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Abdominal vagal afferents excite A1 area neurons antidromically activated from the region of the supraoptic nucleus in the rabbit
181
Brain Research, 616 (1993) 181-187 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 18992
Abdominal vagal afferents excite A1 area neurons antidromically activated from the region of the supraoptic nucleus in the rabbit Z.J. G i e r o b a a n d W . W . B l e s s i n g Departments of Medicine and Physiology, Centre for Neuroscience, Flinders University, Bedford Park, SA (Australia) (Accepted 2 February 1993)
Key words: A1 catecholamine-synthesizingneuron; Abdominal vagus; Vasopressin; Ventrolateral medulla; Electrophysiology
We made extracellular recordings from 113 spontaneously active neurons in the A1 area, after identifying the cells by antidromically activating them from the region of the supraoptic nucleus in urethane-anesthetized rabbits. We tested the response of these neurons to inputs from abdominal vagal, renal and somatic nerves. Electrical stimulation of the abdominal vagus nerve activated 64/85 neurons tested (75%), and had no effect on the remaining 25%. Latency was 195_+25 ms, (conduction velocity 0.7 m / s ) . Stimulation of renal afferents had no effect on the discharge rate of 4 neurons tested. Stimulation of branches of the sciatic nerve inhibited 7/17 A1 area neurons tested, excited 4 and had no effect on 6 neurons. Stimulation of the central ear nerve inhibited 4 / 1 7 neurons tested, excited 6 and had no effect on 7 neurons. Gastric distension had no effect on 20/24 neurons tested. Lightly touching the animals back and legs had no effect on the discharge of 45/49 neurons tested. Similarly, painful stimuli failed to affect 44/49 neurons tested. Our results indicate that A1 area neurons, with projections to the region of the supraoptic nucleus, receive excitatory inputs from the abdominal vagus nerve. The visceral information transmitted to A1 cells by these abdominal vagal afferents is not yet determined, but acute gastric distention does not appear to be a physiological stimulus. A1 area neurons seem not to be involved in transmitting somatic information to the hypothalamus.
INTRODUCTION
Plasma levels of vasopressin or oxytocin are elevated after systemic administration of a number of agents whose action is dependent on the integrity of the afferent abdominal vagus nerve 15'23. In certain species, these agents cause nausea and vomiting, also by an action mediated by the afferent abdominal vagus 24'25'36, and in humans the sensation of nausea may be accompanied by elevation of plasma vasopressin 3°. Electrical stimulation of the afferent abdominal vagus causes release of either vasopressin or oxytocin, as well as inducing emesis in species capable of this response 15. The discharge rate of the vasopressin- or oxytocinsecreting magnocellular hypothalamic neurons is also affected by vagally mediated abdominal stimuli 29'34. Since vagal afferents terminate in the nucleus tractus solitarius (nTS), the central pathway to the relevant hypothalamic neurons must originate in this nucleus. Rat nTS neurons project directly to oxytocin-secreting magnocellular hypothalamic cells 8'28'33, but the func-
tional pathway to vasopressin-secreting neurons is likely to be indirect because nTS neurons do not project directly to vasopressin-secreting hypothalamic cells. In the case of vasopressin secretion in response to hemorrhage, baroreceptor-derived information is relayed from the nTS to the A1 noradrenaline-synthesizing neurons in the caudal ventroiateral medulla, and then to the hypothalamus4,13'21. In the present electrophysiological study, we determined whether rabbit A1 area neurons, antidromically activated from the region of the supraoptic nucleus, alter their discharge rate in response to electrical stimulation of the afferent abdominal vagus. Since gastric distention alters the activity of magnocellular neurons z9'34, we determined whether this more physiological stimulus could reproduce the effects on the discharge of the A1 area neurons of electrical stimulation of the abdominal vagus. We also determined whether neurons which respond to afferent vagal stimulation also respond to baroreceptor and chemoreceptor inputs.
Correspondence: Z.J. Gieroba, Department of Medicine, Flinders Medical Centre, Bedford Park, SA 5042, Australia. Fax: (61) (8) 2045450.
182 Magnocellular
vasopressin-secreting neurons,
mmHg. Arterial pCO2, p O 2 and pH were measured using a pH/Blood Gas Analyser 813 (Instrumentation Laboratory, USA), and resting values were pH 7.35-7.45, pO 2 250-350 mmHg (ventilation with O2-enriched air), pCO 2 35-40 mmHg. Body temperature was monitored by a rectal thermistor and maintained at 38-39°C by a heating pad. The trachea was cannulated, the animal was paralyzed with pancuronium bromide (0.5 mg/kg, i.v. initially, with supplemental doses as necessary) and artificially ventilated with oxygen-enriched air using a Harvard model 681 rodent ventilator. A bilateral pneumothorax was induced. The head of the rabbit was fixed in a Kopf stereotaxic frame. The medulla was exposed by incision and retraction of the atlanto-occipital membrane. Neck flexion was adjusted so that the dorsal surface of the medulla was horizontal. The rostral edge of the area postrema in the midline (obex) served as the stereotaxic zero for rostro-caudal and medio-lateral coordinates.
and
s e c r e t i o n of v a s o p r e s s i n , may b e a f f e c t e d by s o m a t i c a f f e r e n t s 14, p o s s i b l y a c t i n g via t h e A 1 r e g i o n n , a n d b y o t h e r v i s c e r a l a f f e r e n t s , i n c l u d i n g s o m e w h i c h m a y act through
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tested the effect on discharge of A1 area neurons of e l e c t r i c a l s t i m u l a t i o n o f s o m a t i c a n d r e n a l a f f e r e n t s , as w e l l as t h e e f f e c t o f l i g h t t o u c h a n d p a i n s t i m u l i . MATERIALS AND METHODS
General procedures Experiments were performed on 30 male New Zealand White rabbits (2.5-3.0 kg), anesthetised with urethane (1.5 g/kg, infused into a marginal ear vein over 30 min). Scopolamine methylbromide (50/zg/kg, Sigma Chemical Co, USA) was given to minimize airway secretions. Catheters were inserted into the marginal ear vein for administering drugs, and into the left femoral artery for recording arterial pressure (AP) and for arterial blood gas analysis. A Statham P23ID transducer connected to a Grass Model 7 polygraph was used to record AP. Mean AP was obtained by filtering the phasic signal. Heart rate was measured with a Grass 7P4G tachograph, triggered by the phasic arterial signal. End-expiratory CO 2 was monitored (Datex Normocap CO 2 monitor, Finland) and maintained at 35-40
Electrical stimulation of the supraoptic nucleus A burr hole was drilled in the parietal bone of the skull, 2 mm rostral to bregma. A monopolar stainless steel stimulating electrode (0.5 mm bare tip) was lowered perpendicular to a line passing through lambda and 1.5 mm above bregma. The tip of the electrode was positioned 2 mm rostral to bregma, 2 mm lateral to the midline and 12.0-13.5 mm below the cerebral surface. The optic tract field potential was evoked by flash stimulation of the contralateral eye, and the electrode was retracted until the potential almost disappeared, indicating that the tip was in or close to the supraoptic nucleus. The stimulus intensity was then set at 0.5-1.2 mA (0.5 ms duration, 1 Hz) for antidromic activation of neurons in the A1 area.
