Hyperexcitability of bladder afferent neurons associated with reduction of Kv1.4 α-subunit in rats with spinal cord injury

Abstract

Purpose: To clarify the functional and molecular mechanisms inducing hyperexcitability of C-fiber bladder afferent pathways after spinal cord injury we examined changes in the electrophysiological properties of bladder afferent neurons, focusing especially on voltage-gated K channels.

Materials and methods: Freshly dissociated L6-S1 dorsal root ganglion neurons were prepared from female spinal intact and spinal transected (T9-T10 transection) Sprague Dawley® rats. Whole cell patch clamp recordings were performed on individual bladder afferent neurons. Kv1.2 and Kv1.4 α-subunit expression levels were also evaluated by immunohistochemical and real-time polymerase chain reaction methods.

Results: Capsaicin sensitive bladder afferent neurons from spinal transected rats showed increased cell excitability, as evidenced by lower spike activation thresholds and a tonic firing pattern. The peak density of transient A-type K+ currents in capsaicin sensitive bladder afferent neurons from spinal transected rats was significantly less than that from spinal intact rats. Also, the KA current inactivation curve was displaced to more hyperpolarized levels after spinal transection. The protein and mRNA expression of Kv1.4 α-subunits, which can form transient A-type K+ channels, was decreased in bladder afferent neurons after spinal transection.

Conclusions: Results indicate that the excitability of capsaicin sensitive C-fiber bladder afferent neurons is increased in association with reductions in transient A-type K+ current density and Kv1.4 α-subunit expression in injured rats. Thus, the Kv1.4 α-subunit could be a molecular target for treating overactive bladder due to neurogenic detrusor overactivity.

Keywords: 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; A-type K(+); DRG; DTX; DiI; FITC; GAPDH; K(A); K(DR); Kv; NDO; PBS; PCR; SCI; TTX; V(h); afferent pathways; delayed rectifier-type K(+); dorsal root ganglion; fluorescein isothiocyanate; glyceraldehyde-3-phosphate dehydrogenase; half-maximal conductance; nerve fibers; neurogenic detrusor overactivity; overactive; phosphate buffered saline; polymerase chain reaction; potassium channels; spinal cord injuries; spinal cord injury; tetrodotoxin; unmyelinated; urinary bladder; voltage-gated; voltage-gated K(+); α-dendrotoxin.

Figure 1

Representative action potential recordings in capsaicin sensitive bladder afferent neurons from spinal intact and transected rats. A, action potentials evoked by 50-millisecond (ms) depolarizing current pulses injected through patch pipettes during current clamp recording. B, firing patterns during sustained 800-millisecond membrane depolarization.

Figure 2

Changes in capsaicin sensitive bladder afferent neuron K⁺ currents in rats without vs with spinal cord transection. A, representative recordings show superimposed outward K⁺ currents evoked by voltage steps to 0 mV from −120 and −40 mV holding potentials (HP). B, K_A currents were obtained by subtracting K⁺ currents evoked by depolarization to 0 mV from −40 and −120 mV holding potentials. C, mean ± SEM K_A and K_DR current-voltage relationships in 20 cells from 12 spinal intact rats and 17 from 10 spinal transected rats. Asterisk indicates p <0.05 vs spinal intact.

Figure 3

Mean ± SEM steady-state activation and inactivation characteristics of K_A currents in capsaicin sensitive bladder afferent neurons, shown as relative peak conductance of K_A currents normalized to maximal K_A current conductance (G/G_max) plotted against membrane potentials. A, inactivation characteristics in 8 cells from 6 spinal intact rats and 7 from 7 spinal transected rats. B, activation characteristics in 7 cells from 7 spinal intact rats and 12 from 10 spinal transected rats.

Figure 4

Kv α-subunit immunoreactivity in L6 DRGs after spinal cord transection. A, photomicrographs show same Fast Blue, Kv1.2 and Kv1.4 stained sections from spinal intact and transected rats under fluorescence illumination with ultraviolet (blue areas) and FITC (green areas) filters. Arrows indicate neuronal profiles identified by Fast Blue labeling. Rectangles show insets at higher magnification. Scale bars indicate 50 μm. B, mean ± SEM expression shown as ratio of FITC immunoreactivity intensity in Fast Blue labeled vs unlabeled cells, including Kv1.2 in 121 cells from 3 spinal intact rats and 114 from 3 spinal transected rats, and Kv1.4 in 132 cells from 3 spinal intact rats and 112 from 3 spinal transected rats. Asterisk indicates p <0.05 vs spinal intact.

Figure 5

Kv α-subunit mRNA expression after spinal cord transection. A to C, photomicrographs show single L6 DRG section during laser capture microdissection of DiI labeled bladder afferent neurons. Arrows indicate neurons positively stained with DiI. Scale bars represent 100 μm. A, before microdissection. B, before microdissection. Green circles indicate laser captured areas. C, after microdissection. D to G, mean ± SEM levels in 30 neurons from each of 5 spinal intact and 5 spinal transected rats. ns, not significant. Asterisks indicate p <0.01 vs spinal intact.