Loose ligation of the rat sciatic nerve elicits early accumulation of Shank1 protein in the post-synaptic density of spinal dorsal horn neurons

Abstract

Plasticity in the spinal dorsal horn may contribute to the development of pain following peripheral nerve injury. Shank proteins are a constituent family of the post-synaptic density (PSD), and they may play a role in synaptic plasticity through activity-dependent synaptic remodeling and growth. In this study we examined the early consequences of the loose ligation of the sciatic nerve on Shank1 protein and message levels in the PSD of spinal dorsal horn neurons.Four hours after sciatic ligation, the protein levels of Shank1 increased in the ipsilateral PSD of ligated animals. In contrast, no changes were detected in the contralateral PSD of these ligated animals, or either the ipsilateral or contralateral PSD of sham-operated animals. Shank1 was linked to the PSD marker protein PSD-95 and the NR2B subunit of NMDA receptors. The ligated animals also exhibited two early signs of pain behavior, a shift in weight bearing and thermal hyperalgesia. There was no overall change in Shank1 message in either ligated or sham-operated animals. The accumulation of Shank1 in the PSD was abolished by intrathecal pre-treatment with anisomycin or Shank1 siRNA but not non-target siRNA. The same pre-treatment prevented both of the early signs of pain behavior. Intrathecal pre-treatment with either MK-801 or U0126 similarly prevented the Shank1 accumulation and alleviated both of the behavioral signs of pain.The early accumulation of Shank1 in the PSD of dorsal horn neurons may be a necessary step in the injury-associated plasticity that in time leads to the development of persistent pain.

Protein kinase C gamma-mediated phosphorylation of GluA1 in the postsynaptic density of spinal dorsal horn neurons accompanies neuropathic pain, and dephosphorylation by calcineurin is associated with prolonged analgesia

“…We focused on the 7 days post-ligation period because by this time neuropathic pain is well-established [11,12]. …”

“…Calcineurin, the specific PKA peptide inhibitor PKI-tide, the specific PKCγ peptide inhibitor PKC (19-31) and the specific CaMKII inhibitor autocamtide-2-related inhibitory peptide (AIP-2) were all purchased from Enzo Life Sciences (Plymouth Meeting, PA) and injected intrathecally in a volume of 10μl [11,12]. The same volume of saline served as the vehicle control.…”

“…Tissues were homogenized and sequentially centrifuged in HEPES buffer containing sucrose, NaF, EDTA and a protein inhibitor cocktail at 4°C to yield the PSD-containing LP1 fractions [11]. Briefly, samples were first spun at 1,500g for 10min to yield a pellet (P1) and supernatant (S1) fraction.…”

“…After assaying for total protein content, the LP1 fraction was processed for Western immunoblots [11,12]. Each blot was a single individual animal sample.…”

Activation of a Gq-coupled membrane estrogen receptor rapidly attenuates α2-adrenoceptor-induced antinociception via an ERK I/II-dependent, non-genomic mechanism in the female rat

“…ICI 182,780 (an ER antagonist; 10 µM; Tocris) was co-injected with E2BSA or STX. Anisomycin (a protein synthesis inhibitor; 125 µg; Sigma; Miletic et al, 2010) was injected 15 min before clonidine. U0126 (20 µg/10 µl; Wang et al, 2011) that inhibits ERK I/II via MEK inhibition, or U0124, an inactive analogue of U0126 was injected 30 min before clonidine.…”