K+ Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca2+ into Growth Cones.

Membrane excitability in the axonal growth cones of embryonic neurons influences axon growth. Voltage-gated K+ (Kv) channels are key factors in controlling membrane excitability, but whether they regulate axon growth remains unclear. Here, we report that Kv3.4 is expressed in the axonal growth cones of embryonic spinal commissural neurons, motoneurons, dorsal root ganglion neurons, retinal ganglion cells, and callosal projection neurons during axon growth. Our in vitro (cultured dorsal spinal neurons of chick embryos) and in vivo (developing chick spinal commissural axons and rat callosal axons) findings demonstrate that knockdown of Kv3.4 by a specific shRNA impedes axon initiation, elongation, pathfinding, and fasciculation. In cultured dorsal spinal neurons, blockade of Kv3.4 by blood depressing substance II suppresses axon growth via an increase in the amplitude and frequency of Ca2+ influx through T-type and L-type Ca2+ channels. Electrophysiological results show that Kv3.4, the major Kv channel in the axonal growth cones of embryonic dorsal spinal neurons, is activated at more hyperpolarized potentials and inactivated more slowly than it is in postnatal and adult neurons. The opening of Kv3.4 channels effectively reduces growth cone membrane excitability, thereby limiting excessive Ca2+ influx at subthreshold potentials or during Ca2+-dependent action potentials. Furthermore, excessive Ca2+ influx induced by an optogenetic approach also inhibits axon growth. Our findings suggest that Kv3.4 reduces growth cone membrane excitability and maintains [Ca2+]i at an optimal concentration for normal axon growth.SIGNIFICANCE STATEMENT Accumulating evidence supports the idea that impairments in axon growth contribute to many clinical disorders, such as autism spectrum disorders, corpus callosum agenesis, Joubert syndrome, Kallmann syndrome, and horizontal gaze palsy with progressive scoliosis. Membrane excitability in the growth cone, which is mainly controlled by voltage-gated Ca2+ (Cav) and K+ (Kv) channels, modulates axon growth. The role of Cav channels during axon growth is well understood, but it is unclear whether Kv channels control axon outgrowth by regulating Ca2+ influx. This report shows that Kv3.4, which is transiently expressed in the axonal growth cones of many types of embryonic neurons, acts to reduce excessive Ca2+ influx through Cav channels and thus permits normal axon outgrowth.

Reduced expression of A-type potassium channels in primary sensory neurons induces mechanical hypersensitivity.

A-type K+ channels (A-channels) are crucial in controlling neuronal excitability, and their downregulation in pain-sensing neurons may increase pain sensation. To test this hypothesis, we first characterized the expression of two A-channels, Kv3.4 and Kv4.3, in rat dorsal root ganglion (DRG) neurons. Kv3.4 was expressed mainly in the nociceptive DRG neurons, in their somata, axons, and nerve terminals innervating the dorsal horn of spinal cord. In contrast, Kv4.3 appeared selectively in the somata of a subset of nonpeptidergic nociceptive DRG neurons. Most Kv4.3(+) DRG neurons also expressed Kv3.4. In a neuropathic pain model induced by spinal nerve ligation in rats, the protein levels of Kv3.4 and Kv4.3 in the DRG neurons were greatly reduced. After Kv3.4 or Kv4.3 expression in lumbar DRG neurons was suppressed by intrathecal injections of antisense oligodeoxynucleotides, mechanical but not thermal hypersensitivity developed. Together, our data suggest that reduced expression of A-channels in pain-sensing neurons may induce mechanical hypersensitivity, a major symptom of neuropathic pain.

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

Subthreshold A-type K(+) currents (ISA s) 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 ISA s. 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 Aß 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.

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

Subthreshold A-type K(+) currents (ISA s) 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 ISA s. 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.

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

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.

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

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-α) 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-α 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-α 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.