Sacro-lumbar intersegmental spinal reflex in autonomic pathways mediating female sexual function.

INTRODUCTION: Autonomic neurons in paracervical ganglia mediating vasodilation in the female reproductive tract receive inputs from both midlumbar and sacral spinal levels. However, it is not known how the lumbar pathways are activated. AIM: This study tested whether stimulation of pudendal sensory nerve could activate lumbar spinal outflows to paracervical ganglia via a spinal reflex pathway. METHODS: Isolated spinal cords with attached peripheral nerves were removed from urethane-anesthetized female guinea pigs and perfused via the aorta with physiological salt solution. Spinal pathways to midlumbar preganglionic neurons were tested by recording extracellular compound action potentials (CAPs) in lumbar splanchnic or distal hypogastric nerves after electrical stimulation of thoracic spinal cord or the pudendal nerve. CAPs also were recorded from pelvic nerves after pudendal nerve stimulation. Sensory neurons were retrogradely traced from the pudendal nerve and characterized immunohistochemically. MAIN OUTCOME MEASURES: Activation of preganglionic neurons projecting from midlumbar spinal cord to paracervical ganglia following stimulation of pudendal sensory nerves in isolated preparations. RESULTS: Thoracic spinal cord stimulation produced CAPs in hypogastric nerves that were abolished by transection of L3 lumbar splanchnic nerves. Pudendal nerve stimulation produced CAPs in L3 lumbar splanchnic, hypogastric, and pelvic nerves, demonstrating an ascending intersegmental spinal circuit to midlumbar levels in addition to the sacral spinal circuit. These CAPs in hypogastric nerves were enhanced by bicuculline (10 µM), blocked by tetrodotoxin (1 µM) but were not affected by hexamethonium (200 µM). Retrograde axonal tracing revealed four groups of sensory neurons in S3 dorsal root ganglia that were distinguished immunohistochemically. CONCLUSION: Midlumbar preganglionic neurons projecting to paracervical ganglia regulating blood flow and motility in the female reproductive tract can be activated by an ascending intersegmental spinal pathway from pudendal sacral inputs, which is inhibited by local spinal circuits. This pathway will help understand pathological conditions affecting reproductive function.

Most peptide-containing sensory neurons lack proteins for exocytotic release and vesicular transport of glutamate.

We used multiple-labeling immunohistochemistry and confocal microscopy to examine co-expression of immunoreactivity for vesicular glutamate transporters (VGluTs), synaptic vesicle proteins, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in peptide-containing sensory neurons of guinea pigs, mice, and toads. Axon terminals in the superficial layers of the dorsal horn of the spinal cord with immunoreactivity (IR) for both substance P (SP) and calcitonin gene-related peptide (CGRP) lacked IR for synaptosome-associated protein of 25 kDa (SNAP-25), syntaxin, synaptotagmin, synaptophysin, and synapsin, although adjacent varicosities without neuropeptides had IR for these synaptic proteins. Similarly, peptide-containing axon terminals in the superficial dorsal horn lacked IR for VGluT1 and VGluT2, despite the presence of VGluT2-IR in nearby nonpeptide varicosities. VGluT3-IR was sparse in the dorsal horn of the mouse spinal cord and was not present in peptide-containing axons. Most peripheral terminals of sensory neurons with both SP-IR and CGRP-IR in the skin, viscera, and autonomic ganglia of guinea pigs and mice also lacked IR for synaptic vesicle proteins, SNARE proteins, VGluT1, and VGluT2. In dorsal root ganglia from guinea pigs and mice, most small neurons with IR for both SP and CGRP lacked IR for SNAP-25, VGluT1, and VGluT2. Thus, proteins considered essential for vesicular uptake and exocytotic release of glutamate are not expressed at detectable levels by most sensory neurons containing SP and CGRP in rodents and toads. These data raise the possibility that most peptide-containing sensory neurons may not normally release glutamate as a transmitter.

Functional organization of vasodilator neurons in pelvic ganglia of female guinea pigs: comparison with uterine motor neurons.

