Ocular retardation (or) is a murine eye mutation causing microphthalmia, a thin hypocellular retina and optic nerve aplasia. Here we show that mice carrying the OrJ allele have a premature stop codon in the homeobox of the Chx10 gene, a gene expressed at high levels in uncommitted retinal progenitor cells and mature bipolar cells. No CHX10 protein was detectable in the retinal neuroepithelium of orJ homozygotes. The loss of CHX10 leads both to reduced proliferation of retinal progenitors and to a specific absence of differentiated bipolar cells. Other major retinal cell types were present and correctly positioned in the mutant retina, although rod outer segments were short and retinal lamination was incomplete. These results indicate that Chx10 is an essential component in the network of genes required for the development of the mammalian eye, with profound effects on retinal progenitor proliferation and bipolar cell specification or differentiation. off

BackgroundATP-sensitive potassium (KATP) channels in neurons regulate excitability, neurotransmitter release and mediate protection from cell-death. Furthermore, activation of KATP channels is suppressed in DRG neurons after painful-like nerve injury. NO-dependent mechanisms modulate both KATP channels and participate in the pathophysiology and pharmacology of neuropathic pain. Therefore, we investigated NO modulation of KATP channels in control and axotomized DRG neurons.ResultsCell-attached and cell-free recordings of KATP currents in large DRG neurons from control rats (sham surgery, SS) revealed activation of KATP channels by NO exogenously released by the NO donor SNAP, through decreased sensitivity to [ATP]i.This NO-induced KATP channel activation was not altered in ganglia from animals that demonstrated sustained hyperalgesia-type response to nociceptive stimulation following spinal nerve ligation. However, baseline opening of KATP channels and their activation induced by metabolic inhibition was suppressed by axotomy. Failure to block the NO-mediated amplification of KATP currents with specific inhibitors of sGC and PKG indicated that the classical sGC/cGMP/PKG signaling pathway was not involved in the activation by SNAP. NO-induced activation of KATP channels remained intact in cell-free patches, was reversed by DTT, a thiol-reducing agent, and prevented by NEM, a thiol-alkylating agent. Other findings indicated that the mechanisms by which NO activates KATP channels involve direct S-nitrosylation of cysteine residues in the SUR1 subunit. Specifically, current through recombinant wild-type SUR1/Kir6.2 channels expressed in COS7 cells was activated by NO, but channels formed only from truncated isoform Kir6.2 subunits without SUR1 subunits were insensitive to NO. Further, mutagenesis of SUR1 indicated that NO-induced KATP channel activation involves interaction of NO with residues in the NBD1 of the SUR1 subunit.ConclusionNO activates KATP channels in large DRG neurons via direct S-nitrosylation of cysteine residues in the SUR1 subunit. The capacity of NO to activate KATP channels via this mechanism remains intact even after spinal nerve ligation, thus providing opportunities for selective pharmacological enhancement of KATP current even after decrease of this current by painful-like nerve injury.

BackgroundATP-sensitive potassium (KATP) channels in neurons mediate neuroprotection, they regulate membrane excitability, and they control neurotransmitter release. Because loss of DRG neuronal KATP currents is involved in the pathophysiology of pain after peripheral nerve injury, we characterized the distribution of the KATP channel subunits in rat DRG, and determined their alterations by painful axotomy using RT-PCR, immunohistochemistry and electron microscopy.ResultsPCR demonstrated Kir6.1, Kir6.2, SUR1 and SUR2 transcripts in control DRG neurons. Protein expression for all but Kir6.1 was confirmed by Western blots and immunohistochemistry. Immunostaining of these subunits was identified by fluorescent and confocal microscopy in plasmalemmal and nuclear membranes, in the cytosol, along the peripheral fibers, and in satellite glial cells. Kir6.2 co-localized with SUR1 subunits. Kir6.2, SUR1, and SUR2 subunits were identified in neuronal subpopulations, categorized by positive or negative NF200 or CGRP staining. KATP current recorded in excised patches was blocked by glybenclamide, but preincubation with antibody against SUR1 abolished this blocking effect of glybenclamide, confirming that the antibody targets the SUR1 protein in the neuronal plasmalemmal membrane.In the myelinated nerve fibers we observed anti-SUR1 immunostaining in regularly spaced funneled-shaped structures. These structures were identified by electron microscopy as Schmidt-Lanterman incisures (SLI) formed by the Schwann cells. Immunostaining against SUR1 and Kir6.2 colocalized with anti-Caspr at paranodal sites.DRG excised from rats made hyperalgesic by spinal nerve ligation exhibited similar staining against Kir6.2, SUR1 or SUR2 as DRG from controls, but showed decreased prevalence of SUR1 immunofluorescent NF200 positive neurons. In DRG and dorsal roots proximal to axotomy SLI were smaller and showed decreased SUR1 immunofluorescence.ConclusionsWe identified Kir6.2/SUR1 and Kir6.2/SUR2 KATP channels in rat DRG neuronal somata, peripheral nerve fibers, and glial satellite and Schwann cells, in both normal state and after painful nerve injury. This is the first report of KATP channels in paranodal sites adjacent to nodes of Ranvier and in the SLI of the Schwann cells. After painful axotomy KATP channels are downregulated in large, myelinated somata and also in SLI, which are also of smaller size compared to controls.Because KATP channels may have diverse functional roles in neurons and glia, further studies are needed to explore the potential of KATP channels as targets of therapies against neuropathic pain and neurodegeneration.

