Post-stimulus potentiation of transmission in pelvic ganglia enhances sympathetic dilatation of guinea-pig uterine artery in vitro
1 Centre for Neuroscience, Flinders University, Adelaide, SA 5001, Australia
Abstract
Vasodilatation produced by stimulation of preganglionic neurones in lumbar and sacral pathways to pelvic ganglia was studied using an in vitro preparation of guinea-pig uterine artery and associated nerves in a partitioned bath allowing selective drug application to the ganglia or artery. Arterial diameter was monitored using real time video imaging. Vasodilatations produced by hypogastric nerve stimulation (HN; 300 pulses, 10 Hz) were significantly larger and longer in duration than with pelvic nerve stimulation (N = 18). Stimulation of ipsilateral lumbar splanchnic nerves or ipsilateral third lumbar ventral roots also produced prolonged vasodilatations. Blockade of ganglionic nicotinic receptors (0.1–1 mM hexamethonium) delayed the onset and sometimes reduced the peak amplitude of dilatations, but slow dilatations persisted in 16 of 18 preparations. These dilatations were not reduced further by 3 μM capsaicin applied to the artery and ganglia, or ganglionic application of 1 μM hyoscine, 30–100 μM suramin or 10 μM CNQX. Dilatations were reduced slightly by ganglionic application of NK1 and NK3 receptor antagonists (SR140333, SR142801; 1 μM), but were reduced significantly by bathing the ganglia in 0.5 mM Ca2+ and 10 mM Mg2+. Intracellular recordings of paracervical ganglion neurones revealed fast excitatory postsynaptic potentials (EPSPs) in all neurones on HN stimulation (300 pulses, 10 Hz), and slow EPSPs (3–12 mV amplitude) in 25 of 37 neurones. Post-stimulus action potential discharge associated with slow EPSPs occurred in 16 of 37 neurones (firing rate 9.4 ± 1.5 Hz). Hexamethonium (0.1–1 mM) abolished fast EPSPs. Hexamethonium and hyoscine (1 μM) did not reduce slow EPSPs and associated post-stimulus firing in identified vasodilator neurones (with VIP immunoreactivity) or non-vasodilator paracervical neurones. These results demonstrate a predominantly sympathetic origin of autonomic pathways producing pelvic vasodilatation in females. Non-cholinergic mediators of slow transmission in pelvic ganglia produce prolonged firing of postganglionic neurones and long-lasting dilatations of the uterine artery. This mechanism would facilitate maintenance of pelvic vasodilatation on stimulation of preganglionic neurones during sexual activity.
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Introduction
Neurogenic vasodilatation in the genitalia and internal reproductive organs of both males and females is essential for erection and fluid secretion during copulation (Meston & Frohlich, 2000). Preganglionic neurones leaving the lumbo-sacral spinal cord synapse with vasodilator neurones in the anterior pelvic ganglia (de Groat & Booth, 1993b; Keast, 1999). The vasodilator neurones release nitric oxide (NO) and vasoactive intestinal peptide (VIP) to dilate the pelvic vasculature by a direct action on vascular smooth muscle (Polak & Bloom, 1984; Morris, 1993; Simonsen et al. 2001). Pelvic vasodilator nerve pathways are activated periodically rather than tonically. The preganglionic neurones are normally under descending inhibitory control from the brainstem, but are stimulated during sexual activity by sensory input from the genitalia combined with a descending net excitation from supraspinal levels (de Groat & Booth, 1993b; McKenna & Marson, 1997). In recent electrophysiological studies we found that the major synaptic input to vasodilator neurones in guinea-pig anterior pelvic (paracervical) ganglia runs in the hypogastric nerve rather than the pelvic nerve (Jobling et al. 2003, 2004).
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Autonomic vasodilatation of the guinea-pig uterine artery is mediated primarily by neurally derived nitric oxide at low levels of nerve stimulation. Additionally, release of peptide co-transmitters provides an efficient mechanism for producing long-lasting vasodilatation in response to stimulation with trains of pulses at frequencies of 5 Hz and above (Morris, 1993). Release of VIP into the penile circulation during erection indicates that the physiological firing rate of pelvic vasodilator neurones during sexual activity is sufficient to release neuropeptides (Virag et al. 1982). Furthermore, studies of pelvic nerve pathways to the cat bladder have shown that preganglionic neurones can fire at rates up to 5–10 Hz for the duration of micturition (de Groat & Booth, 1980). The prolonged activation of postganglionic neurones at high frequencies occurs by temporal facilitation within the vesical ganglia that act as high pass filters. This facilitation seems to be due primarily to enhanced transmitter release from preganglionic neurones (Booth & de Groat, 1979; de Groat & Booth, 1980). However, postsynaptic actions of acetylcholine acting on muscarinic receptors, amines such as 5-hydroxytryptamine or co-transmitters such as neuropeptides also can facilitate transmission to postganglionic neurones in autonomic ganglia (Jan et al. 1979; Jnig et al. 1982, 1983; Dun et al. 1984; Wang & Ma, 1990; Alkadhi et al. 1996; Niel et al. 1996; Ivanoff & Smith, 1997). These forms of slow synaptic transmission enhance nicotinic transmission from subthreshold synaptic inputs, a phenomenon termed synaptic gain (Karila & Horn, 2000; Wheeler et al. 2004).
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Many of the preganglionic inputs to vasodilator neurones in guinea-pig pelvic ganglia contain the peptide substance P (Morris & Gibbins, 1987), which facilitates synaptic transmission in many other autonomic ganglia (Peters & Kreulen, 1986; Kawatani et al. 1989; Mawe, 1995; Canning et al. 2002). However, pelvic neurones have only one or two synaptic inputs (Jobling et al. 2003). These inputs are suprathreshold for action potential generation and are regarded as ‘strong’ inputs. It is not clear how slow synaptic events such as those produced by neuropeptides can modulate ganglionic transmission mediated by fast, suprathreshold inputs (de Groat & Booth, 1993a; McLachlan, 2003). Nevertheless, non-cholinergic transmission to vasoconstrictor neurones in cat sympathetic ganglia produces prolonged firing of neurones that normally receive strong synaptic inputs (Jnig et al. 1983). This study set out to determine whether slow synaptic events in anterior pelvic ganglia of female guinea-pigs can modulate the firing of postganglionic neurones and, hence, the long-lasting vasodilatation produced by neuropeptides acting on arterial smooth muscle. As synaptic transmission from lumbar preganglionic axons onto neurones in anterior pelvic (paracervical) ganglia utilizes a different profile of voltage-dependent calcium channels compared with sacral preganglionic neurones (Jobling et al. 2004), we compared the effects of stimulating the lumbar and sacral pathways on modulation of ganglionic transmission and dilatation of the uterine artery.
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Methods
Female guinea-pigs (Hartley IMVS, Institute for Medical and Veterinary Science, Adelaide, Australia; 150–250 g) were killed by stunning and exsanguination via the carotid arteries, as approved by the Flinders University Animal Welfare Committee in accordance with guidelines of the National Health and Medical Research Council of Australia. The anterior pelvic (paracervical) ganglia, posterior pelvic ganglia, hypogastric nerve and pelvic nerve together with the internal iliac, uterine and vaginal arteries were removed unilaterally and placed in Hepes-buffered balanced salt solution (composition (mM): 146 NaCl, 4.7 KCl, 0.6 MgSO4, 1.6 NaHCO3, 0.13 NaH2PO4, 2.5 CaCl2, 7.8 glucose, 20 Hepes, adjusted to pH 7.3 with NaOH and bubbled with 100% O2). In some experiments the connection of the hypogastric nerve to the inferior mesenteric ganglion, lumbar splanchnic nerves and dorsal and ventral roots of the mid lumbar spinal cord (L3) were included in the isolated nerve–artery preparation (Fig. 1A).
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A, photograph of entire nerve–artery preparation pinned out at the conclusion of an experiment. APG, anterior pelvic (paracervical) ganglia; CN, colonic nerves; HN, hypogastric nerve; IIA, internal iliac artery; IMG, inferior mesenteric ganglion; IN, intermesenteric nerve; LSN, lumbar splanchnic nerves; L3DRG, 3rd lumbar dorsal root ganglion; L3VR, 3rd lumbar ventral roots; PN, pelvic nerve; SC, paravertebral sympathetic chain; UA, main uterine artery. Boxed area shows region of uterine artery imaged for diameter measurement. B, image of uterine artery mounted in an organ bath and superfused with solution containing guanethidine (1 μM) and phenylephrine (3 μM) prior to stimulation of vasodilator nerves. Note longitudinal ridges formed by endothelial folds during arterial constriction. The high contrast outer edges of the artery are tracked by DIAMTRAK for a continuous readout of diameter.
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Measurement of arterial dilatation
Isolated nerve–artery preparations (N = 87) were pinned to the base of a dish (2 ml volume) coated with silicone elastomer (Sylgard, Dow Corning, Midland, MI, USA) and mounted in a PDM1-2 micro incubator (Harvard Apparatus Ltd, Edenbridge, Kent, UK) on the stage of an Olympus BH2 microscope. The caudal portion of the main uterine artery was slightly stretched and pinned flat, taking care not to damage the paravascular nerves connecting to the anterior pelvic ganglia. Preparations were superfused at 1–2 ml min–1 with Hepes-buffered solution maintained at 36°C by a TC-202A temperature controller (Harvard Apparatus Ltd). Agonist or antagonist drugs were added to the reservoir of superfusate. In some preparations a thin plastic partition sealed with high vacuum grease (Dow Corning, Ajax Chemicals, Auburn, NSW, Australia) was placed in the bath towards the caudal end of the uterine artery to separate the solution superfusing the uterine artery from that superfusing the pelvic ganglia and attached nerve trunks. Suction electrodes were placed on one or more of the pelvic nerve, hypogastric nerve, intermesenteric nerve, colonic nerves, lumbar splanchnic nerves, L3 ventral roots or L3 dorsal roots (see McLachlan, 1985). Nerves were stimulated with trains of square wave pulses (0.3 ms duration) delivered at 2–20 Hz by an S88 stimulator (Grass Instruments, Quincy, MA, USA) connected to a stimulus isolation unit (Grass Instruments) and low impedance interface (BioMedical Engineering, Flinders Medical Centre, Bedford Park, SA, Australia). The uterine artery was imaged using a 4 x long working distance objective and a Watek WAT-902B CCD video camera (Fig. 1B). Arterial outer diameter was monitored continuously on-line using DIAMTRAK v 3.1 (T.O. Neild, Flinders University) and was recorded using a MacLab 4S running Chart v 4.2 software (AD Instruments, Castle Hill, NSW, Australia). Neurogenic dilatations of the uterine artery were recorded after preconstriction with phenylephrine (3 μM) or prostaglandin F2 (3 μM). In most experiments guanethidine (1 μM) also was present in the artery superfusate to block release of transmitter from any noradrenergic axons in the stimulated nerve trunks (Morris & Murphy, 1988). When partitioned baths were used, phenylephrine and guanethidine were present only in the solution superfusing the uterine artery. The maximum amplitude of dilatations during the stimulation period (μm), latency (s) from start of stimulation period to commencement of dilatation, and integrated dilator response (area under the curve to a line joining two vertical cursors marking the period of dilatation; μm s) were measured using Chart v 4.2 and NIH Image v 1.62 (NIH, Bethesda, MD, USA).