Neurons producing vasodilation during reproductive activity constitute a large population of neurons in pelvic autonomic ganglia. We used intracellular recording, dye-filling and multiple-labeling immunohistochemistry to determine the morphology and electrophysiological properties of, and number of synaptic inputs to, vasodilator pelvic neurons in female guinea pigs. Vasodilator neurons, identified by their immunoreactivity for vasoactive intestinal peptide (VIP) and their location in paracervical ganglia, had simple dendritic arbors (1 primary dendrite) compared with nonvasodilator neurons (3 dendrites). Vasodilator neurons had more depolarized resting membrane potentials (-47 mV) than other paracervical neurons (-55 mV) and had smaller apparent cell capacitances (65 pF vs. 110 pF). Vasodilator and nonvasodilator neurons could not be distinguished on the basis of their action potential discharge characteristics or current voltage relationships. Most pelvic neurons ( approximately 70%) had tonic (slowly adapting) discharges. Fifty-five percent of vasodilator and 60% of nonvasodilator neurons showed inward rectification when hyperpolarized below -90 mV. Around 65% of neurons showed evidence of M-current. Both vasodilator and nonvasodilator neurons ( approximately 80%) expressed an A-like current. Vasodilator neurons and nonvasodilator neurons received 1-2 fast synaptic inputs following stimulation of pelvic or hypogastric nerve trunks. Most neurons received a least one strong synaptic input. These results indicate that vasodilator neurons and neighboring neurons projecting to other pelvic targets, primarily in the myometrium, express a similar range of ionic conductances and integrate few synaptic inputs. The similarities between these two populations of neurons may be related to their coactivation as part of spinal somato-pelvic reflexes. Vasodilation and uterine contraction during reproductive behavior in female guinea pigs are likely to involve input from preganglionic neurons at both lumbar and sacral spinal levels.

Synaptic density, convergence, and dendritic complexity of prevertebral sympathetic neurons.

Prevertebral sympathetic ganglia contain a unique population of final motor neurons receiving convergent synaptic inputs not only from spinal preganglionic neurons, but also from peripheral intestinofugal neurons projecting from the gut. We used quantitative confocal and ultrastructural immunohistochemistry to determine how this increased synaptic convergence is accommodated by sympathetic final motor neurons in the celiac ganglion of guinea pigs. Terminals of intestinofugal neurons were identified by their immunoreactivity to vasoactive intestinal peptide. Stereologic analyses were based on transects and point counts at confocal and ultrastructural levels. The relative amount of dendritic neuropil in the medial regions of the ganglion was approximately 2.5 times greater than in the lateral regions of the ganglion, consistent with the 2 to 3 times difference in average dendritic field size of neurons in these regions. The total numbers of boutons and synaptic profiles showed significant positive correlations with the relative amount of neuropil in a region. However, the overall density of synaptic boutons was twice as high in the medial region of the ganglion compared with the lateral regions. Because the relative density of preganglionic synapses was similar in each region, this difference was due to the selective projection of intestinofugal inputs to neurons in the medial celiac ganglion, where they provided 45% of synaptic contacts. These results show that, compared with vasoconstrictor neurons, sympathetic neurons regulating gastrointestinal activity support a higher number of convergent inputs in two ways: in addition to having larger dendritic fields, they also have a twofold higher density of synapses.

Heterogeneous expression of SNAP-25 and synaptic vesicle proteins by central and peripheral inputs to sympathetic neurons.

Neurons in prevertebral sympathetic ganglia receive convergent synaptic inputs from peripheral enteric neurons in addition to inputs from spinal preganglionic neurons. Although all inputs are functionally cholinergic, inputs from these two sources have distinctive neurochemical and functional profiles. We used multiple-labeling immunofluorescence, quantitative confocal microscopy, ultrastructural immunocytochemistry, and intracellular electrophysiologic recordings to examine whether populations of inputs to the guinea pig coeliac ganglion express different levels of synaptic proteins that could influence synaptic strength. Boutons of enteric intestinofugal inputs, identified by immunoreactivity to vasoactive intestinal peptide, showed considerable heterogeneity in their immunoreactivity to synaptosome-associated protein of 25 kDa (SNAP-25), synapsin, synaptophysin, choline acetyltransferase, and vesicular acetylcholine transporter. Mean levels of immunoreactivity to these proteins were significantly lower in terminals of intestinofugal inputs compared with terminals of spinal preganglionic inputs. Nevertheless, many boutons with undetectable levels of SNAP-25 immunoreactivity formed morphologically normal synapses with target neurons. Treatment with botulinum neurotoxin type A (20-50 nM for 2 hours in vitro) generated significant cleavage of SNAP-25 and produced similar dose- and time-dependent inhibitions of synaptic transmission from all classes of inputs, regardless of their mean level of SNAP-25 expression. The simplest interpretation of these results is that only synaptic boutons with detectable levels of SNAP-25 immunoreactivity contribute significantly to fast cholinergic transmission. Consequently, the low synaptic strength of intestinofugal inputs to final motor neurons in sympathetic pathways may be due in part to the low proportion of their boutons that express SNAP-25 and other synaptic proteins.

