Elsevier

Experimental Neurology

Volume 232, Issue 2, December 2011, Pages 222-233
Experimental Neurology

Rac1-regulated dendritic spine remodeling contributes to neuropathic pain after peripheral nerve injury

https://doi.org/10.1016/j.expneurol.2011.08.028Get rights and content

Abstract

Although prior studies have implicated maladaptive remodeling of dendritic spines on wide-dynamic range dorsal horn neurons as a contributor to pain after spinal cord injury, there have been no studies on dendritic spines after peripheral nerve injury. To determine whether dendritic spine remodeling contributes to neuronal hyperexcitability and neuropathic pain after peripheral nerve injury, we analyzed dendritic spine morphology and functional influence in lamina IV–V dorsal horn neurons after sham, chronic constriction injury (CCI) of the sciatic nerve, and CCI treatment with NSC23766, a selective inhibitor of Rac1, which has been implicated in dendritic spine development. 10 days after CCI, spine density increased with mature, mushroom-shaped spines preferentially distributed along dendritic branch regions closer to the cell body. Because spine morphology is strongly correlated with synaptic function and transmission, we recorded the response of single units to innocuous and noxious peripheral stimuli and performed behavioral assays for tactile allodynia and thermal hyperalgesia. Wide dynamic range dorsal horn neurons of CCI animals exhibited hyperexcitable responses to a range of stimuli. They also showed reduced nociceptive thresholds in the ipsilateral hind paw. 3-day treatment with NSC23766 significantly reduced post-CCI spine dimensions and densities, and attenuated injury-induced hyperexcitability. Drug treatment reduced behavioral measures of tactile allodynia, but not for thermal hyperalgesia. Together, our results demonstrate that peripheral nerve injury induces Rac1-regulated remodeling of dendritic spines on dorsal horn neurons, and suggest that this spine remodeling contributes to neuropathic pain.

Highlights

► Dendritic spines on dorsal horn neurons remodel after peripheral nerve injury ► Dendritic spines change in density, distribution, and shape after nerve injury ► Inhibiting Rac1 activity disrupts nerve injury-induced dendritic spine remodeling ► Disrupting dendritic spine remodeling reduces signs of neuropathic pain

Introduction

Neuropathic pain is broadly defined as chronic pain arising as a consequence of disease or dysfunction of the somatosensory system, and may arise from a spectrum of traumatic insults to the nervous system, including peripheral nerve injury (Treede et al., 2008). Neuropathic pain is often refractory to clinical therapies, and a major challenge of pain research today is to identify the underlying pathological mechanisms of neuropathic pain. A wide body of evidence has shown that many factors can contribute to the development of neuropathic pain, including the loss of inhibitory neurotransmission (Cordero-Erausquin et al., 2005, Coull et al., 2003), inflammation (Hains and Waxman, 2006), and aberrant sodium ion channel expression (Hains et al., 2004, Waxman et al., 1999). Although primary afferent plasticity has been implicated in pain after peripheral nerve injury (Woolf and Salter, 2000, Woolf et al., 1992), it is unknown whether second-order dorsal horn nociceptive neurons also exhibit maladaptive structural plasticity.

Dendritic spines are micron-sized protrusions on dendritic branches, which represent modifiable sites of synaptic contact. In the normal nervous system, dendritic spines subserve several adaptive functions, including experience-dependent circuit reorganization and memory formation (Calabrese et al., 2006). In pathology, emerging evidence has suggested that dendritic spines have an important role, and change shape, size, and number in response to various injury and disease insults (Halpain et al., 2005). In a model for Fragile X mental retardation, mice with Fmr1 protein knockout show impaired long-term potentiation (Wilson and Cox, 2007), decreased neuropathic allodynia (Price et al., 2007), and malformed dendritic spines due to dysfunctional Rac1 signaling (Chen et al., 2010). Similarly, spinal cord injury (SCI) results in dendritic spine alterations on motor cortex neurons (Kim et al., 2006), and induces Rac1-regulated spine remodeling on nociceptive dorsal horn neurons that contributes to neuropathic pain (Tan et al., 2008, Tan et al., 2009a).

In the present study we show that dendritic spine remodeling occurs within dorsal horn neurons after chronic constriction injury (CCI), a well-established peripheral nerve injury model of neuropathic pain. We reasoned that if dendritic spine remodeling has a role in neuropathic pain states, then disrupting spine structure should attenuate injury-induced pain. Because in vitro and in vivo studies have shown that Rac1, a small kinase, can modulate dendritic spine structure and function (Tashiro and Yuste, 2004, Tashiro and Yuste, 2008), we treated peripheral nerve injured animals with the Rac1-specific inhibitor, NSC23766 (Gao et al., 2004). Targeted inhibition of Rac1 signaling reduced injury-induced spine malformation, decreased neuronal excitability, and improved nociceptive behavioral thresholds. These findings demonstrate that peripheral nerve injury can promote dendritic spine remodeling on nociceptive dorsal horn neurons, and suggest a role for Rac1-dendritic spine remodeling in chronic neuropathic pain after nerve injury.

Section snippets

Animals

Experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All animal protocols were approved by the Yale University Institutional Animal Use Committee. Adult male Sprague–Dawley rats (200–225 g) were used for this study. Animals were housed under a 12 h light/dark cycle in a pathogen-free area with water and food provided ad libitum.

Peripheral nerve injury

Chronic constriction injury (CCI) was performed as described previously (Zhao et al., 2006

NC23766 decreases dendritic spine expression on cultured dorsal horn neurons

To determine whether the Rac1 inhibitor NSC23766 perturbs dendritic spines on spinal cord dorsal horn neurons in vitro, we treated neurons in culture (Figs. 1A, B). NSC23766 treatment reduced dendritic spine density on dorsal horn neurons compared with untreated neurons (p < 0.05, 5.08 ± 2.1 untreated vs. 1.26 ± 0.4 spines/10 μm dendrite branch treated with NSC23766) (Fig. 1C). As spine morphology directly contributes to spine physiology (i.e., mature mushroom-shaped spine associates with a more

Discussion

Our data support a model of dendritic spine remodeling underlying neuropathic pain in the spinal cord after peripheral nerve injury, and identify Rac1 signaling as an important regulator of this injury-induced plasticity. After peripheral nerve injury, dendritic spine density increases in dorsal horn neurons and spines redistribute toward more proximal locations to cell bodies. Spine length and head diameters also increased, evident in the greater number of mushroom-shaped, mature spine type.

Conflict of interest statement

None.

Acknowledgments

The work was supported in part by grants from the Medical Research Service and Rehabilitation Research Service, Department of Veterans Affairs. The Center for Neuroscience and Regeneration Research is a Collaboration of the Paralyzed Veterans of America and the United Spinal Association with Yale University.

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