Removing a single neuron in a vertebrate brain forever abolishes an essential behavior

The giant Mauthner (M) cell is the largest neuron known in the vertebrate brain. It has enabled major breakthroughs in neuroscience but its ultimate function remains surprisingly unclear: An actual survival value of M cell-mediated escapes has never been supported experimentally and ablating the cell repeatedly failed to eliminate all rapid escapes, suggesting that escapes can equally well be driven by smaller neurons. Here we applied techniques to simultaneously measure escape performance and the state of the giant M axon over an extended period following ablation of its soma. We discovered that the axon survives remarkably long and remains still fully capable of driving rapid escape behavior. By unilaterally removing one of the two M axons and comparing escapes in the same individual that could or could not recruit an M axon, we show that the giant M axon is essential for rapid escapes and that its loss means that rapid escapes are also lost forever. This allowed us to directly test the survival value of the M cell-mediated escapes and to show that the absence of this giant neuron directly affects survival in encounters with a natural predator. These findings not only offer a surprising solution to an old puzzle but demonstrate that even complex brains can trust vital functions to individual neurons. Our findings suggest that mechanisms must have evolved in parallel with the unique significance of these neurons to keep their axons alive and connected.

Mechanistic insights into the interactions of dynein regulator Ndel1 with neuronal ankyrins and implications in polarity maintenance

Ankyrin-G (AnkG), a highly enriched scaffold protein in the axon initial segment (AIS) of neurons, functions to maintain axonal polarity and the integrity of the AIS. At the AIS, AnkG regulates selective intracellular cargo trafficking between soma and axons via interaction with the dynein regulator protein Ndel1, but the molecular mechanism underlying this binding remains elusive. Here we report that Ndel1’s C-terminal coiled-coil region (CT-CC) binds to giant neuron-specific insertion regions present in both AnkG and AnkB with 2:1 stoichiometry. The high-resolution crystal structure of AnkB in complex with Ndel1 CT-CC revealed the detailed molecular basis governing the AnkB/Ndel1 complex formation. Mechanistically, AnkB binds with Ndel1 by forming a stable 5-helix bundle dominated by hydrophobic interactions spread across 6 distinct interaction layers. Moreover, we found that AnkG is essential for Ndel1 accumulation at the AIS. Finally, we found that cargo sorting at the AIS can be disrupted by blocking the AnkG/Ndel1 complex formation using a peptide designed based on our structural data. Collectively, the atomic structure of the AnkB/Ndel1 complex together with studies of cargo sorting through the AIS establish the mechanistic basis for AnkG/Ndel1 complex formation and for the maintenance of axonal polarity. Our study will also be valuable for future studies of the interaction between AnkB and Ndel1 perhaps at distal axonal cargo transport.

Differential Contributions of Shaker and Shab K+ Currents to Neuronal Firing Patterns in Drosophila

Different K+ currents participate in generating neuronal firing patterns. The Drosophila embryonic “giant” neuron culture system has facilitated current- and voltage-clamp recordings to correlate distinct excitability patterns with the underlying K+ currents and to delineate the mutational effects of identified K+ channels. Mutations of Sh and Shab K+ channels removed part of inactivating IA and sustained IK, respectively, and the remaining IA and IK revealed the properties of their counterparts, e.g., Shal and Shaw channels. Neuronal subsets displaying the delayed, tonic, adaptive, and damping spike patterns were characterized by different profiles of K+ current voltage dependence and kinetics and by differential mutational effects. Shab channels regulated membrane repolarization and repetitive firing over hundreds of milliseconds, and Shab neurons showed a gradual decline in repolarization during current injection and their spike activities became limited to high-frequency, damping firing. In contrast, Sh channels acted on events within tens of milliseconds, and Sh mutations broadened spikes and reduced firing rates without eliminating any categories of firing patterns. However, removing both Sh and Shal IA by 4-aminopyridine converted the delayed to damping firing pattern, demonstrating their actions in regulating spike initiation. Specific blockade of Shab IK by quinidine mimicked the Shab phenotypes and converted tonic firing to a damping pattern. These conversions suggest a hierarchy of complexity in K+ current interactions underlying different firing patterns. Different lineage-defined neuronal subsets, identifiable by employing the GAL4-UAS system, displayed different profiles of spike properties and K+ current compositions, providing opportunities for mutational analysis in functionally specialized neurons.

Nitric Oxide Potentiates cAMP-Gated Cation Current in Feeding Neurons of Pleurobranchaea californica Independent of cAMP and cGMP Signaling Pathways

Critical roles for nitric oxide (NO) in regulating cell and tissue physiology are broadly appreciated, but aspects remain to be explored. In the mollusk Pleurobranchaea, NO synthase activity is high in CNS ganglia containing motor networks for feeding and locomotion, where a cAMP-gated cation current ( INa,cAMP) is also prominent in many neurons. We examined effects of NO on INa,cAMP using voltage-clamp methods developed to analyze cAMP signaling in the live neuron, focusing on the identified metacerebral giant neuron of the feeding network. NO donors enhanced the INa,cAMP response to injected cAMP by an averaged 85%. In dose-response measures, NO increased the current stimulated by cAMP injection without altering either apparent cAMP binding affinity or cooperativity of current activation. NO did not detectably alter levels of native cAMP or synthesis or degradation rates as observable in both current saturation and decay rate of INa,cAMP responses to cAMP injection. NO actions were not exerted by cGMP signaling, as they were not mimicked by cGMP analogue nor blocked by inhibitors of guanylate cyclase and protein kinase G. NO potentiation of INa,cAMP was broadly distributed among many other neurons of the feeding motor network in the buccal ganglion. However, NO did not affect a second type of INa,cAMP found in locomotor neurons of the pedal ganglia. These results suggest that NO acts through a novel mechanism to regulate the gain of cAMP-dependent neuromodulatory pathways that activate INa,cAMP and may thereby affect the set points of feeding network excitability and reactivity to exogenous input.