Max Larsson

Translocation of GluR1-containing AMPA receptors to a spinal nociceptive synapse during acute noxious stimulation.

Potentiation of spinal nociceptive transmission by synaptic delivery of AMPA receptors, via an NMDA receptor- and Ca2+/calmodulin-dependent 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.

Spinal HMGB1 induces TLR4-mediated long-lasting hypersensitivity and glial activation and regulates pain-like behavior in experimental arthritis.

Summary Spinal injection of disulfide extracellular high mobility group box‐1 protein (HMGB1) induces mechanical hypersensitivity and spinal glial activation, and inhibition of spinal HMGB1 resolves mechanical hypersensitivity induced by collagen antibody‐induced arthritis. ABSTRACT Extracellular high mobility group box‐1 protein (HMGB1) plays important roles in the pathogenesis of nerve injury‐ and cancer‐induced pain. However, the involvement of spinal HMGB1 in arthritis‐induced pain has not been examined previously and is the focus of this study. Immunohistochemistry showed that HMGB1 is expressed in neurons and glial cells in the spinal cord. Subsequent to induction of collagen antibody‐induced arthritis (CAIA), Hmgb1 mRNA and extranuclear protein levels were significantly increased in the lumbar spinal cord. Intrathecal (i.t.) injection of a neutralizing anti‐HMGB1 monoclonal antibody or recombinant HMGB1 box A peptide (Abox), which each prevent extracellular HMGB1 activities, reversed CAIA‐induced mechanical hypersensitivity. This occurred during ongoing joint inflammation as well as during the postinflammatory phase, indicating that spinal HMGB1 has an important function in nociception persisting beyond episodes of joint inflammation. Importantly, only HMGB1 in its partially oxidized isoform (disulfide HMGB1), which activates toll‐like receptor 4 (TLR4), but not in its fully reduced or fully oxidized isoforms, evoked mechanical hypersensitivity upon i.t. injection. Interestingly, although both male and female mice developed mechanical hypersensitivity in response to i.t. HMGB1, female mice recovered faster. Furthermore, the pro‐nociceptive effect of i.t. injection of HMGB1 persisted in Tlr2‐ and Rage‐, but was absent in Tlr4‐deficient mice. The same pattern was observed for HMGB1‐induced spinal microglia and astrocyte activation and cytokine induction. These results demonstrate that spinal HMGB1 contributes to nociceptive signal transmission via activation of TLR4 and point to disulfide HMGB1 inhibition as a potential therapeutic strategy in treatment of chronic inflammatory pain.

Distribution of vesicular glutamate transporters 1 and 2 in the rat spinal cord, with a note on the spinocervical tract

To evaluate whether the organization of glutamatergic fibers systems in the lumbar cord is also evident at other spinal levels, we examined the immunocytochemical distribution of vesicle glutamate transporters 1 and 2 (VGLUT1, VGLUT2) at several different levels of the rat spinal cord. We also examined the expression of VGLUTs in an ascending sensory pathway, the spinocervical tract, and colocalization of VGLUT1 and VGLUT2. Mainly small VGLUT2‐immunoreactive varicosities occurred at relatively high densities in most areas, with the highest density in laminae I–II. VGLUT1 immunolabeling, including small and medium‐sized to large varicosities, was more differentiated, with the highest density in the deep dorsal horn and in certain nuclei such as the internal basilar nucleus, the central cervical nucleus, and the column of Clarke. Lamina I and IIo displayed a moderate density of small VGLUT1 varicosities at all spinal levels, although in the spinal enlargements a uniform density of such varicosities was evident throughout laminae I–II in the medial half of the dorsal horn. Corticospinal tract axons displayed VGLUT1, indicating that the corticospinal tract is an important source of small VGLUT1 varicosities. VGLUT1 and VGLUT2 were cocontained in small numbers of varicosities in laminae III–IV and IX. Anterogradely labeled spinocervical tract terminals in the lateral cervical nucleus were VGLUT2 immunoreactive. In conclusion, the principal distribution patterns of VGLUT1 and VGLUT2 are essentially similar throughout the rostrocaudal extension of the spinal cord. The mediolateral differences in VGLUT1 distribution in laminae I–II suggest dual origins of VGLUT1‐immunoreactive varicosities in this region. J. Comp. Neurol. 497:683–701, 2006.

