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Study of possible transmitters in the solitary tract nucleus of the cat involved in the carotid sinus baro- and chemoreceptor reflex
Journalofthe Autonomic Nervo~s" System, 19 (1987) 179-188
179
Elsevier
JAN 00721
Research Papers
Study of possible transmitters in the solitary tract nucleus of the cat involved in the carotid sinus baro- and chemoreceptor reflex Mitsuhiko Miura, Kiyoshige T a k a y a m a and Junichi Okada Department of Physiology, 1st Division, Gunma Unit,ersity School o/Medicine, Showa-machi. Maebashi (Japan) (Received 11 July 1986) (Revised version received 19 December 1986) (Accepted 19 January 1987)
Key words: Solitary tract nucleus; Carotid sinus nerve; Opiate; Substance P: 7-Aminobutyric acid; a-Adrenergic receptor Summary By using a multibarrelled microelectrode, the possibility that putative transmitters may influence on the field potential evoked in the solitary tract nucleus by electrical stimulation of the carotid sinus nerve (the NTS response) was examined electrophysiologically in the cat. After iontophoretic application of a selective antagonist to the putative transmitter, it was determined whether or not the NTS response was influenced. Both substance P antagonist and naloxone altered the NTS response recorded in the depressor and apneic (or hypopneic) response zone as well as in the pressor and apneustic (or inspiratory) response zone throughout the rostral, intermediate and commissure regions, suggesting that substance P and opioid peptide may play the role of excitatory transmitters. Under the polarizing cathodal current which may activate inhibitory inputs to the site of the NTS response, bicuculline and prazosin strongly enhanced the NTS response recorded in the pressor and apneustic zone, while naloxone weakly enhanced the NTS response recorded in the depressor and apneic zone. These results suggest that y-aminobutyric acid, a-adrenergic agonist and opioid peptide may have an inhibitory influence on the NTS response.
Introduction The carotid sinus nerve (CSN) contains baroreceptor and chemorecptor afferents and projects to the solitary tract nucleus (NTS) of the caudal medulla oblongata as an important reflex input for regulation of circulation and respiration [101. Recently we identified functional subdivisions of the NTS in the cat by analyzing field potentials evoked in the NTS following electrical stimulation Correspondence: M. Miura. Department of Physiology, 1st Division, G u n m a University School of Medicine, 3-39-22 Showa-machi, Maebashi 371. Japan.
of the CSN ('NTS response') and by relating them to the respiratory and circulatory effects which were produced by stimulation of these 'NTS response' sites [19]. Pressor and apneustic (or inspiratory) responses, like the CSN chemoreceptor reflex, represent the effect of stimulating the rostral regions, the lateral portions of the intermediate regions and the ventral portions of the commissure regions of the NTS. On the other hand, depressor and apneic (or hypopneic) responses, like the CSN baroreceptor reflex, represent the effect of stimulating the medial portions of the intermediate regions and the dorsal portions of the commissure regions of the NTS. On the basis of these functional subdivisions of the NTS, we
0165-1838/87/$03.50 a,; 1987 Elsevier Science Publishers B.V. (Biomedical Division)
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attempted to find effects of antagonists to possible transmitters on the NTS response involved in the carotid baro- and chemoreceptor reflex. A preliminary report has already been published [20,21].
Materials and Methods
Preparation of animals Experiments were performed on 31 adult cats of either sex weighing 2.5-4.6 kg. Anesthesia was induced by ketamine hydrochloride (30 mg/kg, i.m.) and maintained by chloralose (initially 30 mg/kg, i.v.; additionally 5 m g / k g every 2 h). The trachea was cannulated with vinyl tubing (length 7 cm, diameter 7 mm) for sampling airway gas and monitoring CO 2 concentration in airway gas (Beckman, LB2) and for measuring the respiratory gas flow rate and tidal volume (Nihon Kohden, AQ-601G and AR-601G). The femoral artery was cannulated for measurement of the arterial blood pressure. The heart rate was counted with a cardiotachometer triggered by the R wave of the ECG. The arterial blood pressure, heart rate, mean arterial blood pressure, CO 2 concentration in airway gas, respiratory gas flow rate and tidal volume were continuously displayed on a polygraph. The rectal temperature was maintained at 3 7 ° C by means of a heating lamp. Each animal was placed in a stereotaxic frame with the head flexed at 45 °. The floor of the 4th ventricle was exposed by removing the caudal vermis of the cerebellum and covered with 4% agar and 0.9% NaC1 solution to prevent evaporative cooling of the surface of the brain. The side to be subjected to an operation for exposure of the CSN was determined after ascertaining which side of the surface of the NTS was not overlain by large vessels. Then, the CSN was exposed from the back (right side, 5 animals; left side, 26 animals), cut at the peripheral end, and mounted under mineral oil on a bipolar platinum electrode with an interelectrode distance of 2 mm.
