Postnatal Restriction of Activity-Induced Ca 2+ Responses to Schwann Cells at the Neuromuscular Junction Are Caused by the Proximo-Distal Loss of Axonal Synaptic Vesicles during Development.

Terminal or perisynaptic Schwann cells (TPSCs) are nonmyelinating, perisynaptic glial cells at the neuromuscular junction (NMJ) that respond to neural activity by increasing intracellular calcium (Ca ²⁺ ) and regulate synaptic function. The onset of activity-induced TPSC Ca ²⁺ responses, as well as whether axonal Schwann cells (ASCs) along the nerve respond to nerve stimulation during development, is unknown. Here, we show that phrenic nerve stimulation in developing male and female mice elicited Ca ²⁺ responses in both ASCs and TPSCs at embryonic day 14. ASC responses were lost in a proximo-distal gradient over time, but could continue to be elicited by bath application of neurotransmitter, suggesting that a loss of release rather than a change in ASC competence accounted for this response gradient. Similar to those of early postnatal TPSCs, developing ASC/TPSC responses were mediated by purinergic P2Y ₁ receptors. The loss of ASC Ca ²⁺ responses was correlated to the proximo-distal disappearance of synaptophysin immunoreactivity and synaptic vesicles in phrenic axons. Accordingly, developing ASC Ca ²⁺ responses were blocked by botulinum toxin. Interestingly, the loss of ASC Ca ²⁺ responses was also correlated to the proximo-distal development of myelination. Finally, compared with postnatal TPSCs, neonatal TPSCs and ASCs displayed Ca ²⁺ signals in response to lower frequencies and shorter durations of nerve stimulation. Together, these results with GCaMP3-expressing Schwann cells provide ex vivo evidence that both axons and presynaptic terminals initially exhibit activity-induced vesicular release of neurotransmitter, but that the subsequent loss of axonal synaptic vesicles accounts for the postnatal restriction of vesicular release to the NMJ. SIGNIFICANCE STATEMENT Neural activity regulates multiple aspects of development, including myelination. Whether the excitation of developing neurons in vivo results in the release of neurotransmitter from both axons and presynaptic terminals is unclear. Here, using mice expressing the genetically encoded calcium indicator GCaMP3 in Schwann cells, we show that both terminal/perisynaptic Schwann cells at the diaphragm neuromuscular junction and axonal Schwann cells along the phrenic nerve exhibit activity-induced calcium responses early in development, mediated by the vesicular release of ATP from the axons of motor neurons acting on P2Y ₁ receptors. These ex vivo findings corroborate classic in vitro studies demonstrating transmitter release by developing axons, and thus represent a tool to study the mechanisms and significance of this process during embryonic development.
(Copyright © 2018 the authors 0270-6474/18/388650-16$15.00/0.)