Intrathecal substance p–saporin attenuates operant escape from nociceptive thermal stimuli

An intensely painful stimulus may lead to hyperalgesia, the enhanced sensation of subsequent painful stimuli. This is commonly believed to involve facilitated transmission of sensory signals in the spinal cord, possibly by a long-term potentiation-like mechanism. However, plasticity of identified synapses in intact hyperalgesic animals has not been reported. Here, we show, using neuronal tracing and postembedding immunogold labeling, that after acute noxious stimulation (hindpaw capsaicin injections), immunolabeling of Ca 2ϩ / calmodulin-dependent protein kinase II (CaMKII) and of CaMKII phosphorylated at Thr 286/287 (pCaMKII) are upregulated postsynaptically at synapses established by peptidergic primary afferent fibers in the superficial dorsal horn of intact rats. In contrast, postsynaptic pCaMKII immunoreactivity was instead downregulated at synapses of nonpeptidergic primary afferent C-fibers; this loss of pCaMKII immunolabel occurred selectively at distances greater than ϳ20 nm from the postsynaptic membrane and was accompanied by a smaller reduction in total CaMKII contents of these synapses. Both pCaMKII and CaMKII immunogold labeling were unaffected at synapses formed by presumed low-threshold mechanosensitive afferent fibers. Thus, distinct molecular modifications, likely indicative of plasticity of synaptic strength, are induced at different populations of presumed nociceptive primary afferent synapse by intense noxious stimulation, suggesting a complex modulation of parallel nociceptive pathways in inflammatory hyperalgesia. Furthermore, the activityinduced loss of certain postsynaptic pools of autophosphorylated CaMKII at previously unmanipulated synapses supports a role for the kinase in basal postsynaptic function.

Section: Discussionsupporting

confidence: 73%

Pathway-Specific Bidirectional Regulation of Ca²⁺/Calmodulin-Dependent Protein Kinase II at Spinal Nociceptive Synapses after Acute Noxious Stimulation

Larsson¹,

Broman²

2006

“…We have previously reported (Wiley and Lappi, 2005) two observations that further support the conclusion that intrathecal Derm-sap does not damage primary afferent neurons: (1) cell counts from lumbar DRGs taken 24 -96 h after 750 ng of intrathecal Derm-sap failed to reveal any evidence of the typical acute, severe chromatolysis produced by saporin; and (2) assessment of MOR staining from rats with multilevel lumbar dorsal rhizotomies in the absence or presence of intrathecal Derm-sap showed that rhizotomies or Derm-sap alone each produced ϳ50% loss of MOR staining in the superficial lumbar dorsal horn, but combining rhizotomies and Derm-sap completely abolished MOR staining in the dorsal horn consistent with two separate cellular pools of dorsal horn MOR, primary afferent terminals, and lamina II interneurons (Aicher et al, 2000;Abbadie et al, 2002). Because lumbar intrathecal injections of substance P-saporin conjugate have no effect on morphine antinociception in the same hotplate protocols used in the present study (Vierck et al, 2003), we conclude the observed effects of Derm-sap on morphine antinociception are specific for this conjugate and not a common property of all lumbar intrathecal peptide-saporin conjugate injections. We estimate the observed 34% decrease actually represents ϳ68% loss of dorsal horn postsynaptic MOR1, because about half of all MOR1 staining is presynaptic on primary afferent terminals (i.e., lost after rhizotomy) (Wiley and Lappi, 2005).…”

Section: Discussionsupporting

confidence: 54%

“…Spinal cord sections were prepared as described previously (Vierck et al, 2003). Briefly, 2 weeks to 4 months after toxin or vehicle injections, rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with 200 -300 ml of cold normal saline containing 5 mM sodium phosphate, pH 7.5, 1 g/L sodium nitrite (vasodilator), and 1000 U/L sodium heparin (anticoagulant), followed by 4% formaldehyde prepared from paraformaldehyde in 100 mM sodium phosphate, pH 7.5.…”

