“Molecular basis of infrared detection by snakes”

Article link: http://www.nature.com.qe2a-proxy.mun.ca/nature/journal/v464/n7291/full/nature08943.html

The use of infrared information by snakes to detect endothermic (warm-blooded) prey is a characteristic that is unique to only specific snake species (Krochmal et al. 2004). The signature group that have this ability are the crotaline snakes, commonly known as the pit vipers (Krochmal et al. 2004). These snakes contain a specialized sensory organ, known as pit organs, facial pits, or labial pits, which is essentially provides the snake with “heat vision” (Chen et al. 2012). The pit organs are innervated by nerves with a direct connection to the brain, and are able to send thermal profiles of endothermic animals to the brain that are subsequently superimposed with the snake’s visual images (Amemiya et al. 1995). This enables the snake to track its prey with a high degree of precision and speed. Studies have shown that the pit organs effectively allow infrared detection of prey at a distance up to 1 m (Gracheva et al. 2010). 

Figure 1: Rattlesnake head showing location of nostril (black arrow) and pit organ (red arrow) (left image). Pit organ schematic depicting the innervation of the pit membrane by trigeminal nerve fibers (right image).

Although prey acquisition appears to be one of the primary functions of the pit organs, the actual mechanism of how these unique sensory organs detect infrared stimuli and convert them into neural impulses remains elusive (Gracheva et al. 2010). “Molecular basis of infrared detection by snakes” is a journal article that pertains to a study conducted by Gracheva et al. 2010, which attempts to characterize the molecular underlying responsible for thermal detection by the pit organs. The authors propose several questions regarding the mechanism and identity of infrared detection. For instance, it is uncertain whether the infrared sensors are contained within the actual pit membrane, or if these sensors are expressed by the nearby nerve fibers (Gracheva et al. 2010). It is possible that the infrared receptors directly detect photons, which then lead to a photochemical stimulus, or it may be that the receptors are indirectly activated when actual pit tissue is heated (Gracheva et al. 2010). Another question at hand is whether all snakes which contain pit organs (crotaline snakes) and similar structures (boid snakes) use the same method for detecting infrared radiation (Gracheva et al. 2010).

In this particular study, the researchers used transcriptome profiling allowing them to identify a candidate infrared receptor, TRPA1 (transient receptor potential A1). This extremely heat-sensitive ion channel is highly abundant in the trigeminal nuclear masses (TNMs) which innervates the pit organ (Gracheva et al. 2010). In mammals, the TRPA1 receptor acts primarily as a detector of chemical and inflammatory agents, whereas in snakes it appears to have a specialized sensory function. The authors state that snakes, especially venomous pit vipers, are difficult subjects for physiological studies with limited genomic information, impeding molecular studies (Gracheva et al. 2010).

Exploiting specialization of pit vipers

The pit organs are innervated by three branches of the trigeminal ganglia (TG), which serves as a direct connection to the brain (Moon, 2011). In pit-bearing snakes, the trigeminal ganglia is unusually large compared to mammals, and is hypothesized by the authors to express proteins which are specialized for the pit organ function (Gracheva et al. 2010). The expression of these proteins will by convention be less abundant in other ganglia, such as the dorsal root ganglion (DRG). The researchers compared the transcriptomes from rattlesnake (a pit viper) trigeminal ganglia and dorsal root ganglion, which indicated the presence of a single gene (from the TG transcriptome) encoding a TRPA1 orthologue (Gracheva et al. 2010). Other members of the TRP channel family however, showed similar expression profiles in both ganglia. The TRPA1 expression was enhanced 400-fold in the TG (Gracheva et al. 2010).

It is worth noting that if the TRPA1 serves a unique function in pit-bearing snakes then there should be no discrepancy in TRPA1 expression between the TG and the DRG in non-pit species (i.e. non pit-bearing snakes should not show a higher expression of TRPA1 in the TG compared to the DRG). The authors showed that there was indeed no expression difference of TRPA1 in Texas rat and Western coachwhip snakes (both non pit species) (Gracheva et al. 2010). Furthermore, the researchers compared the TG transcriptomes from a rattlesnake versus a non-pit species, which again identified the TRPA1 as the only gene that is expressed differently (Gracheva et al. 2010). No opsin-like sequences were detected in the TG of any of the snake species studied (Gracheva et al. 2010). 

