Mammalian target of rapamycin signaling in the spinal cord is required for neuronal plasticity and behavioral hypersensitivity associated with neuropathy in the rat

Abstract

The protein kinase mammalian target of rapamycin (mTOR) regulates mRNA translation and is inhibited by rapamycin. Signaling pathways involving mTOR are implicated in physiological and pathophysiological processes. We determined the spinal effects of the rapamycin analogue cell cycle inhibitor (CCI)-779 on neuronal responses and behavioral hypersensitivity in a model of persistent neuropathic pain. We also assessed the anatomical distribution of spinal mTOR signaling pathways. Specifically, we ligated rat spinal nerves L5 and L6 to produce a model of neuropathic pain. After confirming neuropathy with behavioral testing, we obtained in vivo single-unit extracellular stimulus-evoked recordings from deep dorsal horn spinal neurons. We applied CCI-779 spinally in electrophysiological and behavioral studies and assessed its effects accordingly. We also used immunohistochemistry to probe for mTOR signaling pathways in dorsal root ganglia (DRG) and the spinal cord. We found that spinally administered CCI-779 rapidly attenuated calibrated mechanically but not thermally evoked neuronal responses and mechanically evoked behavioral responses. Immunohistochemistry showed presence of mTOR signaling pathways in nociceptive-specific C-fiber DRG and in neurons of inner lamina II of the spinal cord. We conclude that alterations in the activity of spinal mTOR signaling pathways are crucial to the full establishment of spinal neuronal plasticity and behavioral hypersensitivity associated with nerve injury.

Perspective: This study is consistent with growing evidence implicating mTOR signaling pathways as important modulators of persistent pain, providing novel insights into the molecular mechanisms of pain maintenance and potential for novel approaches into treating chronic pain.

Figure 1

SNL results in painlike behavior. Paired analysis of difference scores show that after SNL (white, n = 22), there was a significant increase in paw withdrawals from the injured side versus the uninjured side compared to sham rats (black, n = 24). (A) For a 1 g stimulus, this increase in behavioral hypersensitivity was most apparent on days 9 and 14. (B-D) For a 6- and 8-g stimulus as well as acetone, behavioral hypersensitivity was significantly increased as early as day 2 and maintained up to day 14. ∗P < .01. ∗∗∗P < .001.

Figure 2

mTOR signaling pathways are more readily inhibited by CCI-779 in SNL rats. (A) 250 nM CCI-779 inhibited nociceptive-specific C-fiber–mediated transmission onto WDR neurons from SNL rats (n = 12) compared to sham rats (n = 10) and pooled predrug controls (n = 22). CCI-779 also had a significant inhibitory effect on WDR neuronal postdischarge (PD) compared to sham rats. (B) CCI-779 inhibited wind-up, a potentiated response mediated by nociceptive C-fiber activity and a measure of neuronal hyperexcitability from SNL rats compared to sham rats. (C) CCI-779 was effective in inhibiting neuronal responses to mechanical stimuli from SNL rats compared to sham rats (8, 15, 26, and 60 g) and pooled predrug controls (15, 26, and 60 g). (D) The inhibitory effects of CCI-779 were not extended to thermally evoked responses since there were no differences on thermally evoked responses between pooled predrug controls and CCI-779-treated sham or SNL rats. ∗P < .05. ∗∗P <.01. ∗∗∗P < .001.

Figure 3

mTOR signaling pathways contribute to behavioral hypersensitivity displayed by SNL rats. (A) Paired analysis showed that spinal administration of 250 μM CCI-779 (white) resulted in marked (but not significant) attenuation of behavioral hypersensitivity compared to saline (black) from baseline (B) levels to a 1 g von Frey filament. Complete (but not significant) inhibition by CCI-779 was observed at 20 and 40 minutes. (B) CCI-779 significantly attenuated behavioral hypersensitivity to 6 g at 40 minutes, with a return to baseline levels thereafter. (C) CCI-779 significantly attenuated behavioral hypersensitivity to 8 g at 20 minutes, with a return to baseline levels thereafter. (D) CCI-779 significantly attenuated behavioral hypersensitivity to acetone at 40, 60, and 100 minutes with some recovery to baseline levels at 120 minutes. ∗P < .05. ∗∗P < .01. ∗∗∗P < .001.

Figure 4

SNL affects CGRP immunoreactivity and mTOR signaling pathways in L5 spinal neurons. (A) Observation of the stained tissue sections revealed that immunoreactivity of CGRP in the dorsal horn ipsilateral (Ipsi) to the injury (left) appeared to be lower when compared to the dorsal horn contralateral (contra) to the injury (middle). This was confirmed by a paired analysis of the fluorescence of the ipsilateral versus the contralateral side to the injury (right), thus confirming an expected feature of SNL. Scale bars = 100 μM. (B) Likewise, there appeared to be a reduction in phospho p70S6K in the dorsal horn ipsilateral to the injury (left) compared to the side contralateral to the injury (middle), and this was confirmed by paired analysis of the fluorescence of the ipsilateral versus the contralateral side to injury (right). Scale bars = 100 μM. (C) Surprisingly, phospho p70S6K was mainly activated in inner lamina II of the dorsal horn of the spinal cord, an area known to be occupied by PKCγ-expressing interneurons. Double labeling spinal cord sections for phospho p70S6K (top) and PKCγ (middle) revealed that the 2 signals were at least partially colocalized, suggesting mTOR signaling pathway activity in a subpopulation of PKCγ interneurons (bottom). Scale bars = 50 μM. (D) Double labeling DRG sections for phospho p70S6K (left) and CGRP (middle) revealed that the 2 signals were at least partially colocalized, suggesting that some nociceptive-specific fibers utilize mTOR signaling pathways, at least at the level of the DRG (right). Scale bars = 100 μM. ∗P < .05.

Figure 5

SNL does not affect CGRP and p70S6K immunoreactivity in L4 spinal neurons. (A) Observation of the stained tissue sections revealed that immunoreactivity of CGRP in the dorsal horn ipsilateral (Ipsi) to the injury (left) appeared unchanged when compared to the dorsal horn contralateral (contra) to the injury (middle). This was confirmed by a paired analysis of the fluorescence of the ipsilateral versus the contralateral side to the injury (right). (B) Likewise, there appeared to be no change in phospho p70S6K in the dorsal horn ipsilateral to the injury (left) compared to the side contralateral to the injury (middle), and this was confirmed by paired analysis of the fluorescence of the ipsilateral versus the contralateral side to injury (right). Scale bars = 100 μM.