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T i m e (ms) Fig. 1. A: diagram showing positions of stimulating electrode tips, marked by lesions (dots), in and near the supraoptic nucleus, f, fornix; ic internal capsule; ot, optic tract; ox, optic chiasm; SON, supraoptic nucleus. B: photomicrograph of the lesion made by the tip of recording electrode in the vincinity of A1 neurons (FAGLU-fluorescence method). Bar = 2 mm (A) and 120 ~m (B). C,D: collision test. Each trace is a computer average of 20 sweeps triggered by the spontaneous spike; C: supraoptic nucleus stimulus (at arrow) was delivered just greater after the critical delay and produced a constant latency spike response (marked by star) on every occasion; D: supraoptic nucleus stimulus was delivered just before the critical delay, and the antidromic spike response was always cancelled; E: computer average of 20 superimposed sweeps of a negative-positive-going spike (marked by star in C) of the antidromically activated neuron.
183 Electrical stimulation of the abdominal vagus, renal, sciatic and central ear nerues The anterior vagal trunk was dissected at the junction of the oesophagus and stomach, either from above or below the diaphragm. The intact nerve was placed inside a bipolar silver wire cuff electrode, constructed from teflon-coated silver wire and silastic tubing. The tubing was filled with paraffin oil, and surrounded with low melting point wax (BDH Chemicals, Poole, U K ) to insulate the exposed electrode wire tips from tissue fluids. In 1 rabbit, both anterior and posterior vagal trunks were positioned in separate electrodes and separately stimulated. In one rabbit the stimulating hook electrode was placed on the left cervical vagus low in the neck. In a different rabbit the vagi were cut low in the neck after testing the effect of stimulation of the abdominal vagus on an antidromically activated A1 area neuron. In another rabbit, the central ends of all nerves entering the hilus of the kidney were isolated from a retroperitoneal approach, sectioned close to the kidney and protected in paraffin oil. T h e central cut ends of the nerves were placed on a bipolar stainless steel hook electrode. In 3 other rabbits the left sciatic nerve was isolated. The branches were dissected free, cut distally, and positioned in a silver wire cuff electrode. In the same rabbits the left central ear nerve was isolated, cut distally and its central end was positioned in a cuff electrode. Nerves were stimulated by 1 - 3 cathodal pulses (0.5 ms, 200 Hz, 500-1000 p,A). In some experiments, for electrical stimulation of the abdominal vagus, repetitive stimulation was used (10 s train of cathodal pulses, 0.5 ms duration, 50 Hz, 200-600 tzA).
Stomach distension and physiological activation of somatic afferents The stomach was first emptied by an orogastric tube. A latex balloon was connected to a silastic tube and inserted through the oesophagus to the stomach. T h e stomach was distended by injecting 50-100 ml of air within 5 s. Distension was maintained for 30 s. T h e degree of distention was judged by observing and palpating the upper abdomen, to determine the size and firmness of the stomach. For physiological activation of somatic afferent nerves the body was lightly touched for 10 s, or either the hindlimb or the ear was firmly pinched for 5 s with a pair of long-nosed pliers.
Activation o f baroreceptors and chemoreceptors Baroreceptor activity was altered by raising AP by 50-75 m m H g (phenylephrine hydrochloride 50 t z g / k g i.v., Sigma Chemical Co., USA). Chemoreceptors were stimulated by ventilating rabbits for 30-45 s with 10% 0 2 in N 2.
ing aorta and the brain was fixed by perfusion with f o r m a l d e h y d e / glutaraldehyde solution. The brain was removed and 50 ~ m transverse sections were cut on a Vibratome. Sections of the hypothalamus were stained with Perl Prussian blue for demonstration of the position of the tip of the stimulating electrode. The f o r m a l d e h y d e / glutaraldehyde ( F A G L U ) fluorescence histochemical procedure I2 was used to relate recording sites, marked with a lesion, to the A1 group of catecholamine-containing neurons. In some cases, the same sections were rehydrated, stained with Neutral red and coverslipped.
Stat&tical analysis Base-line values of AP, heart rate and unit discharge rate, and changes in these values caused by different procedures were recorded. Data were expressed as mean_+ S.E.M. and changes in values were examined using a paired student t-test. For peristimulus time histograms, changes in discharge probability and duration of effect were analyzed according to Davey et al. 9.
RESULTS
Identification of A1 area neurons antidromically activated from the supraoptic nucleus Approximately 30% of antidromically activated neurons did not discharge spontaneously so that no collision test was possible. These neurons were excluded from further analysis. Recordings were made from 113 neurons which passed the collision test (Fig. 1C,D). The threshold stimulus intensity ranged between 100 and 1000 ~A. The amplitude of the action potential ranged between 150 and 1200/zV (mean 425 _+30/zV). The distance between stimulating and recording electrodes was approximately 24 mm (axonal conduction velocity 0.5-4.8 m/s, median 0.75 m/s, Fig. 2). The frequency of spontaneous action potentials ranged from 0.5 to 20 spikes/s (median 4 spikes/s). Histological determination of recording sites revealed that the le-
Extracellular recording in the A1 area Recordings from neurons in the A1 area were made by inserting a recording electrode vertically into the medulla, from 1 m m rostral to 2 m m caudal to the obex, 3-3.5 m m lateral from the midline and 3-4.5 m m ventral from the dorsal surface of the medulla. Glasscoated tungsten electrodes were used. Unit activities were recorded with an N T l l 4 A differential amplifier (Neomedix), filtered (bandpass 100-5000 Hz), amplified using a NL106 amplifier (Digitimer) and monitored on a Tektronix 7313 oscilloscope. Single unit activity, digitized by a window discriminator (Spike Trigger, NL201), was counted during set intervals (0.2-10 s) and displayed as integrated activity on a Grass Polygraph. The original unit activity was continuously recorded, together with A P and end-expiratory CO2, using a C R C VR-100A Digital Data Recorder (Instrutech Corp., New York, USA) and video casette recorder. Responses to nerve stimulation were assessed with peristimulus time histograms, using an ITC16 interface (Instrutech Corp., New York, USA) and a Macintosh Ilfx computer programmed with I G O R (WaveMetrics, Lake Oswago, USA). M a x i m u m data acquisition resolution was 0.1 ms. Only neurons which passed a collision test z2 after antidromic activation were used for further study.
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Histology At the end of most experiments small electrolytic lesions were made to localize the position of the stimulating (Fig. 1A) and recording electrodes (Fig. 1B). A catheter was placed in the ascend-
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184 SINGLEPULSE
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Fig. 3. Peristimulus time histograms showing responses to stimulation of abdominal vagus, central ear nerve and branches of sciatic nerve (100 sweeps, bin width 0.5 ms). Parameters of stimulation were 800/zA, 0.5 ms, 1 pulse (left side) or 3 pulses (right side).