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Intracellular electrophysiology
Anterior pelvic ganglia in isolated nerve–artery preparations (N = 54) were dissected free of connective tissue and fat and pinned to the base of a recording chamber (1 ml) mounted on the stage of an Olympus IMT2 inverted microscope for intracellular electrophysiology. In some instances these preparations were obtained from the contralateral side of the same animals as used for measurement of arterial diameter. Preparations were superfused at 2.5 ml min–1 with Hepes-buffered solution bubbled with O2 and maintained at 35°C. Neurones were impaled under visual control with glass microelectrodes filled with 0.5 M KCl (resistance 80–200 M). In some experiments Neurobiotin (0.5%, Vector, Burlingame, CA, USA) was included in the electrode filling solution to allow later visualization of the impaled neurones. Data were recorded using an Axoclamp 2B amplifier (Axon Instruments, Union City, CA, USA). Voltage and current records were digitized at 4–20 kHz using a Powerlab running Chart v 4.2 and Scope v 3.6 software (AD Instruments) and were analysed using Igor Pro v 3.16 (WaveMetrics, Lake Oswego, OR, USA). Changes in membrane potential were recorded using bridge mode. Somata were voltage clamped using single electrode voltage clamp (SEVC). During SEVC, the headstage was continuously monitored and the cycling frequency adjusted to minimize the effects of electrode capacitance. The cycling frequency was 2–4 kHz. The hypogastric or pelvic nerve trunks were stimulated with square wave pulses (40 μA to 1 mA amplitude, 0.3–0.5 ms duration) via suction electrodes connected to a WPI A360 (World Precision Instruments Inc, Sarasota, FL, USA) or a WECO SC-100 constant current stimulator (Winston Electronics Co., Millbrae, CA, USA). Agonist and antagonist drugs were added to the reservoir of superfusate. Instantaneous action potential discharge frequency was determined using the ‘Spike Utilities’ procedure for Igor Pro (v 1.5, Neil Berman, University of Ottowa).
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Immunohistochemistry
At the conclusion of electrophysiological experiments where neurones were filled with Neurobiotin, preparations were fixed by immersion in Zamboni's solution (2% formaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.0) for 16–64 h at 4°C. Tissues were processed as whole-mounts by washing in 80% ethanol and clearing in 100% dimethylsulfoxide (DMSO), as previously described (Jobling et al. 2003). After washing in phosphate-buffered saline (PBS), preparations were exposed for 48–72 h to an antiserum raised in a rat against vasoactive intestinal peptide (VIP; antiserum code FI/III supplied by Dr R. Murphy), diluted 1: 200. Preparations were washed in PBS and incubated for 24 h in donkey anti-rat IgG conjugated to Cy3 (Jackson ImunoResearch Laboratories, West Grove, PA, USA) to visualize neurones with VIP immunoreactivity (IR), and streptavidin conjugated to Cy5 to visualize the Neurobiotin-filled neurones. After further washes in PBS, preparations were mounted on slides in carbonate-buffered glycerol (pH 8.6) and were examined with an Olympus AX70 epifluorescence microscope or a Bio-Rad MRC-1024 scanning laser confocal microscope fitted to an Olympus AX70. Images were collected from the epifluorescence microscope with an Orca cooled CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan) using IPLab Spectrum software (Scanalytics Inc., Faifax, VA, USA) or from the confocal microscope using Lasersharp v3.1 software.
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Drugs
Guanethidine sulphate, hexamethonium hydrochloride, hyoscine hydrobromide, NG-nitro-L-arginine methyl ester, phenylephrine hydrochloride, suramin sodium salt were all purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Stock solutions were made up in phosphate-buffered saline and were stored at 4°C. Capsaicin (Sigma-Aldrich) was dissolved in 95% ethanol: 5% Tween 80 and stock solutions (30 mM) were stored at 4°C. Substance P and senktide (Auspep, Parkville, VIC, Australia) were stored frozen as stock solutions of 1 mM in dH2O. Prostaglandin F2 (Cayman Chemical Company, Ann Arbor, MI, USA) was dissolved in PBS and 3 mM stock solutions were stored frozen. Tetrodotoxin (with citrate; Alomone Laboratories, Jerusalem, Israel) was dissolved in dH2O and 1 mM stock solution was stored at 4°C. CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; Sigma-RBI) was dissolved in DMSO and di-Na-CNQX was dissolved in dH2O immediately before use. Kynurenic acid (Sigma-RBI) was dissolved in 0.1 M NaOH immediately before use. NK1, NK2 and NK3 receptor antagonists (SR140333, SR48968 and SR142801), generous gifts of Sanofi Recherche (Montpellier, France), were dissolved in DMSO and stored frozen at a concentration of 10 mM.
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Data analysis
Data are expressed as mean ± S.E.M., with N referring to the number of preparations and n the number of neurones used for electrophysiological analysis. Statistical analysis was performed by repeated measures analysis of variance (ANOVA) using SPSS 10 for Macintosh (SPSS, Chicago, IL, USA). The level of significance was set at P < 0.05.
Results
Source of vasodilator outflow from the spinal cord
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Stimulation of the ipsilateral pelvic nerve or hypogastric nerve with trains of 50–300 pulses produced dilatation of the preconstricted uterine artery. Artery diameter increased progressively and at lower stimulation frequencies (< 5 Hz) the maximum amplitude of the neurogenic dilatation was reached at the end of the stimulation period (Fig. 2A). As the stimulation frequency was increased above 5 Hz for a given number of pulses, the initial dilatation did not increase further in amplitude, but a second, slow vasodilatation was apparent after cessation of stimulation. The amplitude of the slow dilatation sometimes exceeded the maximum amplitude during the stimulation period, and lasted up to 10–20 min (Fig. 2B and C). Hypogastric nerve stimulation at 10–20 Hz always produced larger dilatations than stimulation of the pelvic nerve in the same preparations (N = 18; Fig. 3). The slow phase of the neurogenic dilatation was particularly prominent after hypogastric compared with pelvic nerve stimulation (Fig. 3).
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The same artery is dilated by trains of 300 pulses delivered to the hypogastric nerve at different frequencies, 2 Hz in A, 10 Hz in B and 20 Hz in C. Dashed vertical lines mark the start and end of each stimulation period.
A, dilatations of a uterine artery produced by stimulation of the hypogastric nerve (HN) or the pelvic nerve (PN) in the same preparation with 300 pulses at 10 Hz. B, group data for 18 preparations where the hypogastric nerve and pelvic nerve were stimulated. Bars represent mean + S.E.M. Note significant increase in latency of dilatations (repeated measures ANOVA, F1,17 = 13, P = 0.002) and significantly smaller amplitude of dilatations (F1,17 = 48, P = 0.0001) and integrated dilator response (area; F1,17 = 37, P = 0.001) of dilatations after pelvic nerve stimulation. *P < 0.05.
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The source of axons in the hypogastric nerve producing dilatation of the uterine artery was investigated by stimulating the colonic nerves, intermesenteric nerve, ipsilateral lumbar splanchnic nerves, or ipsilateral ventral roots and dorsal roots at L3 with trains of pulses at 10 Hz or 20 Hz. Large biphasic dilatations always were seen after stimulation of the lumbar splanchnic nerves (9 of 9 preparations; Figs 4 and 5). Smaller dilatations were apparent after stimulation of the intermesenteric nerve (4 of 4 preparations) or the L3 ventral roots (5 of 5 preparations). However, dilatations were not seen after stimulation of the colonic nerves (0 of 2 preparations) or the L3 dorsal roots (0 of 4 preparations).
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Representative traces of vasodilator responses to stimulation of the hypogastric nerve (HN) in A, lumbar splanchnic nerves (LSN) in B, or L3 ventral roots (L3VR) in C with trains of 300 pulses at 10 Hz. Control responses shown in black; responses at least 20 min after addition of hexamethonium (0.3–1 mM) shown in grey.
Group data showing effect of hexamethonium (Hex, 0.3–1 mM) on dilatations produced by stimulation of the hypogastric nerve (HN, N = 6), pelvic nerve (PN, N = 4), lumbar splanchnic nerves (LSN, N = 5), intermesenteric nerve (IN, N = 3) or L3 ventral roots (L3VR, N = 3). Bars represent mean + S.E.M. Filled bars, responses before Hex; open bars, responses at least 20 min after Hex. For all nerves, hexamethonium produced: A, a significant increase in latency of dilatations (repeated measures ANOVA, F1,16 = 275, P = 0.0001); B, a significant decrease in amplitude of dilatations at the end of the stimulation period (F1,16 = 168, P = 0.0001); C, a significant decrease in integrated dilator response (area; F1,16 = 22, P = 0.0001). in B, dilatations to L3VR remaining after hexamethonium did not commence until after cessation of stimulation, so amplitude at the end of stimulation period was zero.
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The nicotinic receptor antagonist hexamethonium (0.1–1 mM), was used to ascertain whether these vasodilator nerve pathways to the uterine artery involved ganglionic synapses. Vasodilatations produced by stimulation of the hypogastric nerve, pelvic nerve, intermesenteric nerve, lumbar splanchnic nerves or L3 ventral roots at 10–20 Hz all were affected significantly by hexamethonium (Figs 4 and 5). The effects of hexamethonium were similar whether it was applied to the whole preparation, or was present only in the solution superfusing the ganglia and nerve trunks. In either case, there was a pronounced increase in latency and a reduction in amplitude of dilatations during the stimulation period. However, a slow dilatation persisted in almost all experiments, even after the concentration of hexamethonium was increased to 1 mM (Figs 4 and 5). With hypogastric nerve stimulation, the hexamethonium-resistant dilatations commenced towards the end of the stimulation period (latency 20 s). The peak amplitude occurring after cessation of stimulation sometimes was reduced (Fig. 6Aa), but often was similar to that of control dilatations (Fig. 4A).
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Aa, dilatation produced by hypogastric nerve stimulation (HN) with 300 pulses at 10 Hz (Control, shown in black) was reduced by 1 mM hexamethonium (+ Hex, shown in grey) and then enhanced by subsequent addition of 10 μM CNQX (+ CNQX, shown in grey). Ab, dilatation produced by L3 ventral root stimulation (L3VR) with 300 pulses delivered at 20 Hz (Control, black) was enhanced by 10 μM CNQX (+ CNQX, shown in grey) and then reduced, but not abolished, by subsequent treatment with 1 mM hexamethonium (+ Hex, shown in grey). B, dilatation produced by hypogastric nerve stimulation (HN) with 300 pulses at 10 Hz was greatly reduced by solution low in calcium and high in magnesium (low Ca/high Mg, shown in grey) then abolished by subsequent treatment with 0.3 mM hexamethonium (+ Hex, shown in grey). C, dilatation produced by hypogastric nerve stimulation (HN) with 300 pulses at 10 Hz (Control, shown in black) was slightly enhanced after application of 10 μM capsaicin then washout after 5 min (+ Capsaicin, shown in grey). Subsequent addition of 1 mM hexamethonium delayed the onset and increased the amplitude of the slow dilatation (+ Hex, shown in grey).
