Spontaneous recovery of the injured higher olfactory center in the terrestrial slug limax

Abstract

Background: Of all organs and tissues in adult mammals, the brain shows the most limited regeneration and recovery after injury. This is one reason why treating neurological damage such as ischemic injury after stroke presents such a challenge. Here we report a novel mode of regeneration which the slug's cognitive center, the procerebrum, shows after surgical lesioning in the adult. It is well known that the land slug Limax possesses the capacity to demonstrate conditioned food aversion. This learning ability critically depends on the procerebrum, which is the higher olfactory center in the brain of the terrestrial mollusk.

Principal findings: In the present study, after a 1-month recovery period post-surgical lesioning of the procerebrum we investigated whether the brain of the slug shows recovery from damage. We found that learning ability, local field potential oscillation, and the number of cells in the procerebrum (PC) all recovered spontaneously within 1 month of bilateral lesioning of the PC. Moreover, neurogenesis was enhanced in the lesioned PC. However, memory acquired before the surgery could not be retrieved 1 month after surgery although the procerebrum had recovered from injury by this time, consistent with the notion that the procerebrum is the storage site of odor-aversion memory, or deeply involved in the memory recall process.

Significance: Our findings are the first to demonstrate that a brain region responsible for the associative memory of an adult organism can spontaneously reconstitute itself, and can recover its function following injury.

Figure 1. Learning ability recovers 1 month but not 1 week after a PC lesion.

(A) PC-lesioned slugs were unable to learn following a 1 week recovery period (n = 28 for sham-operated, n = 34 for PC-lesioned slug) with a significantly smaller percentage of lesioned slugs demonstrating odor-aversion conditioned behavior compared to that of the sham operated slugs. *P<0.001 by χ²-test. (B) PC-lesioned slugs were able to learn following a 1 month recovery period (n = 58 for sham-operated, n = 74 for PC-lesioned slugs) with a percentage of lesioned slugs demonstrating odor-aversion conditioning that was comparable to that of sham operated slugs.

Figure 2. Spontaneous LFP oscillation is restored within a 1 month recovery period.

(A) Periodic LFP oscillation in the sham-operated PC. (B) Infrequent and irregular burstings in the 1 week recovery PC group. (C) Periodic LFP oscillation in the 1 month recovery PC group. In (A)–(C), all the recordings were made from different slugs. (D) Quantitative data on oscillatory frequency (mean ± SE) show that the frequency was significantly lower 1 week following PC lesion as compared to sham-operated slugs, while 1 month following PC lesion LFP frequency was comparable to that of the sham-operated group. (E) Quantitative data on the coefficient of variation (CV) of bursting intervals (mean±SE) in the PC show that the bursting interval 1 week following a lesion was significantly more variable than the sham-operated PC group. While there was some recovery to stable oscillation in the 1 month recovery group, it did not return to a level comparable to that of the sham-operated group. sham, n = 64; 1 week, n = 30; 1 month, n = 64. *P<0.05, **P<0.001 by Student's t-test (two-tailed).

Figure 3. The size and number of cells in the PC is larger in the 1 month recovery group than in the 1 week recovery group.

Representative photographs of toluidine blue-stained sections of brains 1 week after a PC lesion (A–C), 1 month after a PC lesion (D–F), and 1 week after a sham operation (G). Only the cerebral ganglia are shown. Arrows indicate the PC. Scale bar, 500 µm. (H) A schematic drawing of the slug brain. The rectangle surrounds the cerebral ganglia including the PC. (I) Quantification of the sum of the bilateral PC area (1 week recovery, n = 34; 1 month recovery, n = 53; sham, n = 23) showed that the area is significantly diminished one week and one month following lesion, with some recovery in the one month recovery group. (J) The number of cells within the PC is larger in the 1 month recovery group (n = 19) than in the 1 week recovery group (n = 18). *P<0.001 by Student's t-test. A, anterior; P, posterior; R, right; L, left.

Figure 4. The relationship between the size of the PC and learning ability.

(A) The size of the PC is larger in the slugs that showed odor-aversion (n = 45) than in the slugs that did not demonstrate aversion when the recovery period was not considered (n = 30). *P<0.005 by Student's t-test. (B) There is no difference in the size of the PC between slugs that showed odor-aversion (n = 6) and slugs that did not show such aversion (n = 19) in the 1 week recovery group. (C) There is no difference in the size of the PC between the slugs that showed odor-aversion (n = 39) and those that did not show such aversion (n = 11) in the 1 month recovery group.

Figure 5. BrdU incorporation is enhanced in the lesioned PC.

(A) The experimental schedule. (B) Bright field microscopic image of the PC. Scale bar: 500 µm. (C) Nuclear DAPI staining. (D) Immunohistochemical staining of BrdU. An arrow indicates the lesioned (left) PC. (E) Magnified image of the left PC. The BrdU signal and the nuclear staining are green and blue, respectively. Arrows indicate pairs of juxtaposed BrdU labeled cells. Scale bar: 50 µm. (F) Quantification of the densities of the number of BrdU-positive cells in the lesioned (n = 8) and non-lesioned PC (n = 8). *P<0.005 by Student's t-test.

Figure 6. Double staining of BrdU and vGluT mRNA.

(A, B) Confocal images of sections dually stained by anti-BrdU antibody (green) and an antisense riboprobe against vGluT mRNA (red). Nuclei were stained with DAPI (blue). Arrows indicate BrdU-positive nuclei surrounded by a vGluT signal as demonstrated by in situ hybridization. (C, D) No signal is detected by a sense probe against vGluT mRNA. Magnifications of objective lenses were 63× (A, C) or 100× (B, D). Scale bars: 20 µm.

Figure 7. Odor-aversion memory was irreversibly lost after PC lesioning.

(A) Time schedule of the experiment in which the bilateral PC were lesioned 7 days after conditioning. (B) A significantly higher percentage of sham-operated slugs (n = 37) show odor-aversion behavior than do PC-lesioned slugs (n = 38). (C) Time schedule of the experiment in which the PC was bilaterally lesioned 14 days after conditioning. (D) Sham-operated slugs (n = 30) show better memory retrieval than PC-lesioned slugs (n = 31). *P<0.05, **P<0.001 by χ²-test.