LOCALIZATION OF NEURONS INNERVATING THE UPPER PORTION OF THE DUODENUM IN THE MOTOR NUCLEUS OF THE VAGUS NERVE

V. A. Bagaev, F. N. Makarov, V.L. Rybakov, 1~. I~. Granstrem, E. V. Kopylov, E. B. Pluzhnichenko, S. I. Smirnov, and L. V. Filippova

UDC 612.822.8+612.33

Using the method of the retrograde axonal transport of horseradish peroxidase and a microlectrode technique, a population of neurons sending axons to the upper portion of the duodenum was identified in the dorsal motor nucleus of the vagus nerve. It was established that the maximal number of such neurons was located 1.0-2.5 mm rostral to the obex. The effects of their stimulation on the electrical activity of the smooth muscles of the duodenum was studied.

Individual attempts have been undertaken previously [12, 14] to elucidate the localization of neurons innervating various internal organs in the dorsal motor nucleus of the vagus nerve. However, only in recent years has it become possible with the advent of the methods of retrograde axonal transport of markers to investigate sufficiently precisely the location of groups of neurons which send axons not only to particular organs but to their functionally distinct parts as well. Thus, it has been established that a group of preganglionic parasympathetic neurons innervating the pyloroantral region of the stomach is localized within the dorsal motor nucleus (0.5-1.5 mm rostral to the obex), and it has been demonstrated that their microstimulation is accompanied by marked changes in the contractile activity of this portion of the organ [1, 15]. It is known that the coordination of the functioning of the pyloroantral region of the stomach and the upper portion of the duodenum is accomplished not only by local but also by central nervous mechanisms [3]. Both vasovagal motoric and inhibitory reflexes [9, 10], the realization of which may be achieved with the participation of neurons of the dorsal motor nucleus, are evidently among these. Therefore, the detailed study of its structural-functional organization appears necessary for the elucidation of specific neurophysiological mechanisms of the bulbar regulation of the functions of the upper portion of the duodenum. The localization and functional characteristics of neurons of the dorsal motor nucleus of the vagus nerve which send axons to the upper portion of the duodenum were investigated in the present study. METHODS The study was carried out in acute experimental conditions in cats weighing 2.5-3.5 kg. Three series of experiments were carried out. The topographic characteristics of the dorsal motor nucleus of the vagus nerve were studied in the first preliminary series. Using a modified Nissl staining method for nerve ceils [11], the features of the localization of the left and right nuclei in the caudorostral direction were determined. In addition, the total number of neurons in the nuclei was counted. The second series was carried out on seven cats using the method of retrograde axonal transport of horseradish peroxidase. Under nembutal anesthesia, the animals were administered, by means of several injections, 40-50 ~tliters of a 33% solution of horseradish peroxidase (Olaine, Riga; RZ = 2,7) placed in a limited section (1.0 x 1.0 cm) of the wall of the duodenum (1.5 cm distal to the pyloric sphincter). The animals were administered dexamethasone (0.2 mg/kg, intramuscularly) and atropine (0.2 mg/kg, subcutaneously) before laparotomy, prior to the administration of the marker. The animals were reanesthetized Laboratory of Corticovisceral Physiology, Laboratory of the Morphology of the Central Nervous System, and Laboratory of General Physiology of Perception, I. P. Pavlov Institute of Physiology of the Academy of Sciences of the USSR, Leningrad. Translated from Fiziologicheskii Zhumal SSSR imeni I. M. Sechenova, Vol. 77, No. 1, pp. 45-52, January, 1991. Original article submitted April 24, 1990. 230