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Hexamethonium-resistant dilatations to hypogastric nerve stimulation were not reduced further by the muscarinic receptor antagonist hyoscine (1 μM), added to the ganglion side of a partitioned bath. Indeed, the amplitude of dilatations was slightly increased after addition of hyoscine to the hexamethonium (repeated measures ANOVA: dilatation latency, F1,5 = 0.8, P = 0.4; amplitude, F1,5 = 7.3, P = 0.04; integrated dilatation, F1,5 = 0.4, P = 0.6). Addition of the ionotropic glutamate receptor antagonist CNQX (10 μM), did not reduce the neurogenic dilatations, either before or after treatment with hexamethonium. Instead, CNQX applied to the whole preparation or to the ganglia and nerve trunks only, always produced a large potentiation of dilatations produced by stimulation of the hypogastric nerve (N = 10; Fig. 6Aa), pelvic nerve (N = 3) or the L3 ventral roots (N = 2; Fig. 6Ab). The potentiation was apparent as an increase in the amplitude and duration of the slow dilatations (Fig. 6A). A similar potentiation was seen after treatment with kynurenic acid (1 mM; N = 2).
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To determine whether the dilatations remaining after blockade of ganglionic acetylcholine receptors were due to stimulation of axons reaching the uterine artery without synapsing in pelvic ganglia, partitioned baths were used to bathe ganglia in a Hepes-buffered salt solution low in calcium (0.5 mM) and high in magnesium (10 mM; N = 4), or in cadmium (50–100 μM; N = 3). These treatments greatly reduced or abolished the neurogenic dilatations to hypogastric nerve stimulation. Small dilatations remaining in low calcium and high magnesium were abolished by hexamethonium (Fig. 6B). In separate experiments (N = 10), capsaicin (1–10 μM) was used to stimulate and then block transmission from peptide-containing sensory neurones to test involvement of these axons in dilatation of the uterine artery in response to hypogastric nerve stimulation. Capsaicin produced a large (20–60 μm), transient dilatation of the uterine artery, but had very little effect on the amplitude of neurogenic dilatations either in the continued presence or after washout of capsaicin. Furthermore, capsaicin pre-treatment did not reduce the incidence of hexamethonium-resistant vasodilatations to hypogastric nerve stimulation (9 of 9 preparations; Fig. 6C). Addition of the nitric oxide synthase inhibitor L-NAME (30 μM), to the whole bath or to the artery side of a partitioned bath reduced the amplitude and increased the latency of dilatations to hypogastric nerve (N = 6) or L3 ventral root (N = 2) stimulation, before or after treatment with hexamethonium. However, a slow dilatation was still present after L-NAME treatment. The remaining dilatations were abolished by tetrodotoxin (0.3–1 μM).
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Responses of pelvic neurones to repetitive stimulation of the hypogastric nerve
Stimulation of the hypogastric nerve with trains of 50–100 pulses delivered at 10–20 Hz produced fast excitatory postsynaptic potentials (EPSPs) and action potentials in pelvic neurones in paracervical ganglia in response to each pulse. In addition, slow EPSPs were apparent in a subpopulation of neurones (7 of 12 neurones after 50 pulses at 10 Hz; 2 of 4 neurones after 100 pulses at 10 Hz; 6 of 17 neurones after 100 pulses at 20 Hz). Furthermore, 3 of 13 pelvic neurones continued to fire action potentials after cessation of stimulation of the hypogastric nerve with 50–100 pulses at 10 Hz. Increasing the train of hypogastric nerve stimulation at 10 Hz to 300 pulses produced slow EPSPs in 25 of 37 neurones. The amplitude of the slow EPSP ranged from 3 to 12 mV and the duration was 1–6 min. Thirteen of these neurones continued to fire action potentials after the stimulation period (Fig. 7A), and a further three neurones fired continuously until the next stimulation period (10 min; Fig. 8A and B). The maximum instantaneous firing rate ranged from 2 to 24 Hz (mean = 9.4 ± 1.5 Hz). Pelvic nerve stimulation also produced slow EPSPs in 24% of the pelvic neurones (2 of 7 neurones after 50 pulses at 10 Hz; 5 of 18 neurones after 100 pulses at 20 Hz and 1 of 8 neurones after 300 pulses at 10 Hz). However, only one of these neurones fired any action potentials during the slow EPSP. This burst was of less than 7 s duration.
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Intracellular recording of membrane potential in an identified VIP immunoreactive pelvic neurone (middle traces) during and after stimulation of hypogastric nerve with 300 pulses at 10 Hz. Top traces show responses to the first 5 pulses in each train of stimuli. Bottom traces show instantaneous frequency of action potential firing. A, control response; B, 10 min after addition of 0.3 mM hexamethonium (Hex); C, 25 min after addition of hexamethonium.
Intracellular recording of membrane potential (upper traces) in a VIP immunoreactive pelvic neurone showing rate of action potential firing (lower traces) during and after stimulation of the hypogastric nerve with 300 pulses at 10 Hz. A, control response. B, the neurone continues to fire between stimulation periods. Trace starts 450 s after cessation of previous stimulus train; C, response to HN nerve stimulation 10 min after addition of 1 mM hexamethonium and 1 μM hyoscine (Hex + Hyo). Inset Ca, action potentials 1 s before commencement of the stimulation period. Inset Cb, action potentials during the last second of the 30 s stimulation period have increased frequency but are not linked to the stimulus pulses (black dots).
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Some pelvic neurones (n = 11) were filled with Neurobiotin and examined for VIP immunoreactivity at the conclusion of electrophysiological analysis. All six neurones with VIP-IR had slow EPSPs and continued to fire action potentials after hypogastric nerve stimulation with 300 pulses at 10 Hz (Figs 7A and 8A). Of the five neurones without VIP-IR, four had slow EPSPs and three continued firing after the 30 s stimulation period.
In order to investigate further the contribution of non-cholinergic transmission to the action potential discharge observed following long trains of hypogastric nerve stimulation, hexamethonium (0.3–1 mM) alone, or hexamethonium together with hyoscine (1 μM) was used. Hexamethonium markedly reduced or abolished fast EPSPs (Fig. 7B and C). The hexamethonium block was use dependent with the first one or two EPSPs in the train sometimes reaching threshold. This use-dependent block by hexamethonium is similar to that observed in other autonomic ganglia (Skok, 1986). Hexamethonium and hyoscine did not reduce the amplitude of slow EPSPs (Fig. 9). Furthermore, after cholinergic receptor blockade, pelvic neurones with or without VIP-IR commenced firing during or at the end of the stimulation period and continued firing after cessation of stimulation (Fig. 9).
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Group data showing no effect of hexamethonium and hyoscine (+ Hex/Hyo) on the amplitude (repeated measures ANOVA, F1,3 = 0.003, P = 0.96) or duration (F1,3 = 0.03, P = 0.88) of slow EPSPs to hypogastric nerve stimulation with 300 pulses at 10 Hz. There was a significant increase in firing rate (F1,6 = 10.4, P = 0.02) but not duration of firing (F1,4 = 2.3, P = 0.2) of pelvic neurones after hexamethonium and hyoscine. Bars represent mean + S.E.M. *P < 0.05.
CNQX (10 μM) was added to the superfusate before or after blockade of cholinergic receptors. In 2 of 5 neurones, CNQX caused depolarization and initiated bursts of action potential firing at rates up to 5–10 Hz. The firing rate was increased further on stimulation of the hypogastric nerve with trains delivered at 10 Hz. The membrane potential returned to pre-CNQX levels soon after washout and action potential firing ceased.
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Effects of substance P on pelvic neurones
Substance P (SP, 1 μM) was added to the superfusate for 1 min and membrane potential changes were recorded in 38 pelvic neurones from 24 preparations. Half of the neurones (18 of 38) were depolarized by SP by an average of 7.6 ± 0.8 mV. Some neurones (5 of 18) started firing action potentials during the SP-induced depolarization (Fig. 10). In nine neurones that were voltage clamped, SP evoked an inward current of 0.09 ± 0.01 nA. Six of nine neurones also responded to the NK3 receptor agonist senktide (0.5 μM). The mean inward current measured in five of these neurones was 0.14 ± 0.04 nA.
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Membrane potential and instantaneous rate of action potential discharge of pelvic neurone superfused with substance P (SP; 1 μM for 1 min at bar).
Mediators of non-cholinergic ganglionic transmission
To investigate potential mediators of non-cholinergic transmission to vasodilator neurones in pelvic ganglia, receptor antagonists for SP or ATP were applied to the ganglion side of partitioned baths and their effects on hypogastric nerve-mediated vasodilatations were determined. Antagonists for NK1 and NK3 receptors (SR140333 and SR142801), or for NK1, NK2 and NK3 receptors (SR140333, SR48968 and SR142801), all at 1 μM, were added to six preparations after treatment with hexamethonium and hyoscine. In some preparations, dilatations produced by hypogastric nerve stimulation (300 pulses at 10 Hz) appeared to be slower and shorter in duration in the presence of SP antagonists. However, the group effects were only marginally significant (Fig. 11). In a further five preparations, addition of the non-selective purinoceptor antagonist suramin (30–100 μM), did not alter the latency, maximum amplitude or integrated response of the slow, hexamethonium-resistant dilatations to hypogastric nerve stimulation (Fig. 11). Addition of neurokinin receptor antagonists and suramin together produced a marginal reduction in the amplitude and area of slow dilatations in four preparations (Fig. 11). Given the lack of effect of suramin alone, these small reductions produced by combined antagonists are likely to be due to marginal effects of NK receptor blockade that sometimes reach statistical significance and other times do not.
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Group data showing the latency, maximum amplitude and area of slow dilatations to hypogastric nerve stimulation with 300 pulses at 10 Hz in the presence of hexamethonium and hyoscine, before (Control, filled bars) and after treatment with antagonists (open bars) for NK1 and NK3 receptors (NK, 1 μM, N = 6), suramin (Sur, 30–100 μM, N = 5) or NK antagonists and suramin together (NK + Sur, N = 4). Bars show mean values + S.E.M.A, a significant increase in latency of dilatations was produced by NK antagonists (F1,5 = 6.6, P = 0.05), not by suramin (F1,4 = 4.8, P = 0.09) but was increased marginally by NK antagonists plus suramin (F1,3 = 8.4, P = 0.06). B, maximum amplitude of slow dilatations was not affected by NK antagonists (F1,5 = 3.1, P = 0.14) nor by suramin (F1,4 = 1.9, P = 0.24), but was reduced significantly by both NK antagonists and suramin (F1,3 = 18, P = 0.02). C, the integrated vasodilator response (area of dilatation) was reduced marginally by NK antagonists (F1,5 = 5.3, P = 0.07), was not affected by suramin (F1,4 = 0.1, P = 0.75) and was reduced significantly by all antagonists together (F1,3 = 14, P = 0.03). *P < 0.05.