Electrode A multibarrelled microelectrode was used. The tip of the barrel was bevelled obliquely to 16-20 /~m at the minor axis and 40-50/~m at the major
axis. The first barrel was filled with 0.9% NaCl and 20% H R P solution (Toyobo, I-C) and used for recording evoked field potentials, stinmlating a small part of the brain and injecting HRP as a marker. The second barrel was filled with 0.9% NaC1 solution and used for injecting polarizing current. The other 3 barrels were filled with drug solutions and used for iontophoretic injection of a drug to test its effect on the evoked field potentials. The drugs used were 10 mM substance P antagonist [D-Arg 1, I>Pro 2, D-Trpv'9, Leul~]-SP (Peninsula), 250 mM Naloxone (Sankyo), 5 mM bicuculline (Sigma) and 3 mM prazosin (Pfizer). The tip resistances ranged from 1 to 2 MfL
Experimental procedure The CSN was stimulated by a train (6 s) of square-wave pulses (100/~s, 20 Hz). The threshold current was defined as that which produced the first observable change in blood pressure, heart rate or respiration on the polygraph tracings; this ranged between 20 and 50 /~A. The stimulus strength was limited to 10 times the threshold. The CSN was repetitively stimulated at 0.5 Hz, while the NTS was systematically explored for evoked field potentials. The electrode was inserted dorsoventrally at 200-/~m steps to a maximum depth of 2.0-2.2 mm, and the activity was recorded and amplified with a pre-amplifier (band pass 160 H z - 3 kHz). At each step, evoked field potentials elicited by 50 repetitive stimuli applied to the CSN were averaged by a conventional signal averager (NTS response). The NTS response was recorded under 3 different conditions: (1) normal condition without polarizing current; (2) condition of polarizing anodal current; (3) condition of polarizing cathodal current. In order to avoid the record o f the NTS response being interfered with by an electrical artefact associated with the switching of the pulse on and off, the polarizing pulse (3 /~A, 300 ms) was started 200 ms prior to the NTS response. In addition, the effect of electrical stimulation of the site of the NTS response on blood pressure, heart rate and respiration was examined by a train (6 s) of square-wave pulses (1 ms, 5 ~tA, 50 Hz). To test the effect of a drug on the NTS re-
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sponse, the microelectrode was placed at the site of the maximal NTS response in the microelectrode track, and a drug was injected through the barrel by passing a DC anodal current of 5 ffA from a constant current supply (Nihon Kohden) in 30-50 repetitive cycles of 0.3 s on 0.2 s off (45-75 flA. s). At the end of the experiment, the position of the microelectrode was marked by depositing H R P (4 ffA, 30 s, electrode-positive pulse).
Results
The effect of polarizing current on the N T S response As detailed in the previous paper from our laboratory [19], the NTS response is a field potential which is evoked by electrical stimulation of the afferent A6 fibers of the ipsilateral carotid sinus nerve (CSN), and is recorded in confined areas within the NTS. Fig. 1 shows the typical NTS response recorded at different depths and under different conditions in a microelectrode track which penetrated the area lateral to the solitary tract in the rostral regions of the NTS. At depths of 1.6 m m and 1.7 mm, the amplitude of the negative potential of the NTS response reached the maximal level. It was augmented under anodal current, which we called anodal facilita-
Histologo, Each animal was perfused transcardially by a conventional method [29]. The brain was removed, fixed, frozen and sectioned serially at 50 ffm. The sections were then processed for histochemical demonstration of H R P deposition.
