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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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Transmitter release regulates synapse formation but not elimination, causing a gradual divergence of synaptic development between wildtype and mGluR6-YFP/TeNT micea, b, Developmental increase in density (a) and number (b) of PSD95-CFP puncta on ON RGCs (5 -16 cells per time point and genotype). c, First image of a time-lapse series of a P9 RGC labeled with td-Tomato (blue) and glutamatergic postsynaptic densities labeled with PSD95-CFP (red). d, Time series of the region indicated by a yellow box in c in which a PSD95 cluster forms (arrow). YFP expression in BCs shown in green. PSD95-CFP channel isolated in the lower panels. e, Higher magnification of a single plane of the region indicated by the white box in d showing that the new punctum formed at an apposition of BC axon and RGC dendrite. f - h, analogous to c - e for loss of a PSD95 cluster. i, j, Summary data (mean ± s.e.m) for the rate of PSD95-FP cluster formation (i) and elimination (j). * indicates p < 0.05.
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Figure 4: Transmitter release regulates synapse formation but not elimination, causing a gradual divergence of synaptic development between wildtype and mGluR6-YFP/TeNT micea, b, Developmental increase in density (a) and number (b) of PSD95-CFP puncta on ON RGCs (5 -16 cells per time point and genotype). c, First image of a time-lapse series of a P9 RGC labeled with td-Tomato (blue) and glutamatergic postsynaptic densities labeled with PSD95-CFP (red). d, Time series of the region indicated by a yellow box in c in which a PSD95 cluster forms (arrow). YFP expression in BCs shown in green. PSD95-CFP channel isolated in the lower panels. e, Higher magnification of a single plane of the region indicated by the white box in d showing that the new punctum formed at an apposition of BC axon and RGC dendrite. f - h, analogous to c - e for loss of a PSD95 cluster. i, j, Summary data (mean ± s.e.m) for the rate of PSD95-FP cluster formation (i) and elimination (j). * indicates p < 0.05.

Mentions: Because the number of BCs, their axons and the RGC dendrites they contact were normal in TeNT retinas, it remained unclear which developmental mechanisms account for the lower density of BC - RGC synapses observed at maturity (Fig. 2). To begin to address this question, we measured the number and density of excitatory synapses on ON RGC dendrites at different times during development (Fig. 4a and b). At P7, when synaptic transmission between BCs and RGCs is first observed19, synapse numbers and densities were indistinguishable between wildtype and mGluR6-YFP/TeNT mice (p > 0.4) and gradually diverged over the following days toward the differences observed at maturity (Fig. 4a and b). To assess the dynamics which underlie this divergent development, we carried out live imaging experiments of RGCs biolistically labeled with tdTomato and PSD95-CFP in mGluR6-YFP/TeNT mice and wildtype littermates at P920,21. Time-lapse imaging revealed frequent synapse formation (Fig. 4c - e and Supplementary Fig. 9) and elimination (Fig. 4f - h) events. The synaptic turnover rate was 27 ± 5.8% per day with a net increase in synapses of 11 ± 6.5% per day in wildtype mice. Surprisingly, in mGluR6-YFP/TeNT mice the rate of synapse formation was reduced several fold (Fig. 4i), whereas synapse elimination was indistinguishable from wildtype littermates (Fig. 4j). We verified that > 90% of newly formed synapses were present in more than one time-lapse image to make sure that the reduced rate of synapse formation we report was not caused by a shortening of synapse lifetimes below our sampling interval. Moreover, the difference in the rate of synapse formation between TeNT and wildtype retinas (36 ± 13%; TeNT / wt in %, Fig. 4i), was similar to the difference in synapse density increase estimated by linear regression from P7 to P21 (23% ± 13%; TeNT / wt in %, Fig. 4a). Together these results suggest that inhibition of glutamate release from TeNT-expressing ON BCs selectively lowers the rate at which new synapses are established during development, thus accounting for the reduced density of BC - RGC synapses observed in mature mGluR6-YFP/TeNT mice.


Neurotransmission selectively regulates synapse formation in parallel circuits in vivo.

