Dendrite regeneration mediates functional recovery after complete dendrite removal

Abstract

Unlike many cell types, neurons are not typically replaced if damaged. Therefore, regeneration of damaged cellular domains is critical for maintenance of neuronal function. While axon regeneration has been documented for several hundred years, it has only recently become possible to determine whether neurons respond to dendrite removal with regeneration. Regrowth of dendrite arbors has been documented in invertebrate and vertebrate model systems, but whether it leads to functional restoration of a circuit remains unknown. To test whether dendrite regeneration restores function, we used larval Drosophila nociceptive neurons. Their dendrites detect noxious stimuli to initiate escape behavior. Previous studies of Drosophila sensory neurons have shown that dendrites of single neurons regrow after laser severing. We removed dendrites from 16 neurons per animal to clear most of the dorsal surface of nociceptive innervation. As expected, this reduced aversive responses to noxious touch. Surprisingly, behavior was completely restored 24 ​h after injury, at the stage when dendrite regeneration has begun, but the new arbor has only covered a small portion of its former territory. This behavioral recovery required regenerative outgrowth as it was eliminated in a genetic background in which new growth is blocked. We conclude that dendrite regeneration can restore behavior.

Keywords: Dendrite growth; Dendrite regeneration; Functional recovery; Nociception.

Figure 1.

Laser severing of dendrites or axons of 16 ddaC neurons. (A) A dorsal overview of a larva expressing CD4-tdGFP in class IV neurons before injury. (B) Dendrites of 16 ddaC neurons in an animal expressing the control RNAi hairpin were severed and allowed to regenerate for 24 hours; HPD stands for hours post dendrotomy. (C) Axons of 16 ddaC neurons were severed and the animal was imaged 24 hours later; HPA stands for hours post axotomy. Red arrows indicate severed and degenerating axons, green arrows indicate intact axons in the head. (D) Sec6 RNAi hairpins were expressed in Class IV neurons and dendrites were severed from 16 ddaC neurons and imaged 24 hours later as in (B).

Figure 2.

Larval movements in response to 46° probe. (A) Sequence of larval movement during a stereotyped roll. The tail and head curl in (first image on left), and the body rotates in the opposite direction (2^nd, 3^rd, and 4^th images) until right side up again (5^th image). (B) Example images of aversive behaviors quantified in figures 3 and 4. These include: rapid and simultaneous contraction of the head and/or tail (1^st image); repetitive backwards crawling (2^nd image); lifting and flailing of the head or tail (3^rd and 4^th images); or an incomplete roll (last image).

Figure 3.

Class IV dendrites can regenerate and regain nociceptive capability. (A) An example neuron expressing control RNAi before injury (left), 4h post dendrotomy (HPD) (middle), and after 24h of regeneration (right). Red boxes indicate extent of dendrite arbor size at each time point. (B) The latency to roll from the onset of stimulus is graphed for uninjured, dendrite cut 4 and 24 hours after injury and axon cut 6 and 24 hours after injury. Any larvae that did not roll were not included in this graph but are summarized in the table above. (C) The latencies in (B) were binned into <2s, 2–4s, 4–6s, and 6–10s to compare proportions for each condition. (D) % of larvae displaying any immediate aversive reaction to the stimulus is graphed for each condition. Numbers in bars represent numbers of larvae. ***, p<.001 with Mann-Whitney test.

Figure 4.

Sec6 RNAi neurons do not regenerate or regain nociception. (A) An example Sec6 RNAi-expressing neuron before injury (left), 4h post dendrotomy (HPD) (middle), and after 24h of regeneration (right). Red boxes indicate extent of dendrite arbor size at each time point. (B) Latency to roll from stimulus onset is graphed for uninjured, dendrite cut 4 and 24 hours after injury, and axon cut 6 and 24 hours after injury. Any larvae that did not roll are not graphed but are summarized in the table above the graph. (C) The latencies to roll were binned into categories, <2s, 2–4s, 4–6s, and 6–10s, to compare proportions for each condition. (D) % of larvae displaying any immediate aversive reactions to the stimulus are graphed. Numbers in bars represent numbers of larvae. ***, p<.001 with Mann-Whitney test.