Blastemal Depolarization Microbursts Decline during Contractile Episodes and Substrate Uptake in Immobilized Limb Buds

Blastemal depolarization microbursts (BDMs) are intermittent, fast, synchronous bioelectric oscillations that are thought to consolidate pattern memory during appendage regeneration. Previous work reported that BDMs emerge during so-called “consummatory phases,” such as nutrient uptake following a successful morphogenetic maneuver. Yet most studies using freely moving larvae have not rigorously separated the act of substrate ingestion from the mere pause of collective cell migration. Here we quantified BDMs during growth-factor–seeking contraction and subsequent substrate uptake in completely immobilized juvenile salamander limb buds held under a microfluidic sheath. The buds executed a cyclical myocyte hold–release contraction; every alternate release delivered a bolus of mitogenic medium (false versus true assimilation). Surprisingly, BDM incidence fell sharply during both substrate uptake and contraction release. Naïve limb buds exposed only to medium perfusion showed a comparable drop in BDMs during simple ingestion. Conversely, BDM frequency rose progressively during the contraction-hold phase and was potentiated by anticipated mitogen delivery. A subset of blastemal cells exhibited calcium-spike bursts locked to cue/release and mitogen arrival, as previously observed. Another subset showed voltage dynamics mirroring BDMs—decreased activity during contraction release and uptake, and enhancement by expectancy during the hold period. Both cue-responsive and BDM-like populations displayed stronger temporal coupling to field BDMs than cells unrelated to the task. These data indicate that regenerating tissue generates BDMs primarily during relative quiescence between mechanical actions, rather than during the act of nutrient assimilation itself. We propose that BDMs couple contractile behavior to trophic outcome, stabilizing positional information when the blastema is momentarily still.