Brain
Reseurch
Rdkrin.
Vol. 25. pp. X5-371.
t Pergamon Press pk. 1990. Printed m the U.S.,4
Vagus Nerve Afferent and Efferent Innervation of the Rat Uterus: An Electrophysiological and HRP Study’ MARISELA ORTEGA-VILLALOBOS,” MAYDA GARCiA-BAZAN,t LUIS PASTOR SOLANO-FLORES,? JEStiS GUILLERMO NINOMIYA-ALARCON.: ROSALINDA GUEVARA-GUZMAN:’ “Departamento de Ciencius Bioldgicas. Fucultud de Estudios Superiores Cuuutitla’n, Estudo de M&co, MPxico und fDepurtumento de Fisiologiu, Fucultud de Medicinu, U.N.A.M. Apurtudo Postal No. 70-250, 03510 M&co, D.F. 1 Mkxico AND MATTHEW J. WAYNER Division oj’ Life Sciences,
The Universig
oj’ Texas at Sun Antonio,
Received
8 June
Sun Antonio,
TX 78285
1990
ORTEGA-VILLALOBOS. M.. M. GARCIA-BAZAN. L. P. SOLANO-FLORES. J. G. NINOMIYA-ALAR&N. R. GUEVARAGUZMAN AND M. J. WAYNER. Vqu nerve U@YW nnd efferenr innrrvnrion of the rut uterus: An e/rcrroph?~io/oKic,rr/ trntl HRP study. BRAIN RES BULL 25(3) 365-371, 1990.-To determine a possible b&stem connection with the uterus, a study with electrophysiological techniques and horseradish peroxidase (HRP) tracing was performed in the rat. Neurons of the nucleus of the tractus solitarius decreased in discharge frequency during cervicovaginal distension. HRP injections into the uterine walls resulted in the appearance of labelled cells in the nodose ganglion and in the dorsal motor nucleus of the vagus nerve. The rewlts demonstrate a direct bidirectional vagal complex-uterus connection via the vagus nerve. Results are discussed in terms of a complex uterus control system in which the paraventricular nucleus might play an integrative role. Nodose ganglion Vagus nerve Vagina Cervix Uterus Rat Electrophysiology
Dorsal motor nucleus Autonomic system
THE brainstem vagal complex is involved in the integration of respiratory, cardiovascular, and gastrointestinal functions. The nucleus of the tractus solitarius (NTS) seems to be the most important sensory relay structure in this complex. Also, the dorsal motor nucleus of the vagus nerve (DMNX) seems to be the main source of efferent fibers to these related structures (17. 18. 21. 31). With reference to the reproductive organs, the results of several important studies have appeared. Uterine afferent fibers arising from the T,,, L, through S , dorsal root ganglia that travel through the hypogastric and pelvic nerves have been described in the rat by mean\ of anterograde (23) and retrograde (5) HRP tracing. These nerves also supply sympathetic and parasympathetic efferents to the uterine cervical ganglia (4,16). In addition.
Paraventricular Nucleus of the tractus solitarius Reproduction control Parasympathetic system
nucleus HRP
uterine horn and vaginal distension modulates the activity of tonically discharging neurons of the paraventricular nucleus of the hypothalamus (PVN) (2). These PVN neurons project to the spinal cord (30,34). The involvement of the vagus nerve in the rat reproductive process has been studied extensively. Abdominal vagotomy during pregnancy results in a decreased number of live fetuses (20). delay in the onset of puberty (25). and disruption of the estrous cycle (7), probably as a consequence of depressed ovarian function and impairment of luteinizing hormone release (3). An HRP study demonstrated that the rat ovary is innervated by the vagus nerve (8). Even though it has been reported that abdominal vagotomy also reduces uterine weight. probably due to a decreased ovarian function (25). these studies on the role of the
‘This research was supported in part by PADEP: MED-901 and CONACyT Posgrado *Requests for reprints should be addressed to Dr. Rosalinda Guevara-Guzmin.
#33
ORTEGA-VILLALOBOS E7‘ Al..
366
1. Representative peristimulus histogram showing the effect of cervicovaginal distension upon the discharge frequency of a neuron of the nucleus of the tractus solitarius (NTS). Bin= 870 msec. The CD horizontal line indicates the distension period. Arrows signal the onset and the end of the distension. Black dots in the upper histological drawing indicate the sites where the tips of the recording micropipetteswere FIG.
located.