0097-0549]91/2203-0230512.50 9 1992 Plenum Publishing Corporation

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Fig. 1. Topography of the dorsal motor nucleus of the vagus nerve (hatched) in diagrams of frontal sections of the medulla oblongata (from -2.0 to +3.0 mm relative to the obex). 0, the level of the obex. and perfused by the transcardiac route after 72 h with 1.0-1.5 liters of a mixture consisting of a 1% solution of paraformaldehyde in a phosphate buffer. Frontal serLal sections of the medulla oblongata, 50 ~tm in thickness, were obtained on a freezing microtome; these were processed by Mesulam's method [13]. The number of horseradish peroxidase-labeled nerve cells was calculated in percents of the total number of labeled neurons throughout the entire extent of the left and right dorsal motor nuclei. The third series . of experiments was carried out in 12 cats. The feeding of the animals was stopped one day before the beginning of the experiment. In a number of narcosa we used a mixture of ot-chlorase (45 mg/kg) and urethane (400 mg/kg) intraperitoneally. The level of arterial pressure in the femoral artery (80-120 mm Hg) and the dynamics of the heart rate (120-200 beats per minute) were recorded in order to monitor the functional state of the animal. Body temperature was maintained at the 37~ level throughout the entire experiment. In order to gain access to the dorsal surface of the medulla oblongata the animal was fixed in the stereotaxic apparatus. The occipital portion of the skull was removed and the caudal portion of the cerebellum was moved aside and fixed by means of a special blade. Stimulating tungsten microelectrodes in lacquer insulation, with a tip diameter of 2-5 ~tm and a resistance of 150-200 lda, were introduced into the right dorsal motor nucleus (from -1.5 to +3.0 mm relative to the obex). Stimulation was carded out by means of rectangular bipolar pulses, 0.25 msec in duration. The current strength was 30-150 rtA, stimulation duration 14 sec, and frequency of stimulation 10 Hz. Electrocoagulation of the brain tissue was carded out after each experiment, with subsequent histological determination of the site of localization of the tip of the microelectrode.

231

The recording of the electrical activity of the wall of the upper portion of the duodenum was accomplished by means of bipolar silver electrodes [7], which were fixed under the serosa at a distance of 1.0-1.5 cm distal to the pyloric sphincter (i.e., precisely in that portion of the intestine in which the horseradish peroxidase solution was introduced in the preceding series of experiments). The stimulation of the nucleus and the recording of the electrical activity of the duodenum were carded out in six animals with it detached from the stomach (ligature in the region of the pyloric sphincter) with maintenance of the external innervation. The methods utilized in the present study for the recording and analysis of the electroduodenograms are similar to those previously described [2]. The experiments involving the stimulation of sections of the dorsal motor nucleus were carried out under peridural spinal anesthesia. For this purpose trimecaine (1.7 mg/kg) was introduced into the peridural space through the intervertebral ligaments (Thm-Thw), following which a brief decrease (of 10-15 mm Hg) was observed in the level of the systemic arterial pressure, with its subsequent recovery over the course of 1-3 min.

