Stage-dependent regulation of intracellular calcium by spontaneous activity in developing brainstem

Abstract

Spontaneous activity supports developmental processes in many brain regions during embryogenesis, and the spatial extent and frequency of the spontaneous activity are tightly regulated by stage. In the developing mouse hindbrain, spontaneous activity propagates widely and the waves can cover the entire hindbrain at E11.5. The activity then retracts to waves that are spatially restricted to the rostral midline at E13.5, before disappearing altogether by E15.5. However, the mechanism of retraction is unknown. We studied passive membrane properties of cells that are spatiotemporally relevant to the pattern of retraction in mouse embryonic hindbrain using whole-cell patch clamp and Ca_i<super>2+</super> imaging techniques. We find that membrane excitability progressively decreases due to hyperpolarization of resting membrane potential and increased resting conductance density between E11.5-E15.5, in a spatiotemporal pattern correlated with the retraction sequence. Retraction can be acutely reversed by membrane depolarization at E15.5, and the induced events propagate similarly to spontaneous activity at earlier stages, though without involving gap junctional coupling. Manipulation of [K<super>+</super>]_o or [Cl<super>-</super>]_o reveals that membrane potential follows E_K more closely than E_Cl, suggesting a dominant role for K<super>+</super> conductance in the membrane hyperpolarization. Reducing membrane excitability by hyperpolarization of the resting membrane potential and increasing resting conductance are effective mechanisms to desynchronize spontaneous activity in a spatiotemporal manner, while allowing information processing to occur at the synaptic and cellular level. Between embryonic days (E) 11.5 and E13.5, spontaneous events propagate across the mouse brainstem as waves, each of which is driven by membrane depolarization, leading to calcium influx. Most calcium events rise from and return to a defined baseline. However, at E12.5, events rapidly burst with [Ca<super>2+</super>]_i staying close to peak value, well above baseline, for up to tens of minutes; we termed this Bash Bursts (Bash-B). Here, we investigate the mechanism of this unusual activity using calcium imaging and electrophysiology. Bash-B is triggered by an event originating at the midline of the rostral hindbrain, and is usually the result of that event propagating along a defined circular path. The looping circuit can either encompass both the midbrain and hindbrain, or remain in the hindbrain only, and the type of loop determines the duration of a single lap time, 5 or 3 s, respectively. Bash-B is supported by high membrane excitability of midline cells and is regulated by persistent inward “window current” at rest, contributing to spontaneous activity. The looping propagation pattern that underlies Bash-B is inhibited by flupirtine and APV, acting on unknown targets. A looping circuit is an effective mechanism for increasing [Ca<super>2+</super>]_i at regular intervals. Bash-B disappears by E13.5 via alteration of the looping circuit, making Bash-B a short phenomenon. The resulting sustained [Ca<super>2+</super>]_i may influence development of raphe serotonergic and ventral tegmental dopaminergic neurons by modulating gene expression.