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Fallen at the First Gate: New Evidence of Inhibitory Filtering in the DRG

New research in rodents shows that GABA exerts an inhibitory effect on noxious inputs in the dorsal root ganglia. Can it now be considered the first sensory gate?

Fred Schwaller


31 March 2023


PRF News

Recording Paradigm

New research in rodents shows that GABA exerts an inhibitory effect on noxious inputs in the dorsal root ganglia. Can it now be considered the first sensory gate?

The dorsal horn of the spinal cord has long been seen as the major area where sensory information is first modulated. In recent years, however, there’s been increasing evidence that the dorsal root ganglia (DRG) may be an important center for neuromodulation, perhaps gating sensory inputs even before they have reached the spinal cord.

A new study from Xiaona Du (Habei Medical University, Shijiazhuang, China) and Nikita Gamper (University of Leeds, UK) provides further evidence that the DRG is a major site for neuromodulation of nociceptive information.

The study – performed in rodents – shows that the DRG exerts a dynamic control of sensory information coming from the periphery via GABAergic signaling. This filtering seems to be more efficient in C-fiber afferents compared to A-fibers, and occurs at the axonal bifurcation of the T-junction in DRG cells.

“This paper is an incremental addition to the growing line of evidence for sensory gating in the DRG. What’s new here is the neurological evidence for the role of T-junctions of DRG axons as a site of neuromodulation, which other investigators have alluded to in prior studies,” said Srinivasa Raja, a pain researcher at Johns Hopkins University School of Medicine, USA, who was not involved in the study.

The study was published in PLOS Biology on 5 January 2023.

 

Recording peripheral neuron activity

“This paper is an extension of a study published in 2017, where we first showed GABAergic inhibition of the painful input in the DRG,” Gamper said.

Back in the original paper, Gamper’s team found GABA injected into the DRG caused analgesia in behaving mice.

“But these findings lacked mechanistic proof of how GABA acts to modulate action potential firing in the DRG. In this study, we tried to close the gap,” said Gamper.

The team used in vivo electrophysiology to record action potential firing in individual sensory neurons in anesthetized rats in a rather unique way.

“We used two electrodes placed before and after the DRG to record action potential firing at two locations on the neurons – the spinal nerve (the peripheral process), and the dorsal root (the central process). We then compared the firing frequency in response to peripheral stimuli,” said Du.

The thinking behind the dual recordings was to test whether inputs coming from the periphery (triggered by noxious and innocuous stimulation of the paw) are modulated at locations in or around the DRG.

 

GABA filters C-fibers in the DRG

In initial recordings, the team found that the injection of various painful compounds – like capsaicin and bradykinin – into the hindpaw increased firing activity in both the peripheral and central processes of DRG neurons.

What the team was really interested to learn was how GABA changes the filtering of noxious inputs. Here, they applied GABA onto the DRG and tested how it changed the propagation of action potentials along the DRG in response to capsaicin or bradykinin injected into the paw.

Interestingly, GABA significantly reduced nociceptive activity in the dorsal root “after” the DRG, but not in the spinal root “before” the DRG.  Similarly, application of the GABAA receptor antagonist bicuculline onto the DRG increased spontaneous firing rate at the dorsal root but not the spinal root.

This meant that GABA was filtering noxious inputs somewhere between the soma and the recording site farther along the dorsal root, but not upstream toward the distal limb.

“This is consistent with another experiment we did back in 2017, where injection of a GABA reuptake inhibitor into the DRG produced an analgesic effect on behaving animals,” said Gamper.

 

GABA filters noxious but not innocuous inputs in the DRG

The team then compared the ability of GABA to filter in C-fibers versus A-fibers, differentiating them by their conduction velocities. They found that GABA strongly inhibited C-fiber activity – but not A-fiber activity – at the dorsal root.

From these findings, the team hypothesized that GABA was filtering noxious inputs, but not innocuous inputs, in the DRG.

To test this, they turned to sophisticated genetic tools in mice, artificially amplifying GABAergic signaling in the DRG.

Here, they transplanted GABAergic cells from the forebrain into the DRG, which release GABA upon stimulation. These cells contained yellow fluorescent protein so they can be visualized, along with channel rhodopsins so they can be selectively activated.

Initial experiments found that optogenetic activation of the GABAergic cells with blue light reduced firing activity in DRG neurons in response to a variety of acute painful stimuli such as mechanical probes – or noxious heat and cold – applied to the paw.