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Painful axotomy decreases KATP channel current (IKATP) in primary afferent neurons. Because cytosolic Ca 2؉ signaling is depressed in injured dorsal root ganglia (DRG) neurons, we investigated whether Ca 2؉ -calmodulin (CaM)-Ca 2؉ /CaM-dependent kinase II (CaMKII) regulates IK ATP in large DRG neurons. Immunohistochemistry identified the presence of K ATP channel subunits SUR1, SUR2, and Kir6.2 but not Kir6.1, and pCaMKII in neurofilament 200 -positive DRG somata. Single-channel recordings from cell-attached patches revealed that basal and evoked IK ATP by ionomycin, a Ca 2؉ ionophore, is activated by CaMKII. In axotomized neurons from rats made hyperalgesic by spinal nerve ligation (SNL), basal K ATP channel activity was decreased, and sensitivity to ionomycin was abolished. Basal and Ca 2؉ -evoked K ATP channel activity correlated inversely with the degree of hyperalgesia induced by SNL in the rats from which the neurons were isolated. Inhibition of IK ATP by glybenclamide, a selective KATP channel inhibitor, depolarized resting membrane potential (RMP) recorded in perforated whole-cell patches and enhanced neurotransmitter release measured by amperometry. The selective K ATP channel opener diazoxide hyperpolarized the RMP and attenuated neurotransmitter release. Axotomized neurons from rats made hyperalgesic by SNL lost sensitivity to the myristoylated form of autocamtide-2-related inhibitory peptide (AIPm), a pseudosubstrate blocker of CaMKII, whereas axotomized neurons from SNL animals that failed to develop hyperalgesia showed normal IK ATP inhibition by AIPm. AIPm also depolarized RMP in control neurons via K ATP channel inhibition. Unitary current conductance and sensitivity of K ATP channels to cytosolic ATP and ligands were preserved even after painful nerve injury, thus providing opportunities for selective therapeutic targeting against neuropathic pain.calcium ͉ calmodulin ͉ potassium channels ͉ dorsal root ganglia ͉ neuropathic pain A fter peripheral nerve injury, phenotypic changes in axons and the corresponding somata in the dorsal root ganglia (DRG) lead to membrane hyperexcitability, which results in neuropathic pain (1, 2). These alterations include decreased Ca 2ϩ influx via voltage-gated calcium channels (VGCC), reduced [Ca 2ϩ ] i , and diminished Ca 2ϩ -induced Ca 2ϩ release (3, 4). Additionally, ATPsensitive potassium (K ATP ) channels in large axotomized DRG neurons from rats with hyperalgesia exhibit decreased opening (5, 6). K ATP channels in cardiac myocytes and pancreatic ␤-cells are modulated by cytosolic Ca 2ϩ (7,8), but it remains unknown whether Ca 2ϩ affects neuronal K ATP channels.Cytosolic Ca 2ϩ regulates neuronal channels and other targets via multiple mechanisms (9), including the Ca 2ϩ -calmodulin (CaM)-Ca 2ϩ /CaM-dependent kinase II (CaMKII) pathway (10). CaMKII, which is abundant in neurons (11), assembles into a dodecameric holoenzymatic structure (12, 13). Ca 2ϩ /CaM activates CaMKII subunits, triggering autophosphorylation at T286 residues (14) that increase the affinit...