Zebrafish neurophysiology: 'swimming' in sync with gap junctions

Developmental neurobiologists use a wide variety of invertebrate and vertebrate preparations to explore the mechanisms used by neurons to establish precise, functional connections within identifiable neural circuits. Despite prolific research, there is a paucity of data on the in vivo physiological mechanisms underlying the electrogenesis of patterned neural activity during embryonic stages of vertebrate development. In recent years, whole-cell and single-channel patch-clamp methods have been used successfully to gain insights into some of the mechanisms that contribute to the early formation of functional neural circuits in living zebrafish embryos. These embryos are transparent, and the clear visibility and relative sparsity of the identified spinal motoneurons and interneurons that generate rhythmic movements facilitate the detailed electro-physiological analysis of spinal networks in the living embryo. Zebrafish embryos develop very rapidly: the spinal neural circuits that are necessary and sufficient for generating rhythmic, spontaneous contractions of the embryonic trunk are functional as early as 17 hours post-fertilization. Ultimately, these spinal circuits, and the rhythmic movements generated by their activation, elicit the familiar swimming action of these fish. Synchronous rhythmic activity of neurons is believed to be important for the establishment and consolidation of proper synaptic connections. One fundamental question is whether electrical synapses play important roles in the synchronization of neural activity in vivo during early development. Saint-Amant and Drapeau 1 examined the patterns of electrical activity of identified spinal interneurons and motoneurons at the onset of motor activity in early zebrafish embryos (20?24 hours post-fertilization). Using paired recordings from connected spinal interneurons in vivo, they report that electrotonic coupling (through gap junctions) between some neurons is required to synchronize rhythmic electrical activity in these spinal networks. Pharmacological blockade of glutamatergic synaptic transmission did not affect the spontaneous generation of periodic membrane depolarizations in some of these coupled neurons. Indeed, complete blockade of chemical transmission (by injection of botulinum neurotoxin type B) paralyzed embryos but spared these periodic depolarizations. In contrast, blockers of gap junctions, such as heptanol and cytoplasmic acidification, abolished the periodic depolarizations. More importantly, the periodic depolarizations of some of these pairs of electrotonically coupled neurons were correlated with coactivity in the connected cells, indicating that these depolarizations result from synchronized network activity among these neurons. Further studies by the authors showed that gap-junctional generation of periodic depolarizations involves activation of calcium-dependent potassium channels. These findings reveal the importance of electrotonic coupling in the generation of rhythmic neuronal network activity in an early embryo. They describe, perhaps, the earliest developmental time at which synchronized neural activity has been recorded in identified spinal neurons of a living vertebrate embryo. It will be interesting to record the activities of coupled spinal neurons in mutant zebrafish embryos with defects in rhythmic movements. Such studies might identify the genes and proteins that are important for the in vivo regulation of electrotonic coupling and synapse formation during early neural development.