Meei-Ling Tsaur

Cloning, expression and CNS distribution of Kv4.3, an A‐type K+ channel α subunit

A full‐length K+ channel cDNA of Kv4.3, with an open reading frame of 611 amino acids, was isolated from rat hippocampus. Functional expression of Kv4.3 cDNA in Xenopus oocytes revealed an A‐type K+ channel. In the central nervous system, Kv4.3 is most prominently expressed in the retrosplenial cortex, medial habenula, anterior thalamus, hippocampus, cerebellum, as well as lateral geniculate and superior colliculus, which are important for vision. The abundant expression of Kv4.3 in many CNS neurons supports its important role as a major component of subthreshold A currents in the control of action potentials and thus neuronal excitability.

Mirror-image pain is mediated by nerve growth factor produced from tumor necrosis factor alpha-activated satellite glia after peripheral nerve injury.

Summary After peripheral nerve injury, tumor necrosis factor &agr;‐activated satellite glia in the contralateral dorsal root ganglion produce excess nerve growth factor that, in turn, enhances nociceptor excitability and results in mirror‐image pain. ABSTRACT Mirror‐image pain is characterized by mechanical hypersensitivity on the uninjured mirror‐image side. Recent reports favor central mechanisms, but whether peripheral mechanisms are involved remains unclear. We used unilateral spinal nerve ligation (SNL) to induce mirror‐image pain in rats. On the mirror‐image (contralateral) side, we found that satellite glia in the dorsal root ganglion (DRG) were activated, whereas macrophages/Schwann cells in the DRG and astrocytes/oligodendrocytes/microglia in the dorsal spinal cord were not. Subsequently, an increase in nerve growth factor (NGF) was detected in the contralateral DRG, and NGF immunoreactivity was concentrated in activated satellite glia. These phenomena were abolished if fluorocitrate (a glial inhibitor) was intrathecally injected before SNL. Electrophysiological recordings in cultured small DRG neurons showed that exogenous NGF enhanced nociceptor excitability. Intrathecal injection of NGF into naive rats induced long‐lasting mechanical hypersensitivity, similar to SNL‐evoked mirror‐image pain. Anti‐NGF effectively relieved SNL‐evoked mirror‐image pain. In the contralateral DRG, the SNL‐evoked tumor necrosis factor alpha (TNF‐&agr;) increase, which started later than in the ipsilateral DRG and cerebrospinal fluid, occurred earlier than satellite glial activation and the NGF increase. Intrathecal injection of TNF‐&agr; into naive rats not only activated satellite glia to produce extra NGF in the DRG but also evoked mechanical hypersensitivity, which could be attenuated by anti‐NGF injection. These results suggest that after SNL, satellite glia in the contralateral DRG are activated by TNF‐&agr; that diffuses from the injured side via cerebrospinal fluid, which then activates satellite glia to produce extra NGF to enhance nociceptor excitability, which induces mirror‐image pain.

Expression of A-type K+ channel α subunits Kv4.2 and Kv4.3 in rat spinal lamina II excitatory interneurons and colocalization with pain-modulating molecules

Voltage‐gated K+ channel α subunits Kv4.2 and Kv4.3 are the major contributors of somatodendritic A‐type K+ currents in many CNS neurons. A recent hypothesis suggests that Kv4 subunits may be involved in pain modulation in dorsal horn neurons. However, whether Kv4 subunits are expressed in dorsal horn neurons remains unknown. Using immunohistochemistry, we found that Kv4.2 and Kv4.3 immunoreactivity was concentrated in the superficial dorsal horn, mainly in lamina II. Both Kv4.2 and Kv4.3 appeared on many rostrocaudally orientated dendrites, whereas Kv4.3 could be also detected from certain neuronal somata. Kv4.3(+) neurons were a subset of excitatory inerneurons with calretinin(+)/calbindin(–)/PKCγ(–) markers, and a fraction of them expressed µ‐opioid receptors. Kv4.3(+) neurons also expressed ERK2 and mGluR5, which are molecules related to the induction of central sensitization, a mechanism mediating nociceptive plasticity. Together with the expression of Kv4.3 in VR1(+) DRG neurons, our data suggest that Kv4 subunits could be involved in pain modulation.

