Bladder overactivity and hyperexcitability of bladder afferent neurons after intrathecal delivery of nerve growth factor in rats

Abstract

Nerve growth factor (NGF) has been proposed as an important mediator inducing bladder overactivity under pathological conditions such as spinal cord injury, bladder outlet obstruction, or cystitis. We therefore examined the effects of chronic NGF treatment on bladder activity and the properties of bladder afferent neurons. In adult female rats, NGF (2.5 microg/microl) was infused continuously into the intrathecal space at the L6-S1 level of spinal cord for 1 or 2 weeks using osmotic pumps (0.5 microl/h). Bladder afferent neurons were labeled with axonal transport of Fast Blue injected into the bladder wall. After intrathecal injection of NGF, cystometrograms under an awake condition showed bladder overactivity revealed by time-dependent reductions in intercontraction intervals and voided volume. ELISA analyses showed significant increases in NGF levels in L6-S1 dorsal root ganglia of NGF-treated rats. In patch-clamp recordings, dissociated bladder afferent neurons exhibiting tetrodotoxin (TTX)-resistant action potentials from NGF-treated animals were larger in diameter and had significantly lower thresholds for spike activation compared with sham rats. In addition, the number of TTX-resistant action potentials during 600 ms depolarizing pulses was significantly increased time dependently after 1 or 2 weeks of NGF application. The density of slowly inactivating A-type K+ currents was decreased by 52% in bladder afferent neurons with TTX-resistant spikes after 2 week NGF treatment. These results indicate that increased NGF levels in bladder afferent pathways and NGF-induced reduction in A-type K+ current density could contribute to the emergence of bladder overactivity as well as somal hypertrophy and hyperexcitability of bladder afferent neurons.

Figures

Figure 1.

Representative traces of cystometry. A, Vehicle-treated rat (sham). B, NGF-treated rat for 1 week (NGF 1W). C, NGF-treated rat for 2 weeks (NGF 2W). Note a time-dependent reduction in intercontraction intervals in NGF-treated rats.

Figure 2.

The effects of NGF treatment on cystometric parameters. A, Averaged ICIs in vehicle-treated rats (sham) (n = 7) and rats treated with NGF for 1 week (1W) (n = 5) or 2 weeks (2W) (n = 7). B, Averaged voided volume in vehicle-treated rats (sham) (n = 7) and rats treated with NGF for 1 (1W) (n = 5) or 2 weeks (2W) (n = 7). Values of p < 0.05 compared with the sham group are indicated. Error bars indicate SE.

Figure 3.

ELISA measurements of NGF in DRGs in untreated (normal), vehicle-treated (sham), and NGF-treated rats [1 week (1W) or 2 weeks (2W)]. A, L6 DRGs. B, S1 DRGs. C, L5 DRGs. NGF values (in picograms) were standardized by tissue protein levels (in micrograms) and expressed as picograms per microgram of total protein. Values of p < 0.05 compared with the sham group are indicated. Error bars indicate SE.

Figure 4.

Characteristics of action potentials in capsaicin-sensitive bladder afferent neurons with TTX-resistant action potentials. A, Vehicle-treated rat (sham). B, Rat treated with NGF for 2 weeks (NGF 2W). The left panels are voltage responses and action potentials evoked by 60 ms depolarizing current pulses injected through the patch pipette in current-clamp conditions. Asterisks with dashed lines indicate the thresholds for spike activation (−18 mV in A and −31 mV in B). The middle panels show the effects of TTX application (1 μm) on action potentials. The right panels show firing patterns during membrane depolarization (600 ms duration). The current intensity was set to the threshold value for inducing single spikes with 10 ms current pulses as indicated in middle panels.

Figure 5.

