Histology 3500 Blog 2: Paper Review

Histology 3500 Blog 2: Paper Review

Paper Reviewed: Molecular basis of infrared detection by snakes

Authors: Elena O. Gracheva, Nicholas T. Ingolia, Yvonne M. Kelly, Julio F. Cordero-Morales, Gunther Hollopeter,Alexander T. Chesler, Elda E. Sanchez, John C. Perez, Jonathan S. Weissman & David Julius

Paper can viewed on Pubmed at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855400/

Introduction:

          This paper examines the 'thermal imaging' process that enables snakes to capture their prey in a quick and efficient manner. In particular, the paper examines the Western Diamondback Rattlesnake (Crotalus atrox), a crolatine snake with an unmatched thermal detection ability. This is accomplished through the use of loreal pit organs present between the eye and nostril on the head of the snake (shown in  Figure 1a). Within the pit is a thin membrane that is heavily innervated with trigeminal nerve fibers (as shown in Figure 1b). These fibres transmit the thermoreceptive signal from the pit organ to the optical center of the brain where, in tandem with other signals, forms the 'thermal image' that assists in the snakes ability to hunt for prey. Some groups of snakes (boas and pythons) can detect infrared radiation through the use of labial pits located along the snout of the snake, but are not as sensitive as loreal pits partially due to a lower concentration of trigeminal nerve fibres. This lower sensitivity suggested a molecular difference between loreal pit organs and labial pit organs. This paper attempts to determine the exact molecule that detects thermal stimulus,  whether this molecule is found within the inner membrane of the pit organ, or if it is closer to the nerve, and if there are molecular differences between molecular identity in labial and loreal pit detection.

          To determine the molecule that acts as the thermal receptor transcriptome profiling methods (Illumina Sequencing, In-situ hybridization chemistry, etc.) were used to identify pit-enriched sensory transducers. One such molecule that was derived using these methods was an orthologue to the mammalian "wasabi receptor" TRPA1. This channel was found to be highly upregulated near the trigeminal neurons that innervate the pit organ and show some heat sensitive response. Evolutionary divergance has made this protein into a heat sensitive protein used in the pit organs within snakes, while in mammals, the same protein is used to detect chemical irritants and inflammatory agents. 

Pit Viper Specialisation

          Sensory systems often have specialised cells that detect a stimulus which then transmit the signal to nearby nerve fibres, though this is not true for the somatosensory system, where the nerve endings themselves are detect the stimuli. Trigeminal ganglia (TG) of snakes with pit organs are larger than those of mammals, and were thought to be able to express proteins relevant to the pit function when compared to other ganglia such as the dorsal root ganglia (DRG). Mammals have TG and DRG expression profiles being relatively similar, so any significant differences in the expression profile may be unique to snakes and suggest a functional use in thermal detection. As shown in Figure 1c, a paired comparison of rattlesnake TG and DRG transcriptomes, the gene for TRPA1 is highly expressed within the TG tissue, but other members of the TRP gene family showed normal levels of expression in both TG and DRG tissues. It was also found that when comparisons were done with non-pit species of snakes (Elaphe obsoleta lindheimeri and Masticophis flagellum testaceus, were used in the comparison for Figure 1d), TRPA1 was not upregulated in TG tissue as it was in snakes that use pit organs.

Unique expression of TRPA1 in viper TG

          Ganglia involved with the vertebrate somatosensory system have diverse sub-populations of neurons, all with different functions. Typically, neurons with the largest soma diameter are involved in sensitive detections (lighter touches) while those with smaller soma diameters are involved in the detection of more significant stimuli. TRPA1 in mammals are typically expressed in the same neurons that also express TRPV1 (25% of all neurons in the system), but this is not the case in rattlesnake systems. In rattlesnakes, TRPA1 is expressed within neurons of the TG that are considered a medium or large size, as shown in Figure 2a and 2b. TRPV1 was found to be expressed in lower amounts within the TG and DRG neurons when compared to the mammalian profile (a rodent used in Figure 2), further suggesting that TRPA1 has evolved within the pit organs for the use of thermal detection. 

Snake TRPA1 is a heat-activated channel

          Mammalian TRPA1 is activated by allyl isothiocyanate (AITC), an agent from wasabi (hence the earlier name of wasabi receptor) and other mustard plants. Rattlesnake and rat homologues of TRPA1 share 81% identity with each other and share 63% identity with human TRPA1, and contain three heavily conserved cysteines present on the N-terminus of the protein, which are necessary for activation.