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

Transmitter release regulates synapse formation but not elimination, causing a gradual divergence of synaptic development between wildtype and mGluR6-YFP/TeNT micea, b, Developmental increase in density (a) and number (b) of PSD95-CFP puncta on ON RGCs (5 -16 cells per time point and genotype). c, First image of a time-lapse series of a P9 RGC labeled with td-Tomato (blue) and glutamatergic postsynaptic densities labeled with PSD95-CFP (red). d, Time series of the region indicated by a yellow box in c in which a PSD95 cluster forms (arrow). YFP expression in BCs shown in green. PSD95-CFP channel isolated in the lower panels. e, Higher magnification of a single plane of the region indicated by the white box in d showing that the new punctum formed at an apposition of BC axon and RGC dendrite. f - h, analogous to c - e for loss of a PSD95 cluster. i, j, Summary data (mean ± s.e.m) for the rate of PSD95-FP cluster formation (i) and elimination (j). * indicates p < 0.05.
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Related In: Results  -  Collection

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Figure 4: Transmitter release regulates synapse formation but not elimination, causing a gradual divergence of synaptic development between wildtype and mGluR6-YFP/TeNT micea, b, Developmental increase in density (a) and number (b) of PSD95-CFP puncta on ON RGCs (5 -16 cells per time point and genotype). c, First image of a time-lapse series of a P9 RGC labeled with td-Tomato (blue) and glutamatergic postsynaptic densities labeled with PSD95-CFP (red). d, Time series of the region indicated by a yellow box in c in which a PSD95 cluster forms (arrow). YFP expression in BCs shown in green. PSD95-CFP channel isolated in the lower panels. e, Higher magnification of a single plane of the region indicated by the white box in d showing that the new punctum formed at an apposition of BC axon and RGC dendrite. f - h, analogous to c - e for loss of a PSD95 cluster. i, j, Summary data (mean ± s.e.m) for the rate of PSD95-FP cluster formation (i) and elimination (j). * indicates p < 0.05.
Mentions: Because the number of BCs, their axons and the RGC dendrites they contact were normal in TeNT retinas, it remained unclear which developmental mechanisms account for the lower density of BC - RGC synapses observed at maturity (Fig. 2). To begin to address this question, we measured the number and density of excitatory synapses on ON RGC dendrites at different times during development (Fig. 4a and b). At P7, when synaptic transmission between BCs and RGCs is first observed19, synapse numbers and densities were indistinguishable between wildtype and mGluR6-YFP/TeNT mice (p > 0.4) and gradually diverged over the following days toward the differences observed at maturity (Fig. 4a and b). To assess the dynamics which underlie this divergent development, we carried out live imaging experiments of RGCs biolistically labeled with tdTomato and PSD95-CFP in mGluR6-YFP/TeNT mice and wildtype littermates at P920,21. Time-lapse imaging revealed frequent synapse formation (Fig. 4c - e and Supplementary Fig. 9) and elimination (Fig. 4f - h) events. The synaptic turnover rate was 27 ± 5.8% per day with a net increase in synapses of 11 ± 6.5% per day in wildtype mice. Surprisingly, in mGluR6-YFP/TeNT mice the rate of synapse formation was reduced several fold (Fig. 4i), whereas synapse elimination was indistinguishable from wildtype littermates (Fig. 4j). We verified that > 90% of newly formed synapses were present in more than one time-lapse image to make sure that the reduced rate of synapse formation we report was not caused by a shortening of synapse lifetimes below our sampling interval. Moreover, the difference in the rate of synapse formation between TeNT and wildtype retinas (36 ± 13%; TeNT / wt in %, Fig. 4i), was similar to the difference in synapse density increase estimated by linear regression from P7 to P21 (23% ± 13%; TeNT / wt in %, Fig. 4a). Together these results suggest that inhibition of glutamate release from TeNT-expressing ON BCs selectively lowers the rate at which new synapses are established during development, thus accounting for the reduced density of BC - RGC synapses observed in mature mGluR6-YFP/TeNT mice.

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