Early Changes in Homer1 Proteins in the Spinal Dorsal Horn Are Associated with Loose Ligation of the Rat Sciatic Nerve

Abstract

Background Plasticity in the spinal dorsal horn is thought to underlie at least in part pain behavior following peripheral nerve injury. Homer1 proteins play an important role in synaptic plasticity through an activity-dependent remodeling of the post-synaptic density (PSD). In this study we examined the early consequences of the loose ligation of the sciatic nerve on the levels of Homer1a and Homer1b/c proteins in the PSD of spinal dorsal horn neurons. Methods Male rats were randomly assigned to control, sham-operated or ligated groups. Four hours after sciatic exposure or ligation the animals were anesthetized and euthanized. Dorsal horn ipsilateral and contralateral quadrants were homogenized and centrifuged to obtain a PSD-containing LP1 fraction. Homer1 isoforms were identified in Western immunoblots. In some animals, Homer1 small interfering RNA (siRNA), non-target siRNA, MK-801 or U-01026 were injected intrathecally before surgery to assess the effects of this treatment on the levels of Homer1 isoforms and on two signs of injury-associated pain behavior, a shift in weight-bearing distribution and thermal hyperalgesia. Results In ligated animals the protein levels of Homer1a increased and those of Homer1b/c decreased in the ipsilateral LP1 fraction of the spinal dorsal horn. In contrast, no changes were detected in the contralateral LP1 fraction of ligated animals, or either the ipsilateral or contralateral LP1 fraction of sham-operated animals. Intrathecal injections of Homer1 siRNA, but not non-target siRNA, 2h before the ligation prevented the accumulation of Homer1a and loss of Homer1b/c in the ipsilateral LP1 fraction. The same pre-treatment with Homer1 siRNA also alleviated both a shift in weight-bearing behavior and thermal hyperalgesia in the ligated animals. Intrathecal injections of MK-801 or U0126 15 min before the ligation similarly prevented the injury-associated changes in Homer1 protein levels and the behavioral signs of pain. Conclusion The ligation-associated changes in the protein levels of Homer1a and Homer 1b/c in the ipsilateral PSD of spinal dorsal horn neurons may be an important early reflection of the injury-associated plasticity that in time leads to the development of persistent pain.

Immunostaining for Homer reveals the majority of excitatory synapses in laminae I–III of the mouse spinal dorsal horn

“…Several studies have investigated the role of Homer in neuropathic and inflammatory pain models in both rats and mice. It has been reported that levels of Homer1b/c in the postsynaptic density fraction show an early reduction and then a prolonged increase in the dorsal horn ipsilateral to a chronic constriction (neuropathic) injury ( Miletic et al, 2005 , Miletic et al, 2009 , Obara et al, 2013 ), and a similar prolonged increase was seen following inflammation induced by an intra-articular injection of complete Freund’s adjuvant ( Yao et al, 2011 ). It has also been shown that disrupting the function of Homer1b/c with intrathecally administered antisense oligonucleotides led to a reduction of pain in an inflammatory model ( Yao et al, 2011 , Yao et al, 2014 ), while over-expression of Homer1c/2b in the dorsal horn exacerbated pain in the chronic constriction injury model ( Obara et al, 2013 ).…”

Alleviation of chronic pain following rat spinal cord compression injury with multimodal actions of huperzine A

“…Homer1a is the short form of the scaffolding proteins in the Homer1 family that can disrupt the mGluR signaling complex and negatively regulate nociceptive plasticity through an activity-dependent remodeling of the postsynaptic density in spinal cord sensory neurons (33). Thus, unlike approaches for pain management that aim to increase Homer1a expression in nociceptive relay neurons (33,42), we found that HUP-A reduces the release of primary sensory neurotransmitters and inhibits secondary sensory neurons, preventing up-regulation of Homer1a in the DH (Fig. 6 I and J).…”

“…6 G and H). Similarly, Homer1a, an activity-dependent inducible dominant-negative form of the linking protein that can disrupt the mGluR-signaling complex and regulate nociceptive plasticity at spinal synapses (33), was significantly lower in the cervical and lumbar DH of HUP-A-treated rats than in controls (Fig. 6 I and J).…”

Homers at the Interface between Reward and Pain

“…That both Homer1a deletion and the Grm5 R/R transgene exacerbate neuropathic pain symptoms, while neither Homer1 nor Homer2 deletion influence pain hypersensitivity (Obara et al, 2013), argues that the severity of neuropathic pain symptoms is not a determinant of CCI-induced deficits in heroin CPP (Table 2). CCI-induced neuropathy likely involves temporally dynamic changes in inducible vs. constitutive Homer expression, with early post-injury elevations in inducible Homers facilitating synaptic rearrangement that is later maintained by injury-induced increases in CC-Homer expression (e.g., Miletic et al, 2005, 2009; Miyabe et al, 2006; Tappe et al, 2006; Ma et al, 2009; Obara et al, 2013). Thus, the genetic interruption of the temporal dynamics of the interplay between inducible and CC-Homer protein expression at glutamate receptors, and likely other Homer-interacting molecules, while not always sufficient to prevent neuroplasticity within pain pathways, appears to be sufficient to prevent whatever mesocorticolimbic neuroplasticity mediating CCI-induced deficits in heroin-conditioned reward.…”