sions made by tip of the electrode were located between 2 m m caudal and 1 m m rostral to the obex. The F A G L U fluorescence procedure verified that the lesions were located in the vicinity of A1 catecholamine cells (Fig. 1B). Lesions made by the stimulating electrode in the hypothalamus were located within or close to the supraoptic nucleus (Fig. 1A)
sponses consisted of either a single spike or a burst of 3 - 4 spikes. Increasing the intensity of the stimulus, or increasing the number of pulses from 1 to 3, increased the number of spikes (Fig. 3). Excitation was sometimes followed by inhibition, lasting approximately 130 ms (Fig. 3). Four of 5 tested neurons were activated by stimulating either the anterior or the posterior abdominal vagal trunk (Fig. 4). In one rabbit the effect of electrically stimulating the left cervical vagus was tested. This procedure excited 3 of 4 neurons tested with latency 102 _+ 7 ms (distance 65 mm, conduction velocity 0.6 m / s ) , the other being unaffected. In one rabbit in which we identified A1 area neurons excited by electrical stimulation of the abdominal vagus, subsequent sectioning of the vagus low in the neck completely abolished the excitatory effect of the stimulation even with current as high as 2 mA. Nine neurons, all excited by single pulses to the abdominal vagal trunk, were tested by trains of pulses
Responses to stimulation of the abdominal vagus nerve, renal nerve, and to gastric distension Responses to electrical stimulation of the abdominal vagus nerve of 85 antidromically identified neurons were examined with peristimulus time histograms. Sixty four neurons (75%) were excited with onset latency of 195 _+ 25 ms (distance 130 mm, conduction velocity 0.7 m / s ) . The remaining 25% of neurons were unaffected even with the current as high as 1.5 mA. No cells were inhibited. The threshold current for the effect on A1 area neurons was 200-500 /xA. The excitatory re40-
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Fig. 4. Peristimulus time histograms of the activity of A1 area neuron antidromically activated from the supraoptic nucleus, showing response to stimulation of either anterior or posterior abdominal vagal trunks (200 sweeps, bin width 5 ms). Parameters of stimulation were 800 izA, 0.5 ms, 3 pulses at 200 Hz.
185 C
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Fig. 5. Polygraph traces illustrating arterial pressure, heart rate and discharge rate of A1 area neuron in response to intravenous injection of phenylephrine (A), ventilation of rabbit with hypoxic gas mixture (B) or stimulation of anterior abdominal vagus (C; train of cathodal pulses; 0.5 ms duration, 50 Hz, 500 ~A).
at the same site (10 s train, 50 Hz, 0.5 ms duration, 200-500 /zA). U n d e r these conditions only 3 of the neurons were excited, and the brief excitation was followed by inhibition lasting for the duration of the pulse train (Fig. 5C). T h r e e were unaffected and 3 were inhibited. Single or triple pulse stimulation of the renal nerve had no effect on the discharge rate of 4 antidromically activated cells previously shown to have an excitatory response to stimulation of the abdominal vagus. We studied the effect of gastric distention on 24 A1 area neurons, 18 of which were tested by stimulation of the abdominal vagus, with 13 being excited. Acute gastric distension had no effect on the discharge rate of 20 of these 24 neurons (83%), even when sufficient air was injected to grossly inflate the stomach, as determined by abdominal palpation. One neuron was briefly excited, and 3 neurons (13%) were inhibited. There was no relationship between response to gastric distention and effect of vagal stimulation. Distention of the stomach usually caused a small increase in AP.
Responses to electrical and physiological stimulation of somatic nerves Responses to electrical stimulation of the branches of the sciatic nerve of 17 antidromically identified neurons were examined with peristimulus time histograms. Seven neurons (41%) were inhibited (latency 56 + 6 ms, distance 40 cm, conduction velocity 7.1 m / s ) , for up to 360 ms (Fig. 3), with the cumulative sum diverging by more than 2 standard deviations from the control values. Six neurons (35%) were unaffected and 4 neurons (24%) were excited (latency 35 ms,
conduction velocity 11.4 m / s ) . All these neurons were excited by stimulation of the abdominal vagus nerve. Responses of 17 A1 area neurons to electrical stimulation of the central ear nerve were examined with peristimulus time histograms. Four neurons (24%) were inhibited (latency 43 + 7 ms, distance 7 cm, conduction velocity 1.6 m / s ) , for 100 + 29 ms (Fig. 3), with the cumulative sum diverging by more than 2 standard deviations from the control values. Seven neurons (41%) were unaffected and the remaining 6 (35%) were excited (latency 26 + 9 ms, conduction velocity 2.7 m / s ; Fig. 3). Three of these 6 excited neurons showed a second peak of excitation with a longer latency (207 _+ 7 ms, conduction velocity 0.34 m / s ) . All these neurons were excited by stimulation of the abdominal vagus nerve. Light touch over the body had no effect on 45 of 49 neurons (92%). T h r e e neurons were inhibited and 1 was excited. Forceful pinching of the skin, either an ear or a tiindlimb paw, had no effect on 44 of these neurons (90%). Five neurons (10%) were excited.
Baroreceptor, chemoreceptor and somatic responses of neurons tested by abdominal vagal stimulation Thirty six neurons excited by stimulation of the abdominal vagus were tested by increasing AP with phenylephrine. All these neurons were inhibited. Their discharge rate decreased from 4.4 + 0.2 to 0.6 + 0.2 s p i k e s / s ( P < 0.01; Fig. 5). Of 33 neurons excited by stimulation of the abdominal vagus, 26 were excited by hypoxic stimulation (from 4 _+ 0.3 to 10 _+ 1.1 spikes/s, P < 0.01), 4 were inhibited and 3 were unaffected. Of 36 neurons excited by abdominal vagal stimulation, 35 were unaffected by light touch over the body or by painful pinching of the hindlimb or the ear. Eleven neurons unaffected by abdominal vagal stimulation were tested by increasing AP with phenylephrine. Nine neurons were inhibited, 1 was excited and 1 was unaffected. Of 5 neurons unaffected by abdominal vagal stimulation, 4 were inhibited by hypoxic stimulus and 1 was excited. Of 7 neurons unaffected by abdominal vagal stimulation, 5 were unaffected by light touch over the body and 2 were inhibited. Of 7 neurons unaffected by abdominal vagal stimulation, 3 were unaffected by painful pinching, and 4 were excited. DISCUSSION Peristimulus time histograms indicated that electrical stimulation of the afferent abdominal vagus powerfully excited 75% of 85 neurons identified by collision test following antidromic activation from the region of the supraoptic nucleus. The spontaneous discharge rate