vagus nerve in reproduction processes have emphasized the control of ovarian function. It is not known, however, if the sensory information from the uterus is integrated in the vagus brainstem center alone or if some other higher structures might be involved. HRP as a retrograde tracer to establish autonomic pathways has been successfully used for some years (11). Also, distension of the hollow viscera has been shown to be useful in activating afferent fibers for electrophysiological studies (2,12). Therefore, the present study was carried out to identify and describe the brainstem connections with the uterus using electrophysiological and HRP tracing techniques in the rat. The results demonstrate the existence of a brainstem and uterus circuit which involves the vagus nerve. METHOD
Electrophysiological experiments were carried out in 11 naive female Wistar rats weighing 180-310 g. Only animals showing estrous vaginal smears were used (1). The rats were anesthetized with chloral hydrate (400 mg/kg, IP), the head was immobilized in a stereotaxic frame, and the necessary surgical procedures were performed. Exposed skin and tissue surrounding pressure points were infused with a xylocaine solution. Body temperature was continuously monitored and maintained at 37°C by means of an electric heating pad. Cervicovaginal distension was carried out using a pediatric urinary latex catheter that was inserted through the vagina until it reached the cervix. From 0.4 to 0.6 ml of room temperature tap water was infused into the balloon at the end of the catheter for a 15set period. Once the balloon was filled, it remained distended at least for one minute, afterwards, the balloon was allowed to empty over a 3-set period. Using conventional electrophysiological methods, the extracellular activity of NTS units was recorded with glass micropipettes filled with 2 M NaCl solution saturated with fast green. NTS stereotaxic coordinates were P = 11.4-l 1.7 from bregma in the horizontal skull; L = 1.5 and H = 4.5-7.1 from brain surface (26). The signals were stored on magnetic tape for further processing. The data were fed to a window discriminator which sorted the neuronal spikes and generated square pulses. A PC computer was used to perform the
analysis and to construct peristimulus histograms. Changes in the discharge frequency of the units, associated with cervicovaginal distension, were considered significant only when they were verified at least three times and when they were greater than 50% of the baseline activity. At the end of the experiment, the animal was deeply anesthetized and perfused with 10% formalin. The brain was removed, sectioned, and examined with a microscope to verify the location of the tip of the micropipette marked by a deposit of fast green. HRP experiments were performed on 20 female Wistar rats weighing 190-220 g which were ovariectomized or ligated one week prior to HRP administration. Rats were anesthetized with chloral hydrate (400 mg/kg, IP) and the uterus exposed through a midventral incision. Using a microsyringe fitted with a 26- or 30-gauge needle, 10 ~1 of a saline solution of HRP (10% Sigma lectin conjugate) was injected into the walls of the cervix and/or in the body of the uterus. Care was taken to avoid leakage of HRP from the injection site, therefore, the syringe needle was inserted into the midlayers and parallel to the uterine wall from 5 to 10 millimeters and then the HRP was delivered. After a survival time of 96 hours, the rats were anesthetized with chloral hydrate and perfused intracardially with 0.5 ml heparin solution, then with 250 ml 0.9% saline solution followed by 500 ml 1.25% glutaraldehyde and 1.0% paraformaldehyde in phosphate buffer, pH 7.4, and finally with 500 ml of 10% sucrose in phosphate buffer, pH 7.4. The brains and the nodose ganglion were removed and stored 24 hours in 30% sucrose buffer, pH 7.4 at 4”C, they were then frozen and sliced in 50 p.m thick sections. The sections were processed according to Mesulam’s technique (22) using 3, 3’, 5, 5’ tetramethyl benzidine as chromogen. The tissue was counterstained with neutral red and analyzed using both dark field and bright field illumination. HRP-labelled cells in the DMNX were localized according to Dennison et al. (9). RESULTS
Eleven spontaneously active units were examined in the NTS, one from each animal. All of the units responded to cervicovaginal distension by decreasing in discharge rate. In general, responsive units had a spontaneous discharge frequency of not more than 20
UTERUS VAGAL INNERVATION
spikes per second. The effects of distension were almost immediate and units began to decrease in frequency as can be seen in Fig. 1. In some neurons, the discharge completely ceased. In all cells a clear effect was established within 40 seconds; then, the discharge frequencies increased to baseline levels and continued at those levels even though the cervicovaginal distension was still present. When the cervicovaginal distension was terminated, an off-response was not observed. HRP injected into the wall of the uterine horns and/or the cervix resulted in the retrograde transport of HRP into some of the soma of the DMNX neurons (Part B, Fig. 2) and into several soma of the nodose ganglion of all of the studied rats (Part C, Fig. 2). The area enclosed by a square in Part C of Fig. 2 is enlarged and appears below as Part D. In general, since an insufficient number of labelled soma (Part C, Fig. 2) were observed in the nodose ganglion a topographic relationship could not be established between the location of labelled cells in the ganglion and the site of HRP injection in the uterus. In contrast, HRP administration into the wall of the rostra1 third of the uterine horns yielded labelled cells in the lateral areas of the caudal DMNX (Part I, Fig. 3). When HRP was injected exclusively in the cervix, the lateral parts of almost the full extent of the DMNX and of the nucleus commissuralis displayed labelled cells (Part III, Fig. 3). HRP injections involving the cervix, the caudal and the medial third of the uterine horns, yielded labelled cells throughout the whole extent of the DMNX (Parts II and IV. Fig. 3). DISCUSSION
