (ON and OFF) processing streams even where they converge onto the same postsynaptic cell.

Show MeSH
Related in: MedlinePlus