Contrasting expression of Kv4.3, an A‐type K+ channel, in migrating Purkinje cells and other post‐migratory cerebellar neurons

Kv4.3, an A‐type K+ channel, is the only channel molecule showing anterior–posterior (A–P) compartmentalization in the granular layer of mammalian cerebellum known so far. Kv4.3 mRNA has been detected from the posterior but not anterior granular layer in adult rat cerebellum. To characterize this A‐P compartmentalization further, we examined Kv4.3 protein expression in rat cerebellum by immunohistochemistry at the embryonic, early postnatal and adult stages. Specificity of the Kv4.3 antibody was confirmed by both Western blot and immunoprecipitation analysis. In adulthood, Kv4.3 was detected from the somatodendritic domain of posterior granule cells, with a restriction boundary in the vermal lobule VI extending laterally to the hemispheric crus 1 ansiform lobules. At the early postnatal stage, this A–P pattern first appeared on postnatal day 8, when significant numbers of granule cells had migrated into the posterior granular layer and started to express Kv4.3. Similar Kv4.3 expression in the somatodendritic domain of post‐migratory neurons in the cerebellum was also observed in basket cells, stellate cells, a subset of GABAergic deep neurons, Lugaro cells and, probably, deep Lugaro cells. However, none of them showed A–P compartmentalization. Strikingly, we found Kv4.3 in several clusters of migrating Purkinje cells with mediolateral compartmentalization. These Purkinje cells no longer expressed Kv4.3 after completing the migration. By contrasting the expression in migrating and post‐migratory neurons, our results suggest that Kv4.3 may play an important role in the development of cerebellum, as well as in the mature cerebellum.

Expression of B-type endothelin receptor gene during neural development

Mutations of the B‐type endothelin receptor (ETRB) gene have been found to cause defects in the development of enteric neurons, which resulted in aganglionic megacolon in rodents and humans. To determine the distribution of ETRB mRNA during neural development, mainly in the CNS, in situ hybridization was applied at various developmental stages of rat. ETRB gene was abundantly expressed prenatally in the ventricular and subventricular zones, as well as postnatally in the ependymal and subependymal cells. ETRB mRNA was also strongly detected prenatally in the dorsal root ganglia, as well as postnatally in the cerebellar Bergmann glial cells and epithelial cells of choroid plexus. Our data suggest that ETRB acts as a regulator in the differentiation, proliferation, or migration of neural cells during development.

Nerve growth factor-induced synapse-like structures in contralateral sensory ganglia contribute to chronic mirror-image pain.

Abstract Elevated nerve growth factor (NGF) in the contralateral dorsal root ganglion (DRG) mediates mirror-image pain after peripheral nerve injury, but the underlying mechanism remains unclear. Using intrathecal injection of NGF antibodies, we found that NGF is required for the development of intra-DRG synapse-like structures made by neurite sprouts of calcitonin gene-related peptide (CGRP+) nociceptors and sympathetic axons onto neurite sprouts of Kv4.3+ nociceptors. These synapse-like structures are formed near NGF-releasing satellite glia surrounding large DRG neurons. Downregulation of the postsynaptic protein PSD95 with a specific shRNA largely eliminates these synapse-like structures, suppresses activities of Kv4.3+ but not CGRP+ nociceptors, and attenuates mirror-image pain. Furthermore, neutralizing the neurotransmitter norepinephrine or CGRP in the synapse-like structures by antibodies has similar analgesic effect. Thus, elevated NGF after peripheral nerve injury induces neurite sprouting and the formation of synapse-like structures within the contralateral DRG, leading to the development of chronic mirror-image pain.