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Fig. 1. A map of the NTS response, surface-negative field potential evoked by electrical stimulation of the carotid sinus nerve (CSN) in the solitary tract nucleus (NTS). Records are averaged responses to 50 CSN stimuli at different depths in the rostral regions of the cat NTS and under different conditions consisting of normal, polarizing anodal current and polarizing cathodal current. A: photograph of the dorsal medulla oblongata and a microelectrode track in the NTS. Attached panels show ESB effects on blood pressure (BP, mm Hg), heart rate (HR, bpm) and tidal volume (VT, ml) at a depth of 1.7 mm. B: the NTS response recorded at 0.2or 0.1-mm steps from a depth of 2.3-1.3 mm from the surface. Note that the NTS response reached the maximal level at 1.6 and 1.7 mm. Time and voltage calibration, 200/~V and 5 ms. Ic, nucleus intercalatus; insp, inspiration; Ts, tractus solitarius; X, nucleus nervi vagi dorsalis motorius; XII, nucleus nervi hypoglossi.
182 tion. On the contrary, it was depressed under cathodal current, which we called cathodal inhibition. Electrical stimulation of the site of the maximal NTS response always produced marked effects on circulation and respiration. The insert of Fig. 1A shows the marked effects of the NTS stimulation which were a set of pressor-depressor, cardiac slowing and apneustic responses. Since both anodal facilitation and cathodal inhibition remained unchanged when the 0.9% NaC1 solution of the recording barrel was replaced with 1 M choline chloride solution, it cannot have been due to the effect of sodium ions. Such effects of polarizing current on the NTS response were marked in the pressor and apneustic (or inspiratory) response zone covering the lateral area of the rostral and intermediate regions, but far less in the depressor and apneic (or hypopneic) response zone covering the medial area of the intermediate regions and almost absent in the commissure regions. Since the effect of polarizing current on the NTS response was none or small at depths some distance from the site of the maximal response, the effect of polarizing current may be due to the mechanism working in the site of the maximal response. We suggest that at the site of the maximal response anodal current may suppress the background inhibitory influence on the NTS response and eventually augment the size of the NTS response, while cathodal current may intensify the background inhibitory influence on the NTS response and eventually depress the size of the NTS response.
the maximal NTS response, we determined whether it was decreased in size a n d / o r reversed in polarity. In 11 experiments, it was shown that both SP antagonist and naloxone were effective on the same maximal NTS response recorded at 28 sites. Fig. 2 shows the distribution of 28 sites, consisting of 12 in the rostral regions, I0 in the intermediate regions and 6 in the commissure regions. The effect of an antagonist was strong on the NTS response recorded in the pressor-apneustic response zone of the rostral and intermediate regions, while weak on those in the depressorapneic response zone of the intermediate and commissure regions. Fig. 3 shows the data determining the site of the maximal NTS response and the effect of SP antagonist and naloxone on the maximal NTS response in the rostral regions. The microelectrode track ran through just lateral to the solitary tract. At depths of 1.4-2.0 mm, the effects of the NTS
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The effect of antagonist of the possible excitatory transmitter If the NTS response is mediated through the possible transmitter released from primary afferent terminals of the CSN, an antagonist of the possible transmitter should alter the NTS response. So far, m a n y possible transmitters have been thought to be effective transmitters which mediate the NTS response. In this study, we tested the effect of reliable antagonists like SP antagonist ([D-Arg 1, D-Pro 2, D-Trp7'9, LeuU]-SP) and naloxone on the maximal NTS response. After the injection of the selective antagonist into the site of
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Fig. 2. Distribution of sites where substance P antagonist as well as naloxone blocked the NTS response. Twenty-eight positive sites were superimposed on 3 representative frontal sections of the lower medulla oblongata. Rostrat, intermediate and commissure regions represent levels 0.8-2.5 ram, 0-0.8 mm ahead of the obex and 0-2 mm behind the obex.
183
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Fig. 3. Effects of substance P antagonist and naloxone on the NTS response in the rostral regions. A: diagram of the rostral regions and a microelectrode track in the lateral area of the NTS. B: effects of the NTS stimulation on blood pressure (BP, mm Hg), heart rate (HR, bpm) and tidal volume (VT, ml), and the NTS response at different depths. The site of the maximal NTS response was determined to be at a depth of 1.8 ram. C: change in the NTS response after the injection of substance P antagonist and naloxone. Time and voltage calibration, 100 ~V and 5 ms.