Differential involvement of N-type calcium channels in transmitter release from vasoconstrictor and vasodilator neurons.

1. The effects of calcium channel blockers on co-transmission from different populations of autonomic vasomotor neurons were studied on isolated segments of uterine artery and vena cava from guinea-pigs. 2. Sympathetic, noradrenergic contractions of the uterine artery (produced by 200 pulses at 1 or 10 Hz; 600 pulses at 20 Hz) were abolished by the N-type calcium channel blocker omega-conotoxin (CTX) GVIA at 1-10 nm. 3. Biphasic sympathetic contractions of the vena cava (600 pulses at 20 Hz) mediated by noradrenaline and neuropeptide Y were abolished by 10 nm CTX GVIA. 4. Neurogenic relaxations of the uterine artery (200 pulses at 10 Hz) mediated by neuronal nitric oxide and neuropeptides were reduced <50% by CTX GVIA 10-100 nm. 5. Capsaicin (3 microm) did not affect the CTX GVIA-sensitive or CTX GVIA-resistant neurogenic relaxations of the uterine artery. 6. The novel N-type blocker CTX CVID (100-300 nm), P/Q-type blockers agatoxin IVA (10-100 nm) or CTX CVIB (100 nm), the L-type blocker nifedipine (10 microm) or the 'R-type' blocker SNX-482 (100 nm), all failed to reduce CTX GVIA-resistant relaxations. The T-type channel blocker NiCl(2) (100-300 microm) reduced but did not abolish the remaining neurogenic dilations. 7. Release of different neurotransmitters from the same autonomic vasomotor axon depends on similar subtypes of calcium channels. N-type channels are responsible for transmitter release from vasoconstrictor neurons innervating a muscular artery and capacitance vein, but only partly mediate release of nitric oxide and neuropeptides from pelvic vasodilator neurons.

Cloning of a C-terminally truncated NK-1 receptor from guinea-pig nervous system.

In order to examine the possibility that some actions of substance P may be mediated by a variant of the neurokinin-1 (NK-1) receptor, we isolated and sequenced the cDNA encoding a truncated NK-1 receptor from guinea-pig celiac ganglion and brain mRNA by two-step RT-PCR based on the 3'RACE method. The truncated NK-1 receptor sequence corresponded to a splice variant missing the final exon 5, and encoded a 311-amino acid protein that was truncated just after transmembrane domain 7, in an identical position to a truncated variant of the human NK-1 receptor. Thus, the truncated NK-1 receptor lacked the intracellular C-terminus sequence required for the phosphorylation and internalisation of the full-length NK-1 receptor. Using a sensitive one-step semi-quantitative RT-PCR assay, we detected mRNA for both the full length and truncated NK-1 receptors throughout the brain, spinal cord, sensory and autonomic ganglia, and viscera. Truncated NK-1 receptor mRNA was present in lower quantities than mRNA for the full-length NK-1R in all tissues. Highest levels of mRNA for the truncated NK-1 receptor were detected in coeliac ganglion, spinal cord, basal ganglia and hypothalamus. An antiserum to the N-terminus of the NK-1 receptor labelled dendrites of coeliac ganglion neurons that were not labelled with antisera to the C-terminus of the full length NK-1 receptor. These results show that a C-terminally truncated variant of the NK-1 receptor is likely to be widespread in central and peripheral nervous tissue. We predict that this receptor will mediate actions of substance P on neurons where immunohistochemical evidence for a full-length NK-1 receptor is lacking.

Interleukin-1 receptor immunoreactivity in sympathetic vascular and non-vascular neurons in guinea-pig coeliac ganglion.

Immunoreactivity (IR) for the interleukin-1 receptor type I (IL1RI) was examined in sympathetic neurons in guinea-pig coeliac ganglion using multiple-labelling immunofluorescence. IL1RI-IR was present in 8% of sympathetic neurons in untreated preparations. The proportion of neurons with IL1RI-IR increased significantly after incubation in interleukin-6 (200 ng/ml) for 2-4 h (16-26% neurons), or after incubation for 4 h without cytokine (16%), with interleukin-1beta (IL1beta, 200 ng/ml; 18%) or tumour necrosis factor-alpha (200 ng/ml; 16%). This increase occurred predominantly in neuropeptide Y-IR, vasoconstrictor neurons. IL1RI-IR also was present in varicose axons, some of which projected from the gut, and in vascular smooth muscle cells and endothelium. These potential binding sites for the proinflammatory cytokine, IL1beta, on vasoconstrictor neurons and blood vessels may modulate sympathetic regulation of intestinal blood flow in inflammatory conditions.