RESULTS Morphological Characterization of the Nucleus. It was demonstrated in the first series of experiments that the length of dorsal motor nucleus is 5-9 ram, and depends on animal size. Total number of neurons ranges from 5 to 9 thousand. Large (more than 20 ~m), medium-sized (15-20 #m), and small (7.5-14.5 #m) cells can be distinguished along entire nucleus. Nucleus begins at level 2.0-4.0 mm caudal to obex. In rostral parts 3.5-5.0 mm rostral to obex. Topography of the nucleus is shown in Fig. 1. Initially it is a flattened column of nerve cells located at a depth of 1.5-1.7 mm from the dorsal surface of the medulla oblongata and 0.8-1.2 mm lateral to the midline. Immediately after the obex the nucleus gradually takes on a rounded form and approaches the bottom of the fourth ventricle, lying more superficially. At the +2 mm level relative to the obex, both the left and right nuclei come practically right up against the surface. The rostral portion of the nucleus is traced at a depth of 0.5-0.8 mm. Laterally the nucleus is located after the obex at a distance of from 1.2-1.5 to 1.5-2.0 mm from the midline (+1.0 and +3.0 mm rostral to the obex, respectively). Localization of Neurons Sending Axons to the Upper Portion of the Duodenum, The character of the distribution of the preganglionic parasympathetic neurons in the nucleus under investigation is shown in Fig. 2. The horseradish peroxidase-labeled nerve cells were located practically along the entire nucleus (bilaterally) in a band extending in the caudal-mstral direction, and made up approximately 5% of the total neuronal population. The maximal number of neurons identified was found in the region limited to 1.0-2.5 mm rostral to the obex. It turned out upon computation that the number of stained cells was practically identical in the left and right nuclei. The overwhelming majority of the preganglionic parasympathetic neurons innervating the upper portion of the duodenum are found in the dorsomedial portion of the nucleus. Horseradish peroxidase-labeled neurons were not found by us in this series of experiments in any other structures of the dorsal surface of the medulla oblongata. Changes in the Electrical Activity in the Wall of the Duodenum during Stimulation of Segments of the Nucleus. It is known that the electrical activity of the smooth muscles of the duodenum in the norm is represented by two types of potentials. The first is represented by slow waves at a frequency of 16--18 per rain, and the second by peaked potentials (spike potentials), the appearance of which is associated with intensification of the contractile'activity of the smooth muscles [16]. In our experiments the electrical activity of the wall of the duodenum prior to stimulation was represented in practically all cases only by slow waves. This may apparently be associated with the conditions under which the experiment was carried out and with the influence of the substances we used for anesthesia on the functional state of the smooth muscles of this portion of the gastrointestinal tract, in particular. It should be noted that in a number of experiments we also simultaneously studied the activity of the wall of the stomach (the pyloric region). In this case, spike potentials were also recorded in the baseline initial electrogastrogram. The stimulation of segments of the dorsal motor nucleus, and, in particular, of the region with the maximal number of neurons sending axons to the upper portion of the duodenum, did not lead to any changes in the frequency of the slow waves. When the nucleus was stimulated the occurrence of spike potentials was recorded, i.e., the intensification of the contractile activity of the intestinal wall. The application of stimulation through an electrode located at a distance of 1.5 + 0.5 mm rostral to the obex in the period of the recording only of slow waves led in practically all of the experiments to the appearance of spike potentials (Fig. 3). At the same time the changes in the electrical activity of the smooth muscles of the pyloric region of the stomach were insignificant. It was of interest in this series of experiments to define those segments of the nucleus the

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Fig. 2. Distribution of horseradish peroxidase-labeled neurons sending axons to the upper portion of the duodenum in the dorsal motor nucleus of the vagus nerve (in the caudorostral direction). Along the abscissa: distance relative to the obex (0), mm; along the ordinate: number of labeled neurons, %, in relation to the total number of labeled cells. In A) Left nucleus; in B) right nucleus.

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Fig. 3. Occurrence of spike potentials in response to the electrical stimulation of a segment of the right dorsal motor nucleus of the vagus nerve. 1, 2) Electrical activity of the smooth muscles of the duodenum and of the pyloric region of the stomach, respectively; 3) systemic arterial pressure; 4) heart rate. To the right of each segment of the figure, calibration: amplitude, 250 IxV; arterial pressure, mm Hg; heart rate, beats per min. Time calibration, 4 sec. Arrow directed upward, beginning; downward, ending of stimulation. stimulation of which is accompanied by the maximal changes in the dynamics of the spike potentials. For this purpose, four stimulating microelectrodes were introduced at intervals of 1.5 mm in the rostrocaudal direction into the right nucleus. In this case it was shown that the reaction of the appearance of spike potentials is identically effectively reproduced when all of the indicated segments of the nucleus are stimulated. The changes described were also observed when the duodenum and the stomach were detached. The transection of the ipsilateral vagus nerve completely eliminated all of the effects of the stimulation of the nucleus. We should note that in the period of application of the stimulation and following it the level of the systemic arterial pressure and the heart rate changed insignificantly. 233