Figure 2: in situ hybridization depicting the expression profiles of TRPA1 and TRPV1 in the trigeminal ganglia (TG) and the dorsal root ganglion (DRG) of a rattlesnake.

Unique expression of TRPA1 in viper TG

The TRPA1 ion channel is expressed by 25% of all somatosensory neurons in mammals, preferentially in neurons which act as nociceptors and which also expressed the TRPV1 (transient receptor potential V1 or capsaicin receptor) channel (Gracheva et al. 2010). Generally, the size of the soma of a somatosensory neuron usually indicates what type of sensory function the neuron may have (Gracheva et al. 2010). For example, neurons with large soma diameters are involved in detecting innocuous stimuli, whereas medium to small soma neurons act as nociceptors and detect harmful stimuli. In rattlesnakes, most of the TG neurons had a medium to large soma size, approximately 60% of which expressed the TRPA1 protein. No TRPA1 signal was detected in the rattlesnake DRG during analysis (Gracheva et al. 2010).

Furthermore, the TRPV1 channel was only expressed in 13.6% and 14.6% of neurons in the rattlesnake TG and DRG, respectively (Gracheva et al. 2010). Most of these neurons had a small soma diameter. Therefore, it seems apparent that the pit viper TG is unique among the vertebrates, and reflects an adaption for detecting infrared radiation (Gracheva et al. 2010). 

Figure 3: Neuronal cell size (diameter) quantification determined from histological sections of rattlesnake trigeminal ganglia (left image). Quantitative analysis of cells within the trigeminal ganglia and dorsal root ganglion, that express TRPA1 and TRPV1 transcripts (right image).

Snake TRPA1 is a heat-activated channel

In mammals, the TRPA1 channel is activated by a pungent agent, allyl isothiocyanate, found in wasabi and other mustard plants. This agent, along with other electrophilic irritants, drives the channel through a modification mechanism involving cysteine residues attached to the amino termini within the cytoplasm (Gracheva et al. 2010). Both the rattlesnake and the rat snake TRPA1 sequence displays 63% identity with the human TRPA1, and 81% identity with one another (Gracheva et al. 2010). The researchers show that the TRPA1 sequence contains three conserved N-terminal cyteines, which is required for activation by the electrophilic agents. Expression of TRPA1 from either snake species in HEK293 cells (Human Embryonic Kidney cells) showed that in both cases the channels responded to allyl isothiocynate, signifying the functionality of the cloned channels (Gracheva et al. 2010). 

Figure 4: HEK293 cells expressing cloned TRPA1 channels from rattlesnake and rat snake. Cells response to heat and allyl-isothiocyanate (AITC) were analyzed by observing relative change in fluorescence ratio.

The pit organ detect changes in ambient temperature above ~30°C, thus if the TRPA1 channel plays a role in infrared detection, then it should respond to thermal stimuli consistent with the pit’s thermal sensitivity (Gracheva et al. 2010). In fact, the authors demonstrated that the rattlesnake TRPA1 channel was inactive at room temperature (~25°C), but was strongly active above 28.1°C (Gracheva et al. 2010). Curiously, the rat snake (non-pit species) TRPA1 channel was also heat-sensitive, but had a significantly higher temperature activation threshold (36.4°C). The rattlesnake TRPA1 did not respond to cold temperatures (~12°C) (Gracheva et al. 2010). 

Figure 5: The relative heat response profiles of rattlesnake and rat snake TRPA1 channels  expressed in Xenopus oocytes.

In order to determine the thermal response profiles, the researchers measured heat-triggered membrane currents in Xenopus oocytes expressing the snake TRPA1 channels (Gracheva et al. 2010). The rattlesnake TRPA1 channel showed a stong and highly sensitive response to heat, with a threshold of 27.7°C, whereas the rat snake channel responded to heat above a higher threshold of 37.3°C. These findings indicate that the rat snake TRPA1 channels are in fact heat-sensitive, but the thermal response makes it less suitable to act as an infrared sensor compared to the pit viper’s TRPA1 channel (Gracheva et al. 2010). It is likely that the TRPA1 channels, along with the TRPV1 channels, may be involved in somatic thermo-sensation in non-pit snakes which corresponds with the higher temperature activation thresholds (Gracheva et al. 2010).

