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Spinal Astrocytes: New Players in Noradrenergic Descending Pain Modulation

Dorsal horn astrocytes activated by noradrenergic neurons in the locus coeruleus drive mechanical hypersensitivity in mice, contrary to expectations.

by Fred Schwaller


11 January 2021


PRF News

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Dorsal horn astrocytes activated by noradrenergic neurons in the locus coeruleus drive mechanical hypersensitivity in mice, contrary to expectations.

Pain isn’t just under the dominion of neurons: Glial cells, including microglia and astrocytes, have long been known to modulate pain sensitivity via interactions with neurons in both acute and chronic pain states. A new study now finds a long-range and direct pathway from neurons in the brainstem to astrocytes in the spinal cord dorsal horn, which regulates mechanical pain hypersensitivity.

 

The research, led by Makoto Tsuda, Kyushu University, Fukuoka, Japan, shows that these astrocytes express the transcription factor Hes5 as well as α1A-adrenergic receptors. Further, the cells are activated by noradrenergic neurons in the locus coeruleus that project to the spinal dorsal horn, a pathway that is well known to modulate pain sensitivity. Moreover, the authors find that these astrocytes might reduce the analgesic effect of duloxetine on neuropathic mechanical hypersensitivity.

 

“This is a very interesting study that has furthered our understanding of non-neuronal cells in their regulation of acute pain and chronic pain. What is most exciting is the finding that glial cells – astrocytes in this case – are involved in descending modulation of pain,” said Ru-Rong Ji, Duke University, US, who studies the contribution of non-neuronal cells to pain but was not part of the current investigation.

 

The research was published in the November 2020 issue of Nature Neuroscience.

 

Hes5+ astrocytes in the dorsal horn

Astrocytes were originally thought to provide only metabolic support to neurons in the central nervous system. But evidence now supports a critical role for these cells in the modulation of neural processing, including modulation of nociceptive signaling and chronic pain (Ji et al., 2019).

 

However, astrocytes are numerous and diverse, and Tsuda and colleagues wanted to find out whether there are specific astrocyte populations in the dorsal horn of the spinal cord that modulate pain responses, particularly in adult animals.

 

“When we started this project, there were several reports showing the heterogeneity of spinal astrocytes during development in mice. However, it was completely unknown if there were astrocyte populations in the adult spinal cord with a clear role in modulating the somatosensory system,” Tsuda said.

 

His team aimed to identify these astrocyte populations by comparing expression levels of several known astrocyte genes between the dorsal and ventral horns of the spinal cord. The thinking was that astrocyte genes enriched in the dorsal horn would define populations of astrocytes involved in somatosensory processing.

 

The researchers began with quantitative PCR analysis of 17 astrocyte genes and found that one gene, Hes5, was strongly enriched in the adult mouse spinal dorsal horn. Fluorescent labeling would allow the group to identify the exact location of these Hes5+ astrocytes.

 

“We found that Hes5+ spinal astrocytes were localized specifically in superficial layers of the adult dorsal horn,” according to Yuta Kohro, co-first author along with Tsuyoshi Matsuda and Kohei Yoshihara.

 

The next question was whether the Hes5+ astrocytes were involved in somatosensory processing. The investigators first asked whether these astrocytes could be activated by painful stimuli, by recording the cells’ activity by using in vivo calcium imaging after injection of capsaicin into the hindpaw.

 

“Astrocytic activation in the left dorsal horn was observed after capsaicin injection not only into the left hindpaw, but surprisingly also into the right hindpaw,” according to Kohro.

 

This indicated that Hes5+ astrocytes not only responded to nociceptive inputs from the capsaicin-injected hindpaw, but also from other parts of the nervous system that are involved with bilateral responses to pain. That finding paved the way toward the group’s next set of experiments.

 

Brainstem noradrenergic pathways activate astrocytes

The authors turned immediately to the descending pain modulatory system to find out what other regions of the central nervous system might be involved, since these pathways from the brainstem to the spinal dorsal horn are well known to bilaterally modulate sensory transmission and pain sensitivity.

 

The authors began by testing the responses of Hes5+ astrocytes to noradrenaline (also called norepinephrine) and serotonin, two major neurotransmitters of the descending pathway. Recording astrocyte calcium activation in ex vivo spinal cord slices, the researchers found that astrocytes responded to noradrenaline, but not serotonin.