186 of these neurons was approximately 4 spikes/s, so that excitation from the abdominal vagus was obvious in peristimulus histograms. The excitation latency was approximately 200 ms, indicating a vagal fiber conduction velocity of approximately 0.7 m / s , consistent with C fiber mediation of the excitation. No A1 area neurons were inhibited by stimulation of the abdominal vagus. Although excitation of A1 area neurons was obvious in the peristimulus time histogram studies, it was difficult to document sustained increases in discharge rates in response to trains of electrical stimuli. This may partially reflect post-excitation inhibition which lasted for approximately 200 ms after each neuronal discharge. In addition, trains of stimuli (from 5 to 100 Hz) caused a rapid and sustained increase in AP, as previously reported following stimulation of the abdominal vagus 7. In the present study we found that more than 90% of A1 area neurons (antidromically identified from the region of the supraoptic nucleus) were inhibited by increases in AP, as previously demonstrated 17,21. We suspect that in the present experiments the rise in AP from abdominal vagal stimulation caused baroreceptor-mediated inhibition of A1 cells, thereby counteracting the direct excitatory effect, resulting in the biphasic change in discharge rate illustrated in Fig. 5C. Whether the same or different vagal afferents mediated the rise in AP and the directly mediated increase in A1 neuronal discharge rate is not known. Electrophysiological characteristics of recorded cells and their responses to baroreceptor and chemoreceptor stimulation were generally in agreement with a study of A1 area neurons in the rat ~7, and with previous results in the rabbit from our laboratory 21. We found that individual A1 area neurons excited from the abdominal vagus also receive inputs originating in baroreceptors and chemoreceptors, suggesting that individual cells are affected by varied physiological stimuli. Catecholamine fluorescence examination showed the recording electrode in the A1 cell group. About 8 0 90% of caudal ventrolateral medullary neurons with projections to the hypothalamus belong to this group and the A1 neurons innervate vasopressin-secreting neurons rather than oxytocin-secreting neurons 3'8'28'3~. Obviously some recordings could have been from nonA1 neurons. Our stimulating electrode could also activate A1 fibers passing near the supraoptic nucleus en route to regions such as the median preoptic area and the amygdala. At present it is not known whether the same A1 neurons projects to multiple hypothalamic and basal forebrain sites. Electrical stimulation of the central end of the cut
renal nerve did not affect the discharge of A1 area neurons. Other studies have suggested a relationship between renal afferents and vasopressin secretion 5'1°'37. Our finding suggests that the relevant central pathway does not involve neurons in the A1 area. Stimulation of the central end of a major trunk of the sciatic nerve produced variable effects on A1 area neurons, and no excitation was observed in the C fiber conduction velocity range. Variable effects were also observed with stimulation of the central ear nerve. In about 30% of cases, there was C fiber-mediated excitation but the response was never as vigorous as we observed with abdominal vagal stimulation. No consistent excitation of A1 area neurons was observed when the rabbit's fur was lightly rubbed, or when a paw or an ear was forcefully squeezed. In the rat, approximately 50% of vasopressin-secreting cells in the supraoptic nucleus were reported to be excited by pain, and by stimulation of the sciatic nerve TM. On the basis of GABA-induced blockade of the A1 area, Day and Sibbald 1~ concluded that the rat A1 cell group is involved in transmitting pain stimuli to the hypothalamic neurosecretory vasopressin cells. Our study does not support this conclusion in the rabbit. Whether painful stimulation causes secretion of vasopressin in unanesthetized animals is a controversial issue 2"16'19"2°. Abdominal vagal afferents are activated by gastric distention and by release of cholecystokinin from the upper intestine 26'27'32. These and other gastrointestinal events may cause secretion of vasopressin or oxytocin (depending on the species) via vagal afferents 15'23'29'32. The present finding that electrical activation of abdominal vagal afferents excites A1 area neurons in the rabbit suggests that similar gastrointestinal stimuli might cause secretion of vasopressin. Indeed, recently we found that electrical stimulation of the abdominal vagus markedly increases plasma vasopressin in the rabbit (Gieroba and Blessing, unpublished data). In view of the vigorous effects of gastric distention on hypothalamic neuronal discharge rates in the rat 29'34, we were surprized that this stimulus did not markedly affect the discharge of A1 area neurons in our study. Stimulation of hepatic osmoreceptors may produce secretion of vasopressin, probably via vagal afferents, but possibly via a spinal pathway ~'6'1s'35. Such a vagal pathway could be physiologically relevant to our present results. In conclusion, our electrophysiological results indicate that A1 area neurons, with projections to the region of the supraoptic nucleus, receive powerful excitatory inputs from the abdominal vagus nerve. The visceral information normally transmitted to A1 cells by these abdominal vagal afferents is not yet deter-
187 mined, but acute gastric distention does not appear to be the relevant physiological stimulus. Acknowledgements. Our study was supported by the National Health and Medical Research Council and by the National Heart Foundation of Australia. Mr. Adam Steer provided technical assistance.
REFERENCES 1 Adachi, A., Niijima, A. and Jacobs, H.L., An hepatic osmoreceptor mechanism in the rat: electrophysiological and behavioral studies, Am. J. Physiol., 231 (1976) 1043-1049. 2 Anderson, I.D., Forsling, M.L., Little, R.A. and Pyman, J.A., Acute injury is a potent stimulus for vasopressin release in man, J. Physiol., 416 (1989) 28P. 3 Blessing, W.W., Jaeger, C.B., Ruggiero, D.A. and Reis, D.J., Hypothalamic projections of medullary catecholamine neurons in the rabbit: a combined cateeholamine fluorescence and HRP transport study, Brain Res. Bull., 9 (1982) 279-286. 4 Blessing, W.W. and Willoughby, J.O., Inhibiting the rabbit caudal ventrolateral medulla prevents baroreceptor-initiated secretion of vasopressin, J. PhysioL, 367 (1985) 253-265. 5 Caverson, M.M. and Ciriello, J., Effect of stimulation of afferent renal nerves on plasma levels of vasopressin, Am. J. Physiol., 252 (1987) R801-R807. 6 Chwalbinska-Moneta, J., Role of hepatic portal osmoreception in the control of ADH release, Am. J. PhysioL, 236 (1979) E603E609. 7 Cragg, B.G. and Evans, D.H.L., Some reflexes mediated by the afferent fibers of the abdominal vagus in the rabbit and cat, Exp. Neurol., 2 (1960) 1-12. 8 Cunningham, E.T. JR. and Sawchenko, P.E., Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei of the rat hypothalamus, J. Comp Neurol., 274 (1988) 60-76. 9 Davey, N.J., Ellaway, P.H. and Stein, R.B., Statistical limits for detecting change in the cumulative sum derivative of the peristimulus time histogram, J. Neurosci. Methods, 17 (1986) 153-166. 10 Day, T.A. and Ciriello, J., Afferent renal nerve stimulation excites supraoptic vasopressin neurons, Am. J. Physiol., 249 (1985) R368-R371. 11 Day, T.A. and Sibbald, J.R., Noxious somatic stimuli excite neurosecretory vasopressin cells via A1 cell group, Am. J. PhysioL, 258 (1990) R1516-R1520. 12 Furness, J.B., Costa, M. and Blessing, W.W., Simultaneous fixation and production of catecholamine fluorescence in central nervous tissue by perfusion with aldehydes, Histochem. J., 9 (1977) 745-750. 13 Gieroba, Z.J., Fullerton, M.J., Funder, J.W. and Blessing, W.W., Medullary pathways for adrenocorticotropic hormone and vasopressin secretion in rabbits, Am. J. PhysioL, 262 (1992) R1047R1056. 14 Hamamura, M., Shibuki, K. and Yagi, K., Noxious inputs to supraoptic neurosecretory cells in the rat, Neurosci. Res., 2 (1984) 49-61. 15 Hawthorn, J., Andrews, P.L.R., Ang, V.T.Y. and Jenkins, J.S., Differential release of vasopressin and oxytocin in response to abdominal vagal afferent stimulation or apomorphine in the ferret, Brain Res., 438 (1988) 193-198. 16 Husain, M.K., Manger, W.M., Rock, T.W., Weiss, R.J. and Frantz, A.G., Vasopressin release due to manual restraint in the rat: role of body compression and comparison with other stressful stimuli, Endocrinology, 104 (1979) 641-644.