DISCUSSION OF RESULTS The question of the significance of the efferent fibers of the vagus nerve in the regulation of the contractile activity of the duodenum has been repeatedly subjected to discussion in a large number of investigations ([3] et al.). The interest in this problem is not only explained by the necessity of understanding the mechanisms underlying the formation of different types of motoric activity of the intestine. The fact that vagotomy [5, 6] has been used in recent years in clinical practice in the treatment of ulcerative disease of the duodenum should be borne in mind. For this reason the study of the structural and functional organization of the dorsal motor nucleus neurons which send axons to different segments of the gastroduodenal region within the makeup of the vagus nerve takes on practical significance as well. It should be noted that as far back as 1843, Stilling (quoted in [14]) was perhaps the first to describe a group of cells on the dorsal surface of the medulla oblongata which was then defined as the dorsal motor nucleus of the vagus nerve. Attempts were subsequently made to localize populations of cells within the boundaries of the nucleus which were associated with the innervation of the internal organs [14]. However, the imperfection of the methodological techniques employed did not permit the construction of a clear notion of the site of disposition of the neurons which send axons to various regions, in particular the gastrointestinal tract. The topography of the nucleus has been presented with sufficient precision in a number of anatomical studies [4, 8]. At the same time, it has been of interest to determine, along with the topographical features, certain quantitative characteristics of the neurons of the nucleus as well. The data we have obtained concerning the outlines of the nucleus and its position in relation to the dorsal suface of the medulla oblongata and the obex are presented in Fig. 1, and correspond on the whole to the descriptions of other authors (see [4]). With regard to the quantitative characteristics, it has turned out that both the left and the right nuclei contain an identical number of neurons (five to nine thousand, depending on the size of the animal), which are disposed in a compact group, starting at the level of 2.0-4.0 mm caudal to the obex. The most rostral portion of the nucleus, according to our data, may lie at a distance up to 5.0 mm relative to the obex. What is the functional significance of the neurons of the nucleus? It has been shown that some of them send axons to the organs of the gastrointestinal tract. The neurons which innervate various regions of the stomach are located in the main rostral to the obex in a portion of the nucleus limited to 0.5-1.5 mm [1, 15], Apparently it is precisely this "gastric zone" which is involved first and foremost in the processes of the bulbar regulation of the secretory-motor function of the stomach. It has already been noted above that the so-called vasovagal reflexes are of substantial significance in the coordination of the functions of the stomach and the duodenum. It has been hypothesized [9, 10] that these reactions may be accomplished with the participation of the preganglionic parasympathetic neurons of the dorsal motor nucleus. In our opinion the gastroduodenal motoric and duodenogastral inhibitory reactions may be numbered among the vasovagal reflexes [3]. It can be conjectured that the bulbar preganglionic parasympathetic neurons which innvervate, in particular, the proximal portion of the duodenum, and which participate in the realization of these reactions, should be located within the limits of the dorsal motor nucleus close to its "gastric zone." Using the method of retrograde axonal transport of horseradish peroxidase, we succeeded in the present study in demonstrating that the maximal number of such cells is found at the level of 1.0--2.5 mm rostral to the obex. At the same time, the number of neurons associated with the innervation of the upper portion of the duodenum does not exceed 5% of the total number of cells of the nucleus. Of course, in order to determine the degree of participation of these neurons in the realization of the vasovagal reflexes (those which determine the coordinated functioning of the gastroduodenal zone), special investigations are required. On the other hand, in the present study, on the basis of the data obtained, one can only speak with confidence thus far of the site of localization of the maximal number of cells sending axons to the upper portion of the duodenum. The innervation of this portion of the intestine is accomplished to an equal degree by neurons of both the left and the right nuclei. The fact that the number of peroxidase-labeled cells was practically identical in both nuclei suggests this. The effects of the stimulation of the identified segments of the nucleus on the indices of the electrical activity of the duodenum have been examined in our study. It should be noted that, in keeping with the available data [16] on the frequency of slow waves, it is possible to make a judgment regarding the frequency of the possible contractions of this segment of the intestine. The appearance, on the other hand, of spike potentials on the electroduodenogram reflects the moment of the beginning of the contractions of the smooth muscle wall or their intensification. Taking these notions into account, the third series of our experiments was carried out as well. It was found that the stimulation of the segments of the dorsal motor nucleus at the site of localization of the maxima] number of labeled cells does not change the frequency of the slow waves of the duodenum. In our preceding study [2], when the so-called "gastric zone" of this nucleus was stimulated, the effect of a decrease in the frequency of the slow waves of the wall of the stomach was observed in practically all the experiments. Incidentally, the decrease in the frequency of the slow waves during the stimulation of the neurons of the nucleus (from 0.5 to 2.5 mm rostral to the obex) readily developed in the pyloric region of the stomach, and was also not recorded in the duodenum 234