The present study provides anatomical and electrophysiological evidence which support the existence of a brainstem-uterus circuit which involves the vagus nerve in the rat. Labelled soma were found in the nodose ganglion after uterine HRP injections. Additionally, changes in the discharge frequency of NTS units were evoked by mechanical cervicovaginal distension. On the other hand, HRP-labelled cells were observed in the DMNX after injection of the tracer into the uterus. Labelled cells have been found in the nodose ganglion after HRP injections into the ovary (8). The nodose ganglion HRPlabelled cells described in the present work must have been retrogradely labelled with the tracer captured exclusively by uterine nerve endings since the rats used were spayed or ligated one week before the HRP uterine administration. Intraperitoneal administration of HRP by dropping HRP solution underneath the omentum has resulted in labelled cells in the DMNX; thus, it has been suggested that, in studies of the autonomic supply of the abdominal viscera using retrograde transport of HRP, some HRP leakage can, possibly, penetrate directly into the autonomic neural elements located in the vicinity of the injection site (32). In the present work. to avoid any contamination of HRP to other abdominal structures, special care was taken to inject the HRP solution in the midlayers of the uterine wall by directing the syringe needle parallel to the wall for 5 to 10 millimeters prior to HRP delivery. Although the present HRP study was not designed specifically to search for a quantitative topographical representation of the vagal motor innervation of the uterus, the results indicate that the cervix and the rostra1 third of the uterine horns seem to be innervated preferentially by the lateral areas of the DMNX; the cervix, besides, is innervated by the nucleus commissuralis. Whereas the medial areas of the DMNX do not project to the cervix, they seem to project to the caudal thirds of the uterine horns. Probably, the lateral areas of the DMNX project also to the two caudal thirds of the uterine horns. The discharge frequency of NTS units decreased after cervicovaginal distension. This decrease endured for several set and then, the discharge frequency returned to a baseline level even though
367
the distension was maintained. Also, no off-responses were observed after the cervicovaginal distension was terminated. These facts suggest the existence of a sensory pathway from the reproductive tract to the NTS with stretch receptors localized in the vaginal and uterine walls. It seems that the processes associated with this pathway and/or the receptors themselves are quite adaptable to distension and respond only to the onset of the distension stimulus. Additionally, labelled cells were observed in the nodose ganglion after HRP administration into the uterus. Furthermore, it is well established that the terminal fields of the nodose ganglion sensory fibers are localized in the NTS (18). These findings. as a whole, support the existence of sensory fibers arising from the uterus, with soma localized in the nodose ganglion that project to the NTS. The possibility that other nuclei of the vagal complex might also receive uterine afferents (18). cannot be discarded. Afferent fibers from the T,,, L, ganglia innervate the cranial regions of the reproductive tract, which correspond also to the sympathetic innervated area of this tract. Additionally L,. S, dorsal root ganglia supply afferents to the caudal portion of the reproductive tract, which correspond to the parasympathetic innervated area (23). The hypogastric and the pelvic nerves supply those afferent fibers to the uterus (5,23). as well as sympathetic and parasympathetic efferents to the uterine cervical ganglia (4,16). It is interesting to point out that as long as the reproductive tract displays an autonomic circuitry composed of a dual spinal afferent innervation and of a sympathetic and parasympathetic spinal efferent system, the data presented in this report indicate the existence of a vagal afferent-efferent additional fiber supply to the whole extent of the uterus. Oxytocin might be the most important neural transmitter for the control of uterine function. The magnocellular division of the PVN synthesizes oxytocin and releases it from the posterior lobe of the pituitary gland (6). This hormone and the uterine spinal innervation seem to be necessary for the parturition function of the uterus, thus, distension of the birth canal during parturition is related to an increase of oxytocin levels (13) and with an increase of activity of PVN neurons (33). Additional evidence suggests that fibers in the hypogastric and pelvic nerves are PVN afferents from the uterus contributing, possibly, to the positive feedback on oxytocin release by uterine contractions, since uterine distension alters the firing activity of PVN units (2) and pelvic neurectomy disrupts oxytocin release during parturition (14) and abolishes the reflex of fetus expulsion (15). On the other hand, histological evidence suggests that oxytocinergic cells in the parvocellular division of the PVN project to autonomic cell groups in the spinal cord and/or to the dorsal vagal complex (30). Furthermore, neurons of the DMNX were shown to respond to oxytocin by increases in their firing rate (10). These facts indicate the existence of an additional oxytocinergic system that projects to the dorsal vagal complex, which, as supported from the data presented in this report. is related to the uterus through afferent and efferent vagal fibers. Complementing this circuitry, it has been shown that the NTS sends direct connections to the PVN (19,27), substantially, to its parvocellular division but not to the magnocellular division (29). With regard to the intrinsic connections of the PVN, it has been observed that magnocellular neurons have dendrites ramifying throughout the parvocellular area. Also. parvocellular neurons project axons to the magnocellular region; terminal boutons of these axons contact dendrites of magnocellular neurons (35). In this way, uterine sensory information conveyed via the vagus nerve and relayed in the NTS might influence the magnocellular oxytocin-secreting neurons by way of those magno-parvocellular contacts. Finally, it seems that, at least, two oxytocinergic efferent systems are acting upon the uterus: a magnocellular humoral system and a parvocellular neural system with the DMNX partic-
36X
ORTEGA-VILL.4LOBOS
E7‘ M
UTERUSVAGALINNERVATION
369
FACING PAGE AND ABOVE FIG. 2. HRP evidence of sensory and motor vagal innervation of the uterus. (A) Site of HRP injection in the uterine cervix. (B) Dark field photomicrograph of HRP-labelled cells of the dorsal motor nucleus of the vagus nerve (DMNX). The white circle delineates the central canal; magnification: 380 x (C) Bright field photomicrograph of a nodose ganglion section in which an HRP-labelled cell was found (square area). An amplification of the square area is shown below in panel (D). Magnification: 950 x
370
III
FIG. 3. Histological drawings of brainstem coronal sections encompassing the dorsal motor nucleus of the vagus nerve (DMNX). which depict the relationship between the HRP injection site in the uterus and the DMNX areas where labefled soma were found (black areas). The V-shaped drawings, lateral to each histological set, represent the uterus. (I) HRP injection into the rostra1 third of the uterine horns. (II) HRP injection in the cervix and caudal third of the uterine horns. (III) HRP injection in the cervix. (IV) HRP injection in the cervix and all of the uterine horns.