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Discussion
Sympathetic pathway to pelvic vasodilator neurones
Pelvic ganglia are unusual in receiving preganglionic projections from both lumbar and sacral levels of the spinal cord. Typically, lumbar preganglionic neurones innervate catecholamine-synthesizing pelvic neurones innervating the urinogenital organs, and sacral preganglionic neurones project to non-catecholamine-synthesizing pelvic neurones, many of which synthesize acetylcholine, nitric oxide and neuropeptides (Keast, 1999). Preganglionic input to pelvic vasodilator neurones contributing to erection in males arises predominantly from sacral levels, although outflow from the lumbar cord can mediate erection after damage to sacral spinal cord (Dail et al. 1985; de Groat & Booth, 1993b). In contrast, the present study has extended our previous electrophysiological work (Jobling et al. 2003) to demonstrate directly that neurones projecting from mid-lumbar spinal cord via the lumbar splanchnic and hypogastric nerves provide the major neural pathway for vasodilatation of pelvic arteries supplying the internal reproductive organs of female guinea-pigs. This is consistent with reports of hypogastric nerve stimulation but not pelvic nerve stimulation increasing venous outflow and VIP release from the cat uterus (Fahrenkrug & Ottesen, 1982). The relative large magnitude of vasodilatations produced by stimulation of the ipsilateral hypogastric or lumbar splanchnic nerves compared with the L3 ventral roots is likely to reflect the extensive bilateral projection of lumbar preganglionic neurones down both hypogastric nerves (McLachlan, 1985).
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Our previous studies have shown conclusively that the final motor neurones in pelvic vasodilator pathways are located in the anterior pelvic (paracervical) ganglia and not sympathetic ganglia (Morris & Gibbins, 1987; Morris et al. 1998; Jobling et al. 2003). Furthermore, we have now shown directly that ganglionic synapses exist in the vasodilator pathway between the hypogastric nerve and the uterine artery. The vasodilator neurones in paracervical ganglia supply a dense innervation to the uterine, cervical and vaginal vascular beds (Morris & Gibbins, 1987; Morris et al. 1998). Thus, the lumbar sympathetic pathway to vasodilator neurones in anterior pelvic ganglia is likely to represent the major outflow from the central nervous system that increases blood flow in the vagina, cervix and uterus during copulation. This sympathetic vasodilator pathway also would enhance secretion from the cervical epithelium produced predominantly by stimulation of the hypogastric rather than the pelvic nerve (Hammarstrm, 1989).
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Prolonged firing of pelvic neurones contributes to long-lasting vasodilatation
Volleys of action potentials in pelvic vasodilator neurones innervating the uterine artery can release both nitric oxide and neuropeptides if delivered at frequencies of 5 Hz and above, producing prolonged dilatation of the pelvic arteries (Morris, 1993). However, the current study has demonstrated clearly that vasodilatation can be further enhanced and prolonged by post-stimulus potentiation of transmission within the pelvic ganglia. Many pelvic neurones continued to fire at 5–10 Hz after cessation of a train of stimuli at 10 Hz. Furthermore, some neurones fired at up to 20–25 Hz after a train of stimuli at 10 Hz, and then continued firing at rates of 5–10 Hz for many minutes in the absence of synaptic input. This prolonged firing is associated with slow depolarization of the neurones, and was not seen until after the first train of stimuli was applied to a preparation. Despite vasodilator neurones having a more depolarized resting membrane potential compared with non-vasodilator neurones in paracervical ganglia (Jobling et al. 2003), non-vasodilator neurones (without VIP-IR) as well as vasodilator neurones (VIP-IR) showed post-stimulus action potential discharge in the present in vitro study. Both vasodilator and uterine motor neurones are activated during sexual activity, although it is not clear if the two populations of pelvic neurones are always co-activated in vivo.
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Mediators of fast ganglionic transmission
As expected, the fast transmitter mediating the strong synaptic inputs from preganglionic axons to pelvic vasodilator neurones is acetylcholine acting on nicotinic receptors (Smith & Cunnane, 1999; Bobryshev & Skok, 2002). Relatively high doses of hexamethonium were required to abolish the fast EPSP, consistent with the very high safety factor for transmission at these synapses (Jobling et al. 2003, 2004). However, it is also possible that nicotinic receptors in anterior pelvic ganglia have a low sensitivity to hexamethonium, similar to rat cardiac ganglia (Selyanko & Skok, 1992; Skok, 2002). In addition to our observations in single vasodilator neurones, hexamethonium produced a significant delay in onset of vasodilatation. Thus, action potential firing of vasodilator neurones after blockade of nicotinic receptors does not appear to be associated with another fast ganglionic transmitter.
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Glutamate has been reported to act as a fast neurotransmitter in enteric ganglia (Liu et al. 1997; see, however, Ren et al. 2000). Furthermore, we recently demonstrated vesicular glutamate transporter-1 in preganglionic inputs to pelvic vasodilator neurones (Morris et al. 2005), and ionotropic glutamate receptors were reported to be present on some pelvic neurones (Chambille & Rampin, 2002). However, we found no functional evidence for involvement of the excitatory amino acid transmitter, glutamate, in fast excitatory transmission to vasodilator neurones in pelvic ganglia. Moreover, receptor antagonists such as CNQX or kynurenic acid enhanced rather than reduced uterine artery vasodilatation. This facilitatory effect potentially could be due to blockade of presynaptic glutamate receptors (Carpenedo et al. 2001). However, the depolarization and action potential firing produced in some pelvic neurones by CNQX alone indicates that the compound is likely to be having a direct agonist action, perhaps on a variant form of kainate receptors, as has been reported in some regions of the central nervous system (McBain et al. 1992; Brickley et al. 2001; Maccaferri & Dingledine, 2002; Hashimoto et al. 2004). Although CNQX is likely to be acting primarily as an agonist on pelvic neurones, its potent actions demonstrate that other agonists, including endogenous agents, that increase pelvic neurone excitability also are likely to enhance pelvic vasodilatation.
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Mediators of slow EPSPs and potentiation of vasodilatation
The post-stimulus potentiation of ganglionic transmission is closely linked with the presence of slow depolarizations produced by trains of stimuli. Slow EPSPs produced by non-nicotinic ganglionic transmission are widespread in autonomic ganglia (Jan et al. 1979; Jnig et al. 1982, 1983; Blumberg & Jnig, 1983; Konishi et al. 1983; Dun et al. 1984; Wang & Ma, 1990; Alkadhi et al. 1996; Jobling & Horn, 1996; Niel et al. 1996; Zhao et al. 1996; Ivanoff & Smith, 1997; Thorne & Horn, 1997; Karila & Horn, 2000; Alex et al. 2002; Johnson & Bornstein, 2004; Monro et al. 2004). In many autonomic ganglia, acetylcholine acting on postsynaptic muscarinic receptors produces a slow EPSP and increases the excitability of postganglionic neurones by inhibiting M-current (Brown & Selyanko, 1985). However, in the current study the muscarinic receptor antagonist, hyoscine, did not affect slow EPSPs and neurone firing, or vasodilatation, produced by trains of stimuli. ATP acting on P2X or P2Y receptors mediates fast and slow synaptic events in enteric ganglia (Monro et al. 2004), and purinoceptors are present on guinea-pig pelvic neurones in culture (Zhong et al. 2001). Nevertheless, the non-selective purinoceptor antagonist, suramin, did not affect vasodilatations of the uterine artery remaining after cholinergic receptor blockade.
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Amines such as 5-hydroxytryptamine (5-HT) and noradrenaline also can potentiate neurotransmission in autonomic ganglia, particularly in prevertebral sympathetic ganglia where terminals of preganglionic or intestinofugal neurones appear to take up and store 5-HT (Dun et al. 1984; Wang & Ma, 1990). However, our histochemical studies have demonstrated that there are no varicose terminals with catecholamine or 5-HT fluorescence in paracervical ganglia, even after incubation with amine precursors (Morris & Gibbins, 1987). Furthermore, long-lasting vasodilatations of the uterine artery were unaffected when pelvic ganglia were exposed to guanethidine and phenylephrine. Thus, we have no evidence for endogenous amines modulating synaptic transmission in anterior pelvic ganglia.
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A strong candidate for mediator of the slow depolarization is substance P. Substance P is located in preganglionic neurones projecting selectively to VIP immunoreactive vasodilator neurones in guinea-pig paracervical ganglia (Morris & Gibbins, 1987). Furthermore, immunoreactivity for NK1 receptors has been demonstrated on a subpopulation (12%) of VIP immunoreactive neurones in paracervical ganglia (Messenger et al. 1999). In the current study we found that about half of the paracervical neurones were depolarized and fired volleys of action potentials when exposed to exogenous substance P. This discrepancy in the proportion of neurones responding to substance P compared with that with NK1 receptor immunoreactivity may be due to the presence of other receptor subtypes, such as truncated NK1 receptors that do not show immunoreactivity to C-terminally directed antisera (Baker et al. 2003), or NK3 receptors (Mawe, 1995; Jobling et al. 2001; Johnson & Bornstein, 2004). Indeed, using the NK3 agonist, senktide, we have confirmed the presence of functional NK3 receptors on a subpopulation of paracervical neurones. Nevertheless, a combination of NK1, NK2 and NK3 receptor antagonists had only a marginal effect in reducing vasodilatations produced by stimulating the hypogastric nerve after blockade of ganglionic cholinergic receptors. Thus, release of substance P from nerve terminals in the anterior pelvic ganglia seems to contribute slightly to the prolonged vasodilatation of the uterine artery. As these dilatations were produced by stimulation of the L3 ventral roots but not dorsal roots, and were not affected by capsaicin, any contribution by substance P is most likely from the substance P-immunoreactive preganglionic nerve terminals rather than the rare peptide-containing sensory axons in the ganglia (Morris & Gibbins, 1987; Anderson et al. 1997). However, the major contributor to non-cholinergic ganglionic transmission in the pelvic vasodilator pathways is unlikely to be substance P, and remains to be identified.
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Conclusions
This study has shown that, in females, the vasodilator pathway from the lumbar spinal cord to pelvic arteries predominates over the sacral vasodilator pathway. Release of non-cholinergic neurotransmitters, both at the level of the anterior pelvic ganglia and at the neurovascular junction, act in concert to prolong the period of vasodilatation after volleys of stimuli applied to preganglionic neurones. This modulation of ganglionic transmission occurs despite the presence of only one or two strong synaptic inputs onto each vasodilator neurone. This differs from the phenomena of long-term potentiation or synaptic gain described in other autonomic ganglia with multiple subthreshold synaptic inputs (Booth & de Groat, 1979; Gola & Niel, 1993; Karila & Horn, 2000; Wheeler et al. 2004), but is similar to previous reports of non-cholinergic transmission to vasoconstrictor neurones in cat sympathetic ganglia (Jnig et al. 1982, 1983). The prolonged firing appears to be produced by largely unknown, slow-acting substances released from preganglionic neurones. Thus, the vasodilator pathway from the lumbar spinal cord to the pelvic arteries in females is well designed to maintain a high level of firing of postganglionic neurones and produce maximal vasodilatation for sustained periods. This long-lasting excitation of pelvic neurones might be even further enhanced by prolonged firing of the preganglionic neurones, as has been reported for preganglionic neurones in splanchnic nerves of cats after reflex activation from sacral levels (Bahr et al. 1986). All of these mechanisms are likely to contribute to the increased blood flow to the genitalia and internal reproductive organs during sexual activity, when ongoing inhibitory influences from supraspinal levels are replaced by excitation both from supraspinal levels and from spinal reflexes initiated by sacral sensory neurones (de Groat & Booth, 1993b; McKenna & Marson, 1997).