 

A deeper dive into the pharmacology of the system revealed that Hes5+ astrocyte activity was changed only by α1A-adrenergic receptor (α1A-AR) modulators. Specifically, the cells were activated by the α1-AR agonist phenylephrine and inhibited by the α1A-AR antagonist silodosin. Collectively, the data showed that Hes5+ astrocytes in the dorsal horn were activated by noradrenaline via α1A-ARs.

 

The group was aware that the only candidate brain region known to regulate descending noradrenergic signaling in the dorsal horn was the locus coeruleus, a noradrenergic brainstem nucleus with anti-nociceptive effects. To confirm the involvement of this structure, the investigators deactivated or activated these descending locus coeruleus noradrenergic neurons and examined what effects this had on astrocyte activation.

 

They found that ablating descending noradrenergic neurons by injecting the neurotoxin DSP-4 suppressed astrocytic activity after capsaicin injection. Meanwhile, activating these neurons using chemogenetics increased astrocytic activity. Thus, descending noradrenergic transmission was sufficient to activate Hes5+ astrocytes in the dorsal horn.

 

Astrogliogenic mechanical hypersensitivity

The next issue to address was whether descending noradrenergic activation of Hes5+ astrocytes had an effect on pain behaviors. Here, the group intrathecally injected noradrenaline or the α1-AR agonist phenylephrine into the spinal cords of mice and tested the animals’ mechanical and thermal withdrawal responses. Remarkably, both manipulations caused mice to be more sensitive to mechanical von Frey hair stimulation of the hindpaw, with no effect on thermal sensitivity. The authors referred to this as “astrogliogenic mechanical hypersensitivity.”

 

“The finding that intrathecal injection of noradrenaline or phenylephrine produced mechanical hypersensitivity was totally unexpected. It implied we had discovered an opposite role for noradrenaline in spinal pain processing,” Kohro told PRF.

 

Ji was also surprised by the finding, and highlighted how it challenges previous evidence that noradrenergic modulation could only inhibit pain.

 

“Descending noradrenergic modulation was traditionally considered to be inhibitory. This study shows that activation of spinal cord astrocytes by this descending pathway can actually enhance mechanical pain,” Ji said.

 

From astrocytes to neurons: D-serine is the link

Having identified an upstream activator of Hes5+ astrocytes, the team then wanted to know what happens downstream of astrocyte activation to cause mechanical hypersensitivity. They turned their attention to N-methyl-D-aspartate receptors (NMDARs), a type of glutamate receptor in nerve cells.

 

“In our extended datasets, astrogliogenic mechanical hypersensitivity was inhibited by pre-treatment with NMDAR antagonists,” Tsuda explained. “We speculated that astrocytic factors can selectively modulate NMDAR function, and we focused on D-serine because it modulates NMDARs and can be released from astrocytes.”

 

That spurred experiments in which chemogenetics was used to activate spinal Hes5+ astrocytes while intrathecal injection of the D-serine antagonist DCK was simultaneously used to block D-serine activity in the spinal cord. This reduced astrogliogenic mechanical hypersensitivity, indicating that this process involves astrocytic D-serine signaling via neuronal NMDARs.

 

Getting in the way of analgesia

The antidepressant duloxetine is prescribed to many people with chronic pain conditions such as fibromyalgia, diabetic neuropathy, and low back pain. This drug is thought to ease pain by acting on the descending pain pathway via inhibition of spinal noradrenaline reuptake. Duloxetine, however, fails to provide pain relief for many patients.

 

From their data so far, the authors hypothesized that Hes5+ astrocytes might have a role in reducing the effectiveness of duloxetine analgesia. A final experiment supported the idea, as genetic ablation of α1A-ARs from Hes5+ astrocytes enhanced the suppression of mechanical hypersensitivity by duloxetine, in mice with peripheral nerve injury.

 

Tsuda believes that this finding suggests a possible way to enhance the effectiveness of drugs like duloxetine.

 

“Spinal dorsal horn astrocytes activated by the descending brainstem pathway may disturb the analgesic effect of duloxetine. We think that selective inhibition of descending locus coeruleus noradrenergic signaling to Hes5+ spinal astrocytes can potentiate the effect of existing analgesics that act on the locus coeruleus noradrenergic pathway. This is one thing we’ll be investigating in the future,” Tsuda said.

 

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

 

Image credit: Kohro et al. Nat Neurosci. 2020 Nov;23(11):1376-1387.

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