The use of genetically encoded calcium indicators in vivo reveals polymodality is a rare phenomenon in dorsal root ganglion (DRG) sensory neurons. Instead, most of these neurons respond specifically to a single type of sensation, such as mechanical stimulation, cold, or heat, reports a team of researchers led by Edward Emery and John Wood, University College London, UK.
“This paper applies a new technique to an old question of whether DRG neurons respond to one or more sensory modalities,” says David Yarmolinsky, Boston Children’s Hospital, US, who was not involved in the new study. “Unlike electrophysiological experiments, which examine single neurons, this calcium imaging technique allows researchers to really see how the system as a whole responds, which affords a more meaningful sense of how pain is produced. The approach has distinct advantages over studying pain neurons in a dish.”
The new results were published November 11 in Science Advances.
The question of whether individual DRG neurons can only detect a single sensory modality or more than one has remained open for decades due to conflicting results from two different kinds of experiments. A number of electrophysiological studies have detected polymodal responses from DRG neurons (for the first description of polymodality in these neurons, see Bessou and Perl, 1969). In contrast, the majority of the abundant knockout mouse studies of pain behavior have demonstrated modality-specific deficits (Lacroix-Fralish et al., 2007), Emery explained.
To better understand whether or not DRG neurons have specificity for a single pain modality, the researchers used GCaMPs, genetically encoded fluorescent calcium indicators, to measure neuronal activity in vivo without the electrode-induced cellular damage inherent to electrophysiological studies.
GCaMPs consist of a green fluorescent protein (GFP) to which a calmodulin calcium binding domain has been added. Calcium binding to calmodulin results in GFP fluorescence, which affords researchers an unprecedented “big picture” view of neuronal activity in vivo. The technique has been used in three prior studies of pain in somatosensory neurons (Kim et al., 2014; Yarmolinsky et al., 2016; Kim et al., 2016; see also PRF related news story).
In the new study, the authors first examined the sensitivity of the GCaMPs. Experiments in cultured DRG neurons from mice expressing either of two calcium indicators, GCaMP3 or GCaMP6s, revealed that both were highly sensitive, capable of detecting single action potential activity. Similarly high sensitivity in DRG neurons was observed in vivo following current pulses to the hindpaws of anesthetized mice expressing GCaMP3.
“The fact that the researchers used electrophysiology to quantify what the calcium response is actually showing is a real strength of the paper,” said Yarmolinsky.
The researchers then looked at GCaMP3- or GCaMP6s-expressing DRG neuron responses under basal conditions following a variety of simple stimuli applied to the hindpaw: two noxious mechanical stimuli—pinch and von Frey filament stimulation—as well as heat and cold water immersion.
“We found that, on the whole, distinct neurons responded to each of the three stimuli types, indicating that DRG sensory neurons are largely modality-specific,” said Wood.
Only a small proportion of the cells responded to more than one stimulus type, with less than 12 percent responding to both mechanical and heat stimuli and less than 6 percent responding to all three stimuli types.
Moving beyond studying the naïve state, the research team then looked at DRG neuron responses 10 minutes after acute, local inflammation by injecting the endogenous inflammatory mediator prostaglandin E2 (PGE2) into the same paw of the same animals used under the basal conditions. The researchers observed large increases in the populations of heat-, cold-, and mechanically responsive neurons that responded to a single sensory modality.
“Once we added the PGE2 to the paw, we saw a whole new population of DRG neurons lighting up. Silent nociceptors, which had been previously unresponsive to any sensory modality, were awakened,” said Wood.
The team also observed a broadening of individual neurons’ response profiles, such that neurons that were previously only mechanically sensitive were now also responsive to heat stimulation.
“We found that, in the basal, naïve, undamaged state, the majority of sensory neurons are modality-specific for heat, cold, and mechanical stimulation. Transient, local inflammation changes the situation drastically, inducing a shift to a polymodal phenotype,” said Wood.
The results provide a potential explanation for why polymodality was observed in traditional electrophysiological experiments, he continued. “Implantation of glass electrodes into DRG neurons may cause enough local inflammation to induce a phenotypic switch from single modality sensitivity to polymodality within several minutes.”
Yarmolinsky thinks there is more work to be done to fully understand the question of polymodality in DRG neurons. “This question about the distribution of polymodality is an important one. I don’t know if this paper really solves it, since calcium imaging isn’t as sensitive at the single-neuron level as electrophysiology, but it definitely shows that the way we think the system works might be different from the dogma,” he said.
The researchers are planning to use GCaMP imaging to look at human-relevant disease models to identify the neurons responsible for conditions such as bone cancer pain and painful diabetic neuropathy, said Emery.
Allison Marin, PhD, is a neuroscientist-turned-science writer who resides in Pittsburgh, US.
Image credit: Emery et al., 2016