However, neuronal responses to innocuous stimuli – like air puffs or gentle poking of the paw – were not modulated when GABAergic cells were optogenetically stimulated.

From this, the team concluded that GABA caused analgesia in the DRG by preferentially filtering pro-nociceptive spike activity in C-fiber cell bodies.

“This would imply that touch and proprioception would not be affected by GABA signaling at the DRG. Clinically, that’s an attractive idea,” said Raja.

 

T-junctions: site of neuromodulation in C-fibers

“GABA filtering noxious but not innocuous stimuli was a complete surprise to us. We really wanted to know why only noxious information was being filtered,” Gamper said.

In their search for an answer, Gamper and the team drew inspiration from looking at Ramon y Cajal’s drawings. In particular, they focused on the stem between the cell body and the T-junction, which is a major site of spike filtering.

“Small fibers are drawn with a very short stem, but larger fibers had very long, convoluted stems. We thought if the soma has influence over [action potential propagation at the] T-junction, the stem must be short,” Gamper said.

The team began to investigate this by using light-sheet microscopy. Here, they labeled C-fibers and A-fibers using antibodies, then measured the distance from the soma to the T-junction. Their findings confirmed Cajal’s drawings – C-fibers had short stems (~60 μm) and A-fibers had longer stems (~232 μm).

But how does stem length influence signal filtering? Well, if the membrane potential changes in the soma, it will diffuse along the axon; however, the longer the stem, the more likely the potential will fade before it reaches the T-junction. The T-junction is where the action potential is propagated, so if the signal has faded along the stem, no modulation will take place.

From their imaging data, the researchers hypothesized that the shorter stem of C-fibers allows the soma to have tighter control of electrotonic coupling of the T-junction, hence, the ability of GABA to filter noxious stimuli at the soma.

On the other hand, Gamper explained, GABA acting at A-fiber soma would have less influence on T-junctions because of the long stem.

“If GABA is released somatically, it may not diffuse towards the T-junction in cells with a longer stem. This may contribute to the difference of GABAergic filtering of A- and C-fibers,” said Gamper. “A disclaimer here – we don’t want to claim this is the only mechanism. There could be other mechanisms at play here.”

In part, these findings also provide some evidence for earlier hypotheses by Marshall Devor (Hebrew University of Jerusalem, Israel) that the excitability of the soma influences reliable propagation of impulses past the DRG T-junction to the spinal cord. Gamper’s findings indicate GABA to be a key mediator here.

 

Tonic or evoked GABAergic inhibition?

Their previous paper from 2017 found that some neurons in the DRG are capable of releasing GABA upon stimulation.

Their final experiments focused on measuring exocytosis of GABA-containing vesicles in cultured DRG neurons using antibodies. Depolarizing the cells with potassium chloride caused robust exocytosis of GABA-containing vesicles, indicating that GABA can indeed be released after stimulation.

However, they found that vesicles also underwent exocytosis in the absence of depolarization, hinting at tonic release of GABA in the DRG. 

Live imaging of GABA release in cultured DRG cells using fluorescence methods also corroborated the findings, suggesting GABA could be released both by depolarization of the cells, and in the absence of depolarization.

Overall, these findings indicate GABAergic inhibition in the DRG can be both tonic and evoked.

 

 DRG: the first sensory gate?

According to Gamper and Du, their data shine a light on the DRG as a crucial sensory gate.

 “I think our findings do rethink the classical gate control theory. They suggest the DRG is the first major gate where information is integrated and processed,” said Gamper.

Raja thinks the implications of this hypothesis could have important clinical relevance.

“The impact of the paper is twofold. [The first is] that it provides more direct physiological evidence for clinically used therapies like dorsal root ganglion stimulation. The second is that it provides further incentives for developing novel peripheral therapies,” said Raja.

Gamper agreed, saying their findings could help the field better design drugs that specifically target nociceptive filtering in the DRG.

“DRG is not protected by the blood-brain barrier, so it’s much more accessible with small-molecule drugs,” said Gamper.

However, as always with hypothesis-driven basic science papers like this one, the translational potential of the paper is a long way from being realized.

Fred Schwaller, PhD, is a freelance science writer based in Germany. 

Featured Image:

Schematic of the optogenetic recording paradigm. Hao et al. PLOS Biology. 2023.

Inline Image:

Schematic of electrode placement. Hao et al. PLOS Biology. 2023.

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