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Abstract
Vasodilatation produced by stimulation of preganglionic neurones in lumbar and sacral pathways to pelvic ganglia was studied using an in vitro preparation of guinea-pig uterine artery and associated nerves in a partitioned bath allowing selective drug application to the ganglia or artery. Arterial diameter was monitored using real time video imaging. Vasodilatations produced by hypogastric nerve stimulation (HN; 300 pulses, 10 Hz) were significantly larger and longer in duration than with pelvic nerve stimulation (N = 18). Stimulation of ipsilateral lumbar splanchnic nerves or ipsilateral third lumbar ventral roots also produced prolonged vasodilatations. Blockade of ganglionic nicotinic receptors (0.1–1 mM hexamethonium) delayed the onset and sometimes reduced the peak amplitude of dilatations, but slow dilatations persisted in 16 of 18 preparations. These dilatations were not reduced further by 3 μM capsaicin applied to the artery and ganglia, or ganglionic application of 1 μM hyoscine, 30–100 μM suramin or 10 μM CNQX. Dilatations were reduced slightly by ganglionic application of NK1 and NK3 receptor antagonists (SR140333, SR142801; 1 μM), but were reduced significantly by bathing the ganglia in 0.5 mM Ca2+ and 10 mM Mg2+. Intracellular recordings of paracervical ganglion neurones revealed fast excitatory postsynaptic potentials (EPSPs) in all neurones on HN stimulation (300 pulses, 10 Hz), and slow EPSPs (3–12 mV amplitude) in 25 of 37 neurones. Post-stimulus action potential discharge associated with slow EPSPs occurred in 16 of 37 neurones (firing rate 9.4 ± 1.5 Hz). Hexamethonium (0.1–1 mM) abolished fast EPSPs. Hexamethonium and hyoscine (1 μM) did not reduce slow EPSPs and associated post-stimulus firing in identified vasodilator neurones (with VIP immunoreactivity) or non-vasodilator paracervical neurones. These results demonstrate a predominantly sympathetic origin of autonomic pathways producing pelvic vasodilatation in females. Non-cholinergic mediators of slow transmission in pelvic ganglia produce prolonged firing of postganglionic neurones and long-lasting dilatations of the uterine artery. This mechanism would facilitate maintenance of pelvic vasodilatation on stimulation of preganglionic neurones during sexual activity.
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Introduction
Neurogenic vasodilatation in the genitalia and internal reproductive organs of both males and females is essential for erection and fluid secretion during copulation (Meston & Frohlich, 2000). Preganglionic neurones leaving the lumbo-sacral spinal cord synapse with vasodilator neurones in the anterior pelvic ganglia (de Groat & Booth, 1993b; Keast, 1999). The vasodilator neurones release nitric oxide (NO) and vasoactive intestinal peptide (VIP) to dilate the pelvic vasculature by a direct action on vascular smooth muscle (Polak & Bloom, 1984; Morris, 1993; Simonsen et al. 2001). Pelvic vasodilator nerve pathways are activated periodically rather than tonically. The preganglionic neurones are normally under descending inhibitory control from the brainstem, but are stimulated during sexual activity by sensory input from the genitalia combined with a descending net excitation from supraspinal levels (de Groat & Booth, 1993b; McKenna & Marson, 1997). In recent electrophysiological studies we found that the major synaptic input to vasodilator neurones in guinea-pig anterior pelvic (paracervical) ganglia runs in the hypogastric nerve rather than the pelvic nerve (Jobling et al. 2003, 2004).
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Autonomic vasodilatation of the guinea-pig uterine artery is mediated primarily by neurally derived nitric oxide at low levels of nerve stimulation. Additionally, release of peptide co-transmitters provides an efficient mechanism for producing long-lasting vasodilatation in response to stimulation with trains of pulses at frequencies of 5 Hz and above (Morris, 1993). Release of VIP into the penile circulation during erection indicates that the physiological firing rate of pelvic vasodilator neurones during sexual activity is sufficient to release neuropeptides (Virag et al. 1982). Furthermore, studies of pelvic nerve pathways to the cat bladder have shown that preganglionic neurones can fire at rates up to 5–10 Hz for the duration of micturition (de Groat & Booth, 1980). The prolonged activation of postganglionic neurones at high frequencies occurs by temporal facilitation within the vesical ganglia that act as high pass filters. This facilitation seems to be due primarily to enhanced transmitter release from preganglionic neurones (Booth & de Groat, 1979; de Groat & Booth, 1980). However, postsynaptic actions of acetylcholine acting on muscarinic receptors, amines such as 5-hydroxytryptamine or co-transmitters such as neuropeptides also can facilitate transmission to postganglionic neurones in autonomic ganglia (Jan et al. 1979; Jnig et al. 1982, 1983; Dun et al. 1984; Wang & Ma, 1990; Alkadhi et al. 1996; Niel et al. 1996; Ivanoff & Smith, 1997). These forms of slow synaptic transmission enhance nicotinic transmission from subthreshold synaptic inputs, a phenomenon termed synaptic gain (Karila & Horn, 2000; Wheeler et al. 2004).
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Many of the preganglionic inputs to vasodilator neurones in guinea-pig pelvic ganglia contain the peptide substance P (Morris & Gibbins, 1987), which facilitates synaptic transmission in many other autonomic ganglia (Peters & Kreulen, 1986; Kawatani et al. 1989; Mawe, 1995; Canning et al. 2002). However, pelvic neurones have only one or two synaptic inputs (Jobling et al. 2003). These inputs are suprathreshold for action potential generation and are regarded as ‘strong’ inputs. It is not clear how slow synaptic events such as those produced by neuropeptides can modulate ganglionic transmission mediated by fast, suprathreshold inputs (de Groat & Booth, 1993a; McLachlan, 2003). Nevertheless, non-cholinergic transmission to vasoconstrictor neurones in cat sympathetic ganglia produces prolonged firing of neurones that normally receive strong synaptic inputs (Jnig et al. 1983). This study set out to determine whether slow synaptic events in anterior pelvic ganglia of female guinea-pigs can modulate the firing of postganglionic neurones and, hence, the long-lasting vasodilatation produced by neuropeptides acting on arterial smooth muscle. As synaptic transmission from lumbar preganglionic axons onto neurones in anterior pelvic (paracervical) ganglia utilizes a different profile of voltage-dependent calcium channels compared with sacral preganglionic neurones (Jobling et al. 2004), we compared the effects of stimulating the lumbar and sacral pathways on modulation of ganglionic transmission and dilatation of the uterine artery.
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Methods
Female guinea-pigs (Hartley IMVS, Institute for Medical and Veterinary Science, Adelaide, Australia; 150–250 g) were killed by stunning and exsanguination via the carotid arteries, as approved by the Flinders University Animal Welfare Committee in accordance with guidelines of the National Health and Medical Research Council of Australia. The anterior pelvic (paracervical) ganglia, posterior pelvic ganglia, hypogastric nerve and pelvic nerve together with the internal iliac, uterine and vaginal arteries were removed unilaterally and placed in Hepes-buffered balanced salt solution (composition (mM): 146 NaCl, 4.7 KCl, 0.6 MgSO4, 1.6 NaHCO3, 0.13 NaH2PO4, 2.5 CaCl2, 7.8 glucose, 20 Hepes, adjusted to pH 7.3 with NaOH and bubbled with 100% O2). In some experiments the connection of the hypogastric nerve to the inferior mesenteric ganglion, lumbar splanchnic nerves and dorsal and ventral roots of the mid lumbar spinal cord (L3) were included in the isolated nerve–artery preparation (Fig. 1A).
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A, photograph of entire nerve–artery preparation pinned out at the conclusion of an experiment. APG, anterior pelvic (paracervical) ganglia; CN, colonic nerves; HN, hypogastric nerve; IIA, internal iliac artery; IMG, inferior mesenteric ganglion; IN, intermesenteric nerve; LSN, lumbar splanchnic nerves; L3DRG, 3rd lumbar dorsal root ganglion; L3VR, 3rd lumbar ventral roots; PN, pelvic nerve; SC, paravertebral sympathetic chain; UA, main uterine artery. Boxed area shows region of uterine artery imaged for diameter measurement. B, image of uterine artery mounted in an organ bath and superfused with solution containing guanethidine (1 μM) and phenylephrine (3 μM) prior to stimulation of vasodilator nerves. Note longitudinal ridges formed by endothelial folds during arterial constriction. The high contrast outer edges of the artery are tracked by DIAMTRAK for a continuous readout of diameter.
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Measurement of arterial dilatation
Isolated nerve–artery preparations (N = 87) were pinned to the base of a dish (2 ml volume) coated with silicone elastomer (Sylgard, Dow Corning, Midland, MI, USA) and mounted in a PDM1-2 micro incubator (Harvard Apparatus Ltd, Edenbridge, Kent, UK) on the stage of an Olympus BH2 microscope. The caudal portion of the main uterine artery was slightly stretched and pinned flat, taking care not to damage the paravascular nerves connecting to the anterior pelvic ganglia. Preparations were superfused at 1–2 ml min–1 with Hepes-buffered solution maintained at 36°C by a TC-202A temperature controller (Harvard Apparatus Ltd). Agonist or antagonist drugs were added to the reservoir of superfusate. In some preparations a thin plastic partition sealed with high vacuum grease (Dow Corning, Ajax Chemicals, Auburn, NSW, Australia) was placed in the bath towards the caudal end of the uterine artery to separate the solution superfusing the uterine artery from that superfusing the pelvic ganglia and attached nerve trunks. Suction electrodes were placed on one or more of the pelvic nerve, hypogastric nerve, intermesenteric nerve, colonic nerves, lumbar splanchnic nerves, L3 ventral roots or L3 dorsal roots (see McLachlan, 1985). Nerves were stimulated with trains of square wave pulses (0.3 ms duration) delivered at 2–20 Hz by an S88 stimulator (Grass Instruments, Quincy, MA, USA) connected to a stimulus isolation unit (Grass Instruments) and low impedance interface (BioMedical Engineering, Flinders Medical Centre, Bedford Park, SA, Australia). The uterine artery was imaged using a 4 x long working distance objective and a Watek WAT-902B CCD video camera (Fig. 1B). Arterial outer diameter was monitored continuously on-line using DIAMTRAK v 3.1 (T.O. Neild, Flinders University) and was recorded using a MacLab 4S running Chart v 4.2 software (AD Instruments, Castle Hill, NSW, Australia). Neurogenic dilatations of the uterine artery were recorded after preconstriction with phenylephrine (3 μM) or prostaglandin F2 (3 μM). In most experiments guanethidine (1 μM) also was present in the artery superfusate to block release of transmitter from any noradrenergic axons in the stimulated nerve trunks (Morris & Murphy, 1988). When partitioned baths were used, phenylephrine and guanethidine were present only in the solution superfusing the uterine artery. The maximum amplitude of dilatations during the stimulation period (μm), latency (s) from start of stimulation period to commencement of dilatation, and integrated dilator response (area under the curve to a line joining two vertical cursors marking the period of dilatation; μm s) were measured using Chart v 4.2 and NIH Image v 1.62 (NIH, Bethesda, MD, USA).