Coexpression of high-voltage-activated ion channels Kv3.4 and Cav1.2 in pioneer axons during pathfinding in the developing rat forebrain

Precise axon pathfinding is crucial for establishment of the initial neuronal network during development. Pioneer axons navigate without the help of preexisting axons and pave the way for follower axons that project later. Voltage‐gated ion channels make up the intrinsic electrical activity of pioneer axons and regulate axon pathfinding. To elucidate which channel molecules are present in pioneer axons, immunohistochemical analysis was performed to examine 14 voltage‐gated ion channels (Kv1.1–Kv1.3, Kv3.1–Kv3.4, Kv4.3, Cav1.2, Cav1.3, Cav2.2, Nav1.2, Nav1.6, and Nav1.9) in nine axonal tracts in the developing rat forebrain, including the optic nerve, corpus callosum, corticofugal fibers, thalamocortical axons, lateral olfactory tract, hippocamposeptal projection, anterior commissure, hippocampal commissure, and medial longitudinal fasciculus. We found A‐type K+ channel Kv3.4 in both pioneer axons and early follower axons and L‐type Ca2+ channel Cav1.2 in pioneer axons and early and late follower axons. Spatially, Kv3.4 and Cav1.2 were colocalized with markers of pioneer neurons and pioneer axons, such as deleted in colorectal cancer (DCC), in most fiber tracts examined. Temporally, Kv3.4 and Cav1.2 were expressed abundantly in most fiber tracts during axon pathfinding but were downregulated beginning in synaptogenesis. By contrast, delayed rectifier Kv channels (e.g., Kv1.1) and Nav channels (e.g., Nav1.2) were absent from these fiber tracts (except for the corpus callosum) during pathfinding of pioneer axons. These data suggest that Kv3.4 and Cav1.2, two high‐voltage‐activated ion channels, may act together to control Ca2+‐dependent electrical activity of pioneer axons and play important roles during axon pathfinding. J. Comp. Neurol. 520:3650–3672, 2012.

Coexpression of auxiliary subunits KChIP and DPPL in potassium channel Kv4-positive nociceptors and pain-modulating spinal interneurons.

Subthreshold A‐type K+ currents (ISAs) have been recorded from the somata of nociceptors and spinal lamina II excitatory interneurons, which sense and modulate pain, respectively. Kv4 channels are responsible for the somatodendritic ISAs. Accumulative evidence suggests that neuronal Kv4 channels are ternary complexes including pore‐forming Kv4 subunits and two types of auxiliary subunits: K+ channel‐interacting proteins (KChIPs) and dipeptidyl peptidase‐like proteins (DPPLs). Previous reports have shown Kv4.3 in a subset of nonpeptidergic nociceptors and Kv4.2/Kv4.3 in certain spinal lamina II excitatory interneurons. However, whether and which KChIP and DPPL are coexpressed with Kv4 in these ISA‐expressing pain‐related neurons is unknown. In this study we mapped the protein distribution of KChIP1, KChIP2, KChIP3, DPP6, and DPP10 in adult rat dorsal root ganglion (DRG) and spinal cord by immunohistochemistry. In the DRG, we found colocalization of KChIP1, KChIP2, and DPP10 in the somatic surface and cytoplasm of Kv4.3(+) nociceptors. KChIP3 appears in most Aβ and Aδ sensory neurons as well as a small population of peptidergic nociceptors, whereas DPP6 is absent in sensory neurons. In the spinal cord, KChIP1 is coexpressed with Kv4.3 in the cell bodies of a subset of lamina II excitatory interneurons, while KChIP1, KChIP2, and DPP6 are colocalized with Kv4.2 and Kv4.3 in their dendrites. Within the dorsal horn, besides KChIP3 in the inner lamina II and lamina III, we detected DPP10 in most projection neurons, which transmit pain signal to brain. The results suggest the existence of Kv4/KChIP/DPPL ternary complexes in ISA‐expressing nociceptors and pain‐modulating spinal interneurons. J. Comp. Neurol. 524:846–873, 2016.

