Spontaneous rhythmic activity is a ubiquitous phenomenon in developing neural networks and is assumed to play an important role in the elaboration of mature circuitry. Here we describe the day-by-day evolution of spontaneous activity in the embryonic mouse spinal cord and show that, at a specific developmental stage, 2 distinct rhythms coexist. On embryonic days E12.5 and E13.5, we observed a single type of regularly recurring short spike-episodes synchronized across cervical, thoracic, and lumbar levels. By E14.5, in addition to this motor rhythm, another type of spontaneous synchronous activity appeared, characterized by much longer lasting episodes separated by longer time intervals. On E15.5, these long episodes disappeared. Short episodes were less numerous and more irregular except at the cervical level where a rhythm was occasionally observed. By E16.5, this cervical rhythm became more robust, whereas the lumbar level fell almost silent. Surprisingly, at E17.5, spontaneous activity resumed at caudal levels, now characterized by numerous erratic short episodes. A striking ontogenetic feature of spontaneous activity was the occurrence of long episodes only at E14.5. Although concomitant at all levels of the spinal cord, long episodes displayed different patterns along the spinal cord, with tonic firing at the thoracic level and rhythmic discharge with occasional sequences of left/right alternation at the lumbar level. Thus at E14.5, the originally synchronized network has started to segregate into more specialized subnetworks. In conclusion, this work suggests that ongoing spontaneous rhythms do not follow a smooth evolution during maturation, but rather undergo profound changes at very specific stages.

Spontaneous synchronous rhythmic activities are a common feature of immature neuronal networks. Although the mechanisms underlying such activities have been studied extensively, whether they might be controlled by modulatory information remains questionable. Here, we investigated the role of descending serotonergic (5-HT) inputs from the medulla to the spinal cord in the maturation of rhythmic activity. We found that in spinal cords maintained, as a whole, in organotypic culture without the medulla, the maturation of spontaneous activity is similar to that found in spinal cords developedin utero. Interestingly, in organotypic cultures without the medulla (i.e., devoid of descending inputs), numerous intraspinal neurons expressed 5-HT, unlike in spinal cords cultivated in the presence of the medulla or maturedin utero. We demonstrated that this 5-HT expression was specifically dependent on the absence of 5-HT fibers and was repressed by 5-HT itself via activation of 5-HT_1Areceptors. Finally, to verify whether the expression of 5-HT intraspinal neurons could compensate for the lack of descending 5-HT fibers and play a role in the development of spontaneous activity, we blocked the 5-HT synthesis usingp-chlorophenylalanine methyl ester in cultures devoid of the medulla. Surprisingly, we found that this pharmacological treatment did not prevent the development of spontaneous activity but accelerated the maturation of intraspinal inhibition at the studied stages. Together, our data indicate that descending 5-HT raphe inputs (1) repress the expression of spinal serotonergic neurons and (2) slow the maturation of inhibitory systems in mouse spinal cord.

Rhythmic electrical activity is a hallmark of the developing embryonic CNS and is required for proper development in addition to genetic programs. Neurotransmitter release contributes to the genesis of this activity. In the mouse spinal cord, this rhythmic activity occurs after embryonic day 11.5 (E11.5) as waves spreading along the entire cord. At E12.5, blocking glycine receptors alters the propagation of the rhythmic activity, but the cellular source of the glycine receptor agonist, the release mechanisms, and its function remain obscure. At this early stage, the presence of synaptic activity even remains unexplored. Using isolated embryonic spinal cord preparations and whole-cell patch-clamp recordings of identified motoneurons, we find that the first synaptic activity develops at E12.5 and is mainly GABAergic. Using a multiple approach including direct measurement of neurotransmitter release (i.e., outside-out sniffer technique), we also show that, between E12.5 and E14.5, the main source of glycine in the embryonic spinal cord is radial cell progenitors, also known to be involved in neuronal migration. We then demonstrate that radial cells can release glycine during synaptogenesis. This spontaneous non-neuronal glycine release can also be evoked by mechanical stimuli and occurs through volume-sensitive chloride channels. Finally, we find that basal glycine release upregulates the propagating spontaneous rhythmic activity by depolarizing immature neurons and by increasing membrane potential fluctuations. Our data raise the question of a new role of radial cells as secretory cells involved in the modulation of the spontaneous electrical activity of embryonic neuronal networks.