17 Kannan, H., Osaka, T., Kasai, M., Okuya, S. and Yamashita, H., Electrophysiological properties of neurons in the caudal ventrolateral medulla projecting to the paraventricular nucleus of the hypothalamus in rats, Brain Res., 376 (1986) 342-350. 18 King, M.S. and Baertschi, A.J., Central neural pathway mediating splachnic osmosensation, Brain Res., 550 (1991) 268-278. 19 Knepel, W., Nutto, D. and Hertting, G., Evidence for inhibition by/3-endorphin of vasopressin release during foot shock-induced stress in the rat, Neuroendocrinology, 34 (1982) 353-356. 20 Lang, R.E., Heil, J.W.E., Ganten, D., Hermann, K., Unger, T. and Rascher, W., Oxytocin unlike vasopressin is a stress hormone in the rat, Neuroendocrinology, 37 (1983) 314-316. 21 Li, Y.W., Gieroba, Z.J. and Blessing, W.W., Chemoreceptor and baroreceptor responses of A1 area neurons projecting to supraoptic nucleus, Am. J. PhysioL, 263 (1992) R310-R317. 22 Lipski, J., Antidromic activation of neurons as an analytic tool in the study of the central nervous system, J. Neurosci. Methods, 4 (1981) 1-32. 23 Miaskiewicz, S.L., Stricker, E.M. and Verbalis, J.G., Neurohypophyseal secretion in response to cholecystokinin but not mealinduced gastric distension in humans, J. Clin. Endocrinol. Metab., 68 (1989) 837-843. 24 Miller, F.R., On gastric sensation, J. Physiol., 41 (1910) 409-415. 25 Miller, A.D. and Nonaka, S., Mechanisms of vomiting induced by serotonin-3 receptor agonists in the cat: effect of vagotomy, splanchnicectomy or area postrema lesion, J. Pharmacol. Exp. Ther., 260 (1992) 509-517. 26 Moran, T.H., Smith, G.P., Hostetler, A.M. and McHugh, P.R., Transport of cholecystokinin (CCK) binding sites in subdiaphragmatic vagal branches, Brain Res., 415 (1987) 149-152. 27 Paintal, A.S., A study of gastric stretch receptors. Their role in the peripheral mechanism of satiation of hunger and thirst, J. Physiol., 126 (1954) 255-270. 28 Raby, W.N. and Renaud, L.P., Dorsomedial medulla stimulation activates rat supraoptic oxytocin and vasopressin neurones through different pathways, J. Physiol., 417 (1989) 279-294. 29 Renaud, L.P., Tang, M., McCann, M.J., Stricker, E.M. and Verbalis, J.G., Cholecystokinin and gastric distention activate oxytocinergic cells in rat hypothalamus, Am. J. Physiol., 253 (1987) R661-R665. 30 Rowe, J.W., Shelton, R.L., Helderman, J.H., Vestal, R.E. and Robertson, G.L., Influence of the emetic reflex on vasopressin release in man, Kidney Int., 16 (1979) 729-735. 31 Sawchenko, P.E. and Swanson, L.W., Central noradrenergic pathways for the integration of hypothalamic neuroendocrine and autonomic responses, Science, 214 (1981) 685-687. 32 Smith, G.P., Jerome, C. and Norgren, R., Afferent axons in abdominal vagus mediate satiety effect of cholecystokinin in rats, Am. J. Physiol., 249 (1985) R638-R641. 33 Ter Horst, G.J., De Boer, P., Luiten, P.G.M. and Van Willigen, J.D., Ascending projections from the solitary tract nucleus to the hypothalamus. A Phaseolus vulgaris lectin tracing study in the rat, Neuroscience, 31 (1989) 785-797. 34 Ueta, Y., Kannan, H. and Yamashita, H., Gastric afferents to the paraventricular nucleus in the rat, Exp. Brain Res., 84 (1991) 487-494. 35 Vallet, P.G. and Baertschi, A.J., Spinal afferents for peripheral osmoreceptors in the rat, Brain Res., 239 (1982) 271-274. 36 Wang, S.C. and Borison, H.L., Copper sulphate emesis: a study of afferent pathways from the gastrointestinal tract, Am. J. Physiol., 164 (1951) 520-526. 37 Yamamoto, A., Keil, L.C. and Reid, I.A., Effect of intrarenal bradykinin infusion on vasopressin release in rabbits, Hypertension, 19 (1992) 799-803.
Brain Research, 616 (1993) 181-187 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 18992
Abdominal vagal afferents excite A1 area neurons antidromically activated from the region of the supraoptic nucleus in the rabbit Z.J. G i e r o b a a n d W . W . B l e s s i n g Departments of Medicine and Physiology, Centre for Neuroscience, Flinders University, Bedford Park, SA (Australia) (Accepted 2 February 1993)
Key words: A1 catecholamine-synthesizingneuron; Abdominal vagus; Vasopressin; Ventrolateral medulla; Electrophysiology
We made extracellular recordings from 113 spontaneously active neurons in the A1 area, after identifying the cells by antidromically activating them from the region of the supraoptic nucleus in urethane-anesthetized rabbits. We tested the response of these neurons to inputs from abdominal vagal, renal and somatic nerves. Electrical stimulation of the abdominal vagus nerve activated 64/85 neurons tested (75%), and had no effect on the remaining 25%. Latency was 195_+25 ms, (conduction velocity 0.7 m / s ) . Stimulation of renal afferents had no effect on the discharge rate of 4 neurons tested. Stimulation of branches of the sciatic nerve inhibited 7/17 A1 area neurons tested, excited 4 and had no effect on 6 neurons. Stimulation of the central ear nerve inhibited 4 / 1 7 neurons tested, excited 6 and had no effect on 7 neurons. Gastric distension had no effect on 20/24 neurons tested. Lightly touching the animals back and legs had no effect on the discharge of 45/49 neurons tested. Similarly, painful stimuli failed to affect 44/49 neurons tested. Our results indicate that A1 area neurons, with projections to the region of the supraoptic nucleus, receive excitatory inputs from the abdominal vagus nerve. The visceral information transmitted to A1 cells by these abdominal vagal afferents is not yet determined, but acute gastric distention does not appear to be a physiological stimulus. A1 area neurons seem not to be involved in transmitting somatic information to the hypothalamus.
INTRODUCTION
Plasma levels of vasopressin or oxytocin are elevated after systemic administration of a number of agents whose action is dependent on the integrity of the afferent abdominal vagus nerve 15'23. In certain species, these agents cause nausea and vomiting, also by an action mediated by the afferent abdominal vagus 24'25'36, and in humans the sensation of nausea may be accompanied by elevation of plasma vasopressin 3°. Electrical stimulation of the afferent abdominal vagus causes release of either vasopressin or oxytocin, as well as inducing emesis in species capable of this response 15. The discharge rate of the vasopressin- or oxytocinsecreting magnocellular hypothalamic neurons is also affected by vagally mediated abdominal stimuli 29'34. Since vagal afferents terminate in the nucleus tractus solitarius (nTS), the central pathway to the relevant hypothalamic neurons must originate in this nucleus. Rat nTS neurons project directly to oxytocin-secreting magnocellular hypothalamic cells 8'28'33, but the func-
tional pathway to vasopressin-secreting neurons is likely to be indirect because nTS neurons do not project directly to vasopressin-secreting hypothalamic cells. In the case of vasopressin secretion in response to hemorrhage, baroreceptor-derived information is relayed from the nTS to the A1 noradrenaline-synthesizing neurons in the caudal ventroiateral medulla, and then to the hypothalamus4,13'21. In the present electrophysiological study, we determined whether rabbit A1 area neurons, antidromically activated from the region of the supraoptic nucleus, alter their discharge rate in response to electrical stimulation of the afferent abdominal vagus. Since gastric distention alters the activity of magnocellular neurons z9'34, we determined whether this more physiological stimulus could reproduce the effects on the discharge of the A1 area neurons of electrical stimulation of the abdominal vagus. We also determined whether neurons which respond to afferent vagal stimulation also respond to baroreceptor and chemoreceptor inputs.