Functional and Anatomical Identification of a Vesicular Transporter Mediating Neuronal ATP Release

ATP is known to be coreleased with glutamate at certain central synapses. However, the nature of its release is controversial. Here, we demonstrate that ATP release from cultured rat hippocampal neurons is sensitive to RNAi-mediated knockdown of the recently identified vesicular nucleotide transporter (VNUT or SLC17A9). In the intact brain, light microscopy showed particularly strong VNUT immunoreactivity in the cerebellar cortex, the olfactory bulb, and the hippocampus. Using immunoelectron microscopy, we found VNUT immunoreactivity colocalized with synaptic vesicles in excitatory and inhibitory terminals in the hippocampal formation. Moreover, VNUT immunolabeling, unlike that of the vesicular glutamate transporter VGLUT1, was enriched in preterminal axons and present in postsynaptic dendritic spines. Immunoisolation of synaptic vesicles indicated presence of VNUT in a subset of VGLUT1-containing vesicles. Thus, we conclude that VNUT mediates transport of ATP into synaptic vesicles of hippocampal neurons, thereby conferring a purinergic phenotype to these cells.

Synaptic Plasticity and Pain: Role of Ionotropic Glutamate Receptors

Pain hypersensitivity that develops after tissue or nerve injury is dependent both on peripheral processes in the affected tissue and on enhanced neuronal responses in the central nervous system, including the dorsal horn of the spinal cord. It has become increasingly clear that strengthening of glutamatergic sensory synapses, such as those established in the dorsal horn by nociceptive thin-caliber primary afferent fibers, is a major contributor to sensitization of neuronal responses that leads to pain hypersensitivity. Here, the authors review recent findings on the roles of ionotropic glutamate receptors in synaptic plasticity in the dorsal horn in relation to acute and persistent pain.

Ionotropic Glutamate Receptors in Spinal Nociceptive Processing

Glutamate is the predominant excitatory transmitter used by primary afferent synapses and intrinsic neurons in the spinal cord dorsal horn. Accordingly, ionotropic glutamate receptors mediate basal spinal transmission of sensory, including nociceptive, information that is relayed to supraspinal centers. However, it has become gradually more evident that these receptors are also crucially involved in short- and long-term plasticity of spinal nociceptive transmission, and that such plasticity have an important role in the pain hypersensitivity that may result from tissue or nerve injury. This review will cover recent findings on pre- and postsynaptic regulation of synaptic function by ionotropic glutamate receptors in the dorsal horn and how such mechanisms contribute to acute and chronic pain.

Different basal levels of CaMKII phosphorylated at Thr286/287 at nociceptive and low‐threshold primary afferent synapses

Postsynaptic autophosphorylation of Ca2+/calmodulin‐dependent protein kinase II (CaMKII) at Thr286/287 is crucial for the induction of long‐term potentiation at many glutamatergic synapses, and has also been implicated in the persistence of synaptic potentiation. However, the availability of CaMKII phosphorylated at Thr286/287 at individual glutamatergic synapses in vivo is unclear. We used post‐embedding immunogold labelling to quantitatively analyse the ultrastructural localization of CaMKII phosphorylated at Thr286/287 (pCaMKII) at synapses formed by presumed nociceptive and low‐threshold mechanosensitive primary afferent nerve endings in laminae I–IV of rat spinal cord. Immunogold labelling was enriched in the postsynaptic densities of such synapses, consistent with observations in pre‐embedding immunoperoxidase‐stained dorsal horn. Presynaptic axoplasm also exhibited sparse immunogold labelling, in peptidergic terminals partly associated with dense core vesicles. Analysis of single or serial pCaMKII‐immunolabelled sections indicated that the large majority of synapses formed either by presumed peptidergic or non‐peptidergic nociceptive primary afferent terminals in laminae I–II of the spinal cord, or by presumed low‐threshold mechanosensitive primary afferent terminals in laminae IIi–IV, contained pCaMKII in their postsynaptic density. However, the postsynaptic levels of pCaMKII immunolabelling at low‐threshold primary afferent synapses were only ∼ 50% of those at nociceptive synapses. These results suggest that constitutively autophosphorylated CaMKII in the postsynaptic density is a common characteristic of glutamatergic synapses, thus potentially contributing to maintenance of synaptic efficacy. Furthermore, pCaMKII appears to be differentially regulated between high‐ and low‐threshold primary afferent synapses, possibly reflecting different susceptibility to synaptic plasticity between these afferent pathways.