stimulation were a set of pressor, cardiac slowing and apneustic responses. At a depth of 1.8 m m both the NTS response and the stimulation effect reached the maximal level. Immediately after injection of SP antagonist and naloxone (75 ~ A . s) into the site of the maximal NTS response, the negative potential was decreased in size and reversed in polarity. This phenomenon suggests that SP antagonist as well as naloxone may influence the site of the maximal NTS response. The reversal of polarity of the potentials may be due to a shift of the sink from the site of the maximal NTS response to the site of the CSN afferent in which the conduction of a nerve impulse is not interrupted [18]. The NTS response, however, recovered completely from the effect of the antagonist with 10 min. Fig. 4A and B shows the site of the NTS response in the intermediate regions and the effect of SP antagonist and naloxone on the NTS response. The microelectrode track ran through the area just medial to the solitary tract. At depths of 1.0 1.2 ram, the effects of the NTS stimulation were a set of depressor, cardiac slowing and apneic responses, and the amplitude of the NTS response
was low. Fig. 4C shows that SP antagonist as well as naloxone altered the NTS response at a depth of 1.1 mm, but this recovered within 10 min. Although no example is shown, a similar effect of SP antagonist and naloxone on the NTS response was recognized in the commissure regions.
The effect of antagonist of the possible inhibito O, transmitter The effect of polarizing current on the NTS response, i.e. anodal facilitation and cathodal inhibition, suggested that the NTS response may be influenced by the background inhibitory input. Therefore, we attempted to obtain an electrophysiological evidence that the possible inhibitory transmitter influences on the NTS response. Immunohistochemical studies of the NTS suggest that GABA, ~-adrenergic agonist and opioid peptides are candidates as inhibitory transmitters. If the selective antagonists of these possible inhibitory transmitters, like bicuculline, prazosin and naloxone, block the background inhibitory input to the site of the NTS response, the amplitude of the NTS response should be enhanced by removal of the background inhibitory input. In order to
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Fig. 4. Effects of substance P antagonist and naloxone on the NTS response in the intermediate regions. Style of figure is the same as that of Fig. 3. Note that the NTS response and effects of the NTS stimulation reached the maximal level at 1.1 mm. Time and Voltage calibration, 50/~V and 5 ms.
v e r i f y this h y p o t h e s i s , w e i n j e c t e d s e l e c t i v e a n t a g o n i s t i n t o t h e site o f the m a x i m a l N T S res p o n s e a n d d e t e r m i n e d w h e t h e r o r n o t the N T S r e s p o n s e w a s i n c r e a s e d in size. Fig. 5 s h o w s s u c h a d i s i n h i b i t o r y e f f e c t of bicuculline and prazosin on the NTS response. T h e sites o f t h e d i s i n h i b i t o r y effect, c o n s i s t i n g of
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11 for b i c u c u l l i n e a n d 16 for p r a z o s i n , w e r e dist r i b u t e d a r o u n d t h e s o l i t a r y t r a c t in t h e r o s t r a l a n d i n t e r m e d i a t e r e g i o n s (Fig. 5A). In o r d e r to a c c e n t u a t e the d i s i n h i b i t o r y effect, t h e N T S res p o n s e was e x a m i n e d u n d e r c a t h o d a l c u r r e n t . A n e x a m p l e o f the e f f e c t of b i c u c u U i n e was s h o w n in t h e N T S r e s p o n s e r e c o r d e d at the site o f p r e s s o r a n d a p n e u s t i c r e s p o n s e s in t h e v e n t r o l a t e r a l a r e a o f t h e r o s t r a l r e g i o n s ( t i n Fig. 5A).
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Fig. 5. Effects of bicuculfine and prazosin on the NTS response. A: distribution of sites of the maximal NTS response where positive effects of bicuculline (O) and prazosin (rn) were obtained, superimposed on two representative frontal sections of the medulla oblongata (upper, rostral regions, lower, intermediate regions). A representative example of the effect of bicuculline was derived from a site in the ventrolateral area of the rostral regions (O) and an example of the effect of prazosm from a site in the lateral area of the intermediate regions (11). B: stimulation effects at the site of the maximal NTS response on blood pressure (mm Hg), heart rate (bpm] and tidal volume (ml). C: the NTS response recorded under cathodal current. anodal current and normal condition, and effects of antagonists after the application of antagonist recorded under cathodal current in order to accentuate the disinhibitory effect. Insert shows recovery curve of the negative potential. Size of negative potential is measured from the isoelectrical line. Dotted lines represent relative size of negative potential under cathodal current (c.c.) and under anodal current (a.c.) before drug application. Time and voltage calibration, 50 #V and 5 ms.
