Identified motoneurons involved in sexual and eliminative functions in the rat are powerfully excited by vasopressin and tachykinins

Abstract

The pudendal motor system is constituted by striated muscles of the pelvic floor and the spinal motoneurons that innervate them. It plays a role in eliminative functions of the bladder and intestine and in sexual function. Pudendal motoneurons are located in the ventral horn of the caudal lumbar spinal cord and send their axon into the pudendal nerve. In the rat, binding sites for vasopressin and tachykinin are present in the dorsomedial and dorsolateral pudendal nuclei, suggesting that these neuropeptides may affect pudendal motoneurons. The aim of the present study was to investigate possible effects of vasopressin and tachykinins on these motoneurons. Recordings were performed in spinal cord slices of young male rats using the whole-cell patch-clamp technique. Before recording, motoneurons were identified by 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate retrograde labeling. The identification was confirmed, a posteriori, by choline acetyltransferase immunocytochemistry. Vasopressin and tachykinins caused a powerful excitation of pudendal motoneurons. The peptide-evoked depolarization, or the peptide-evoked inward current, persisted in the presence of tetrodotoxin, indicating that these effects were mainly postsynaptic. By using selective receptor agonists and antagonist, we determined that vasopressin acted via vasopressin 1a (V1a), but not V1b, V2, or oxytocin receptors, whereas tachykinins acted via neurokinin 1 (NK1), but not NK2 or NK3, receptors. Vasopressin acted by enhancing a nonselective cationic conductance; in some motoneurons, it also probably suppressed a resting K+ conductance. Our data show that vasopressin and tachykinins can excite pudendal motoneurons and thus influence the force of striated perineal muscles involved in eliminative and sexual functions.

Figure 1.

Identification of pudendal motoneurons in neonate rats. A, Coronal section of the spinal cord from a 10-d-old rat showing the immunostaining of ChAT at segmental level L6. Pudendal motoneurons are grouped in DM and DL nuclei. The RDL, which projects to the hindleg, and the ventral nucleus (V), which projects to the pelvic diaphragm, are also detectable, as are isolated immunoreactive cells scattered in the medial and dorsal central gray. B, Photomicrograph illustrating the Fast DiI retrograde labeling of DM motoneurons. The spinal cord section is from a 10-d-old rat that had received an intraperitoneal injection of Fast DiI 24 h before. The animal was fixed by intracardiac perfusion of a solution containing 4% paraformaldehyde in 0.1 m PBS. Note, by comparing A and B, that Fast DiI-labeled DM nuclei are easily recognizable by their shape and position along the midline. C, D, Identification of a recorded biocytin-loaded DM motoneuron. C, Biocytin was visualized by treatment of the section with FITC-conjugated streptavidin. D, The biocytin-labeled motoneuron was immunoreactive for ChAT, detected with a Cy3-conjugated secondary antibody. Scale bars: A, 200 μm; B, D (for B–D), 100 μm.

Figure 2.

Effect of vasopressin and of selective agonists of V1b and V2 receptors on an identified DM pudendal motoneuron. Current-clamp records obtained in the presence of 0.1 μm vasopressin (AVP; top and bottom traces), 1 μm d[Cha⁴]AVP (second trace), and 1 μm dVDAVP (third trace). In this figure and in Figures 3, 4, and 68, the horizontal bar above a voltage or current trace indicates the time during which an agonist was present in the perfusion solution. Note that vasopressin depolarized the motoneuron and induced action potential firing, whereas neither d[Cha⁴]AVP, a V1b receptor agonist, nor dVDAVP, a V2 receptor agonist, had any effect.

Figure 3.

