In the cricket species Gryllus bimaculatus, removing a foreleg severs the connections between the interneurons of the prothoracic ganglion and the auditory afferents of the hearing organ located on the tibia of the leg, disrupting hearing on that side of the cricket. Loss of input to these interneurons is only temporary, for they are able to extend across the midline of the prothoracic ganglion and form functional synapses with the intact auditory afferents on the contralateral side. To determine potential candidates involved in this regenerative mechanism, a class of guidance molecules called Semaphorins was investigated. Semaphorins are known to be involved in the guidance of growing axons during early development, and could have a potential role in a system’s response to neural injury. The profound morphological changes and functional recovery observed in the prothoracic ganglion after deafferentation makes Semaphorins a good candidate for this mechanism. Thus, expression of Semaphorin 1a and 2a were quantified using quantitative-PCR (Q-PCR) to determine if their levels change in response to deafferentation.
Figure 1: Overall higher expression of Sema1a than Sema2a in the prothoracic ganglion.Sema1a was had higher expression levels than Sema2a, and was significantly higher in non-deafferented crickets and 7 day deafferents. Sema1a levels did not significantly change with deafferentation. Sema2a expression was higher at 24 hours after deafferentation compared to non-deafferents, however this wasn’t significant. The y-axis shows relative mRNA expression for either Sema1a or Sema2a. The non-deafferents and deafferents for each target are indicated on the x-axis. The error bars indicate standard deviation.
Q-PCR was done with cDNA synthesized from the tissue of the prothoracic ganglia in deafferented crickets. Sema1a and Sema2a levels were quantified 24 hours and 7 days after deafferentation in male crickets. It was determined that Sema1a had higher expression levels than Sema2a in both control and deafferent male animals (Fig. 1); however Sema1a levels did not change significantly with deafferentation. Sema2a levels increased 24 hours after deafferentation, however this increase wasn’t significant. A Sema2a timecourse done previously from 30 hours to 7 days after deafferentation found significant upregulation at 30 hours, but it wasn’t controlled well and did not account for potential variability between male and female crickets. Thus, the timecourse was repeated except with better controls and separating cDNA by gender. Sema2a was significantly upregulated in males at 30hours (Fig. 2A) and remained upregulated up to 5 days after deafferentation. Although not significant, Sema2a levels in females increased to similar levels as in males at 30 hours deafferentation and appears to be further upregulated at 3 days (Fig. 2B), however the levels came down faster compared to males, being almost at control levels by 5 days (Fig. 2C).
Thus, Sema2a appears to have a role in the observed compensatory growth after deafferentation due to its upregulation as shown by qPCR. Additionally, it appears that Sema2a remains upregulated longer in males than in females. Sema1a is more highly expressed overall, however a more extensive time course will need to be done to determine whether its levels also change with deafferentation.
Figure 2: Upregulation of Sema2a after deafferentation. A. Sema2a was significantly upregulated 30 hours after deafferentation. Sema2a increased in females as well, but this wasn’t significant due to high variability in the female data. B. Although it appears that Sema2a remains upregulated at 3 days, high variability in the male data resulted in no significant increase in Sema2a levels. There was only one female cDNA sample at 3 days after deafferentation, so no statistical significance could be determined. C. A significant upregulation in Sema2a was seen 5 days after deafferentation in male crickets despite lack of significance at 3 days. Sema2a levels are almost at control levels in 5 day female crickets. The y-axis shows relative Sema2a mRNA expression. The non-deafferent males and females are indicated on the x-axis as black and gray bars respectively, while deafferent males and females are indicated as red and blue respectively.
Faculty Mentor: Hadley Horch
Funded by the Paller Neuroscience Research Fellowship