185
Five minutes after injection, the amplitude of the negative potential began to increase. Twenty minutes after injection, it attained a maximal size which was similar to that of the NTS response recorded under anodal current. The disinhibitory effect of bicuculline lasted for at least 200 rain. An example of the effect of prazosin was shown in the NTS response recorded at the site of pressor and apneustic responses in the area just lateral to the intermediate regions (. in Fig. 5A). Five minutes after injection, the amplitude of the negative potential began to increase. Thirty minutes after injection, it attained a maximal size which was similar to that of the NTS response recorded under anodal current. The disinhibitory effect of prazosin lasted for at least 150 rain. Generally, bicuculline and prazosin had a long-term disinhibitory effect on the NTS response in the pressor and apneustic response zone of the lateral area. Since the recovery from the disinhibitory effect was incomplete, we could not test both drugs on the same NTS response. Thus, it is uncertain whether or not both drugs have a disinhibitory effect on the same NTS response. The disinhibitory effect of naloxone on the
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NTS response was observed only in the depressor and apneic response zone in the medial area of the intermediate regions. Fig. 6 shows the representative example (o in Fig. 6A). Immediately after the application of naloxone, the negative potentials between 5.5 and 9 ms disappeared and the positive potential between 9 and 12.5 ms was reversed in polarity, but the change in these potentials recovered within 8 min. Such a fast onset of, and fast recovery from the effect of naloxone seem very different in quality from the effect of bicuculline as well as that of prazosin.
Discussion
Several types of substances have been proposed as transmitters working at the site between primary afferent terminals of the CSN and the secondary neurons in the NTS: (1) substance P (SP) [6,9,12,15]; (2) glutamate [8,30]; and (3) opioid peptide [3,15]. In addition to these possible transmitters in the afferent fibers, y-aminobutyric acid (GABA) [8,25], noradrenaline [1,7], adrenaline [1,11] and opioid peptide [15] are represented in
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Fig. 6. Effect of naloxone on the NTS response. Style of figure is the same as that of Fig. 5. A: distribution of sites of the maximal NTS response where positive effects of naloxone were obtained (C)), superimposed on a frontal section at the level of intermediate regions of the NTS. A representative example of the effect of naloxone was derived from a site in the medial area of the intermediate regions (e). B: stimulation effects at the site of the maximal NTS response. C: the NTS response recorded under different conditions (before) and effects of naloxone recorded under cathodal current in order to accentuate the disinhibitory effect. Time and voltage calibration, 50 /LV and 5 ms.
186
the NTS neurons, and regarded as transmitters. Since antagonists of SP, opioid peptide, G A B A and a-adrenergic agonist have been well established, these substances were used for testing. It is generally agreed that opioid peptides act as an inhibitory transmitter on most of the central neurons. However, Okada and Miura [23] discovered that small oval-type neurons of the cultured rat brainstem are excited by the application of opioid peptide and blocked by the concomitant application of naloxone. Since the cat NTS contains many oval-type neurons [27], we attempted to determine whether opioid peptide acts as a transmitter in the cat N T K and found that circulatory reflex response as well as evoked field potentials elicited by stimulation of the ipsilateral CSN were inhibited by the application of naloxone (250 mM, 300/~A. s) [18]. This finding suggested that opioid peptide might be an excitatory transmitter of the CSN fibers mediating circulatory reflex. However, in this previous experiment, the NTS response was not explored in relation to functional subdivisions of the NTS. After we had succeeded in subdividing the NTS in relation to the functional properties of the CSN reflex [19], we showed that SP antagonist as well as naloxone had an inhibitory effect on the same NTS response. The effect was strong in the pressor-apneustic response zone of the rostral regions and mild in the depressor-apneic response zone of both intermediate and commissure regions of the NTS [20,21]. This phenomenon has been confirmed in the present study, and suggests that SP as well as opioid peptide may act as an excitatory transmitter at the site of the CSN baroreceptor and chemoreceptor inputs. Recently, it has been shown that SP-immunoreactive terminals and SP binding sites are distributed throughout the whole extent of the rat NTS [14,16] and that SP exhibits a potent excitatory action on the cat NTS neuron [22]. These findings also support the notion that SP is a possible transmitter in the NTS. Bicuculline and prazosin augmented the NTS response which had been depressed by the application of cathodal current. This phenomenon was conspicuous in the pressor-apneustic response zone of the lateral NTS. Since cathodal inhibition of the NTS response may be due to activation of the