ipating, perhaps, as a relay station. In conclusion, both the spinal and the vagal uterine innervating systems are reciprocally integrated by the PVN. Dendrites of the motoneurons of the DMNX which innervate the stomach penetrate areas of the overlying NTS, which receive gastric vagal afferents (28.31). Additionaily, vagai sensory terminal fields have been visualized in the DMNX (1824). Similar vagovagal contacts related to uterine activity might well exist and remain to be determined. During the life span of a female animal, different complex functions as copula, egg maintenance, pregnancy, and parturition, are accomplished by the reproductive tract.
The significance of the vagal-uterine connection in these functions remains to be investigated. It would be interesting to determine the effects of oxytocin microinjections in the vagal complex upon the activity of the uterus during different functional states.
The authors are grateful to Biol. Olga Alejandra Dona~i-Aib~~n and Mr. Carlos de 10s Santos Toledo for their excellent technical assistance. to Mrs. Josefina Bolado-Garza for her patience and care in typewriting, and to Ms. Ingrid Mascher for her editorial assistance.
REFERENCES 1. Aclham, N.; Schenk, E. A. Autonomic innervation of the rat vagina, cervix, and uterus and its cyclic variation. Am. J. Obstet. Gynecol. 104:508-516; 1969. 2. Akaishi, T.; Robbins, A.; Sakuma, Y.; Sato, Y. Neural inputs from the uterus to the paraventricular magnocellular neurons in the rat. Neurosci. Lett. 8457-62; 1988. 3. Allen, L. G.; Lawrence, I. E.; Burden, H. W.; Hodson, C. A. Effects of acne vago~my on serum LH concen~tions in female rats. J. Reprod. Fe& 7487-94; 1985. 4. Baljet, B.; Drukker, J. The extrinsic innervation of the pelvic organs in the female rat. Acta Anat. 107:241-267; 1980. 5. Berkeley, K. J.; Robbins, A.; Sato, Y. Afferent fibers supplying the uterus in the rat. J. Neurophysiol. 59:142-163; 1988. 6. Brownstein, M. J.; Russell, J. T.; Gainer, H. Synthesis, transport, and release of posterior pituitary hormones. Science 207:373-378: 1980. 7. Burden, H. W. ; Lawrence, I. E.; Louis, T. M.; Hodson, C. A. Effects of abdominal vagotomy on the estrous cycle of the rat and the induction of pseudopregnancy. Neuroendocrinology 33:2 18222; 1981.
8. Burden, H, W.; Leonard, M.; Smith, C. P.; Lawrence, I, E. The sensory innervation of the ovary: a horseradish peroxidase study in the rat. Anat. Rec. 207~623-627; 1983. 9. Dennison, S. J.; Merritt, J. E.; Aprison, M. H.; Felten, D. L. Redefinition of the location of the dorsal (motor) nucleus of the vagus in the rat. Brain Res. Bull. 6:77-81; 1981. 10. Dreifuss, J. J.; Raggenbass, M.; Charpak, S.; Du~is-Dauphin, M.; Tribollet, E. A role of central oxytocin in autono~c functions: Its action in the motor nucleus of the vagus nerve. Brain Res. Bull. 20~765-770; 1988. 11. Ellison, J. P.; Clark, G. M. Retrograde axonal transport of horseradish peroxidase in peripheral autonomic nerves. J. Comp. Neuml. 161:103-114; 1975. D. E.; Aguilar-Baturoni, H. U.; Guevara-Aguilar, R.; 12. Garcia-D&, Wayner, M. .I. Olfactory bulb neurons respond to gastric distension. Brain Res. Bull. 15661~ 1985. 13. Higuchi, T.; Honda, K.; Fukuoka, T.; Negoro, H.; Wakabayashi, K. Release of oxytocin during suckling and parturition in the rat. J. Endocrinol. 105:339-346; 1985. 14. Higuchi, T.; Uchide, K.; Honda, K.; Negoro, H. Oxytocin release
UTERUSVAGALINNERVATION
during parturition in the pelvic-neurectomized 109:149-154. 1986.
rat.