TRPA1-like channels are found in a range of vertebrate and invertebrate organisms, including the well-studied Drosophila melanogaster, which contains three TRPA1 orthologues (Gracheva et al. 2010). One of these orthologues, dTrpA1, is heat-sensitive and was shown by the researchers to have a thermal threshold of 33.8°C. Compared to the rat TRPA1 channel, which responds to allyl isothiocyanate with a half-max effective concentration of 11 µM, the rattlesnake and Drosophila orthologues respond at a much higher concentration of 500 µM with a significantly slower rate of activation (Gracheva et al. 2010). This inverse relationship between sensitivity to heat and sensitivity to allyl isothiocyanate demonstrates the relative contribution of the TRPA1 channel to thermo-sensation and chemo-sensation in different organisms (Gracheva et al. 2010). In other words, the TRPA1 channel in pit vipers (and apparently Drosophila) would serve primarily in heat detection whereas in non-pit snakes the channel is more sensitive to chemical detection than heat (Gracheva et al. 2010). The researchers state that their bioinformatics, anatomical, and functional data together strongly indicates that the TRPA1 channel functions in infrared detection in the pit vipers (Gracheva et al. 2010).

Ancient snakes use TRPA1 to sense infrared radiation

The ancient boid snakes (pythons and boas) and modern crotaline snakes are separated by a long evolutionary distance which has contributed to the structural and physiological difference of the pit organs (Gracheva et al. 2010). The authors however, questioned whether both boid and crotaline snakes use the same molecule to detect heat. In a comparison between the royal python (Python regius) and the amazon tree boa (Corallus hortulanus), the TRPA1 channel was found to be expressed 65 and 170 fold more in the TG than the DRG for pythons and boas, respectively (Gracheva et al. 2010). Similarly, comparison of the transcript ratios from rattlesnake and python indicated that the TRPA1 channel is the only molecule which stood out as highly specific to the TG. On the other hand, where TRPA1 was not expressed in the DRG of pit vipers, it was expressed in the DRG of the python and boa, although relatively moderate in view of other TRP channels in the ganglion (Gracheva et al. 2010). The researchers further observed that the TRPV1 channel was not highly expressed in pythons suggesting that either the TRPA1 or another heat-sensitive channel may serve in thermo-sensation in this snake species. 

Figure 6: The relative heat response profiles of boa and python TRPA1 channels expressed in Xenopus oocytes.

According to dendrogram analysis, the position of boa and python TRPA1 sequences supports the notion that these species are part of a more ancient branch of snakes that are relatively independent from the modern snakes, such as pit vipers or rat snakes (Gracheva et al. 2010). When python and boa TRPA1 channels were expressed in Xenopus oocytes, the results indicated that in both species the channel are activated by heat and are moderately sensitive to allyl isothiocyanate. The thermal threshold for the TRPA1 channels in python and boa were observed to be 32.7°C and 29.6°C, compared to rattlesnake’s 27.7°C (Gracheva et al. 2010). This finding is consistent with the understanding that these snakes have different sensitivities to infrared radiation, the rattlesnake having the highest sensitivity, but it does appear that the python and boa channels are more sensitive to heat than they are chemical agents (suggesting they function in thermo-sensation rather than chemo-sensation). 

Figure 7: Expression of TRPA1 or TRPV1 transcripts in python and rat snake trigeminal ganglia.

Endogenous TRPA1 subserves infrared detection

The researchers conducted functional studies using pythons (non-pit snake) in order to ascertain the contribution of TRPA1 to heat sensitivity in neurons. The python TG resembles that of the rattlesnake (pit viper). Most neurons (78.2%) from the python TG were heat-sensitive with a heat threshold of 28.0°C (Gracheva et al. 2010). All heat-sensitive neurons responded to allyl isothiocyanate at a concentration of 500µM (similar to rattlesnake), which confirmed expression of functional TRPA1 channels in these cells (Gracheva et al. 2010).  Lack of TRPV1 channels in python TG was confirmed when no capsaicin-sensitive neurons were detected (Gracheva et al. 2010). On the other hand, it was shown that the rat snake TG contained a moderate amount (27.3%) of TRPV1-positive neurons, which signifies that both TRPA1 and TRPV1 function in heat sensation in this snake species (Gracheva et al. 2010). Similarly, the rat snake TG also exhibited a lower sensitivity to heat and allyl isothiocyanate compared to pythons. Neurons that did respond to these agents possessed a medium/small soma size. In general, the rat snake neurons only responded to heat at higher temperature thresholds (~36-39°C) (Gracheva et al. 2010). 