Effect of vasopressin and of a selective oxytocin receptor agonist on an identified DM pudendal motoneuron. Current-clamp records obtained in the presence of 0.5 and 0.1 μm vasopressin (AVP; top and third traces, respectively), in the presence of 0.5 μm vasopressin and 0.5 μm TTX (fourth trace), and in the presence of 0.5 μm TGOT (second trace). The inset in the top trace shows a detail of the membrane potential at the beginning of the vasopressin-evoked firing. Bottom trace, Voltage-clamp recording showing the inward current elicited by vasopressin at 0.5 μm, in the presence of TTX, in this same motoneuron. Note that, contrary to vasopressin, TGOT, an oxytocin receptor agonist, did not induce any depolarization, although in this motoneuron it caused a slight increase in membrane noise. Note also that the vasopressin-induced depolarization and inward current persisted in the presence of TTX, i.e., in conditions of synaptic uncoupling.

Figure 4.

Effect of vasopressin and of a V1a receptor antagonist on identified DL pudendal motoneurons. A, Current-clamp records obtained in the presence of 0.5 μm vasopressin (AVP; top trace) and in the presence of both vasopressin and 0.1 μm of the V1a selective antagonist VPA (bottom trace). B, Voltage-clamp records obtained in a second DL pudendal motoneuron. They show the inward current evoked by vasopressin at 0.5 μm (AVP; top trace) and by vasopressin coapplied with 50 nm of the V1a receptor antagonist VPA (bottom trace). Note that the V1a receptor antagonist suppressed the vasopressin response.

Figure 5.

Current–voltage relationship of the vasopressin-activated current in identified DM pudendal motoneurons. A, B, Voltage-clamp commands were delivered as described in Results, in both the absence (control) and presence of 0.5 μm vasopressin (AVP). The net vasopressin-evoked current was obtained by subtraction. The motoneurons were recorded using K-gluconate-containing patch pipettes. The perfusion solution was supplemented with 0.5 μm TTX. Note that, in the motoneuron in A, the vasopressin-evoked current reversed in polarity at approximately −49 mV, whereas in the motoneuron in B, it approached zero at approximately −120 mV.

Figure 6.

Effect of tachykinin receptor agonists on identified DM pudendal motoneurons. A, Current-clamp records obtained in the presence of 1 μm substance P (SP; top trace) and in the presence of 1 μm β-ala-NKA (bottom trace). B, Current-clamp records obtained in a second motoneuron, in the presence of 0.1 μm sar⁹-SP (top trace), and in the presence of 0.1 μm senktide (bottom trace). C, Current-clamp recording obtained in a third motoneuron in the presence of 0.2 μm sar⁹-SP. The perfusion solution was supplemented with 0.5 μm TTX. Note that the effect of substance P was mimicked by sar⁹-SP, a selective NK1 receptor agonist, whereas β-ala-NKA and senktide, selective agonists of NK2 and NK3 receptors, respectively, were without effect. Note also that the effect of the NK1 receptor agonist persisted in the presence of TTX, i.e., in conditions of synaptic uncoupling.

Figure 7.

Effect of tachykinin receptor agonists on an identified DL pudendal motoneuron. Current-clamp records obtained in the presence of 0.5 μm sar⁹-SP (top trace), 0.5 μm β-ala-NKA (second trace), 0.5 μm senktide (third trace), and sar⁹-SP again, but now with 0.25 μm TTX added to the perfusion solution (bottom trace). Note that only sar⁹-SP, an NK1 receptor agonist, was effective in exciting the motoneuron and that its effect persisted in the presence of TTX.

Figure 8.

Effect of tachykinin receptor agonists on identified RDL and lumbar motoneurons. A, Current-clamp records obtained in an RDL motoneuron in the presence of 0.5 μm sar⁹-SP (top trace), 0.5 μm β-ala-NKA (second trace), 0.5 μm senktide (third trace), and sar⁹-SP again (bottom trace). B, Current-clamp records obtained in a lumbar motoneuron in the presence of 0.5 μm sar⁹-SP in control conditions (top trace) and after adjunction of 0.25 μm TTX to the perfusion solution (bottom trace). Note that sar⁹-SP, an NK1 receptor agonist, caused excitation of the motoneuron, whereas β-ala-NKA and senktide, NK2 and NK3 receptor agonists, respectively, did not. Note also that the effect of the NK1 receptor agonist persisted in the presence of TTX and that senktide caused a slight increase in membrane noise.