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15. Higuchi, T.; Uchide. K.: Honda. K.; Negoro. H. Pelvic neurectomy abolishes the fetus-expulsion reflex and induces dystocia in the rat. Exp. Neuml. 96:443455: 1987. 16. Hulsebosch, C. E.: Cogpeshall. R. E. An analysis of the axon populations in the nerves to the pelvic viscera in the rat. J. Camp. Neural. 21 l:l-IO: 1982. 17. Kalia. M.: Mesulam. M. M. Brain stem projections of sensory and motor components of the vagus complex in the cat: II. Laryngeal, trachrobronchial, pulmonary, cardiac. and gastrointestinal branches. J. Comp. Neural. 193:467-508; 1980. 18. Kalia. M.: Sullivan. J. M. Brainstem projectlons of sensory and motor components of the vagus nerve in the rat. J. Comp. Neurol. 21 I: 248-264; 19X2. 19. Kannan, H.; Yamashita, H. Connections of neurons in the region of the nucleus tractus solitarius with the hypothalamic paraventricular nucleus: Their possible involvement in neural control of the cardiovascular system in rats. Brain Res. 329:205-212; 1985. 20. Lawrence. I. E.; Burden, H. W.: Louis. T. M. Effect of abdominal vagotomy of the pregnant rat on LH and progesterone concentrations and fetal resorption. J. Reprod. Fertil. 53:13l-136; 1978. 21. Leslie, R. A.. Gwyn. D. G.: Hopkins, D. A. The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat. Brain Res. Bull. 8:37-43: 1982. 22. Mesulam. M Tetramethyl benaidine for horseradish peroxidase neurohisto-chemistry: a non-carcinogenic blue reaction product with superior sensltlwty for visualizmg neural afferents and efferenta. J. Hintochem. C‘ytochem. 26:106-l 17; 1978. 23. Nance. D. M : Bums. J.: Klein, C. M.: Burden. H. W. Afferent fibers in the reproductive system and pelvic viscera of female rats: Anterograde tracinp and immunocytochrmlcal studies. Brain Res. Bull. 21:701-709; 198X. 24. Neuhuber. W. L.: Sandoz. P. A. Vagal primary afferent terminals in the dorsal motor nucleus of the rat: are they making monosynaptic contacts on prcpanglionic efferent neurons? Neurosci. Lett. 69: 126-130: 19h6.
25. Ojeda. S. R.: White.
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M. Abdominal vagotomy delays the onset of puberty and inhibits ovarian function in the female rat. Neuroendocrinology 36:261-267: 1983. Paxino\. G.; Watson, C. The rat braln in \tereotaxic coordinates. Australia: Academic Press; 1986. Ricardo. J. A.; Koh, E. T. Anatomical evidence of du-ect projections from the nucleus of the solitary tract to the hypothalamus. amypdala. and other forebram structures in the rat. Bram Res. 153: l-26; 1978. Rinaman. L.; Card. J. P.; Schwaher, J. S.; Miselis, R. R. Ultrabtructural demonstration of a gastric monosynaptic lagal circuit in the nucleus of the solitary tract in rat. J. Neurow. Y:l985-lY96; lY89. Sawchenko. P. E.: Swanson. L. W. Central noradrenergic pathways for the integration of hypothalamic neuroendocrine and autonom-ic responses. Science 2 14:6X.5-687; 1981. Sawchenko. P. E.; Swanson, L. W. Immunohistochemical idcntlfication of neurons in the paraventrlcular nucleus of the hypothalamus that proJect to the medulla or to the spinal cord In the rat. J. Camp. Neurol. 205:260-272: 1982. Shapiro. R. E.: Miselis. R. R. The central organization of the \agus nerve innervating the stomach of the rat. J. Camp. Ncurol. 23X: 473488: lY85. Shoumura. S.: Shoumura. K.: Iwno. H Evidence for penetration of intraperltoneal horseradish peroxidase into vagal preganglionic neural elements. Acta Anat. I IO:]-6; 1981. Summerlee, A. J. S. Extracellular recordings from oxytocln neurones during the expulsive phase of birth in unaneythetiwd rat\. J. Ph!siol. 32l:l-9: 19x1. Saanson. L. W.: Kuypers. H. G. J. M. The paraventricular nucleus of the hypothalamus: cytoarchitectomc aubdiklsions and organization of projection? to the pituitary. dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. J. Comp. Neural. 193:SSS-570: 1980. Van den Pal. A. N. The magnocollular and parvocellular paraventricular nucleus of mt: lntrinw organization J. Camp. Neurol. 206: 317~315: 1982.
Reseurch
Rdkrin.
Vol. 25. pp. X5-371.