           If TRPA1 is critical to thermal sensing, it should be able to be activated within a temperature range that is similar to that of the pit organ, which detects infrared radiation at temperatures greater than 30 degrees Celsius. As shown in the calcium imaging data from Figure 3a, rattlesnake cells with TRPA1 channels did become active above 30 degree temperatures. It was also found that rat snake (Elaphe obsoleta lindheimeri) was also sensitive to heat, but required a higher threshold in order to be activated. Further examination using heat-evoked membrane currents in voltage-clamped Xenopus eggs expressing TRPA1 (shown in Figure 3b and 3c) also supported this finding with rattlesnake TRPA1 becoming active at lower temperatures than those in the rat snake. This makes sense as with a lower activation temperature, TRPA1 can conduct signals to the brain upon detection to produce the "thermal image" at lower temperatures, making it easier to track and capture prey with lower body temperatures. TRPA1 within non-pit snakes (such as the rat snake), may work alongside TRPV1 to act as somatic or cutaneous thermosensors , rather than be used for hunting (where sensitivity would be critical to have). The transcriptome data, in conjunction with anatomical data gathered, suggest that TRPA1 is indeed the molecule responsible for thermosensation within pit organs. 

Ancient snakes use TRPA1 to sense infrared radiation

          While it has been shown that TRPA1 is used within the loreal pits of Crolatine snakes to detect heat, it was unknown if this was also true for older snake lineages that used labial pits, such as boas (the amazon tree boa Corallus hortulanus in this study) and pythons (the royal python Python regius in this study). As shown in Figure 4a and 4b, TRPA1 was shown to be a significantly expressed transcript in the TG than the DRG of both snake species, though DRG expression was still greater than those found within the rattlesnake. It is also observed in Figure 4a that TRPV1 is not expressed beyond expected background levels in pythons, meaning that another molecule may be involved in somatic thermosensitivity in this group. Figure 4c shows a dendrogram analysis of snake TRPA1 channels, which shows the close relationships between these channels. The close relationship between boas and pythons within this phylogenic tree also supports the hypothesis that these groups split off from other snakes to form their own lineage and share commonalities not present in modern snakes. Figure 4d shows that both boa and python derived TRPA1 proteins retain the sensitivity to AITC observed in rattlesnakes though they have a higher heat threshold than that found in rattlesnakes, matching the lower sensitivities recorded for these snake types.
Endogenous TRPA1 subserves infrared detection

          With determination that TRPA1 does show a similar expression level, location of expression and response to stimuli across snakes with both labial and loreal pits, the study still needed to determine that TRPA1 is involved in infrared detection. To test if this was the case pythons were used, due to their relative safety (non-venomous), and similarity to rattlesnakes in TRPA1 expression as it is done mainly through large and medium sized neurons within the TG, as shown in Figure 5a. Comparing to the rat snake (acting as a control), where TRPA1 was expressed in a smaller proportion of the total TG, and only expressed within the smaller neurons. TRPV1 expression within the rat snake was also found to be significant, suggesting a potential role in heat sensation along with TRPA1.  Rat snake neurons also show lower sensitivity to AITC and heat sensitivity when compared to python species, as shown in Figure 5b, where a higher threshold was needed to induce activation of TRPA1. These results suggest that TRPA1 is responsible for infrared and somatic heat detection not only in rattlesnakes, as described earlier, but for all pit bearing species, while TRPA1 and TRPV1 are responsible for somatic heat detection within non-pit bearing species. Patch clamp recording verified the presence of heat sensitive membrane currents within snake neurons. Figure 5c shows these currents, with sensitivities to heat and AITC confirmed while being blocked by cold and ruthenium red (an agent that typically inhibits calcium ion transportation). This provides further evidence that TRPA1 is the molecule responsible for infrared thermal detection within pit bearing snakes (with some neurons being the exception as shown in Figure 5c on the right, though these neurons were a minority among the population.

Paper Critique

Q: Was the paper an enjoyable read?

A: Yes, I think so. All information necessary to understanding the topic was given clearly in the introduction, and the significance of the discovery (understanding a molecule which relates to a sensory system unknown to humans, yet was still present within our genome, just for a different purpose) was made vary clear. Figures were all colourful and interesting (though I do with that the histological slides in Figure 2 were in colour instead of black and white) and conveyed information clearly. 

Q: Do the results support the authors claims?

A; Yes, they do. With the initial hypothesis of TRPA1 being the molecule that enables thermal detection in snakes, they underwent several rigorous tests in order to prove it, and moving through several logical questions (does it have correct thermal activity, how is it expressed in non-pit snakes, how is it expressed in snakes with different pit types, why might the expression be different, etc.) that someone may ask in order to attempt to disprove their findings.

Q: Are the experiments well conducted and appropriate?

A; Yes, they are. Proper model organisms were chosen for each method, and in depth details (exact sequencer used for next generation sequencing, in-situ hybridization histochemistry, channel cloning, calcium imaging, sensory neuron culture, oocyte electrophysiology and patch clamp recording) were found within an additional appendix at the end of the paper. The methods were appropriate, being able to create figures that were easy to understand and supported the hypothesis. 

Q: Are the figures adequate?

A; Yes, all figures were well made and easy to understand, supporting the results. Figures were also organised well with colour to help make the paper more presentable as a whole.

Q: Negative Critiques

A: One negative of this paper is the reliance on supplementary figures not placed within the paper itself. A second critique that I had within this paper was the lack of histological slides to help describe the normal state of each pit organ type. While some hand drawn diagrams were presented to show the structure of the pit organ, some histological examples would have been beneficial.