background inhibitory influence on the NTS response~ it is possible that the application of an antagonist of possible inhibitory transmitter may augment the NTS response. Recently, GABAergic neurons were detected immunocytochemically in the rat NTS [5,13,17,26]. They were observed in all subdivisions of the NTS with an increasing concentration from the caudal to rostral regions [26], and mostly detected around the solitary tract in the rostral and intermediate regions [17]. Such a characteristic distribution of the NTS GABAergic neurons is consistent with the site of the NTS response on which bicuculline had a disinhibitory effect. On the other hand, a-adrenergic neurons were also detected immunocytochemically in the rat NTS [1,2], and it has been demonstrated that adrenergic neurons are predominant in the C2 group of H~Skfelt [11], and noradrenergic neurons in the A2 group of DahlstrOm and Fuxe [7]. It has been reported in a study of cultured neurons from the rat lower brainstem [24] that GABA always acts as an inhibitory transmitter, while c~-adrenergic agonist acts as an inhibitory transmitter to a majority of the neurons and as an excitatory transmitter to a minority. Since the NTS neurons in the rostral regions are innervated not only by nearby A2 and C2 group neurons [11,28] but by distant A1 group neurons [4], it is possible that the NTS neurons may receive the inhibitory input from A1, A2 and C1 group neurons. In the medial NTS we showed that under cathodal current the early part of the NTS response was inhibited and the late part was disinhibited by the application of naloxone. This finding suggests that opioid peptides act as inhibitory as well as excitatory transmitters. Such a dual transmitter activity of opioid peptides has been observed in cultured neurons from the rat lower brainstem [23]. Recently, enkephalinergic neurons were detected immunocytochemically in the rat NTS [15], being concentrated in the medial area of the intermediate regions. Such a characteristic distribution of enkephalinergic neurons is consistent with the site of the NTS response on which naloxone had a disinhibitory effect. In conclusion, while we have provided an evidence that opioid peptide, substance P, GABA, a-adrenergic agonist may be transmitters working
187
in the NTS, it is important to point out that our study is limited because we have used the field potentials instead of unit potentials as an indicator and we have not tried all the transmitter candidates. Future studies will have to address the problem concerning all the transmitter candidates acting in the NTS in relation to the carotid sinus baro- and chemoreceptor reflex.
Acknowledgement
This work was supported by a Grant-in-Aid for Scientific Research B from the Japanese Ministry of Education, Science and Culture (no. 59480102).
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10 Heymans, C. and Neil, E., Reflexogenic Areas of tht Cardiovascular Systern, Churchill, London, 1958, 271 pp. 11 Hi3kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O. lmmunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Res., 66 (1974 235-251. 12 H/3kfelt, T., Kellerth, J-O., Nilsson, G. and Pernow, B. Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensor\ neurons, Brain Res., 100 (1975) 235-252. 13 Hwang, B.H. and Wu, J.-Y., Ultrastructural studies or catecholaminergic terminals and GABAergic neurons it: nucleus tractus solitarius of the rat medulla oblongata Brain Res., 302 (1984) 57-67. 14 Kawano. H. and Chiba, T., Distribution of substance t' immunoreactive nerve terminals within the nucleus tractu~ solitarius of the rat, Neurosci. Lett., 45 (1984) 175-179. 15 Maley, B. and Elde. R., lmmunohistochemical localizatio~ of putative neurotransmitters within the feline nucleu~ tractus solitarii, Neuroscience. 7 (1982) 2469 2490. 16 Mantyh, P.W., Hunt, S.P., Maggio, J.E.. Substance P recep tors: localization by light microscopic autoradiography i~: rat brain using [3H]SP as the radioligand, Brain Res.. 30(1984) 147-165. 