Repetitive nociceptive stimulation elicits complex behavioral changes in Hirudo: evidence of arousal and motivational adaptations

Abstract

Appropriate responses to real or potential damaging stimuli to the body (nociception) are critical to an animal's short- and long-term survival. The initial goal of this study was to examine habituation of withdrawal reflexes (whole-body and local shortening) to repeated mechanical nociceptive stimuli (needle pokes) in the medicinal leech, Hirudo verbana, and assess whether injury altered habituation to these nociceptive stimuli. While repeated needle pokes did reduce shortening in H. verbana, a second set of behavior changes was observed. Specifically, animals began to evade subsequent stimuli by either hiding their posterior sucker underneath adjacent body segments or engaging in locomotion (crawling). Animals differed in terms of how quickly they adopted evasion behaviors during repeated stimulation, exhibiting a multi-modal distribution for early, intermediate and late evaders. Prior injury had a profound effect on this transition, decreasing the time frame in which animals began to carry out evasion and increasing the magnitude of these evasion behaviors (more locomotory evasion). The data indicate the presence in Hirudo of a complex and adaptive defensive arousal process to avoid noxious stimuli that is influenced by differences in internal states, prior experience with injury of the stimulated areas, and possibly learning-based processes.

Keywords: Arousal; Invertebrate; Leech; Nociception; Pain; Sensitization.

Fig. 1.

Experimental protocols. (A) Hirudo verbana were placed in the testing chamber (Petri dish) for 30 min prior to undergoing testing. During testing, 40 needle pokes at a 1 min intertrial interval were delivered to the posterior sucker. (B) In experiments that tested for the effects of injury, a T-pin was used to pierce the posterior sucker while the animals were lightly anesthetized in an ice-lined dish. The animals were then transferred to the testing chamber for 30 min followed by testing. (C) Four distinct behaviors were observed during testing. Local shortening or whole-body shortening in response to a needle poke, and then sucker evasion or locomotory evasion when the animal was due to be poked (digital art by J.D.).

Fig. 2.

Behavioral effects of repeated nociceptive stimulation. (A) Shortening reflex scores decreased across trial blocks. (B) Evasion scores increased across trial blocks. Asterisks indicate a statistically significant difference relative to trial block 1. Asterisks indicate a significant difference based on one-way ANOVA (**P<0.005, ***P<0.001 and ****P<0.0001). (C) Frequency distribution of the number of pokes delivered during testing indicates how quickly evasion behaviors were initiated across the 58 animals tested. A multi-modal distribution was observed indicating the presence of three sub-populations that utilize a distinct behavioral strategy: early, intermediate and late evaders. (D) Raster plot showing the pattern of pokes per trial block for all 58 animals with the proposed early, intermediate and late evader designations indicated on the right. Red shades indicate fewer or no pokes per trial block and blue shades indicate more pokes per trial block. (E) Raster plot showing the distribution of shortening (blue shades) and evasion (red shades) responses over all 40 trials for each of the 58 animals tested. White indicates no response for that trial. The rectangle at trial 20 indicates the data used for the frequency distribution analysis in F. (F) Frequency distribution of behavioral responses for early, intermediate and late evaders during trial 20 (indicated by the rectangle in E). Asterisks indicate a significant difference in distribution based on Kolmogorov–Smirnov statistics (***P<0.001); n.s., not significant. LE, locomotory evasion; SE, sucker evasion; NR, no response; LS, local shortening; and WS, whole-body shortening.

Fig. 3.

Distribution of behaviors from all 58 animals tested. (A) Non-injured trial block 1. (B) Non-injured trial block 8. LE, locomotory evasion; SE, sucker evasion; NR, no response; LS, local shortening; and WS, whole-body shortening.

Fig. 4.

Effect of injury on shortening and evasion behaviors. (A) H. verbana tested on the same day as injury (injured–day 1) exhibited significantly greater evasion scores compared with non-injured animals. This effect persisted 7 days later (injured–day 7), although the increase in evasion scores was reduced compared with that for the injured–day 1 group. (B) Pattern of decreases in the shortening behavior scores between the three groups. (C) Injured–day 1 animals exhibited early-evader behavior based on the distribution of the number of needle pokes delivered in that group compared with the subset of non-injured animals analyzed. The distribution in the injured group was different from that of the non-injured group based on Kolmogorov–Smirnov analysis (see Results). (D) Injured–day 1 animals received fewer needle pokes (because they engaged in evasion earlier) compared with non-injured animals based on Mann–Whitney analysis. (E) The distribution of needle pokes in the injured–day 1 group was not different from that of the injured–day 7 group based on Kolmogorov–Smirnov analysis (see Results). (F) There was no difference in the number of needle pokes delivered between the injured–day 1 and injured–day 7 animals. (G–I) Raster plots comparing the distribution of shortening and evasion behaviors over time (trial) for the non-injured (G), injured–day 1 (H) and injured–day 7 (I) animals. (G) In non-injured animals, a range of early, intermediate and late evaders was observed. (H) In injured–day 1 animals, it appeared that only early evaders were observed. (I) In the injured–day 7 animals, most appeared to be early evaders with a small number of intermediate evaders. LE, locomotory evasion; SE, sucker evasion; NR, no response; LS, local shortening; and WS, whole-body shortening. Asterisks indicate a significant difference (*P<0.05, **P<0.005 and ****P<0.0001).

Fig. 5.

Proportion of different reflexive shortening and persistent evasion behaviors observed in injured–day 1 and injured–day 7 groups. (A) In injured–day 1 leeches during trial block 1, a mix of behaviors was observed, with the shortening reflexes being more predominant. (B) By trial block 8, only evasion behaviors were observed and there was an equal proportion of sucker evasion and locomotory evasion. (C) In injured–day 7 animals during trial block 1, a similar mixture of shortening and evasion behaviors was observed. (D) During trial block 8, these animals again exhibited more evasion behaviors, but shortening responses were also observed, unlike in the injured–day1 group. LE, locomotory evasion; SE, sucker evasion; NR, no response; LS, local shortening; and WS, whole-body shortening.

Fig. 6.

Effects of injury plus retesting. (A) H. verbana were injured and tested on day 1 and then retested on day 7. (B) The evasion scores were reduced in the injured–retested day 7 group compared with the injured–day 1 animals. (C) No differences were observed in the shortening reflex scores between the two groups. (D) No differences were observed in the frequency distribution of pokes delivered between the injured–day 1 and injured–retested day 7 groups. (E) There were no differences in the number of stimuli delivered on days 1 and 7. (F) In the injured–retested day 7 group during trial block 1, shortening behaviors dominated, but there were noticeably more ‘no responses’ and few evasion behaviors. (G) By trial block 8, only evasion behaviors were observed, with sucker evasion being the most prominent. LE, locomotory evasion; SE, sucker evasion; NR, no response; LS, local shortening; and WS, whole-body shortening.