Properties of the major classes of mechanoreceptors in the guinea pig bladder

Abstract

Sensory neurons represent an attractive target for pharmacological treatment of various bladder disorders. However the properties of major classes of mechano-sensory neurons projecting to the bladder have not been systematically established. An in vitro bladder preparation was used to examine the effects of a range of mechanical stimuli (stretch, von Frey hair stroking and focal compression of receptive fields) and chemical stimuli (1 mm alpha,beta-methylene ATP, hypertonic solutions (500 mm NaCl) and 3 microm capsaicin) during electrophysiological recordings from guinea pig bladder afferents. Four functionally distinct populations of bladder sensory neurons were distinguished by these stimuli. The first class, muscle mechanoreceptors, were activated by stretch but not by mucosal stroking with light (0.05-0.1 mN) von Frey hairs or by hypertonic saline, alpha,beta-methylene ATP or capsaicin. Removal of the urothelium did not affect their stretch-induced firing. The second class, muscle-mucosal mechanoreceptors, were activated by both stretch and mucosal stroking with light von Frey hairs or by hypertonic saline and by alpha,beta-methylene ATP, but not by capsaicin. Removal of the urothelium reduced their stretch- and stroking-induced firing. The third class, mucosal high-responding mechanoreceptors, were stretch-insensitive but could be activated by mucosal stroking with light von Frey hairs or by hypertonic saline, alpha,beta-methylene ATP and capsaicin. Stroking-induced firing was significantly reduced by removal of the urothelium. The fourth class, mucosal low-responding mechanoreceptors, were stretch insensitive but could be weakly activated by mucosal stroking with light von Frey hairs but not by hypertonic saline, alpha,beta-methylene ATP or capsaicin. Removal of the urothelium reduced mucosal stroking-induced firing. All four populations of afferents conducted in the C-fibre range and showed class-dependent differences in spike amplitude and duration. At least four functional classes of bladder mechanoreceptors can be readily distinguished by different mechanisms of activation and are likely to transmit different types of information to the central nervous system.

Figure 2

Mechanosensitivity of four classes of bladder mechanoreceptors A, stimulus–response function of muscle (Mus, n = 6, N = 6), muscle-mucosal (MusM, n = 8, N = 8)), mucosal high-responding mechanoreceptors (MHR, n = 5, N = 5 responses only to 0.1 and 0.5 mN probing) and mucosal low-responding mechanoreceptors (MLR, n = 9, N = 9) to graded probing of their receptive fields with von Frey hairs. Note that responses to probing of muscle-mucosal mechanoreceptors were significantly larger than those for muscle and mucosal low-responding mechanoreceptors, respectively (*P < 0.01 and †P < 0.001, 2-way ANOVA, Bonferroni post hoc tests). B, stimulus–response function of muscle mechanoreceptors (Mus, n = 6, N = 6), muscle-mucosal mechanoreceptors (MusM, n = 10, N = 10), mucosal high-responding mechanoreceptors (MHR, n = 7, N = 7) and mucosal low-responding (MLR) mechanoreceptors (n = 9, N = 9) to graded stroking of their receptive fields with von Frey hairs. Note that responses to stroking of mucosal high-responding and muscle-mucosal mechanoreceptors were significantly larger than muscle and mucosal low-responding mechanoreceptors (* and †P < 0.001, 2-way ANOVA, Bonferroni post hoc tests). There were also significant differences in responses to stroking, between mucosal high-responding mechanoreceptors and muscle-mucosal mechanoreceptors (‡P < 0.001). Note that nicardipine (3 μm) was present throughout all experiments with probing and stroking of receptive fields with von Frey hairs.

Figure 1

Low threshold, distension-sensitive muscle mechanoreceptors in the bladder Responses of muscle mechanoreceptors to fast 5 mm stretch (at 1 mm s⁻¹, held for 10 s) and to 0.5 mN von Frey hair compressing of the receptive field (indicated by dot). Note that stroking of the receptive field with a 0.1 mN von Frey hair (3 strokes, indicated by bars) did not activate this afferent. # shows stepper motor artefacts.

Figure 3

The effect of removing the urothelium with or without lamina propria on stretch-induced firing of muscle mechanoreceptors A and B, lack of the effects of removal of urothelium on stretch-induced firing (n = 4, N = 4) and muscle contractile responses (N = 4). C and D, removal of the urothelium together with lamina propria reduced but did not abolish stretch-induced firing (n = 5, N = 5, *P < 0.005) and contractile responses (N = 5, †P < 0.0001, 2-way ANOVA). Note that when baseline tone was re-adjusted to control values, stretch-evoked muscle responses and firing recovered to control levels.