17 Meeley, M., Ruggiero, D.A., lshitsuka, T. and Reis, D.J. Intrinsic y-aminobutyric acid neurons in the nucleus of the solitary tract and the rostral ventrolateral medulla of the rat: an immunocytochemical and biochemical study, Neurosci. Lett.. 58 (1985) 83-89. 18 Miura, M. and Takayama, K., Naloxone inhibition of the carotid sinus nerve reflex in the nucleus of the solitary trac: of the cat, Brain Res., 288 (1983) 389 392. 19 Miura, M. and Takayama, K., The functional subdivision~ of the nucleus tractus solitarii of the cat in relation to the carotid sinus nerve reflex, J. Auton. Nert:. Svst.. 15 (1986~ 79-92. 20 Miura, M. and Takayama, K., Neurotransmitters used fo~ relaying the cardiovascular and pulmonary reflex in the nucleus tractus solitarii of the cat. Neurosci. Res.. Suppl. 3 (1986) $16. 21 Miura, M. and Takayama, K., Neurotransmitter in the nucleus tractus solitarii in carotid baroreceptor and chemoreceptor reflex. In K. N a k a m u r a (Ed.), Brain and Bloo, l Pressure Control, Elsevier, Amsterdam, 1986, pp. 89-97. 22 Morin-Surun, M.P., Jordan, D., Champagnat, J., Spyef. K.M. and Denavit-Saubie, M., Excitatory effects of iontophoretically applied substance P on neurons in the nucleu, tractus solitarius of the cat: lack of interaction with opiate, and opioids, Brain Res., 307 (1984) 388 392. 23 Okada, J. and Miura, M., Transmitter sensitivities in oval neurons from rat brainstem cultures, Brain Res., 268 (1983) 377 381. 24 Saji, M. and Miura, M., Transmitter sensitivities a m o n g various types of cultured brain stem neurons, Brain Res.. 233 (1982) 83 96. 25 Siemers, E.R., Rea, M.A., Fehon, D.L. and Aprison, M . H . Distribution and uptake of glycine, glutamate and gamma-
188 aminobutyric acid in the vagal nuclei and eight other regions of the rat medulla oblongata, Neurochem~ Res., 7 (1982) 455-468. 26 Simon, J.R., DiMicco, S.K. and Aprison, M.H., Neurochemical studies of the nucleus of the solitary tract, dorsal motor nucleus of the vagus and the hypoglossal nucleus in rat: topographical distribution of glutamate uptake, GABA uptake, and glutamic acid decarboxylase activity, Brain Res. Bull., 14 (1985) 49-53. 27 Taber, E., The cytoarchitecture of the brain stem of the cat. I. Brain stem nuclei of cat, J. Comp. Neurol., 116 (1961) 27-69.
28 Takahashi, Y., Satoh, K., Sakumoto, T., Tohyama, M. and Shimizu, N., A major source of catecholamine terminals in the nucleus tractus solitarii, Brain Res., 172 (1979) 372 377. 29 Takayama, K. and Miura, M., An o-dianisidme method of horseradish peroxidase neurohistochemistry, Jpn. J. Pllvsiol., 31 (1981) 269-272. 30 Talman, W.T., Perrone, M.H. and Reis, D.J., Evidence for L-glutamate as the neurotransmitter of baroreceptor afferent nerve fibers, Science, 209 (1980) 813-815.
179
Elsevier
JAN 00721
Research Papers
Study of possible transmitters in the solitary tract nucleus of the cat involved in the carotid sinus baro- and chemoreceptor reflex Mitsuhiko Miura, Kiyoshige T a k a y a m a and Junichi Okada Department of Physiology, 1st Division, Gunma Unit,ersity School o/Medicine, Showa-machi. Maebashi (Japan) (Received 11 July 1986) (Revised version received 19 December 1986) (Accepted 19 January 1987)
Key words: Solitary tract nucleus; Carotid sinus nerve; Opiate; Substance P: 7-Aminobutyric acid; a-Adrenergic receptor Summary By using a multibarrelled microelectrode, the possibility that putative transmitters may influence on the field potential evoked in the solitary tract nucleus by electrical stimulation of the carotid sinus nerve (the NTS response) was examined electrophysiologically in the cat. After iontophoretic application of a selective antagonist to the putative transmitter, it was determined whether or not the NTS response was influenced. Both substance P antagonist and naloxone altered the NTS response recorded in the depressor and apneic (or hypopneic) response zone as well as in the pressor and apneustic (or inspiratory) response zone throughout the rostral, intermediate and commissure regions, suggesting that substance P and opioid peptide may play the role of excitatory transmitters. Under the polarizing cathodal current which may activate inhibitory inputs to the site of the NTS response, bicuculline and prazosin strongly enhanced the NTS response recorded in the pressor and apneustic zone, while naloxone weakly enhanced the NTS response recorded in the depressor and apneic zone. These results suggest that y-aminobutyric acid, a-adrenergic agonist and opioid peptide may have an inhibitory influence on the NTS response.