t Pergamon Press pk. 1990. Printed m the U.S.,4
Vagus Nerve Afferent and Efferent Innervation of the Rat Uterus: An Electrophysiological and HRP Study’ MARISELA ORTEGA-VILLALOBOS,” MAYDA GARCiA-BAZAN,t LUIS PASTOR SOLANO-FLORES,? JEStiS GUILLERMO NINOMIYA-ALARCON.: ROSALINDA GUEVARA-GUZMAN:’ “Departamento de Ciencius Bioldgicas. Fucultud de Estudios Superiores Cuuutitla’n, Estudo de M&co, MPxico und fDepurtumento de Fisiologiu, Fucultud de Medicinu, U.N.A.M. Apurtudo Postal No. 70-250, 03510 M&co, D.F. 1 Mkxico AND MATTHEW J. WAYNER Division oj’ Life Sciences,
The Universig
oj’ Texas at Sun Antonio,
Received
8 June
Sun Antonio,
TX 78285
1990
ORTEGA-VILLALOBOS. M.. M. GARCIA-BAZAN. L. P. SOLANO-FLORES. J. G. NINOMIYA-ALAR&N. R. GUEVARAGUZMAN AND M. J. WAYNER. Vqu nerve U@YW nnd efferenr innrrvnrion of the rut uterus: An e/rcrroph?~io/oKic,rr/ trntl HRP study. BRAIN RES BULL 25(3) 365-371, 1990.-To determine a possible b&stem connection with the uterus, a study with electrophysiological techniques and horseradish peroxidase (HRP) tracing was performed in the rat. Neurons of the nucleus of the tractus solitarius decreased in discharge frequency during cervicovaginal distension. HRP injections into the uterine walls resulted in the appearance of labelled cells in the nodose ganglion and in the dorsal motor nucleus of the vagus nerve. The rewlts demonstrate a direct bidirectional vagal complex-uterus connection via the vagus nerve. Results are discussed in terms of a complex uterus control system in which the paraventricular nucleus might play an integrative role. Nodose ganglion Vagus nerve Vagina Cervix Uterus Rat Electrophysiology
Dorsal motor nucleus Autonomic system
THE brainstem vagal complex is involved in the integration of respiratory, cardiovascular, and gastrointestinal functions. The nucleus of the tractus solitarius (NTS) seems to be the most important sensory relay structure in this complex. Also, the dorsal motor nucleus of the vagus nerve (DMNX) seems to be the main source of efferent fibers to these related structures (17. 18. 21. 31). With reference to the reproductive organs, the results of several important studies have appeared. Uterine afferent fibers arising from the T,,, L, through S , dorsal root ganglia that travel through the hypogastric and pelvic nerves have been described in the rat by mean\ of anterograde (23) and retrograde (5) HRP tracing. These nerves also supply sympathetic and parasympathetic efferents to the uterine cervical ganglia (4,16). In addition.
Paraventricular Nucleus of the tractus solitarius Reproduction control Parasympathetic system
nucleus HRP
uterine horn and vaginal distension modulates the activity of tonically discharging neurons of the paraventricular nucleus of the hypothalamus (PVN) (2). These PVN neurons project to the spinal cord (30,34). The involvement of the vagus nerve in the rat reproductive process has been studied extensively. Abdominal vagotomy during pregnancy results in a decreased number of live fetuses (20). delay in the onset of puberty (25). and disruption of the estrous cycle (7), probably as a consequence of depressed ovarian function and impairment of luteinizing hormone release (3). An HRP study demonstrated that the rat ovary is innervated by the vagus nerve (8). Even though it has been reported that abdominal vagotomy also reduces uterine weight. probably due to a decreased ovarian function (25). these studies on the role of the
‘This research was supported in part by PADEP: MED-901 and CONACyT Posgrado *Requests for reprints should be addressed to Dr. Rosalinda Guevara-Guzmin.
#33
ORTEGA-VILLALOBOS E7‘ Al..
366
1. Representative peristimulus histogram showing the effect of cervicovaginal distension upon the discharge frequency of a neuron of the nucleus of the tractus solitarius (NTS). Bin= 870 msec. The CD horizontal line indicates the distension period. Arrows signal the onset and the end of the distension. Black dots in the upper histological drawing indicate the sites where the tips of the recording micropipetteswere FIG.
located.
vagus nerve in reproduction processes have emphasized the control of ovarian function. It is not known, however, if the sensory information from the uterus is integrated in the vagus brainstem center alone or if some other higher structures might be involved. HRP as a retrograde tracer to establish autonomic pathways has been successfully used for some years (11). Also, distension of the hollow viscera has been shown to be useful in activating afferent fibers for electrophysiological studies (2,12). Therefore, the present study was carried out to identify and describe the brainstem connections with the uterus using electrophysiological and HRP tracing techniques in the rat. The results demonstrate the existence of a brainstem and uterus circuit which involves the vagus nerve. METHOD
Electrophysiological experiments were carried out in 11 naive female Wistar rats weighing 180-310 g. Only animals showing estrous vaginal smears were used (1). The rats were anesthetized with chloral hydrate (400 mg/kg, IP), the head was immobilized in a stereotaxic frame, and the necessary surgical procedures were performed. Exposed skin and tissue surrounding pressure points were infused with a xylocaine solution. Body temperature was continuously monitored and maintained at 37°C by means of an electric heating pad. Cervicovaginal distension was carried out using a pediatric urinary latex catheter that was inserted through the vagina until it reached the cervix. From 0.4 to 0.6 ml of room temperature tap water was infused into the balloon at the end of the catheter for a 15set period. Once the balloon was filled, it remained distended at least for one minute, afterwards, the balloon was allowed to empty over a 3-set period. Using conventional electrophysiological methods, the extracellular activity of NTS units was recorded with glass micropipettes filled with 2 M NaCl solution saturated with fast green. NTS stereotaxic coordinates were P = 11.4-l 1.7 from bregma in the horizontal skull; L = 1.5 and H = 4.5-7.1 from brain surface (26). The signals were stored on magnetic tape for further processing. The data were fed to a window discriminator which sorted the neuronal spikes and generated square pulses. A PC computer was used to perform the
