Expression and functional role of BK channels in chronically injured spinal cord white matter.

Spinal cord injury (SCI) causes neuronal death, demyelination of surviving axons, and altered ion channel functioning, resulting in impaired axonal conduction. The large-conductance, voltage and Ca(2+)-activated K(+) (BK or Maxi K(+)) channels contribute to the repolarization phase of action potentials. Therefore, they may play a significant role in regulating axonal conduction in SCI. In this paper, using combined electrophysiological and molecular approaches, we tested the hypothesis that the deficit in axonal conduction in chronic SCI is partially due to the activation of axonal BK channels. BK channels were found to be expressed in spinal cord white matter axons. These channels are not sensitive to BK channel blocker iberiotoxin in uninjured cords, likely reflecting their juxtaparanodal localization. After chronic injury, BK channels were exposed due to axonal demyelination at the injured site and their activation was found to depend on calcium influx, likely through N-type voltage-dependent calcium channels. Activation of BK channels introduced a reduction in the size of the compound action potentials (CAPs) and in axonal response to high frequency stimulation (HFS). Administration of BK channel blocker iberiotoxin significantly enhanced axonal conduction in the injured cords. Thus, pharmacological targeting of axonal BK channels may provide a therapeutic strategy for the treatment of chronic SCI, by restoring conduction to the remaining functional axons.

Contribution of fast and slow conducting myelinated axons to single-peak compound action potentials in rat spinal cord white matter preparations.

Unlike recordings derived from optic nerve or corpus callosum, compound action potentials (CAPs) recorded from rodent spinal cord white matter (WM) have a characteristic single-peak shape despite the heterogeneity of axonal populations. Using a double sucrose gap technique, we analyzed the CAPs recorded from dorsal, lateral, and ventral WM from mature rat spinal cord. The CAP decay was significantly prolonged with increasing stimulus intensities suggesting a recruitment of higher threshold, slower conducting axons. At 3.5 mm conduction distance, a hidden higher threshold, slower conducting component responsible for prolongation of CAP decay was uncovered in 22 of 25 of dorsal WM strips by analyzing the stimulus-response relationships and a normalization-subtraction procedure. This component had a peak conduction velocity (CV) of 5.0 ± 0.2 (SE) m/s as compared with 9.3 ± 0.5 m/s for the lower threshold peak (P < 0.0001). Oxygen-glucose deprivation (OGD), along with its known effects on CAP amplitude, significantly (P < 0.015) shortened the CAP decay. The hidden higher threshold, slower conducting component showed greater sensitivity to OGD compared with the lower threshold, faster conducting component, suggesting a differential sensitivity of axonal populations of spinal cord WM. At longer conduction distances and lower temperatures (9.8 mm, 22-24°C), the slower peak could be directly visualized in CAPs at higher stimulation intensities. A detailed analysis of single-peak CAPs to identify their fast and slow conducting components may be of particular importance for studies of axonal physiology and pathophysiology in small animals where the conduction distance is not sufficiently long to separate the CAP peaks.

D-aspartate and NMDA, but not L-aspartate, block AMPA receptors in rat hippocampal neurons.

1 The amino acid, D-aspartate, exists in the mammalian brain and is an agonist at the N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors. Here, for the first time, we studied the actions of D-aspartate on alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors (AMPARs) in acutely isolated rat hippocampal neurons. 2 In the presence of the NMDA receptor channel blocker, MK801, D-aspartate inhibited kainate-induced AMPAR current in hippocampal neurons. The inhibitory action of D-aspartate on kainate-induced AMPAR current was concentration-dependent and was voltage-independent in the tested voltage range (-80 to +60 mV). 3 The estimated EC50 of the L-glutamate-induced AMPAR current was increased in the presence of D-aspartate, while the estimated maximum L-glutamate-induced AMPAR current was not changed. D-aspartate concentration-dependently shifted the dose-response curve of kainate to the right. Schild plot analysis indicated that D-aspartate acts competitively to block AMPARs. The K(b) for D-aspartate was estimated to be 0.93 mM. 4 D-aspartate also blocked L-glutamate-induced current in Xenopus laevis oocytes that expressed recombinant homomeric AMPARs. 5 NMDA possessed similar inhibitory action on AMPARs. However, L-aspartate had little inhibitory action on AMPARs. 6 D-Aspartate, but not L-aspartate, was found to reduce the amplitude of miniature excitatory postsynaptic current in cultured hippocampal neurons. 7 Our data are consistent with a model in which D-aspartate directly competes with kainate and L-glutamate in binding to the agonist binding site of AMPARs. The prevalence of D-aspartate in the brain suggests a possible role of D-aspartate in modulating AMPAR-mediated fast excitatory synaptic transmission.