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

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 Ca2+/calmodulin-dependent protein kinase II (CaMKII) and of CaMKII phosphorylated at Thr286/287 (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 ∼20 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 activity-induced loss of certain postsynaptic pools of autophosphorylated CaMKII at previously unmanipulated synapses supports a role for the kinase in basal postsynaptic function.

Quantitative analysis of immunogold labeling indicates low levels and non-vesicular localization of L-aspartate in rat primary afferent terminals.

The role of L‐aspartate as an excitatory neurotransmitter in primary afferent synapses in the spinal cord dorsal horn is disputed. To further investigate this issue, we examined the presence of aspartate‐like immunoreactivity in primary afferent nerve terminals and other tissue components of the dorsal horn. We also examined the relationship between aspartate and glutamate immunogold labeling density and the density of synaptic vesicles in primary afferent terminals and presumed inhibitory terminals forming symmetric synapses. Weak aspartate immunosignals, similar to or lower than those displayed by presumed inhibitory terminals, were detected in both C‐fiber primary afferent terminals in lamina II (dense sinusoid axon terminals, identified by morphological criteria) and in A‐fiber primary afferent terminals in laminae III–IV (identified with anterograde transport of choleragenoid‐horseradish peroxidase conjugate). The aspartate immunogold signal in primary afferent terminals was only about one‐fourth of that in deep dorsal horn neuronal cell bodies. Further, whereas significant positive correlations were evident between synaptic vesicle density and glutamate immunogold labeling density in both A‐ and C‐fiber primary afferent terminals, none of the examined terminal populations displayed a significant correlation between synaptic vesicle density and aspartate immunogold labeling density. Thus, our results indicate relatively low levels and a non‐vesicular localization of aspartate in primary afferent terminals. It is therefore suggested that aspartate, rather than being a primary afferent neurotransmitter, serves a role in the intermediary metabolism in primary afferent terminals. J. Comp. Neurol. 430:147–159, 2001.

Vesicular uptake and exocytosis of l-aspartate is independent of sialin

The mechanism of release and the role of l‐aspartate as a central neurotransmitter are controversial. A vesicular release mechanism for l‐aspartate has been difficult to prove, as no vesicular l‐aspartate transporter was identified until it was found that sialin could transport l‐aspartate and l‐glutamate when reconstituted into liposomes. We sought to clarify the release mechanism of l‐aspartate and the role of sialin in this process by combining l‐aspartate uptake studies in isolated synaptic vesicles with immunocyotchemical investigations of hippocampal slices. We found that radiolabeled l‐aspartate was taken up into synaptic vesicles. The vesicular l‐aspartate uptake, relative to the l‐glutamate uptake, was twice as high in the hippocampus as in the whole brain, the striatum, and the entorhinal and frontal cortices and was not inhibited by l‐glutamate. We further show that sialin is not essential for exocytosis of l‐aspartate, as there was no difference in ATP‐dependent l‐aspartate uptake in synaptic vesicles from sialin‐knockout and wild‐type mice. In addition, expression of sialin in PC12 cells did not result in significant vesicle uptake of l‐aspartate, and depolarization‐induced depletion of l‐aspartate from hippocampal nerve terminals was similar in hippocampal slices from sialin‐knockout and wild‐type mice. Further, there was no evidence for nonvesicular release of l‐aspartate via volume‐regulated anion channels or plasma membrane excitatory amino acid transporters. This suggests that l‐aspartate is exocytotically released from nerve terminals after vesicular accumulation by a transporter other than sialin.—Morland, C., Nordengen, K., Larsson, M., Prolo, L. M., Farzampour, Z., Reimer, R. J., Gundersen, V. Vesicular uptake and exocytosis of L‐aspartate is independent of sialin. FASEB J. 27, 1264–1274 (2013). www.fasebj.org