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Intracellular electrophysiology
Anterior pelvic ganglia in isolated nerve–artery preparations (N = 54) were dissected free of connective tissue and fat and pinned to the base of a recording chamber (1 ml) mounted on the stage of an Olympus IMT2 inverted microscope for intracellular electrophysiology. In some instances these preparations were obtained from the contralateral side of the same animals as used for measurement of arterial diameter. Preparations were superfused at 2.5 ml min–1 with Hepes-buffered solution bubbled with O2 and maintained at 35°C. Neurones were impaled under visual control with glass microelectrodes filled with 0.5 M KCl (resistance 80–200 M). In some experiments Neurobiotin (0.5%, Vector, Burlingame, CA, USA) was included in the electrode filling solution to allow later visualization of the impaled neurones. Data were recorded using an Axoclamp 2B amplifier (Axon Instruments, Union City, CA, USA). Voltage and current records were digitized at 4–20 kHz using a Powerlab running Chart v 4.2 and Scope v 3.6 software (AD Instruments) and were analysed using Igor Pro v 3.16 (WaveMetrics, Lake Oswego, OR, USA). Changes in membrane potential were recorded using bridge mode. Somata were voltage clamped using single electrode voltage clamp (SEVC). During SEVC, the headstage was continuously monitored and the cycling frequency adjusted to minimize the effects of electrode capacitance. The cycling frequency was 2–4 kHz. The hypogastric or pelvic nerve trunks were stimulated with square wave pulses (40 μA to 1 mA amplitude, 0.3–0.5 ms duration) via suction electrodes connected to a WPI A360 (World Precision Instruments Inc, Sarasota, FL, USA) or a WECO SC-100 constant current stimulator (Winston Electronics Co., Millbrae, CA, USA). Agonist and antagonist drugs were added to the reservoir of superfusate. Instantaneous action potential discharge frequency was determined using the ‘Spike Utilities’ procedure for Igor Pro (v 1.5, Neil Berman, University of Ottowa).
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Immunohistochemistry
At the conclusion of electrophysiological experiments where neurones were filled with Neurobiotin, preparations were fixed by immersion in Zamboni's solution (2% formaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.0) for 16–64 h at 4°C. Tissues were processed as whole-mounts by washing in 80% ethanol and clearing in 100% dimethylsulfoxide (DMSO), as previously described (Jobling et al. 2003). After washing in phosphate-buffered saline (PBS), preparations were exposed for 48–72 h to an antiserum raised in a rat against vasoactive intestinal peptide (VIP; antiserum code FI/III supplied by Dr R. Murphy), diluted 1: 200. Preparations were washed in PBS and incubated for 24 h in donkey anti-rat IgG conjugated to Cy3 (Jackson ImunoResearch Laboratories, West Grove, PA, USA) to visualize neurones with VIP immunoreactivity (IR), and streptavidin conjugated to Cy5 to visualize the Neurobiotin-filled neurones. After further washes in PBS, preparations were mounted on slides in carbonate-buffered glycerol (pH 8.6) and were examined with an Olympus AX70 epifluorescence microscope or a Bio-Rad MRC-1024 scanning laser confocal microscope fitted to an Olympus AX70. Images were collected from the epifluorescence microscope with an Orca cooled CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan) using IPLab Spectrum software (Scanalytics Inc., Faifax, VA, USA) or from the confocal microscope using Lasersharp v3.1 software.
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Drugs
Guanethidine sulphate, hexamethonium hydrochloride, hyoscine hydrobromide, NG-nitro-L-arginine methyl ester, phenylephrine hydrochloride, suramin sodium salt were all purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Stock solutions were made up in phosphate-buffered saline and were stored at 4°C. Capsaicin (Sigma-Aldrich) was dissolved in 95% ethanol: 5% Tween 80 and stock solutions (30 mM) were stored at 4°C. Substance P and senktide (Auspep, Parkville, VIC, Australia) were stored frozen as stock solutions of 1 mM in dH2O. Prostaglandin F2 (Cayman Chemical Company, Ann Arbor, MI, USA) was dissolved in PBS and 3 mM stock solutions were stored frozen. Tetrodotoxin (with citrate; Alomone Laboratories, Jerusalem, Israel) was dissolved in dH2O and 1 mM stock solution was stored at 4°C. CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; Sigma-RBI) was dissolved in DMSO and di-Na-CNQX was dissolved in dH2O immediately before use. Kynurenic acid (Sigma-RBI) was dissolved in 0.1 M NaOH immediately before use. NK1, NK2 and NK3 receptor antagonists (SR140333, SR48968 and SR142801), generous gifts of Sanofi Recherche (Montpellier, France), were dissolved in DMSO and stored frozen at a concentration of 10 mM.
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Data analysis
Data are expressed as mean ± S.E.M., with N referring to the number of preparations and n the number of neurones used for electrophysiological analysis. Statistical analysis was performed by repeated measures analysis of variance (ANOVA) using SPSS 10 for Macintosh (SPSS, Chicago, IL, USA). The level of significance was set at P < 0.05.
Results
Source of vasodilator outflow from the spinal cord
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Stimulation of the ipsilateral pelvic nerve or hypogastric nerve with trains of 50–300 pulses produced dilatation of the preconstricted uterine artery. Artery diameter increased progressively and at lower stimulation frequencies (< 5 Hz) the maximum amplitude of the neurogenic dilatation was reached at the end of the stimulation period (Fig. 2A). As the stimulation frequency was increased above 5 Hz for a given number of pulses, the initial dilatation did not increase further in amplitude, but a second, slow vasodilatation was apparent after cessation of stimulation. The amplitude of the slow dilatation sometimes exceeded the maximum amplitude during the stimulation period, and lasted up to 10–20 min (Fig. 2B and C). Hypogastric nerve stimulation at 10–20 Hz always produced larger dilatations than stimulation of the pelvic nerve in the same preparations (N = 18; Fig. 3). The slow phase of the neurogenic dilatation was particularly prominent after hypogastric compared with pelvic nerve stimulation (Fig. 3).
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The same artery is dilated by trains of 300 pulses delivered to the hypogastric nerve at different frequencies, 2 Hz in A, 10 Hz in B and 20 Hz in C. Dashed vertical lines mark the start and end of each stimulation period.
A, dilatations of a uterine artery produced by stimulation of the hypogastric nerve (HN) or the pelvic nerve (PN) in the same preparation with 300 pulses at 10 Hz. B, group data for 18 preparations where the hypogastric nerve and pelvic nerve were stimulated. Bars represent mean + S.E.M. Note significant increase in latency of dilatations (repeated measures ANOVA, F1,17 = 13, P = 0.002) and significantly smaller amplitude of dilatations (F1,17 = 48, P = 0.0001) and integrated dilator response (area; F1,17 = 37, P = 0.001) of dilatations after pelvic nerve stimulation. *P < 0.05.
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The source of axons in the hypogastric nerve producing dilatation of the uterine artery was investigated by stimulating the colonic nerves, intermesenteric nerve, ipsilateral lumbar splanchnic nerves, or ipsilateral ventral roots and dorsal roots at L3 with trains of pulses at 10 Hz or 20 Hz. Large biphasic dilatations always were seen after stimulation of the lumbar splanchnic nerves (9 of 9 preparations; Figs 4 and 5). Smaller dilatations were apparent after stimulation of the intermesenteric nerve (4 of 4 preparations) or the L3 ventral roots (5 of 5 preparations). However, dilatations were not seen after stimulation of the colonic nerves (0 of 2 preparations) or the L3 dorsal roots (0 of 4 preparations).
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Representative traces of vasodilator responses to stimulation of the hypogastric nerve (HN) in A, lumbar splanchnic nerves (LSN) in B, or L3 ventral roots (L3VR) in C with trains of 300 pulses at 10 Hz. Control responses shown in black; responses at least 20 min after addition of hexamethonium (0.3–1 mM) shown in grey.
Group data showing effect of hexamethonium (Hex, 0.3–1 mM) on dilatations produced by stimulation of the hypogastric nerve (HN, N = 6), pelvic nerve (PN, N = 4), lumbar splanchnic nerves (LSN, N = 5), intermesenteric nerve (IN, N = 3) or L3 ventral roots (L3VR, N = 3). Bars represent mean + S.E.M. Filled bars, responses before Hex; open bars, responses at least 20 min after Hex. For all nerves, hexamethonium produced: A, a significant increase in latency of dilatations (repeated measures ANOVA, F1,16 = 275, P = 0.0001); B, a significant decrease in amplitude of dilatations at the end of the stimulation period (F1,16 = 168, P = 0.0001); C, a significant decrease in integrated dilator response (area; F1,16 = 22, P = 0.0001). in B, dilatations to L3VR remaining after hexamethonium did not commence until after cessation of stimulation, so amplitude at the end of stimulation period was zero.
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The nicotinic receptor antagonist hexamethonium (0.1–1 mM), was used to ascertain whether these vasodilator nerve pathways to the uterine artery involved ganglionic synapses. Vasodilatations produced by stimulation of the hypogastric nerve, pelvic nerve, intermesenteric nerve, lumbar splanchnic nerves or L3 ventral roots at 10–20 Hz all were affected significantly by hexamethonium (Figs 4 and 5). The effects of hexamethonium were similar whether it was applied to the whole preparation, or was present only in the solution superfusing the ganglia and nerve trunks. In either case, there was a pronounced increase in latency and a reduction in amplitude of dilatations during the stimulation period. However, a slow dilatation persisted in almost all experiments, even after the concentration of hexamethonium was increased to 1 mM (Figs 4 and 5). With hypogastric nerve stimulation, the hexamethonium-resistant dilatations commenced towards the end of the stimulation period (latency 20 s). The peak amplitude occurring after cessation of stimulation sometimes was reduced (Fig. 6Aa), but often was similar to that of control dilatations (Fig. 4A).
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Aa, dilatation produced by hypogastric nerve stimulation (HN) with 300 pulses at 10 Hz (Control, shown in black) was reduced by 1 mM hexamethonium (+ Hex, shown in grey) and then enhanced by subsequent addition of 10 μM CNQX (+ CNQX, shown in grey). Ab, dilatation produced by L3 ventral root stimulation (L3VR) with 300 pulses delivered at 20 Hz (Control, black) was enhanced by 10 μM CNQX (+ CNQX, shown in grey) and then reduced, but not abolished, by subsequent treatment with 1 mM hexamethonium (+ Hex, shown in grey). B, dilatation produced by hypogastric nerve stimulation (HN) with 300 pulses at 10 Hz was greatly reduced by solution low in calcium and high in magnesium (low Ca/high Mg, shown in grey) then abolished by subsequent treatment with 0.3 mM hexamethonium (+ Hex, shown in grey). C, dilatation produced by hypogastric nerve stimulation (HN) with 300 pulses at 10 Hz (Control, shown in black) was slightly enhanced after application of 10 μM capsaicin then washout after 5 min (+ Capsaicin, shown in grey). Subsequent addition of 1 mM hexamethonium delayed the onset and increased the amplitude of the slow dilatation (+ Hex, shown in grey).