Correspondence: Z.J. Gieroba, Department of Medicine, Flinders Medical Centre, Bedford Park, SA 5042, Australia. Fax: (61) (8) 2045450.
182 Magnocellular
vasopressin-secreting neurons,
mmHg. Arterial pCO2, p O 2 and pH were measured using a pH/Blood Gas Analyser 813 (Instrumentation Laboratory, USA), and resting values were pH 7.35-7.45, pO 2 250-350 mmHg (ventilation with O2-enriched air), pCO 2 35-40 mmHg. Body temperature was monitored by a rectal thermistor and maintained at 38-39°C by a heating pad. The trachea was cannulated, the animal was paralyzed with pancuronium bromide (0.5 mg/kg, i.v. initially, with supplemental doses as necessary) and artificially ventilated with oxygen-enriched air using a Harvard model 681 rodent ventilator. A bilateral pneumothorax was induced. The head of the rabbit was fixed in a Kopf stereotaxic frame. The medulla was exposed by incision and retraction of the atlanto-occipital membrane. Neck flexion was adjusted so that the dorsal surface of the medulla was horizontal. The rostral edge of the area postrema in the midline (obex) served as the stereotaxic zero for rostro-caudal and medio-lateral coordinates.
and
s e c r e t i o n of v a s o p r e s s i n , may b e a f f e c t e d by s o m a t i c a f f e r e n t s 14, p o s s i b l y a c t i n g via t h e A 1 r e g i o n n , a n d b y o t h e r v i s c e r a l a f f e r e n t s , i n c l u d i n g s o m e w h i c h m a y act through
spinal
p a t h w a y s 5"m'35'37. W e
therefore
also
tested the effect on discharge of A1 area neurons of e l e c t r i c a l s t i m u l a t i o n o f s o m a t i c a n d r e n a l a f f e r e n t s , as w e l l as t h e e f f e c t o f l i g h t t o u c h a n d p a i n s t i m u l i . MATERIALS AND METHODS
General procedures Experiments were performed on 30 male New Zealand White rabbits (2.5-3.0 kg), anesthetised with urethane (1.5 g/kg, infused into a marginal ear vein over 30 min). Scopolamine methylbromide (50/zg/kg, Sigma Chemical Co, USA) was given to minimize airway secretions. Catheters were inserted into the marginal ear vein for administering drugs, and into the left femoral artery for recording arterial pressure (AP) and for arterial blood gas analysis. A Statham P23ID transducer connected to a Grass Model 7 polygraph was used to record AP. Mean AP was obtained by filtering the phasic signal. Heart rate was measured with a Grass 7P4G tachograph, triggered by the phasic arterial signal. End-expiratory CO 2 was monitored (Datex Normocap CO 2 monitor, Finland) and maintained at 35-40
Electrical stimulation of the supraoptic nucleus A burr hole was drilled in the parietal bone of the skull, 2 mm rostral to bregma. A monopolar stainless steel stimulating electrode (0.5 mm bare tip) was lowered perpendicular to a line passing through lambda and 1.5 mm above bregma. The tip of the electrode was positioned 2 mm rostral to bregma, 2 mm lateral to the midline and 12.0-13.5 mm below the cerebral surface. The optic tract field potential was evoked by flash stimulation of the contralateral eye, and the electrode was retracted until the potential almost disappeared, indicating that the tip was in or close to the supraoptic nucleus. The stimulus intensity was then set at 0.5-1.2 mA (0.5 ms duration, 1 Hz) for antidromic activation of neurons in the A1 area.
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T i m e (ms) Fig. 1. A: diagram showing positions of stimulating electrode tips, marked by lesions (dots), in and near the supraoptic nucleus, f, fornix; ic internal capsule; ot, optic tract; ox, optic chiasm; SON, supraoptic nucleus. B: photomicrograph of the lesion made by the tip of recording electrode in the vincinity of A1 neurons (FAGLU-fluorescence method). Bar = 2 mm (A) and 120 ~m (B). C,D: collision test. Each trace is a computer average of 20 sweeps triggered by the spontaneous spike; C: supraoptic nucleus stimulus (at arrow) was delivered just greater after the critical delay and produced a constant latency spike response (marked by star) on every occasion; D: supraoptic nucleus stimulus was delivered just before the critical delay, and the antidromic spike response was always cancelled; E: computer average of 20 superimposed sweeps of a negative-positive-going spike (marked by star in C) of the antidromically activated neuron.
183 Electrical stimulation of the abdominal vagus, renal, sciatic and central ear nerues The anterior vagal trunk was dissected at the junction of the oesophagus and stomach, either from above or below the diaphragm. The intact nerve was placed inside a bipolar silver wire cuff electrode, constructed from teflon-coated silver wire and silastic tubing. The tubing was filled with paraffin oil, and surrounded with low melting point wax (BDH Chemicals, Poole, U K ) to insulate the exposed electrode wire tips from tissue fluids. In 1 rabbit, both anterior and posterior vagal trunks were positioned in separate electrodes and separately stimulated. In one rabbit the stimulating hook electrode was placed on the left cervical vagus low in the neck. In a different rabbit the vagi were cut low in the neck after testing the effect of stimulation of the abdominal vagus on an antidromically activated A1 area neuron. In another rabbit, the central ends of all nerves entering the hilus of the kidney were isolated from a retroperitoneal approach, sectioned close to the kidney and protected in paraffin oil. T h e central cut ends of the nerves were placed on a bipolar stainless steel hook electrode. In 3 other rabbits the left sciatic nerve was isolated. The branches were dissected free, cut distally, and positioned in a silver wire cuff electrode. In the same rabbits the left central ear nerve was isolated, cut distally and its central end was positioned in a cuff electrode. Nerves were stimulated by 1 - 3 cathodal pulses (0.5 ms, 200 Hz, 500-1000 p,A). In some experiments, for electrical stimulation of the abdominal vagus, repetitive stimulation was used (10 s train of cathodal pulses, 0.5 ms duration, 50 Hz, 200-600 tzA).
Stomach distension and physiological activation of somatic afferents The stomach was first emptied by an orogastric tube. A latex balloon was connected to a silastic tube and inserted through the oesophagus to the stomach. T h e stomach was distended by injecting 50-100 ml of air within 5 s. Distension was maintained for 30 s. T h e degree of distention was judged by observing and palpating the upper abdomen, to determine the size and firmness of the stomach. For physiological activation of somatic afferent nerves the body was lightly touched for 10 s, or either the hindlimb or the ear was firmly pinched for 5 s with a pair of long-nosed pliers.
Activation o f baroreceptors and chemoreceptors Baroreceptor activity was altered by raising AP by 50-75 m m H g (phenylephrine hydrochloride 50 t z g / k g i.v., Sigma Chemical Co., USA). Chemoreceptors were stimulated by ventilating rabbits for 30-45 s with 10% 0 2 in N 2.
ing aorta and the brain was fixed by perfusion with f o r m a l d e h y d e / glutaraldehyde solution. The brain was removed and 50 ~ m transverse sections were cut on a Vibratome. Sections of the hypothalamus were stained with Perl Prussian blue for demonstration of the position of the tip of the stimulating electrode. The f o r m a l d e h y d e / glutaraldehyde ( F A G L U ) fluorescence histochemical procedure I2 was used to relate recording sites, marked with a lesion, to the A1 group of catecholamine-containing neurons. In some cases, the same sections were rehydrated, stained with Neutral red and coverslipped.