Section: Methodsmentioning

confidence: 99%

Spinal μ-Opioid Receptor-Expressing Dorsal Horn Neurons: Role in Nociception and Morphine Antinociception

Kline¹,

Wiley²

2008

The role of spinal cord -opioid receptor (MOR)-expressing dorsal horn neurons in nociception and morphine analgesia is incompletely understood. Using intrathecal dermorphin-saporin (Derm-sap) to selectively destroy MOR-expressing dorsal horn neurons, we sought to determine the role of these neurons in (1) normal baseline reflex nocifensive responses to noxious thermal stimulation (hotplate, tail flick) and to persistent noxious chemical stimulation (formalin) and (2) the antinociceptive activity of intrathecal and systemic morphine in the same tests. Lumbar intrathecal Derm-sap (500 ng) produced (1) partial loss of lamina II MOR-expressing dorsal horn neurons, (2) no effect on MOR-expressing dorsal root ganglion neurons, and (3) no change in baseline tail-flick and hotplate reflex nocifensive responses. Derm-sap treatment attenuated the antinociceptive action of both intrathecal and systemic morphine on hotplate responses. Derm-sap treatment had two effects in the formalin test: (1) increased baseline nocifensive responding and (2) reduced antinociceptive action of systemic morphine. We conclude that MOR-expressing dorsal horn neurons (1) are not essential for determining nocifensive reflex responsiveness to noxious thermal stimuli, (2) are necessary for full antinociceptive action of morphine (intrathecal or systemic) in these tests, and (3) play a significant role in the endogenous modulation of reflex nocifensive responses to persistent pain in the formalin test. Thus, one would predict that altering the activity of MOR-expressing dorsal horn neurons would be antinociceptive and of interest in the search for new approaches to management of chronic pain.

“…That we did not observe substantially changed levels of AMPA receptor subunits at SP ϩ /CGRP ϩ synapses after capsaicin stimulation is surprising, especially because lamina I projection neurons that receive preferential, potentiatable primary afferent input from SP ϩ /CGRP ϩ fibers (McLeod et al, 1998;Todd et al, 2002;Ikeda et al, 2003Ikeda et al, , 2006 are essential for the full expression of hyperalgesia (Mantyh et al, 1997;Nichols et al, 1999;Vierck et al, 2003). In addition, levels of autophosphorylated, presumably active CaMKII are increased at SP ϩ /CGRP ϩ synapses after peripheral capsaicin stimulation (Larsson and Broman, 2006), confirming that these synapses are activated in this model of inflammatory pain.…”

Section: Discussionmentioning

confidence: 81%

Translocation of GluR1-Containing AMPA Receptors to a Spinal Nociceptive Synapse during Acute Noxious Stimulation

Larsson¹,

Broman²

2008

/calmodulindependent protein kinase II (CaMKII)-dependent pathway, has been proposed to underlie certain forms of hyperalgesia, the enhanced pain sensitivity that may accompany inflammation or tissue injury. However, the specific synaptic populations that may be subject to such plasticity have not been identified. Using neuronal tracing and postembedding immunogold labeling, we show that a model of acute inflammatory hyperalgesia is associated with an elevated density of GluR1-containing AMPA receptors, as well as an increased synaptic ratio of GluR1 to GluR2/3 subunits, at synapses established by C-fibers that lack the neuropeptide substance P. A more subtle increase in GluR1 immunolabeling was noted at synapses formed by substance P-containing nociceptors. No changes in either GluR1 or GluR2/3 contents were observed at synapses formed by low-threshold mechanosensitive primary afferent fibers. These results contrast with our previous observations in the same pain model of increased and decreased levels of activated CaMKII at synapses formed by peptidergic and nonpeptidergic nociceptive fibers, respectively, suggesting that the observed redistribution of AMPA receptor subunits does not depend on postsynaptic CaMKII activity. The present ultrastructural evidence of topographically specific, activity-dependent insertion of GluR1-containing AMPA receptors at a central synapse suggests that potentiation of nonpeptidergic C-fiber synapses by this mechanism contributes to inflammatory pain.