Our sense of temperature can tell us whether an environment is hospitable or harmful. Researchers have spent over half a century deciphering how nerve fibers in the skin represent the full range of temperatures, from searing heat to freezing cold. But much less is known about how neurons at the next stage of processing, in the spinal cord, encode temperature.

Now, thanks to high-throughput calcium imaging, senior author Xiaoke Chen and lead author Chen Ran, Stanford University, US, and co-author Mark Hoon, National Institutes of Health, Bethesda, US, report that dorsal horn neurons from anesthetized mice use different codes for cutaneous temperature. While spinal neurons that respond to heat encode absolute temperature, those activated by cold instead encode changes in temperature. The researchers also show that many spinal neurons are “broadly tuned” to temperature, able to encode both heat and cold. This is likely because they receive information from several types of neurons in the dorsal root ganglia (DRG).

“[The study] adds a nice dimension to our understanding of innocuous and noxious information processing,” said Michael Caterina, Johns Hopkins University, Baltimore, US, who was not involved in the study. “By looking at a large population of neurons in the dorsal horn, the authors were able to pick up patterns and complexity that would have taken a million years with electrophysiological recordings,” he said.

But, Caterina thinks it’s too early to know what the coding strategies revealed in the study mean for pain. “It’s difficult to assign the phenomena that were being measured to painful versus non-painful sensations. That’s something that will play out over time.”

The findings were published online July 25 in Nature Neuroscience.

Hot versus cold

Ambient temperature is first detected by free nerve endings of primary sensory neurons in the skin, whose cell bodies lie in the DRG. These cells have been extensively studied for their use of ion channels of the transient receptor potential (TRP) family to sense temperature of the skin and other tissues (Vriens et al., 2014). But in the spinal cord, where dorsal horn neurons receive signals from DRG neurons, temperature processing has remained more of a mystery, said Ran. “The spinal cord has long been regarded as a black box.”

To peer inside, Ran and colleagues knew they needed an approach unlike those of past studies. “When people have examined neurons’ responses to a mild temperature using electrophysiology, only a very tiny proportion of neurons respond,” said Ran. For that reason, and because electrophysiological recordings can only listen in on a small number of neurons to begin with, the researchers developed a high-throughput method to survey a large swath of neurons in anesthetized mice.

Using two-photon imaging, the researchers optically recorded calcium influx in hundreds of superficial dorsal horn neurons simultaneously, after laminectomy at spinal level L4. To elicit responses in these neurons to varying temperatures, they removed the hair from the hind limb, which is innervated by the L4 nerve, and then bathed the limb with temperature-controlled water. At baseline, the water was held at a physiological skin temperature of 32° Celsius.

The investigators found that cooling the skin to temperatures ranging from 29° to 5°C (spanning innocuous and noxious temperatures) activated groups of neurons in the superficial dorsal horn. And, compared to cooling to milder temperatures, cooling to lower temperatures activated more neurons and gave rise to larger calcium signals. However, these neurons did not seem to represent the absolute temperature of the skin. By dropping the temperature at a constant rate and then holding it at a stable target temperature, the researchers found that neuronal responses reached their height during the change in temperature stage, with decreased responses during the stable temperature stage. Further experiments showed that greater changes in temperature activated more neurons and produced larger calcium signals, regardless of the absolute temperature. Together, the results showed that, with cooling, spinal neurons appeared to code only for the change in temperature rather than for the absolute temperature.

Interestingly, the researchers found the opposite when they heated the skin. Neurons were activated by temperatures ranging from 37° to 50°C (43°C and above is generally considered to be noxious), and more neurons responded to more intense heating. But these cells stayed active once the temperature became stable. Moreover, when the authors heated the skin to the same target temperature from different starting temperatures, the same group of neurons was activated, with similar calcium responses. In contrast, when heating by a constant change in temperature, but from different starting temperatures, the researchers found stronger responses for higher target temperatures. In sum, the findings suggested that neurons representing heat process absolute temperature, but not the change in temperature. “It’s very striking to me that these coding properties are so diametrically different,” said Caterina.

But why were they different? Ran thinks it may have to do with the risk of injury from high temperatures. “Heat is more dangerous for an animal than cold,” he said. “If a temperature can damage a tissue, you want neurons to provide a very constant warning signal.” The code identified for heat, but not cold, may be that kind of signal.

A number of inputs from the DRG

The authors went on to examine which temperature-sensitive DRG neurons fed into the coding of heat versus cold in the spinal cord. Here, they turned to transgenic mice, using diphtheria toxin to ablate DRG neurons expressing transient receptor potential vanilloid type 1 (TRPV1), and engineered to carry the diphtheria toxin receptor. In the TRPV1- animals, spinal neurons showed reduced responses to heat, relative to neurons from wild-type mice. The same effect was found across temperatures (37° to 50°C), which implied that TRPV1+ neurons not only convey noxious heat, but also innocuous warmth. The researchers also provided evidence that DRG neurons expressing a TRP channel commonly associated with cold, TRPM8, contributed to how heat is represented in the spinal cord, as ablation of TRPM8+ neurons enhanced the spinal response to warmth.

Similarly, ablating TRPM8+ DRG neurons reduced the number of spinal neurons activated by mild cooling, but not by lower temperatures, compared to neurons from wild-type mice. These remaining responses to more intense cooling were blunted only with the loss of TRPV1+ DRG neurons, thus pinpointing a role for those cells in detecting strong cold. Together, the results from the transgenic mice experiments support the idea that spinal neurons synthesize information from many types of DRG neurons.

The authors also found that some spinal neurons coded for both heat and cold, especially at more extreme temperatures. DRG neurons with bimodal activity have been described before, so Caterina isn’t surprised that spinal neurons further along in the temperature processing pathway do the same (LaMotte and Campbell, 1978). “The existence of single fibers that are responsive to both intense heat and intense cold has been recognized for a long time,” he said.

Overall, the authors show that spinal neurons can represent heat and cold in opposing ways, and that some neurons process both. How the findings relate to chronic pain, however, remains unclear, but the researchers are now exploring this issue.

There are other caveats, too. For instance, the researchers recorded spinal neuron activity in anesthetized mice, when it is known that anesthesia can depress body temperature. “There are data already to suggest that electrical activity under anesthesia versus in an awake animal can be dramatically different,” said Caterina. Nevertheless, Ran thinks that the coding strategies uncovered in the current study are fundamental processes that will still hold true in awake animals.

Matthew Soleiman is a science writer currently residing in Nashville, Tennessee. Follow him on Twitter @MatthewSoleiman

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