analysis and to construct peristimulus histograms. Changes in the discharge frequency of the units, associated with cervicovaginal distension, were considered significant only when they were verified at least three times and when they were greater than 50% of the baseline activity. At the end of the experiment, the animal was deeply anesthetized and perfused with 10% formalin. The brain was removed, sectioned, and examined with a microscope to verify the location of the tip of the micropipette marked by a deposit of fast green. HRP experiments were performed on 20 female Wistar rats weighing 190-220 g which were ovariectomized or ligated one week prior to HRP administration. Rats were anesthetized with chloral hydrate (400 mg/kg, IP) and the uterus exposed through a midventral incision. Using a microsyringe fitted with a 26- or 30-gauge needle, 10 ~1 of a saline solution of HRP (10% Sigma lectin conjugate) was injected into the walls of the cervix and/or in the body of the uterus. Care was taken to avoid leakage of HRP from the injection site, therefore, the syringe needle was inserted into the midlayers and parallel to the uterine wall from 5 to 10 millimeters and then the HRP was delivered. After a survival time of 96 hours, the rats were anesthetized with chloral hydrate and perfused intracardially with 0.5 ml heparin solution, then with 250 ml 0.9% saline solution followed by 500 ml 1.25% glutaraldehyde and 1.0% paraformaldehyde in phosphate buffer, pH 7.4, and finally with 500 ml of 10% sucrose in phosphate buffer, pH 7.4. The brains and the nodose ganglion were removed and stored 24 hours in 30% sucrose buffer, pH 7.4 at 4”C, they were then frozen and sliced in 50 p.m thick sections. The sections were processed according to Mesulam’s technique (22) using 3, 3’, 5, 5’ tetramethyl benzidine as chromogen. The tissue was counterstained with neutral red and analyzed using both dark field and bright field illumination. HRP-labelled cells in the DMNX were localized according to Dennison et al. (9). RESULTS
Eleven spontaneously active units were examined in the NTS, one from each animal. All of the units responded to cervicovaginal distension by decreasing in discharge rate. In general, responsive units had a spontaneous discharge frequency of not more than 20
UTERUS VAGAL INNERVATION
spikes per second. The effects of distension were almost immediate and units began to decrease in frequency as can be seen in Fig. 1. In some neurons, the discharge completely ceased. In all cells a clear effect was established within 40 seconds; then, the discharge frequencies increased to baseline levels and continued at those levels even though the cervicovaginal distension was still present. When the cervicovaginal distension was terminated, an off-response was not observed. HRP injected into the wall of the uterine horns and/or the cervix resulted in the retrograde transport of HRP into some of the soma of the DMNX neurons (Part B, Fig. 2) and into several soma of the nodose ganglion of all of the studied rats (Part C, Fig. 2). The area enclosed by a square in Part C of Fig. 2 is enlarged and appears below as Part D. In general, since an insufficient number of labelled soma (Part C, Fig. 2) were observed in the nodose ganglion a topographic relationship could not be established between the location of labelled cells in the ganglion and the site of HRP injection in the uterus. In contrast, HRP administration into the wall of the rostra1 third of the uterine horns yielded labelled cells in the lateral areas of the caudal DMNX (Part I, Fig. 3). When HRP was injected exclusively in the cervix, the lateral parts of almost the full extent of the DMNX and of the nucleus commissuralis displayed labelled cells (Part III, Fig. 3). HRP injections involving the cervix, the caudal and the medial third of the uterine horns, yielded labelled cells throughout the whole extent of the DMNX (Parts II and IV. Fig. 3). DISCUSSION
The present study provides anatomical and electrophysiological evidence which support the existence of a brainstem-uterus circuit which involves the vagus nerve in the rat. Labelled soma were found in the nodose ganglion after uterine HRP injections. Additionally, changes in the discharge frequency of NTS units were evoked by mechanical cervicovaginal distension. On the other hand, HRP-labelled cells were observed in the DMNX after injection of the tracer into the uterus. Labelled cells have been found in the nodose ganglion after HRP injections into the ovary (8). The nodose ganglion HRPlabelled cells described in the present work must have been retrogradely labelled with the tracer captured exclusively by uterine nerve endings since the rats used were spayed or ligated one week before the HRP uterine administration. Intraperitoneal administration of HRP by dropping HRP solution underneath the omentum has resulted in labelled cells in the DMNX; thus, it has been suggested that, in studies of the autonomic supply of the abdominal viscera using retrograde transport of HRP, some HRP leakage can, possibly, penetrate directly into the autonomic neural elements located in the vicinity of the injection site (32). In the present work. to avoid any contamination of HRP to other abdominal structures, special care was taken to inject the HRP solution in the midlayers of the uterine wall by directing the syringe needle parallel to the wall for 5 to 10 millimeters prior to HRP delivery. Although the present HRP study was not designed specifically to search for a quantitative topographical representation of the vagal motor innervation of the uterus, the results indicate that the cervix and the rostra1 third of the uterine horns seem to be innervated preferentially by the lateral areas of the DMNX; the cervix, besides, is innervated by the nucleus commissuralis. Whereas the medial areas of the DMNX do not project to the cervix, they seem to project to the caudal thirds of the uterine horns. Probably, the lateral areas of the DMNX project also to the two caudal thirds of the uterine horns. The discharge frequency of NTS units decreased after cervicovaginal distension. This decrease endured for several set and then, the discharge frequency returned to a baseline level even though
367