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Hexamethonium-resistant dilatations to hypogastric nerve stimulation were not reduced further by the muscarinic receptor antagonist hyoscine (1 μM), added to the ganglion side of a partitioned bath. Indeed, the amplitude of dilatations was slightly increased after addition of hyoscine to the hexamethonium (repeated measures ANOVA: dilatation latency, F1,5 = 0.8, P = 0.4; amplitude, F1,5 = 7.3, P = 0.04; integrated dilatation, F1,5 = 0.4, P = 0.6). Addition of the ionotropic glutamate receptor antagonist CNQX (10 μM), did not reduce the neurogenic dilatations, either before or after treatment with hexamethonium. Instead, CNQX applied to the whole preparation or to the ganglia and nerve trunks only, always produced a large potentiation of dilatations produced by stimulation of the hypogastric nerve (N = 10; Fig. 6Aa), pelvic nerve (N = 3) or the L3 ventral roots (N = 2; Fig. 6Ab). The potentiation was apparent as an increase in the amplitude and duration of the slow dilatations (Fig. 6A). A similar potentiation was seen after treatment with kynurenic acid (1 mM; N = 2).
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To determine whether the dilatations remaining after blockade of ganglionic acetylcholine receptors were due to stimulation of axons reaching the uterine artery without synapsing in pelvic ganglia, partitioned baths were used to bathe ganglia in a Hepes-buffered salt solution low in calcium (0.5 mM) and high in magnesium (10 mM; N = 4), or in cadmium (50–100 μM; N = 3). These treatments greatly reduced or abolished the neurogenic dilatations to hypogastric nerve stimulation. Small dilatations remaining in low calcium and high magnesium were abolished by hexamethonium (Fig. 6B). In separate experiments (N = 10), capsaicin (1–10 μM) was used to stimulate and then block transmission from peptide-containing sensory neurones to test involvement of these axons in dilatation of the uterine artery in response to hypogastric nerve stimulation. Capsaicin produced a large (20–60 μm), transient dilatation of the uterine artery, but had very little effect on the amplitude of neurogenic dilatations either in the continued presence or after washout of capsaicin. Furthermore, capsaicin pre-treatment did not reduce the incidence of hexamethonium-resistant vasodilatations to hypogastric nerve stimulation (9 of 9 preparations; Fig. 6C). Addition of the nitric oxide synthase inhibitor L-NAME (30 μM), to the whole bath or to the artery side of a partitioned bath reduced the amplitude and increased the latency of dilatations to hypogastric nerve (N = 6) or L3 ventral root (N = 2) stimulation, before or after treatment with hexamethonium. However, a slow dilatation was still present after L-NAME treatment. The remaining dilatations were abolished by tetrodotoxin (0.3–1 μM).
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Responses of pelvic neurones to repetitive stimulation of the hypogastric nerve
Stimulation of the hypogastric nerve with trains of 50–100 pulses delivered at 10–20 Hz produced fast excitatory postsynaptic potentials (EPSPs) and action potentials in pelvic neurones in paracervical ganglia in response to each pulse. In addition, slow EPSPs were apparent in a subpopulation of neurones (7 of 12 neurones after 50 pulses at 10 Hz; 2 of 4 neurones after 100 pulses at 10 Hz; 6 of 17 neurones after 100 pulses at 20 Hz). Furthermore, 3 of 13 pelvic neurones continued to fire action potentials after cessation of stimulation of the hypogastric nerve with 50–100 pulses at 10 Hz. Increasing the train of hypogastric nerve stimulation at 10 Hz to 300 pulses produced slow EPSPs in 25 of 37 neurones. The amplitude of the slow EPSP ranged from 3 to 12 mV and the duration was 1–6 min. Thirteen of these neurones continued to fire action potentials after the stimulation period (Fig. 7A), and a further three neurones fired continuously until the next stimulation period (10 min; Fig. 8A and B). The maximum instantaneous firing rate ranged from 2 to 24 Hz (mean = 9.4 ± 1.5 Hz). Pelvic nerve stimulation also produced slow EPSPs in 24% of the pelvic neurones (2 of 7 neurones after 50 pulses at 10 Hz; 5 of 18 neurones after 100 pulses at 20 Hz and 1 of 8 neurones after 300 pulses at 10 Hz). However, only one of these neurones fired any action potentials during the slow EPSP. This burst was of less than 7 s duration.
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Intracellular recording of membrane potential in an identified VIP immunoreactive pelvic neurone (middle traces) during and after stimulation of hypogastric nerve with 300 pulses at 10 Hz. Top traces show responses to the first 5 pulses in each train of stimuli. Bottom traces show instantaneous frequency of action potential firing. A, control response; B, 10 min after addition of 0.3 mM hexamethonium (Hex); C, 25 min after addition of hexamethonium.
Intracellular recording of membrane potential (upper traces) in a VIP immunoreactive pelvic neurone showing rate of action potential firing (lower traces) during and after stimulation of the hypogastric nerve with 300 pulses at 10 Hz. A, control response. B, the neurone continues to fire between stimulation periods. Trace starts 450 s after cessation of previous stimulus train; C, response to HN nerve stimulation 10 min after addition of 1 mM hexamethonium and 1 μM hyoscine (Hex + Hyo). Inset Ca, action potentials 1 s before commencement of the stimulation period. Inset Cb, action potentials during the last second of the 30 s stimulation period have increased frequency but are not linked to the stimulus pulses (black dots).
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Some pelvic neurones (n = 11) were filled with Neurobiotin and examined for VIP immunoreactivity at the conclusion of electrophysiological analysis. All six neurones with VIP-IR had slow EPSPs and continued to fire action potentials after hypogastric nerve stimulation with 300 pulses at 10 Hz (Figs 7A and 8A). Of the five neurones without VIP-IR, four had slow EPSPs and three continued firing after the 30 s stimulation period.
In order to investigate further the contribution of non-cholinergic transmission to the action potential discharge observed following long trains of hypogastric nerve stimulation, hexamethonium (0.3–1 mM) alone, or hexamethonium together with hyoscine (1 μM) was used. Hexamethonium markedly reduced or abolished fast EPSPs (Fig. 7B and C). The hexamethonium block was use dependent with the first one or two EPSPs in the train sometimes reaching threshold. This use-dependent block by hexamethonium is similar to that observed in other autonomic ganglia (Skok, 1986). Hexamethonium and hyoscine did not reduce the amplitude of slow EPSPs (Fig. 9). Furthermore, after cholinergic receptor blockade, pelvic neurones with or without VIP-IR commenced firing during or at the end of the stimulation period and continued firing after cessation of stimulation (Fig. 9).
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Group data showing no effect of hexamethonium and hyoscine (+ Hex/Hyo) on the amplitude (repeated measures ANOVA, F1,3 = 0.003, P = 0.96) or duration (F1,3 = 0.03, P = 0.88) of slow EPSPs to hypogastric nerve stimulation with 300 pulses at 10 Hz. There was a significant increase in firing rate (F1,6 = 10.4, P = 0.02) but not duration of firing (F1,4 = 2.3, P = 0.2) of pelvic neurones after hexamethonium and hyoscine. Bars represent mean + S.E.M. *P < 0.05.
CNQX (10 μM) was added to the superfusate before or after blockade of cholinergic receptors. In 2 of 5 neurones, CNQX caused depolarization and initiated bursts of action potential firing at rates up to 5–10 Hz. The firing rate was increased further on stimulation of the hypogastric nerve with trains delivered at 10 Hz. The membrane potential returned to pre-CNQX levels soon after washout and action potential firing ceased.
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Effects of substance P on pelvic neurones
Substance P (SP, 1 μM) was added to the superfusate for 1 min and membrane potential changes were recorded in 38 pelvic neurones from 24 preparations. Half of the neurones (18 of 38) were depolarized by SP by an average of 7.6 ± 0.8 mV. Some neurones (5 of 18) started firing action potentials during the SP-induced depolarization (Fig. 10). In nine neurones that were voltage clamped, SP evoked an inward current of 0.09 ± 0.01 nA. Six of nine neurones also responded to the NK3 receptor agonist senktide (0.5 μM). The mean inward current measured in five of these neurones was 0.14 ± 0.04 nA.
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Membrane potential and instantaneous rate of action potential discharge of pelvic neurone superfused with substance P (SP; 1 μM for 1 min at bar).
Mediators of non-cholinergic ganglionic transmission
To investigate potential mediators of non-cholinergic transmission to vasodilator neurones in pelvic ganglia, receptor antagonists for SP or ATP were applied to the ganglion side of partitioned baths and their effects on hypogastric nerve-mediated vasodilatations were determined. Antagonists for NK1 and NK3 receptors (SR140333 and SR142801), or for NK1, NK2 and NK3 receptors (SR140333, SR48968 and SR142801), all at 1 μM, were added to six preparations after treatment with hexamethonium and hyoscine. In some preparations, dilatations produced by hypogastric nerve stimulation (300 pulses at 10 Hz) appeared to be slower and shorter in duration in the presence of SP antagonists. However, the group effects were only marginally significant (Fig. 11). In a further five preparations, addition of the non-selective purinoceptor antagonist suramin (30–100 μM), did not alter the latency, maximum amplitude or integrated response of the slow, hexamethonium-resistant dilatations to hypogastric nerve stimulation (Fig. 11). Addition of neurokinin receptor antagonists and suramin together produced a marginal reduction in the amplitude and area of slow dilatations in four preparations (Fig. 11). Given the lack of effect of suramin alone, these small reductions produced by combined antagonists are likely to be due to marginal effects of NK receptor blockade that sometimes reach statistical significance and other times do not.
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Group data showing the latency, maximum amplitude and area of slow dilatations to hypogastric nerve stimulation with 300 pulses at 10 Hz in the presence of hexamethonium and hyoscine, before (Control, filled bars) and after treatment with antagonists (open bars) for NK1 and NK3 receptors (NK, 1 μM, N = 6), suramin (Sur, 30–100 μM, N = 5) or NK antagonists and suramin together (NK + Sur, N = 4). Bars show mean values + S.E.M.A, a significant increase in latency of dilatations was produced by NK antagonists (F1,5 = 6.6, P = 0.05), not by suramin (F1,4 = 4.8, P = 0.09) but was increased marginally by NK antagonists plus suramin (F1,3 = 8.4, P = 0.06). B, maximum amplitude of slow dilatations was not affected by NK antagonists (F1,5 = 3.1, P = 0.14) nor by suramin (F1,4 = 1.9, P = 0.24), but was reduced significantly by both NK antagonists and suramin (F1,3 = 18, P = 0.02). C, the integrated vasodilator response (area of dilatation) was reduced marginally by NK antagonists (F1,5 = 5.3, P = 0.07), was not affected by suramin (F1,4 = 0.1, P = 0.75) and was reduced significantly by all antagonists together (F1,3 = 14, P = 0.03). *P < 0.05.