A, Superimposed K+ currents activated by depolarizing voltage pulses from −80 to 0 mV from a holding potential of −120 mV in a bladder afferent neuron with TTX-resistant spikes (diameter, 24 μm). B, Superimposed sustained delayer rectifier K+ currents (KDR) activated by depolarizing voltage pulses from −80 to 0 mV from holding potentials of −40 mV in the same neuron as in A. C, Isolated slowly inactivating transient K+ currents (slow KA) obtained by the subtraction of currents in A and B. D, E, I–V relationships of KDR currents (B) and slow KA currents (C), respectively, in vehicle (control) (n = 15) (■) and NGF-treated (n = 20) (▵) bladder afferent neurons. *p < 0.05 compared with control neurons (Student's t test). Error bars indicate SE.

Figure 6.

Steady-state activation and inactivation characteristics of slow KA currents in bladder afferent neurons with TTX-resistant spikes from vehicle-treated and NGF-treated rats (2 weeks). A, Inactivation characteristics of slow KA currents in vehicle-treated animals (n = 15) (▴) and NGF-treated animals (n = 20) (▵). Relative peak amplitude of slow KA currents normalized to the maximal amplitude of slow KA currents (I/Imax) were plotted against membrane potentials. Vh and k obtained by fitting curves using the modified Boltzmann equation were −70.7 mV and −12.3 for vehicle-treated rats and −72.4 mV and −12.3 for NGF-treated rats. B, Activation characteristics of slow KA currents obtained in the neurons from vehicle-treated animals (n = 15) (■) and NGF-treated animals (n = 20) (□). Slow KA conductances normalized to the maximal slow KA conductance (G/Gmax) were plotted against membrane potentials. Vh and k obtained by fitting curves using the modified Boltzmann equation were −39.1 mV and 10.5 for vehicle-treated rats and −40.4 mV and 13.6 for NGF-treated rats. Error bars indicate SE.

Figure 7.

A, Superimposed traces of Na+ currents elicited by depolarizing voltage steps from a holding potential of −60 mV in a bladder afferent neuron with TTX-resistant spikes from a vehicle-treated rat. B, I–V relationships of Na+ currents in vehicle (control) (n = 15) (■) and NGF-treated (n = 20) (▵) bladder afferent neurons. Current amplitudes at −20 to +35 mV in NGF-treated neurons are significantly (p < 0.05) larger compared with control neurons (Student's t test). Error bars indicate SE. C, Superimposed traces of Na+ currents elicited by a depolarizing voltage step to 10 mV from the holding potential of −60 mV in a bladder afferent neuron with TTX-resistant spikes from a NGF-treated rat before (control) and after 1 μm TTX application (TTX). D, Superimposed traces of Na+ currents elicited by a depolarizing voltage step to 10 mV from the holding potential of −120 mV in the same neuron as in C before (control) and after 1 μm TTX application (TTX). The TTX-sensitive component of Na+ currents obtained by the subtraction of currents before and after TTX application is indicated by a thick line (subtraction). Note that TTX-sensitive Na+ currents were negligible when activated from the holding potential of −60 mV (C), whereas TTX-sensitive Na+ currents became apparent when activated from the holding potential of −120 mV (D).

Figure 8.

Steady-state activation and inactivation characteristics of TTX-resistant Na+ currents in bladder afferent neurons with TTX-resistant spikes from vehicle-treated and NGF-treated rats (2 weeks). A, Inactivation characteristics of TTX-resistant Na+ currents in vehicle-treated animals (n = 15) (▴) and NGF-treated animals (n = 20) (▵). Peak amplitude of TTX-resistant Na+ currents normalized to the maximal amplitude of TTX-resistant Na+ currents (I/Imax) were plotted against membrane potentials. Vh and k obtained by fitting curves using the modified Boltzmann equation were −33.3 mV and −6.0 for vehicle-treated rats and −34.1 mV and −6.2 for NGF-treated rats. B, Activation characteristics of TTX-resistant Na+ currents obtained in the neurons from vehicle-treated animals (n = 15) (■) and NGF-treated animals (n = 20) (□). Na+ conductances normalized to the maximal Na+ conductance (G/Gmax) were plotted against membrane potentials. Vh and k obtained by fitting curves using the modified Boltzmann equation were −11.9 mV and 8.2 for vehicle-treated rats and −10.1 mV and 8.7 for NGF-treated rats. Error bars indicate SE.