the distension was maintained. Also, no off-responses were observed after the cervicovaginal distension was terminated. These facts suggest the existence of a sensory pathway from the reproductive tract to the NTS with stretch receptors localized in the vaginal and uterine walls. It seems that the processes associated with this pathway and/or the receptors themselves are quite adaptable to distension and respond only to the onset of the distension stimulus. Additionally, labelled cells were observed in the nodose ganglion after HRP administration into the uterus. Furthermore, it is well established that the terminal fields of the nodose ganglion sensory fibers are localized in the NTS (18). These findings. as a whole, support the existence of sensory fibers arising from the uterus, with soma localized in the nodose ganglion that project to the NTS. The possibility that other nuclei of the vagal complex might also receive uterine afferents (18). cannot be discarded. Afferent fibers from the T,,, L, ganglia innervate the cranial regions of the reproductive tract, which correspond also to the sympathetic innervated area of this tract. Additionally L,. S, dorsal root ganglia supply afferents to the caudal portion of the reproductive tract, which correspond to the parasympathetic innervated area (23). The hypogastric and the pelvic nerves supply those afferent fibers to the uterus (5,23). as well as sympathetic and parasympathetic efferents to the uterine cervical ganglia (4,16). It is interesting to point out that as long as the reproductive tract displays an autonomic circuitry composed of a dual spinal afferent innervation and of a sympathetic and parasympathetic spinal efferent system, the data presented in this report indicate the existence of a vagal afferent-efferent additional fiber supply to the whole extent of the uterus. Oxytocin might be the most important neural transmitter for the control of uterine function. The magnocellular division of the PVN synthesizes oxytocin and releases it from the posterior lobe of the pituitary gland (6). This hormone and the uterine spinal innervation seem to be necessary for the parturition function of the uterus, thus, distension of the birth canal during parturition is related to an increase of oxytocin levels (13) and with an increase of activity of PVN neurons (33). Additional evidence suggests that fibers in the hypogastric and pelvic nerves are PVN afferents from the uterus contributing, possibly, to the positive feedback on oxytocin release by uterine contractions, since uterine distension alters the firing activity of PVN units (2) and pelvic neurectomy disrupts oxytocin release during parturition (14) and abolishes the reflex of fetus expulsion (15). On the other hand, histological evidence suggests that oxytocinergic cells in the parvocellular division of the PVN project to autonomic cell groups in the spinal cord and/or to the dorsal vagal complex (30). Furthermore, neurons of the DMNX were shown to respond to oxytocin by increases in their firing rate (10). These facts indicate the existence of an additional oxytocinergic system that projects to the dorsal vagal complex, which, as supported from the data presented in this report. is related to the uterus through afferent and efferent vagal fibers. Complementing this circuitry, it has been shown that the NTS sends direct connections to the PVN (19,27), substantially, to its parvocellular division but not to the magnocellular division (29). With regard to the intrinsic connections of the PVN, it has been observed that magnocellular neurons have dendrites ramifying throughout the parvocellular area. Also. parvocellular neurons project axons to the magnocellular region; terminal boutons of these axons contact dendrites of magnocellular neurons (35). In this way, uterine sensory information conveyed via the vagus nerve and relayed in the NTS might influence the magnocellular oxytocin-secreting neurons by way of those magno-parvocellular contacts. Finally, it seems that, at least, two oxytocinergic efferent systems are acting upon the uterus: a magnocellular humoral system and a parvocellular neural system with the DMNX partic-
36X
ORTEGA-VILL.4LOBOS
E7‘ M
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FACING PAGE AND ABOVE FIG. 2. HRP evidence of sensory and motor vagal innervation of the uterus. (A) Site of HRP injection in the uterine cervix. (B) Dark field photomicrograph of HRP-labelled cells of the dorsal motor nucleus of the vagus nerve (DMNX). The white circle delineates the central canal; magnification: 380 x (C) Bright field photomicrograph of a nodose ganglion section in which an HRP-labelled cell was found (square area). An amplification of the square area is shown below in panel (D). Magnification: 950 x
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III
FIG. 3. Histological drawings of brainstem coronal sections encompassing the dorsal motor nucleus of the vagus nerve (DMNX). which depict the relationship between the HRP injection site in the uterus and the DMNX areas where labefled soma were found (black areas). The V-shaped drawings, lateral to each histological set, represent the uterus. (I) HRP injection into the rostra1 third of the uterine horns. (II) HRP injection in the cervix and caudal third of the uterine horns. (III) HRP injection in the cervix. (IV) HRP injection in the cervix and all of the uterine horns.
ipating, perhaps, as a relay station. In conclusion, both the spinal and the vagal uterine innervating systems are reciprocally integrated by the PVN. Dendrites of the motoneurons of the DMNX which innervate the stomach penetrate areas of the overlying NTS, which receive gastric vagal afferents (28.31). Additionaily, vagai sensory terminal fields have been visualized in the DMNX (1824). Similar vagovagal contacts related to uterine activity might well exist and remain to be determined. During the life span of a female animal, different complex functions as copula, egg maintenance, pregnancy, and parturition, are accomplished by the reproductive tract.
The significance of the vagal-uterine connection in these functions remains to be investigated. It would be interesting to determine the effects of oxytocin microinjections in the vagal complex upon the activity of the uterus during different functional states.
The authors are grateful to Biol. Olga Alejandra Dona~i-Aib~~n and Mr. Carlos de 10s Santos Toledo for their excellent technical assistance. to Mrs. Josefina Bolado-Garza for her patience and care in typewriting, and to Ms. Ingrid Mascher for her editorial assistance.
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