Figure 10

Electrophysiological parameters and plot of the canonical discriminant functions used to classify sensory neurons on the bases of their spike duration and amplitude, and responses to 4 mm stretch and to 0.1 mN von Frey hair stroking A, the amplitude of spikes of mucosal low-responding (MLR) mechanoreceptors was significantly larger (n = 30, N = 29, *P < 0.0001, univariate ANOVA, Ryan–Einot–Gabriel–Welsch post hoc tests) than that of all other classes. Duration of mucosal high-responding (MHR) mechanoreceptors (n = 13, N = 12) was significantly (†P < 0.0001) longer than that of all other others; duration of muscle-mucosal (MusM) mechanoreceptors (n = 42, N = 40) was significantly (‡P < 0.0001) longer than that of mucosal low-responding mechanoreceptors (n = 30, N = 29). B, conduction velocity of mucosal low-responding mechanoreceptors (n = 7, N = 7) was significantly (*P < 0.05, univariate ANOVA, Ryan–Einot–Gabriel–Welsch post hoc tests) faster than that of mucosal high-responding mechanoreceptors (n = 4, N = 4). C, plot (‘territorial map’) of the canonical discriminant functions used to classify sensory neurons based on four parameters: spike duration and amplitude, and responses to 4 mm stretch and light (0.1 mN) von Frey hair stroking. The value for each neuron was calculated according to the two canonical discriminant functions F₁ and F₂. Canonical discriminant function coefficients were for function 1: F₁ = (0.253 × stroke) + (3.20 × duration) − (0.002 × amplitude) − 2.646; and for function 2: F₂ = (−0.013 × stroke) − (0.446 × duration) − (0.003 × amplitude) + (0.047 × stretch) + 0.225, respectively. The group centroids are the values of the discriminant functions calculated for the means of these four variables in each class of bladder sensory neurons.

Figure 4

Typical responses of low threshold, distension-sensitive muscle-mucosal mechanoreceptors to stretch and mucosal stroking before and after removal of the urothelium and the lamina propria A, responses of low threshold muscle-mucosal mechanoreceptors to fast 4 mm stretch (at 1 mm s⁻¹, held for 10 s) and to a 0.1 mN von Frey hair stroking of the receptive field (three strokes, indicated by bars). B, removal of both the urothelium and the lamina propria reduced stretch-induced firing. After removal the urothelium and the lamina propria, baseline tone was re-adjusted to control values. A and B, no nicardipine in Krebs solution. C, removal of the urothelium reduced stroking-induced firing of muscle-mucosal mechanoreceptors (n = 8, N = 8, *P < 0.0001, 2-way ANOVA, 3 μm nicardipine in Krebs solution).

Figure 5

The effect of removal the urothelium with or without lamina propria on stretch-induced firing of muscle-mucosal mechanoreceptors A and B, the removal of the urothelium significantly reduced stretch-induced firing (n = 10, N = 10, *P < 0.0005) but contractile responses were only slightly reduced to 4 mm stretch (N = 10, ‡P < 0.01, 2-way ANOVA, Bonferroni post hoc tests). C and D, removing the urothelium together with lamina propria significantly reduced both stretch-induced firing (n = 9, N = 9, *P < 0.0001) and contractile responses (N = 9, *P < 0.0001). Note that after baseline tone was re-adjusted to control values, stretch-evoked contractile responses recovered to control levels but stretch-induced firing remained significantly reduced (†P < 0.001, 2-way ANOVA, Bonferroni post hoc tests).

Figure 6

The effect of application of α,β-methylene ATP and hypertonic NaCl on muscle-mucosal mechanoreceptors Muscle-mucosal mechanoreceptor was activated by both stretch and stroking its receptive field with a 0.1 mN von Frey hair. Spritzing both 1 mm α,β-methylene ATP and 500 mm NaCl on the mucosa activated this mechanoreceptor. Note that the latency and duration of the α,β-methylene ATP-induced response were significantly shorter than those for hypertonic NaCl. Note that nicardipine (3 μm) was present throughout the experiment.

Figure 7

Mucosal high-responding mechanoreceptors in the bladder A, responses of low threshold muscle mechanoreceptors (Mus) to fast 5 mm stretch (at 1 mm s⁻¹, held for 10 s) and activation of mucosal high-responding mechanoreceptor (MHR) unit by 0.1 mN and 1 mN stroking of the receptive field (continuous recordings from the same nerve trunk). # indicates stepper motor artefacts. B and C, removal of the urothelium significantly reduced stroking-induced firing of MHR mechanoreceptors. For C, n = 4, N = 4, *P < 0.0001, 2-way ANOVA. D, the shape of superimposed seven action potentials for a muscle mechanoreceptor and mucosal high-responding mechanoreceptor unit. Note that nicardipine (3 μm) was present throughout all experiments in A, B and C.

Figure 9

Discrimination of three units from three different classes of sensory neurons recorded in a single nerve trunk A, muscle mechanoreceptor unit (Mus) is activated by fast 3 mm stretch (at 1 mm s⁻¹, held for 10 s) but not by 0.1 mN mucosal stroking of their receptive field (hotspot (hs) 1). Activation of mucosal high-responding mechanoreceptor unit (MHR) and mucosal low-responding mechanoreceptor (MLR) unit by 0.1 mN stroking of their receptive field (hs2 and hs3, respectively) but not by stretch. B, shape of superimposed seven action potentials for muscle mechanoreceptor and mucosal high-responding and mucosal low-responding mechanoreceptors. C, discriminator window for recordings from a single nerve trunk presented in A shows three clusters of units (Mus, MHR and MLR) discriminated by spike amplitude and duration. Note that nicardipine (3 μm) was present throughout all experiments in A.

Figure 8

Mucosal low-responding mechanoreceptors in the bladder A, responses of low threshold muscle mechanoreceptors (Mus) to fast 5 mm stretch (at 1 mm s⁻¹, held for 10 s) and activation of mucosal low-responding mechanoreceptors (MLR) by 0.1 mN stroking of the receptive field (continuous recordings from the same nerve trunk). B and C, removal of the urothelium significantly reduced stroking-induced firing. For C, n = 8, N = 8, *P < 0.0001, 2-way ANOVA. D, the shape of superimposed seven action potentials for muscle mechanoreceptors and mucosal low-responding mechanoreceptors. Note that nicardipine (3 μm) was present throughout all experiments in A, B and C.