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Discussion
Sympathetic pathway to pelvic vasodilator neurones
Pelvic ganglia are unusual in receiving preganglionic projections from both lumbar and sacral levels of the spinal cord. Typically, lumbar preganglionic neurones innervate catecholamine-synthesizing pelvic neurones innervating the urinogenital organs, and sacral preganglionic neurones project to non-catecholamine-synthesizing pelvic neurones, many of which synthesize acetylcholine, nitric oxide and neuropeptides (Keast, 1999). Preganglionic input to pelvic vasodilator neurones contributing to erection in males arises predominantly from sacral levels, although outflow from the lumbar cord can mediate erection after damage to sacral spinal cord (Dail et al. 1985; de Groat & Booth, 1993b). In contrast, the present study has extended our previous electrophysiological work (Jobling et al. 2003) to demonstrate directly that neurones projecting from mid-lumbar spinal cord via the lumbar splanchnic and hypogastric nerves provide the major neural pathway for vasodilatation of pelvic arteries supplying the internal reproductive organs of female guinea-pigs. This is consistent with reports of hypogastric nerve stimulation but not pelvic nerve stimulation increasing venous outflow and VIP release from the cat uterus (Fahrenkrug & Ottesen, 1982). The relative large magnitude of vasodilatations produced by stimulation of the ipsilateral hypogastric or lumbar splanchnic nerves compared with the L3 ventral roots is likely to reflect the extensive bilateral projection of lumbar preganglionic neurones down both hypogastric nerves (McLachlan, 1985).
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Our previous studies have shown conclusively that the final motor neurones in pelvic vasodilator pathways are located in the anterior pelvic (paracervical) ganglia and not sympathetic ganglia (Morris & Gibbins, 1987; Morris et al. 1998; Jobling et al. 2003). Furthermore, we have now shown directly that ganglionic synapses exist in the vasodilator pathway between the hypogastric nerve and the uterine artery. The vasodilator neurones in paracervical ganglia supply a dense innervation to the uterine, cervical and vaginal vascular beds (Morris & Gibbins, 1987; Morris et al. 1998). Thus, the lumbar sympathetic pathway to vasodilator neurones in anterior pelvic ganglia is likely to represent the major outflow from the central nervous system that increases blood flow in the vagina, cervix and uterus during copulation. This sympathetic vasodilator pathway also would enhance secretion from the cervical epithelium produced predominantly by stimulation of the hypogastric rather than the pelvic nerve (Hammarstrm, 1989).
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Prolonged firing of pelvic neurones contributes to long-lasting vasodilatation
Volleys of action potentials in pelvic vasodilator neurones innervating the uterine artery can release both nitric oxide and neuropeptides if delivered at frequencies of 5 Hz and above, producing prolonged dilatation of the pelvic arteries (Morris, 1993). However, the current study has demonstrated clearly that vasodilatation can be further enhanced and prolonged by post-stimulus potentiation of transmission within the pelvic ganglia. Many pelvic neurones continued to fire at 5–10 Hz after cessation of a train of stimuli at 10 Hz. Furthermore, some neurones fired at up to 20–25 Hz after a train of stimuli at 10 Hz, and then continued firing at rates of 5–10 Hz for many minutes in the absence of synaptic input. This prolonged firing is associated with slow depolarization of the neurones, and was not seen until after the first train of stimuli was applied to a preparation. Despite vasodilator neurones having a more depolarized resting membrane potential compared with non-vasodilator neurones in paracervical ganglia (Jobling et al. 2003), non-vasodilator neurones (without VIP-IR) as well as vasodilator neurones (VIP-IR) showed post-stimulus action potential discharge in the present in vitro study. Both vasodilator and uterine motor neurones are activated during sexual activity, although it is not clear if the two populations of pelvic neurones are always co-activated in vivo.
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Mediators of fast ganglionic transmission
As expected, the fast transmitter mediating the strong synaptic inputs from preganglionic axons to pelvic vasodilator neurones is acetylcholine acting on nicotinic receptors (Smith & Cunnane, 1999; Bobryshev & Skok, 2002). Relatively high doses of hexamethonium were required to abolish the fast EPSP, consistent with the very high safety factor for transmission at these synapses (Jobling et al. 2003, 2004). However, it is also possible that nicotinic receptors in anterior pelvic ganglia have a low sensitivity to hexamethonium, similar to rat cardiac ganglia (Selyanko & Skok, 1992; Skok, 2002). In addition to our observations in single vasodilator neurones, hexamethonium produced a significant delay in onset of vasodilatation. Thus, action potential firing of vasodilator neurones after blockade of nicotinic receptors does not appear to be associated with another fast ganglionic transmitter.
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Glutamate has been reported to act as a fast neurotransmitter in enteric ganglia (Liu et al. 1997; see, however, Ren et al. 2000). Furthermore, we recently demonstrated vesicular glutamate transporter-1 in preganglionic inputs to pelvic vasodilator neurones (Morris et al. 2005), and ionotropic glutamate receptors were reported to be present on some pelvic neurones (Chambille & Rampin, 2002). However, we found no functional evidence for involvement of the excitatory amino acid transmitter, glutamate, in fast excitatory transmission to vasodilator neurones in pelvic ganglia. Moreover, receptor antagonists such as CNQX or kynurenic acid enhanced rather than reduced uterine artery vasodilatation. This facilitatory effect potentially could be due to blockade of presynaptic glutamate receptors (Carpenedo et al. 2001). However, the depolarization and action potential firing produced in some pelvic neurones by CNQX alone indicates that the compound is likely to be having a direct agonist action, perhaps on a variant form of kainate receptors, as has been reported in some regions of the central nervous system (McBain et al. 1992; Brickley et al. 2001; Maccaferri & Dingledine, 2002; Hashimoto et al. 2004). Although CNQX is likely to be acting primarily as an agonist on pelvic neurones, its potent actions demonstrate that other agonists, including endogenous agents, that increase pelvic neurone excitability also are likely to enhance pelvic vasodilatation.
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Mediators of slow EPSPs and potentiation of vasodilatation
The post-stimulus potentiation of ganglionic transmission is closely linked with the presence of slow depolarizations produced by trains of stimuli. Slow EPSPs produced by non-nicotinic ganglionic transmission are widespread in autonomic ganglia (Jan et al. 1979; Jnig et al. 1982, 1983; Blumberg & Jnig, 1983; Konishi et al. 1983; Dun et al. 1984; Wang & Ma, 1990; Alkadhi et al. 1996; Jobling & Horn, 1996; Niel et al. 1996; Zhao et al. 1996; Ivanoff & Smith, 1997; Thorne & Horn, 1997; Karila & Horn, 2000; Alex et al. 2002; Johnson & Bornstein, 2004; Monro et al. 2004). In many autonomic ganglia, acetylcholine acting on postsynaptic muscarinic receptors produces a slow EPSP and increases the excitability of postganglionic neurones by inhibiting M-current (Brown & Selyanko, 1985). However, in the current study the muscarinic receptor antagonist, hyoscine, did not affect slow EPSPs and neurone firing, or vasodilatation, produced by trains of stimuli. ATP acting on P2X or P2Y receptors mediates fast and slow synaptic events in enteric ganglia (Monro et al. 2004), and purinoceptors are present on guinea-pig pelvic neurones in culture (Zhong et al. 2001). Nevertheless, the non-selective purinoceptor antagonist, suramin, did not affect vasodilatations of the uterine artery remaining after cholinergic receptor blockade.
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Amines such as 5-hydroxytryptamine (5-HT) and noradrenaline also can potentiate neurotransmission in autonomic ganglia, particularly in prevertebral sympathetic ganglia where terminals of preganglionic or intestinofugal neurones appear to take up and store 5-HT (Dun et al. 1984; Wang & Ma, 1990). However, our histochemical studies have demonstrated that there are no varicose terminals with catecholamine or 5-HT fluorescence in paracervical ganglia, even after incubation with amine precursors (Morris & Gibbins, 1987). Furthermore, long-lasting vasodilatations of the uterine artery were unaffected when pelvic ganglia were exposed to guanethidine and phenylephrine. Thus, we have no evidence for endogenous amines modulating synaptic transmission in anterior pelvic ganglia.
, 百拇医药
A strong candidate for mediator of the slow depolarization is substance P. Substance P is located in preganglionic neurones projecting selectively to VIP immunoreactive vasodilator neurones in guinea-pig paracervical ganglia (Morris & Gibbins, 1987). Furthermore, immunoreactivity for NK1 receptors has been demonstrated on a subpopulation (12%) of VIP immunoreactive neurones in paracervical ganglia (Messenger et al. 1999). In the current study we found that about half of the paracervical neurones were depolarized and fired volleys of action potentials when exposed to exogenous substance P. This discrepancy in the proportion of neurones responding to substance P compared with that with NK1 receptor immunoreactivity may be due to the presence of other receptor subtypes, such as truncated NK1 receptors that do not show immunoreactivity to C-terminally directed antisera (Baker et al. 2003), or NK3 receptors (Mawe, 1995; Jobling et al. 2001; Johnson & Bornstein, 2004). Indeed, using the NK3 agonist, senktide, we have confirmed the presence of functional NK3 receptors on a subpopulation of paracervical neurones. Nevertheless, a combination of NK1, NK2 and NK3 receptor antagonists had only a marginal effect in reducing vasodilatations produced by stimulating the hypogastric nerve after blockade of ganglionic cholinergic receptors. Thus, release of substance P from nerve terminals in the anterior pelvic ganglia seems to contribute slightly to the prolonged vasodilatation of the uterine artery. As these dilatations were produced by stimulation of the L3 ventral roots but not dorsal roots, and were not affected by capsaicin, any contribution by substance P is most likely from the substance P-immunoreactive preganglionic nerve terminals rather than the rare peptide-containing sensory axons in the ganglia (Morris & Gibbins, 1987; Anderson et al. 1997). However, the major contributor to non-cholinergic ganglionic transmission in the pelvic vasodilator pathways is unlikely to be substance P, and remains to be identified.
, 百拇医药
Conclusions
This study has shown that, in females, the vasodilator pathway from the lumbar spinal cord to pelvic arteries predominates over the sacral vasodilator pathway. Release of non-cholinergic neurotransmitters, both at the level of the anterior pelvic ganglia and at the neurovascular junction, act in concert to prolong the period of vasodilatation after volleys of stimuli applied to preganglionic neurones. This modulation of ganglionic transmission occurs despite the presence of only one or two strong synaptic inputs onto each vasodilator neurone. This differs from the phenomena of long-term potentiation or synaptic gain described in other autonomic ganglia with multiple subthreshold synaptic inputs (Booth & de Groat, 1979; Gola & Niel, 1993; Karila & Horn, 2000; Wheeler et al. 2004), but is similar to previous reports of non-cholinergic transmission to vasoconstrictor neurones in cat sympathetic ganglia (Jnig et al. 1982, 1983). The prolonged firing appears to be produced by largely unknown, slow-acting substances released from preganglionic neurones. Thus, the vasodilator pathway from the lumbar spinal cord to the pelvic arteries in females is well designed to maintain a high level of firing of postganglionic neurones and produce maximal vasodilatation for sustained periods. This long-lasting excitation of pelvic neurones might be even further enhanced by prolonged firing of the preganglionic neurones, as has been reported for preganglionic neurones in splanchnic nerves of cats after reflex activation from sacral levels (Bahr et al. 1986). All of these mechanisms are likely to contribute to the increased blood flow to the genitalia and internal reproductive organs during sexual activity, when ongoing inhibitory influences from supraspinal levels are replaced by excitation both from supraspinal levels and from spinal reflexes initiated by sacral sensory neurones (de Groat & Booth, 1993b; McKenna & Marson, 1997).
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