even after the separation of the duodenum from the stomach (with maintenance of the vagal innervation). Thus, according to our data changes do not take place in the rhythm of the contractions of the wall of the duodenum during the electrical stimulation of the neurons of the dorsal motor nucleus. Evidently, the formation of the rhythm of the contractions is entirely controlled by the', corresponding pacemaker, the existence of which has been hypothesized at the site of the mouth of the pancreatic duct (see [3]), i. e., precisely in that segment of the intestine where the recording electrode was located in our experiments. Moreover, the hypothesis of the possible participation of the neurons of the dorsal motor nucleus in the realization of the vasovagal motor reactions, and, in particular, of the gastroduodenal motor reflexes has already been stated above. In that connection it seemed of interest to assess the effects of the stimulation of the identified populations of neurons of the nucleus on the dynamic,~ of the spike potentials. It was found that the electrical stimulation of these neurons leads to the appearance of spike potentials, i. e., to the contraction of a segment of the duodenum at the site of the recording electrode. In our opinion the reaction described is associated with the activation of efferent fibers only of the vagus nerve. It was not eliminated when the portion of the dorsal motor nucleus was stimulated against the background of spinal anesthesia, and disappeared only after the transection of the ipsilateral vagus nerve. Thus, the results obtained in the present study make it possible to draw the following conclusion. Preganglionic parasympathetic neurons which send neurons to the proximal portion of the duodenum are localized in the mediodorsal portion of the dorsal motor nucleus of the vagus nerve from -2.0 to +3.0 mm (relative to the obex). The maximal number of such neurons is found in the segment from +1.0 to +2.5 mm. The stimulation of this zone of the nucleus is ineffective in relation to the frequency of the slow waves, i. e., it does not change the rhythm of the contractions of the upper portion of the duodenum. The appearance of spike potentials under these conditions (the contraction of the segment of the intestine) is recorded without significant changes in the indices of the activity of the cardiovascular system. The results presented, as well as the data of other authors [10], make it possible to regard the dorsal motor nucleus neurons we studied as one of the functional blocks which accomplish the realization of the gastroduodenal motor reflexes. L I T E R A T U R E CITED 1. V. A. Bagaev, F. N. Makarov, V. L. Rybakov, et al., "Localization of neurons in the dorsal motor nucleus of the vagus nerve innervating the pyloric region of the stomach," Doklady Akad. Nauk SSSR, 304, No. 4, 985-987 (1989). 2. V. A. Bagaev, E. V. Kopylov, and S. I. Smirnov, "Effects of electrostimulation of various segments of the dorsal motor nucleus of the vagus nerve on the electrical activity of the stomach wall," Fiziol. Zh. SSSR, 76, No. 4, 492-501 (1990). 3. P. G. Bogach, "The motoric activity of the small intestine," in: The Physiology of Digestion. The "Manual on Physiology" Series [in Russian], Leningrad (1974), pp. 474-523. 4. A. A. Grantyn', "The morphology, topography, and connections of the medulla oblongata and the pons varolii of the cat," in: Current Problems of the Pharmacology of the Reticular Formation and Synaptic Transmission [in Russian], Leningrad (1963), pp. 165-189. 5. E. M. Matrosova, A. A. Kurygin, and S. D. Groisman, Vagotomy (Sequelae and Their Mechanisms) [in Russian], Leningrad (1981). 6. Yu. M. Pantsyrev and A. A. Grinberg, Vagotomy in Complicated Duodenal Ulcers [in Russian], Leningrad (1979). 7. M. Papazova and K. Milenov, "A method for the recording of biopotentials from stomach musculature in the cat with chronically implanted electrodes," [rough translation from Bulgarian- Tr.] Izv. In-ta BAN, 9, 187-191 (1965). 8. A.L. Berman, The Brain Stem of the Cat. A Cytoarchitectonic Atlas with Stereotaxic Coordinates, Madison (1968). 9. L.A. Blackshaw, D. Grundy, and Th. Scratcherd, "Involvement of gastrointestinal mechano- and chemoreceptors in vagal reflexes: an electrophysiological study," J. Autonom. Nerv. System, 18, No. 3,225-234 (1987). 10. J. S. Davison and D. Grundy, "An electrophysiological investigation of vasovagal reflexes," in: Gastrointestinal Motility, New York (1980), pp. 139-144. 11. R. C. Fernstrom, "A durable Nissl stain for frozen and paraffin sections," Stain Technol., 33, No. 4, 175-176 (1958). 12. B. Gets and T. Sirnes, "The localization within the dorsal motor vagal nucleus," J. Comp. Neurol., 90, No. 1, 95-110 (1949). 13. M.-M. Mesulam, "Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic reactionproduct with superior sensitivity for visualizing neural afferents and efferents," J. Histochem. Cytochem., 26, No. 2, 106-117 (1978). 235