Structure of peripheral synapses: autonomic ganglia.

Final motor neurons in sympathetic and parasympathetic ganglia receive synaptic inputs from preganglionic neurons. Quantitative ultrastructural analyses have shown that the spatial distribution of these synapses is mostly sparse and random. Typically, only about 1%-2% of the neuronal surface is covered with synapses, with the rest of the neuronal surface being closely enclosed by Schwann cell processes. The number of synaptic inputs is correlated with the dendritic complexity of the target neuron, and the total number of synaptic contacts is related to the surface area of the post-synaptic neuron. Overall, most neurons receive fewer than 150 synaptic contacts, with individual preganglionic inputs providing between 10 and 50 synaptic contacts. This variation is probably one determinant of synaptic strength in autonomic ganglia. Many neurons in prevertebral sympathetic ganglia receive additional convergent synaptic inputs from intestinofugal neurons located in the enteric plexuses. The neurons support these additional inputs via larger dendritic arborisations together with a higher overall synaptic density. There is considerable neurochemical heterogeneity in presynaptic boutons. Some synapses apparently lack most of the proteins normally required for fast transmitter release and probably do not take part in conventional ganglionic transmission. Furthermore, most preganglionic boutons in the ganglionic neuropil do not form direct synaptic contacts with any neurons. Nevertheless, these boutons may well contribute to slow transmission processes that need not require conventional synaptic structures.

Post-stimulus potentiation of transmission in pelvic ganglia enhances sympathetic dilatation of guinea-pig uterine artery in vitro.

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 microM capsaicin applied to the artery and ganglia, or ganglionic application of 1 microM hyoscine, 30-100 microM suramin or 10 microM CNQX. Dilatations were reduced slightly by ganglionic application of NK1 and NK3 receptor antagonists (SR140333, SR142801; 1 microM), 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 microM) 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.

Functional organization of peripheral vasomotor pathways.

AIM: In this article, we review the functional organization of the peripheral autonomic pathways regulating the vasculature. RESULTS: The final motor neurones in vasomotor pathways tend to be smaller than neurones in other autonomic pathways. This suggests that they have relatively smaller target territories and receive fewer pre-ganglionic inputs than non-vasomotor neurones. Nevertheless, single vasomotor neurones project to large areas of the vasculature separated by up to 7 mm. Different functional pools of vasomotor neurones project to specific segments of the vasculature, allowing for the selective neural control of resistance in vessels in proximal or distal regions of the vascular bed. In many cases, each functional pool of vasomotor neurones utilizes a characteristic combination of cotransmitters. The various pools of final motor neurones in vasomotor pathways receive convergent synaptic input from different pools of pre-ganglionic neurones, many of which also contain neuropeptides which enhance the excitability of the final motor neurones. The excitability of vasomotor neurones regulating gastrointestinal and mesenteric blood flow, also can be increased by the actions of peptides such as substance P that are released from visceral nociceptors. CONCLUSIONS: We propose that autonomic pathways regulating the vasculature are organized into 'vasomotor units'. Each vasomotor unit consists of a pre-ganglionic neurone, the final motor neurones it innervates, and the blood vessels that they regulate. The vasomotor units are likely to be grouped into functional pools that can be recruited as necessary to provide highly specific, graded control of blood flow both within and between vascular beds.

Botulinum neurotoxin A attenuates release of norepinephrine but not NPY from vasoconstrictor neurons.

We examined effects of botulinum neurotoxin A (BoNTA) on sympathetic constrictions of the vena cava and uterine artery from guinea pigs to test the role of soluble NSF attachment protein receptor (SNARE) proteins in release of the cotransmitters norepinephrine (NE) and neuropeptide Y (NPY). Protein extracts of venae cavae and uterine arteries showed partial cleavage of synaptosomal associated protein of 25 kDa (SNAP-25) after treatment in vitro with BoNTA (50-100 nM). The rising phase of isometric contractions of isolated venae cavae to field stimulation at 20 Hz, mediated by NE acting on alpha-adrenoceptors, was reduced significantly by 100 nM BoNTA. However, sustained sympathetic contractions mediated by NPY were not affected by BoNTA. In uterine arteries, noradrenergic contractions to 1-Hz stimulation were almost abolished by BoNTA, and contractions at 10 Hz were reduced by 50-60%. We conclude that SNARE proteins are involved in exocytosis of NE from synaptic vesicles at low frequencies of stimulation but may not be essential for exocytosis of NPY and NE from large vesicles at high stimulation frequencies.