Modular double sucrose gap apparatus for improved recording of compound action potentials from rat and mouse spinal cord white matter preparations.

Compound action potential (CAP) recording is a powerful tool for studying the conduction properties and pharmacology of axons in multi-axonal preparations. The sucrose gap technique improves CAP recording by replacing the extracellular solution between the recording electrodes with a non-conductive sucrose solution to minimize extracellular shunting. The double sucrose gap (DSG), conferring similar advantages at the stimulation site, has been extensively used on guinea pig spinal cord white matter (WM) in vitro. Establishing the DSG methodology for WM preparations from smaller animals such as rats and mice is appealing due to their extensive use in basic and translationally oriented research. Here we describe a versatile modular DSG apparatus with rubber membrane separation of the compartments, suitable for WM strips from rat and mouse spinal cord. The small volumes of compartments (<0.1 ml) and the air-tight design allow perfusion rates of 0.5-1 ml/min with faster refreshment rates compared to commonly used 2-3 ml/min and larger compartments, providing economical usage of expensive pharmacological drugs. Our improved DSG design is particularly efficient for uncovering slower conducting, higher threshold CAP components, as demonstrated by recordings of C-wave (non-myelinated axons) in rat dorsal WM. In myelin-deficient Shiverer mice with genetically dysmyelinated axons, our DSG apparatus recordings revealed a multi-peak C-wave without preceding faster components. The improved stimulation and recording with our DSG apparatus, lowering the range of required stimulus intensities and reducing the artifact interference with recorded CAPs provide for critical technical advantages that allow for more detailed analysis of CAPs in relatively short preparations.

Versican isoforms modulate expression and function of nicotinic acetylcholine receptors.

Versican is a chondroitin sulfate proteoglycan whose isoforms are differentially expressed, but little is known of their functions in the neuronal system. Here we show that isoforms of versican play different roles in neuronal differentiation and neurite outgrowth. Expression of versican V1 isoform in PC12 cells induced complete neuronal differentiation and increased the expression of nicotinic acetylcholine receptor in NGF-independent manners. The V1-induced neuronal differentiation was different from the NGF-induced differentiation, showing a specific profile of nAChR subunit expression and distinct kinetics of receptor-gated channel activity. Our results have implications for understanding how versican regulates neuronal development, function and repair.

Versican G3 domain regulates neurite growth and synaptic transmission of hippocampal neurons by activation of epidermal growth factor receptor.

Versican is one of the major extracellular matrix (ECM) proteins in the brain. ECM molecules and their cleavage products critically regulate the growth and arborization of neurites, hence adjusting the formation of neural networks. Recent findings have revealed that peptide fragments containing the versican C terminus (G3 domain) are present in human brain astrocytoma. The present study demonstrated that a versican G3 domain enhanced cell attachment, neurite growth, and glutamate receptor-mediated currents in cultured embryonic hippocampal neurons. In addition, the G3 domain intensified dendritic spines, increased the clustering of both synaptophysin and the glutamate receptor subunit GluR2, and augmented excitatory synaptic activity. In contrast, a mutated G3 domain lacking the epidermal growth factor (EGF)-like repeats (G3deltaEGF) had little effect on neurite growth and glutamatergic function. Treating the neurons with the G3-conditioned medium rapidly increased the levels of phosphorylated EGF receptor (pEGFR) and phosphorylated extracellular signal-regulated kinase (pERK), indicating an activation of EGFR-mediated signaling pathways. Blockade of EGFR prevented the G3-induced ERK activation and suppressed the G3-provoked enhancement of neurite growth and glutamatergic function but failed to block the G3-mediated enhancement of cell attachment. These combined results indicate that the versican G3 domain regulates neuronal attachment, neurite outgrowth, and synaptic function of hippocampal neurons via EGFR-dependent and -independent signaling pathway(s). Our findings suggest a role for ECM proteolytic products in neural development and regeneration.