ex-vivo-imaging-cell-specific-calcium-signaling-at-tripartite-synapse

<em>Ex Vivo</em> Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

applications-spatio-temporal-mapping-particle-analysis-techniques-to

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca<sup>2+</sup> Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

University of Vermont

Terminal or perisynaptic Schwann cells (TPSCs) are non-myelinating, perisynaptic glial cells at the neuromuscular junction (NMJ) that respond to neural activity by increasing intracellular calcium (Ca) and regulate synaptic function. The onset of activity-induced TPSC Ca responses, as well as whether 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 responses in both ASCs and TPSCs at embryonic day 14 (E14). 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 receptors. The loss of ASC Ca responses was correlated to the proximo-distal disappearance of synaptophysin immunoreactivity and synaptic vesicles in phrenic axons. Accordingly, developing ASC Ca responses were blocked by botulinum toxin. Interestingly, the loss of ASC Ca responses was also correlated to the proximo-distal development of myelination. Finally, compared to postnatal TPSCs, neonatal TPSCs and ASCs displayed Ca signals in response to lower frequencies and shorter durations of nerve stimulation. Together, these results with GCaMP3-expressing Schwann cells provide  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.Neural activity regulates multiple aspects of development, including myelination. Whether the excitation of developing neurons  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 receptors. These  findings corroborate classic  studies demonstrating transmitter release by developing axons, and thus represent a tool to study the mechanisms and significance of this process during embryonic development.

Postnatal restriction of activity-induced Ca responses to Schwann cells at the neuromuscular junction are caused by the proximo-distal loss of axonal synaptic vesicles during development.

Mutations in GJC2 and GJA1, encoding Cxs (connexins) 47 and 43, respectively, are linked to lymphedema, but the underlying mechanisms are unknown. Because efficient lymph transport relies on the coordinated contractions of lymphatic muscle cells (LMCs) and their electrical coupling through Cxs, Cx-related lymphedema is proposed to result from dyssynchronous contractions of lymphatic vessels.

Grant W. Hennig

Department of Pharmacology,
Larner College of Medicine,
The Robert Larner,
M.D. College of Medicine,
Department of Pharmacology, Larner College of Medicine,
Department of Pharmacology, The Robert Larner, M.D. College of Medicine

University of Vermont

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca²⁺ Signaling In Situ

Grant W. Hennig

University of Vermont

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca²⁺ Signaling In Situ