14. G. A. G. Mitchel and R. Warwick, "The dorsal vagal nucleus," Acta Anat., 25, No. 1,371-395 (1955). 15. F. D. Pagani, W. P. Norman, and R. A. Gillis, "Medullary parasympathetic projections innervate specific sites in the feline stomach," Gastroenterology, 95, No. 2, 277-288 (1988). 16. M. Papasova, "Gastrointestinal motility. Electrophysiological data," in: Frontiers in Physiological Research, Canberra (1984), pp. 36-49.

PERCEPTION OF T E M P E R A T U R E ELEVATION IN HUMAN SEASONAL HEAT ADAPTATION

M. D. Khudaiberdiev, F. F. Sultanov, and L. M. Pokormyakha

UDC 612.014.43:577.4

Higher values of the exponent power function of the perception of temperature elevation (1.66, 1.32, and 1.50) in the summer than in the winter (1.40, 1.08, and 0.84) were established in seasonal investigations in people at room air temperatures of 20, 28, and 40~ Consequently, following adaptation to the seasonal high temperature a person can feel a smaller temperature increment than in the winter. This suggests a reorganization in the functional activity of the temperature analyzer under the prolonged influence of seasonal high temperature.

It is has been established that seasonal adaptation to high temperature is accompanied by distinct changes in the functional activity of central and peripheral heat-sensitive structures of the organism [5-7]. An attempt has been made in the present study to characterize the operation of the temperature analyzer as a whole in human adaptation to seasonal high temperature using a psychological temperature scaling technique. METHODS The subjects were 15 men ranging in age from 21-27 years in the winter and summer seasons at air temperatures of 20, 28, and 40~ and a relative humidities respectively of 47-50, 35--40, and 25-30% [22]. The speed of air movement in the immediate vicinity of the subjects was 0.02 m/sec. After the unclothed subjects were in one of the indicated temperatures for 30-45 min, instructions were given to assessed as 1 point the stimulus presented with the lowest temperature (30--33~ and at 50 points the stimulus with the highest temperature (47-50~ and to assign an estimate in accordance with their sensation to the temperatures with intermediate values. Copper cylinders, diameter 3.5 cm z [sic] and height of 70 mm filled with paraffin which were applied in random order to the internal surface of the middle third of the forearm, were used as the stimulators. The geometric means of the point estimates (E) and the ambient temperature (T) which represents the difference between the temperature of the stimulus and the temperature surface of the skin of the forearm, were plotted on logarithmic paper. The strength of sensation was determined by the formula [22] logE = logk + nlogT, where k is the coefficient of proportionality and n is the exponent of the power function. The body temperature in the rectum and of skin in the region of the forehead, chest, and hand, and the external temperature of the thigh and calf were measured with an accuracy of_+0.01 and +0.1 ~ respectively, by means of copper-constantan thermocouples, the EMF of which, after preliminary amplification by an F 116/1 photoelectric amplifier, was recorded by a 12-point t~PP-09 M 3 automatically recording potentiometer [9]. The weighted mean skin temperature (WMST) was computed as described in [1]. Laboratory of Neurophysiology and Thermal Regulation, Institute of Arid Zone Physiology and Experimental Pathology of the Academy of Sciences of the Turkmen SSR, Ashkhabad. Translated from Fiziologicheskii Zhurnal SSSR imeni I. M. Sechenova, Vol. 77, No. 1, pp. 116-121, January, 1991. Original article submitted December 13, 1989. 236

0097-0549/91/2203-023 6512.50 9 1992 Plenum Publishing Corporation

Localization of neurons innervating the upper portion of the duodenum in the motor nucleus of the vagus nerve.

Using the method of the retrograde axonal transport of horseradish peroxidase and a microelectrode technique, a population of neurons sending axons to...
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