“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
