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

Abstract

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.

Keywords: ablation phenotype; axon initial segment; grandmother neuron; predator–prey; startle response.

Fig. 1.

The giant axon of the M neuron is essential for escape performance. (A) Outline of the idea to use unilateral ablations: An action potential in one of the two M cells elicits powerful contraction of the contralateral body side, causing it to bend (Left). After unilateral ablation of the soma and axon (red), only bending commanded by the ablated contralateral cell body should be affected, but bending to the other side should not. Unspecific side effects of ablation could thus be tested by comparing escapes made toward that side in the unilaterally ablated larvae with escapes made by untreated siblings L, left; R, right. (B and C) Escape latency (B) and release probability (C) in untreated control larvae (n = 862 escapes from 18 larvae) and in ablated larvae for escapes that either could use the remaining cell (ipsi; n = 138 escapes from 5 larvae) or not (contra; n = 43 escapes from 5 larvae). Significance is as indicated (one-way ANOVA; latency: ***P < 0.0001 intact and ipsi vs. contra; P = 1 intact vs. ipsi; probability: ***P < 0.0001 intact vs. contra; *P = 0.02 ipsi vs. contra; P = 0.2 intact vs. ipsi). n.s., not significant. (B, Inset) Side-specific increase in latency in each individual unilaterally ablated larva.

Fig. 2.

All inputs that can rescue escape performance after loss of the M cell soma are sampled at the axon initial segment. (A) Stages of a degenerating M cell: still completely intact; soma absent but axon initial segment and axon intact (+AIS); without initial segment (−AIS) but remaining axon intact; also axon gone (−axon). (B–D) Latency (B), angular speed (C), and release probability (D) for escapes of larvae in which the escape would have recruited the (contralateral) cell in one of the stages shown in A. Boxplots are based on (from left to right) n = 258, 15, 22, 77 escapes from n = 9, 4, 7, 13 larvae for escape performance and n = 9, 6, 13, 15 larvae for probability, respectively. (E) Cumulative distribution function (CDF) of latency (Top) and of average angular speed (Bottom) of escapes with (+AIS; n = 406 escapes, n = 15 larvae) and without axon initial segment (−AIS; n = 100 escapes, n = 15 larvae) to show how axon state predicted escape performance. *P < 0.05; **P < 0.01 and ***P < 0.001, one-way ANOVA with Bonferroni-corrected t tests. n.s., not significant.

Fig. 3.

Direct test of the survival value of having the giant M neuron. (A–C) After a preparation phase, individual last-instar damselfly nymphs (a predator of zebrafish larvae) were combined with eight zebrafish larvae. Ablated larvae had either both M axons (A) or two cerebellar neurons (B) ablated. (A) The experimental groups (n = 11 groups) were mixed groups each of four M neuron-ablated larvae and four casper larvae. (B) Each procedural control group (n = 13 groups) contained four sham-ablated larvae and four nonablated casper larvae. (C) Additional background control groups contained either eight untreated Ca-Tol-056 larvae (n = 15 groups) or eight casper larvae (n = 8 groups). (D) Significant difference between capture rates in M cell-ablated larvae versus each of the three controls (***P < 0.0001, log-rank test) and no significant difference among all control, background, and sham-ablated fish (P > 0.12, log-rank test). (E and F) Analysis of the survivors at the end of the experiment (i.e., after 7 h) using their fluorescence signals confirms higher capture rates of M neuron-ablated fish (E; **P = 0.008, Mann–Whitney U test) but no higher capture rate of sham-ablated fish (F; P = 0.64, Mann–Whitney U test). Boxplots show capture rates as determined in the individual groups (medians are indicated by thick black horizontal lines).

Fig. 4.

Early loss of the M axon can never be compensated in later life. (A) Larvae with unilaterally ablated M axons (Fig. 1) grew up for 5 mo together with unablated siblings. After double-blind examination of their escape behavior, the fish were killed to check which, if any, M axon was missing. (B) As in the larvae, escapes were still predominantly to the side that could use the intact neuron (Student’s t test, ***P < 0.0001; n = 1,880 stimulations from 13 fish; see Fig. 1 for a definition of ipsi and contra). (C) Latency was also always significantly higher in those escapes, performed by the same individual zebrafish, that could not recruit an M cell (Mann–Whitney U test, ***P < 0.0001; n = 515 escapes from 13 fish). Circles with connected lines show median latency in the same individual fish when an M cell could be recruited or not. (D) Mean angular speed in escapes that could not recruit an M cell was significantly lower (Student’s t test, **P = 0.0089; n = 515 escapes from 13 fish). (E) Cumulative distribution function (CDF) of escape latency (<100 ms) for escapes that either could (n = 353 escapes) or could not (n = 162 escapes) recruit the remaining M axon clearly shows a shift in minimum latency even in the adult fish.