K+ channel modulatory subunits KChIP and DPP participate in Kv4-mediated mechanical pain control

The K+ channel pore-forming subunit Kv4.3 is expressed in a subset of nonpeptidergic nociceptors within the dorsal root ganglion (DRG), and knockdown of Kv4.3 selectively induces mechanical hypersensitivity, a major symptom of neuropathic pain. K+ channel modulatory subunits KChIP1, KChIP2, and DPP10 are coexpressed in Kv4.3+ DRG neurons, but whether they participate in Kv4.3-mediated pain control is unknown. Here, we show the existence of a Kv4.3/KChIP1/KChIP2/DPP10 complex (abbreviated as the Kv4 complex) in the endoplasmic reticulum and cell surface of DRG neurons. After intrathecal injection of a gene-specific antisense oligodeoxynucleotide to knock down the expression of each component in the Kv4 complex, mechanical hypersensitivity develops in the hindlimbs of rats in parallel with a reduction in all components in the lumbar DRGs. Electrophysiological data further indicate that the excitability of nonpeptidergic nociceptors is enhanced. The expression of all Kv4 complex components in DRG neurons is downregulated following spinal nerve ligation (SNL). To rescue Kv4 complex downregulation, cDNA constructs encoding Kv4.3, KChIP1, and DPP10 were transfected into the injured DRGs (defined as DRGs with injured spinal nerves) of living SNL rats. SNL-evoked mechanical hypersensitivity was attenuated, accompanied by a partial recovery of Kv4.3, KChIP1, and DPP10 surface levels in the injured DRGs. By showing an interdependent regulation among components in the Kv4 complex, this study demonstrates that K+ channel modulatory subunits KChIP1, KChIP2, and DPP10 participate in Kv4.3-mediated mechanical pain control. Thus, these modulatory subunits could be potential drug targets for neuropathic pain. SIGNIFICANCE STATEMENT Neuropathic pain, a type of moderate to severe chronic pain resulting from nerve injury or disorder, affects 6.9%–10% of the global population. However, less than half of patients report satisfactory pain relief from current treatments. K+ channels, which act to reduce nociceptor activity, have been suggested to be novel drug targets for neuropathic pain. This study is the first to show that K+ channel modulatory subunits KChIP1, KChIP2, and DPP10 are potential drug targets for neuropathic pain because they form a channel complex with the K+ channel pore-forming subunit Kv4.3 in a subset of nociceptors to selectively inhibit mechanical hypersensitivity, a major symptom of neuropathic pain.

Immunohistochemical localization of DPP10 in rat brain supports the existence of a Kv4/KChIP/DPPL ternary complex in neurons.

Subthreshold A‐type K+ currents (ISAs) have been recorded from the cell bodies of hippocampal and neocortical interneurons as well as neocortical pyramidal neurons. Kv4 channels are responsible for the somatodendritic ISAs. It has been proposed that neuronal Kv4 channels are ternary complexes including pore‐forming Kv4 subunits, K+ channel‐interacting proteins (KChIPs), and dipeptidyl peptidase‐like proteins (DPPLs). However, colocalization evidence was still lacking. The distribution of DPP10 mRNA in rodent brain has been reported but its protein localization remains unknown. In this study, we generated a DPP10 antibody to label DPP10 protein in adult rat brain by immunohistochemistry. Absent from glia, DPP10 proteins appear mainly in the cell bodies of DPP10(+) neurons, not only at the plasma membrane but also in the cytoplasm. At least 6.4% of inhibitory interneurons in the hippocampus coexpressed Kv4.3, KChIP1, and DPP10, with the highest density in the CA1 strata alveus/oriens/pyramidale and the dentate hilus. Colocalization of Kv4.3/KChIP1/DPP10 was also detected in at least 6.9% of inhibitory interneurons scattered throughout the neocortex. Both hippocampal and neocortical Kv4.3/KChIP1/DPP10(+) inhibitory interneurons expressed parvalbumin or somatostatin, but not calbindin or calretinin. Furthermore, we found colocalization of Kv4.2/Kv4.3/KChIP3/DPP10 in neocortical layer 5 pyramidal neurons and olfactory bulb mitral cells. Together, although DPP10 is also expressed in some brain neurons lacking Kv4 (such as parvalbumin‐ and somatostatin‐positive Golgi cells in the cerebellum), colocalization of DPP10 with Kv4 and KChIP at the plasma membrane of ISA‐expressing neuron somata supports the existence of Kv4/KChIP/DPPL ternary complex in vivo. J. Comp. Neurol. 523:608–628, 2015.