Stat&tical analysis Base-line values of AP, heart rate and unit discharge rate, and changes in these values caused by different procedures were recorded. Data were expressed as mean_+ S.E.M. and changes in values were examined using a paired student t-test. For peristimulus time histograms, changes in discharge probability and duration of effect were analyzed according to Davey et al. 9.
RESULTS
Identification of A1 area neurons antidromically activated from the supraoptic nucleus Approximately 30% of antidromically activated neurons did not discharge spontaneously so that no collision test was possible. These neurons were excluded from further analysis. Recordings were made from 113 neurons which passed the collision test (Fig. 1C,D). The threshold stimulus intensity ranged between 100 and 1000 ~A. The amplitude of the action potential ranged between 150 and 1200/zV (mean 425 _+30/zV). The distance between stimulating and recording electrodes was approximately 24 mm (axonal conduction velocity 0.5-4.8 m/s, median 0.75 m/s, Fig. 2). The frequency of spontaneous action potentials ranged from 0.5 to 20 spikes/s (median 4 spikes/s). Histological determination of recording sites revealed that the le-
Extracellular recording in the A1 area Recordings from neurons in the A1 area were made by inserting a recording electrode vertically into the medulla, from 1 m m rostral to 2 m m caudal to the obex, 3-3.5 m m lateral from the midline and 3-4.5 m m ventral from the dorsal surface of the medulla. Glasscoated tungsten electrodes were used. Unit activities were recorded with an N T l l 4 A differential amplifier (Neomedix), filtered (bandpass 100-5000 Hz), amplified using a NL106 amplifier (Digitimer) and monitored on a Tektronix 7313 oscilloscope. Single unit activity, digitized by a window discriminator (Spike Trigger, NL201), was counted during set intervals (0.2-10 s) and displayed as integrated activity on a Grass Polygraph. The original unit activity was continuously recorded, together with A P and end-expiratory CO2, using a C R C VR-100A Digital Data Recorder (Instrutech Corp., New York, USA) and video casette recorder. Responses to nerve stimulation were assessed with peristimulus time histograms, using an ITC16 interface (Instrutech Corp., New York, USA) and a Macintosh Ilfx computer programmed with I G O R (WaveMetrics, Lake Oswago, USA). M a x i m u m data acquisition resolution was 0.1 ms. Only neurons which passed a collision test z2 after antidromic activation were used for further study.
20M e d i a n 0.75 m / s n = 113 16
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Histology At the end of most experiments small electrolytic lesions were made to localize the position of the stimulating (Fig. 1A) and recording electrodes (Fig. 1B). A catheter was placed in the ascend-
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Conduction velocity (m/s) Fig. 2. Histogram of axonal conduction velocities of 113 collision test-positive neurons.
184 SINGLEPULSE
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sions made by tip of the electrode were located between 2 m m caudal and 1 m m rostral to the obex. The F A G L U fluorescence procedure verified that the lesions were located in the vicinity of A1 catecholamine cells (Fig. 1B). Lesions made by the stimulating electrode in the hypothalamus were located within or close to the supraoptic nucleus (Fig. 1A)
sponses consisted of either a single spike or a burst of 3 - 4 spikes. Increasing the intensity of the stimulus, or increasing the number of pulses from 1 to 3, increased the number of spikes (Fig. 3). Excitation was sometimes followed by inhibition, lasting approximately 130 ms (Fig. 3). Four of 5 tested neurons were activated by stimulating either the anterior or the posterior abdominal vagal trunk (Fig. 4). In one rabbit the effect of electrically stimulating the left cervical vagus was tested. This procedure excited 3 of 4 neurons tested with latency 102 _+ 7 ms (distance 65 mm, conduction velocity 0.6 m / s ) , the other being unaffected. In one rabbit in which we identified A1 area neurons excited by electrical stimulation of the abdominal vagus, subsequent sectioning of the vagus low in the neck completely abolished the excitatory effect of the stimulation even with current as high as 2 mA. Nine neurons, all excited by single pulses to the abdominal vagal trunk, were tested by trains of pulses
Responses to stimulation of the abdominal vagus nerve, renal nerve, and to gastric distension Responses to electrical stimulation of the abdominal vagus nerve of 85 antidromically identified neurons were examined with peristimulus time histograms. Sixty four neurons (75%) were excited with onset latency of 195 _+ 25 ms (distance 130 mm, conduction velocity 0.7 m / s ) . The remaining 25% of neurons were unaffected even with the current as high as 1.5 mA. No cells were inhibited. The threshold current for the effect on A1 area neurons was 200-500 /xA. The excitatory re40-
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Fig. 4. Peristimulus time histograms of the activity of A1 area neuron antidromically activated from the supraoptic nucleus, showing response to stimulation of either anterior or posterior abdominal vagal trunks (200 sweeps, bin width 5 ms). Parameters of stimulation were 800 izA, 0.5 ms, 3 pulses at 200 Hz.
185 C
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Fig. 5. Polygraph traces illustrating arterial pressure, heart rate and discharge rate of A1 area neuron in response to intravenous injection of phenylephrine (A), ventilation of rabbit with hypoxic gas mixture (B) or stimulation of anterior abdominal vagus (C; train of cathodal pulses; 0.5 ms duration, 50 Hz, 500 ~A).
at the same site (10 s train, 50 Hz, 0.5 ms duration, 200-500 /zA). U n d e r these conditions only 3 of the neurons were excited, and the brief excitation was followed by inhibition lasting for the duration of the pulse train (Fig. 5C). T h r e e were unaffected and 3 were inhibited. Single or triple pulse stimulation of the renal nerve had no effect on the discharge rate of 4 antidromically activated cells previously shown to have an excitatory response to stimulation of the abdominal vagus. We studied the effect of gastric distention on 24 A1 area neurons, 18 of which were tested by stimulation of the abdominal vagus, with 13 being excited. Acute gastric distension had no effect on the discharge rate of 20 of these 24 neurons (83%), even when sufficient air was injected to grossly inflate the stomach, as determined by abdominal palpation. One neuron was briefly excited, and 3 neurons (13%) were inhibited. There was no relationship between response to gastric distention and effect of vagal stimulation. Distention of the stomach usually caused a small increase in AP.