Figure 8: Thermal sensitivity of python and rat snake trigeminal neurons as measured by calcium imaging. Red colour indicates high sensitivity, whereas purple indicates low sensitivity.

In conclusion, the researchers state their findings are indicative that the TRPA1 ion channel is responsible for detecting infrared radiation in pit-bearing snakes, whereas both the TRPA1 and TRPV1 receptors are responsible for heat sensation in the rat snake (Gracheva et al. 2010).

Critique

This study provides a possible molecular explanation regarding the link between the pit organs and infrared detection by snakes. Essentially, the researchers in this study set out to prove that the TRPA1 ion channel is the receptor that is found in neurons near the pit membrane, and is responsible for detecting changes in ambient temperature which would subsequently produce a thermal profile of the surroundings for the snake.

I believe the study was conducted very well despite the complex nature of the topic, and this is further reflected in the language of the article. One of the reasons why I enjoyed the article (and partly why I picked it) was the ease with which I could follow the authors and their various experiments.  The introduction is more than sufficient, and provided a straightforward explanation of the origin, physiology, and anatomy of the pit organs and related structures.

Although only a summary of the methods were included with the paper, which seemed less than adequate to explain the various details of the experiments, articles from high impact journals such as Nature and Science generally do not include the full methods within the actual article (only as supplementary information) in order to save space. The full materials and methods seemed to include all relevant information, and were sufficiently detailed. 

The results were initially easy to follow but as the experiments became more and more complex, so did the results. However, it does appear that the results support the authors’ claims; the TRPA1 ion channel was identified, TRPA1 was predominant in the TG in pit vipers but was equally distributed in non-pit bearing snakes, the channel responded to temperature changes and not photons, and it was more prevalent in pit bearing snakes than non-pit bearing species. It is worth noting that the researchers seem to repeat their findings and conclusions for some parts (like beating a proverbial dead horse). Furthermore, whenever the researchers stated temperature thresholds, they included the standard errors of their calculations which serve as a measure of the accuracy of their results to the true values.

All things considered, article was well-written, despite some images appearing cramped (containing too much information), all graphs were clearly labeled and easily interpreted. Moreover, the micrograph images were also very clear and of high quality. It appears that the researchers have finally identified the receptor responsible for infrared detection by the pit organs.    

Disclaimer: All figures displayed in this post were borrowed from the scientific paper indicated above, and all rights are the property of the article and/or journal, not this website. 

References

- Amemiya, F., Goris, R., Masuda, Y., Kishida, R., Atobe, Y., Ishii, N., & Kusunoki, T. (1995). The surface architecture of snake infrared receptor organs. Biomedical Research -Tokyo. 16(6): 411-421

- Chen, Q., Deng, H., Brauth, S. E., Ding, L., & Tang, Y. (2012). Reduced Performance of Prey Targeting in Pit Vipers with Contralaterally Occluded Infrared and Visual Senses. PLoS ONE. 7(5)

- Gracheva, E. O., Ingolia, N. T., Kelly, Y. M., Cordero-Morales, J. F., Hollopeter, G., Chesler, A. T., Sanchez, E. E., Perez, J. C., Weissman, J. S., & Julius, D. (2010). Molecular basis of infrared detection by snakes. Nature. 464(7291): 1006-1011

- Krochmal, A. R., Bakken, G. S., & LaDuc, T. J. (2004). Heat in evolution's kitchen: Evolutionary perspectives on the functions and origin of the facial pit of pitvipers (Viperidae: Crotalinae). Journal of Experimental Biology. 207(24): 4231-4238

- Moon, C. (2011). Infrared-sensitive pit organ and trigeminal ganglion in the crotaline snakes. Anatomy & Cell Biology. 44(1): 8-13