Responses to electrical and physiological stimulation of somatic nerves Responses to electrical stimulation of the branches of the sciatic nerve of 17 antidromically identified neurons were examined with peristimulus time histograms. Seven neurons (41%) were inhibited (latency 56 + 6 ms, distance 40 cm, conduction velocity 7.1 m / s ) , for up to 360 ms (Fig. 3), with the cumulative sum diverging by more than 2 standard deviations from the control values. Six neurons (35%) were unaffected and 4 neurons (24%) were excited (latency 35 ms,
conduction velocity 11.4 m / s ) . All these neurons were excited by stimulation of the abdominal vagus nerve. Responses of 17 A1 area neurons to electrical stimulation of the central ear nerve were examined with peristimulus time histograms. Four neurons (24%) were inhibited (latency 43 + 7 ms, distance 7 cm, conduction velocity 1.6 m / s ) , for 100 + 29 ms (Fig. 3), with the cumulative sum diverging by more than 2 standard deviations from the control values. Seven neurons (41%) were unaffected and the remaining 6 (35%) were excited (latency 26 + 9 ms, conduction velocity 2.7 m / s ; Fig. 3). Three of these 6 excited neurons showed a second peak of excitation with a longer latency (207 _+ 7 ms, conduction velocity 0.34 m / s ) . All these neurons were excited by stimulation of the abdominal vagus nerve. Light touch over the body had no effect on 45 of 49 neurons (92%). T h r e e neurons were inhibited and 1 was excited. Forceful pinching of the skin, either an ear or a tiindlimb paw, had no effect on 44 of these neurons (90%). Five neurons (10%) were excited.
Baroreceptor, chemoreceptor and somatic responses of neurons tested by abdominal vagal stimulation Thirty six neurons excited by stimulation of the abdominal vagus were tested by increasing AP with phenylephrine. All these neurons were inhibited. Their discharge rate decreased from 4.4 + 0.2 to 0.6 + 0.2 s p i k e s / s ( P < 0.01; Fig. 5). Of 33 neurons excited by stimulation of the abdominal vagus, 26 were excited by hypoxic stimulation (from 4 _+ 0.3 to 10 _+ 1.1 spikes/s, P < 0.01), 4 were inhibited and 3 were unaffected. Of 36 neurons excited by abdominal vagal stimulation, 35 were unaffected by light touch over the body or by painful pinching of the hindlimb or the ear. Eleven neurons unaffected by abdominal vagal stimulation were tested by increasing AP with phenylephrine. Nine neurons were inhibited, 1 was excited and 1 was unaffected. Of 5 neurons unaffected by abdominal vagal stimulation, 4 were inhibited by hypoxic stimulus and 1 was excited. Of 7 neurons unaffected by abdominal vagal stimulation, 5 were unaffected by light touch over the body and 2 were inhibited. Of 7 neurons unaffected by abdominal vagal stimulation, 3 were unaffected by painful pinching, and 4 were excited. DISCUSSION Peristimulus time histograms indicated that electrical stimulation of the afferent abdominal vagus powerfully excited 75% of 85 neurons identified by collision test following antidromic activation from the region of the supraoptic nucleus. The spontaneous discharge rate
186 of these neurons was approximately 4 spikes/s, so that excitation from the abdominal vagus was obvious in peristimulus histograms. The excitation latency was approximately 200 ms, indicating a vagal fiber conduction velocity of approximately 0.7 m / s , consistent with C fiber mediation of the excitation. No A1 area neurons were inhibited by stimulation of the abdominal vagus. Although excitation of A1 area neurons was obvious in the peristimulus time histogram studies, it was difficult to document sustained increases in discharge rates in response to trains of electrical stimuli. This may partially reflect post-excitation inhibition which lasted for approximately 200 ms after each neuronal discharge. In addition, trains of stimuli (from 5 to 100 Hz) caused a rapid and sustained increase in AP, as previously reported following stimulation of the abdominal vagus 7. In the present study we found that more than 90% of A1 area neurons (antidromically identified from the region of the supraoptic nucleus) were inhibited by increases in AP, as previously demonstrated 17,21. We suspect that in the present experiments the rise in AP from abdominal vagal stimulation caused baroreceptor-mediated inhibition of A1 cells, thereby counteracting the direct excitatory effect, resulting in the biphasic change in discharge rate illustrated in Fig. 5C. Whether the same or different vagal afferents mediated the rise in AP and the directly mediated increase in A1 neuronal discharge rate is not known. Electrophysiological characteristics of recorded cells and their responses to baroreceptor and chemoreceptor stimulation were generally in agreement with a study of A1 area neurons in the rat ~7, and with previous results in the rabbit from our laboratory 21. We found that individual A1 area neurons excited from the abdominal vagus also receive inputs originating in baroreceptors and chemoreceptors, suggesting that individual cells are affected by varied physiological stimuli. Catecholamine fluorescence examination showed the recording electrode in the A1 cell group. About 8 0 90% of caudal ventrolateral medullary neurons with projections to the hypothalamus belong to this group and the A1 neurons innervate vasopressin-secreting neurons rather than oxytocin-secreting neurons 3'8'28'3~. Obviously some recordings could have been from nonA1 neurons. Our stimulating electrode could also activate A1 fibers passing near the supraoptic nucleus en route to regions such as the median preoptic area and the amygdala. At present it is not known whether the same A1 neurons projects to multiple hypothalamic and basal forebrain sites. Electrical stimulation of the central end of the cut
renal nerve did not affect the discharge of A1 area neurons. Other studies have suggested a relationship between renal afferents and vasopressin secretion 5'1°'37. Our finding suggests that the relevant central pathway does not involve neurons in the A1 area. Stimulation of the central end of a major trunk of the sciatic nerve produced variable effects on A1 area neurons, and no excitation was observed in the C fiber conduction velocity range. Variable effects were also observed with stimulation of the central ear nerve. In about 30% of cases, there was C fiber-mediated excitation but the response was never as vigorous as we observed with abdominal vagal stimulation. No consistent excitation of A1 area neurons was observed when the rabbit's fur was lightly rubbed, or when a paw or an ear was forcefully squeezed. In the rat, approximately 50% of vasopressin-secreting cells in the supraoptic nucleus were reported to be excited by pain, and by stimulation of the sciatic nerve TM. On the basis of GABA-induced blockade of the A1 area, Day and Sibbald 1~ concluded that the rat A1 cell group is involved in transmitting pain stimuli to the hypothalamic neurosecretory vasopressin cells. Our study does not support this conclusion in the rabbit. Whether painful stimulation causes secretion of vasopressin in unanesthetized animals is a controversial issue 2"16'19"2°. Abdominal vagal afferents are activated by gastric distention and by release of cholecystokinin from the upper intestine 26'27'32. These and other gastrointestinal events may cause secretion of vasopressin or oxytocin (depending on the species) via vagal afferents 15'23'29'32. The present finding that electrical activation of abdominal vagal afferents excites A1 area neurons in the rabbit suggests that similar gastrointestinal stimuli might cause secretion of vasopressin. Indeed, recently we found that electrical stimulation of the abdominal vagus markedly increases plasma vasopressin in the rabbit (Gieroba and Blessing, unpublished data). In view of the vigorous effects of gastric distention on hypothalamic neuronal discharge rates in the rat 29'34, we were surprized that this stimulus did not markedly affect the discharge of A1 area neurons in our study. Stimulation of hepatic osmoreceptors may produce secretion of vasopressin, probably via vagal afferents, but possibly via a spinal pathway ~'6'1s'35. Such a vagal pathway could be physiologically relevant to our present results. In conclusion, our electrophysiological results indicate that A1 area neurons, with projections to the region of the supraoptic nucleus, receive powerful excitatory inputs from the abdominal vagus nerve. The visceral information normally transmitted to A1 cells by these abdominal vagal afferents is not yet deter-
187 mined, but acute gastric distention does not appear to be the relevant physiological stimulus. Acknowledgements. Our study was supported by the National Health and Medical Research Council and by the National Heart Foundation of Australia. Mr. Adam Steer provided technical assistance.
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