Introduction The carotid sinus nerve (CSN) contains baroreceptor and chemorecptor afferents and projects to the solitary tract nucleus (NTS) of the caudal medulla oblongata as an important reflex input for regulation of circulation and respiration [101. Recently we identified functional subdivisions of the NTS in the cat by analyzing field potentials evoked in the NTS following electrical stimulation Correspondence: M. Miura. Department of Physiology, 1st Division, G u n m a University School of Medicine, 3-39-22 Showa-machi, Maebashi 371. Japan.
of the CSN ('NTS response') and by relating them to the respiratory and circulatory effects which were produced by stimulation of these 'NTS response' sites [19]. Pressor and apneustic (or inspiratory) responses, like the CSN chemoreceptor reflex, represent the effect of stimulating the rostral regions, the lateral portions of the intermediate regions and the ventral portions of the commissure regions of the NTS. On the other hand, depressor and apneic (or hypopneic) responses, like the CSN baroreceptor reflex, represent the effect of stimulating the medial portions of the intermediate regions and the dorsal portions of the commissure regions of the NTS. On the basis of these functional subdivisions of the NTS, we
0165-1838/87/$03.50 a,; 1987 Elsevier Science Publishers B.V. (Biomedical Division)
180
attempted to find effects of antagonists to possible transmitters on the NTS response involved in the carotid baro- and chemoreceptor reflex. A preliminary report has already been published [20,21].
Materials and Methods
Preparation of animals Experiments were performed on 31 adult cats of either sex weighing 2.5-4.6 kg. Anesthesia was induced by ketamine hydrochloride (30 mg/kg, i.m.) and maintained by chloralose (initially 30 mg/kg, i.v.; additionally 5 m g / k g every 2 h). The trachea was cannulated with vinyl tubing (length 7 cm, diameter 7 mm) for sampling airway gas and monitoring CO 2 concentration in airway gas (Beckman, LB2) and for measuring the respiratory gas flow rate and tidal volume (Nihon Kohden, AQ-601G and AR-601G). The femoral artery was cannulated for measurement of the arterial blood pressure. The heart rate was counted with a cardiotachometer triggered by the R wave of the ECG. The arterial blood pressure, heart rate, mean arterial blood pressure, CO 2 concentration in airway gas, respiratory gas flow rate and tidal volume were continuously displayed on a polygraph. The rectal temperature was maintained at 3 7 ° C by means of a heating lamp. Each animal was placed in a stereotaxic frame with the head flexed at 45 °. The floor of the 4th ventricle was exposed by removing the caudal vermis of the cerebellum and covered with 4% agar and 0.9% NaC1 solution to prevent evaporative cooling of the surface of the brain. The side to be subjected to an operation for exposure of the CSN was determined after ascertaining which side of the surface of the NTS was not overlain by large vessels. Then, the CSN was exposed from the back (right side, 5 animals; left side, 26 animals), cut at the peripheral end, and mounted under mineral oil on a bipolar platinum electrode with an interelectrode distance of 2 mm.
Electrode A multibarrelled microelectrode was used. The tip of the barrel was bevelled obliquely to 16-20 /~m at the minor axis and 40-50/~m at the major
axis. The first barrel was filled with 0.9% NaCl and 20% H R P solution (Toyobo, I-C) and used for recording evoked field potentials, stinmlating a small part of the brain and injecting HRP as a marker. The second barrel was filled with 0.9% NaC1 solution and used for injecting polarizing current. The other 3 barrels were filled with drug solutions and used for iontophoretic injection of a drug to test its effect on the evoked field potentials. The drugs used were 10 mM substance P antagonist [D-Arg 1, I>Pro 2, D-Trpv'9, Leul~]-SP (Peninsula), 250 mM Naloxone (Sankyo), 5 mM bicuculline (Sigma) and 3 mM prazosin (Pfizer). The tip resistances ranged from 1 to 2 MfL
Experimental procedure The CSN was stimulated by a train (6 s) of square-wave pulses (100/~s, 20 Hz). The threshold current was defined as that which produced the first observable change in blood pressure, heart rate or respiration on the polygraph tracings; this ranged between 20 and 50 /~A. The stimulus strength was limited to 10 times the threshold. The CSN was repetitively stimulated at 0.5 Hz, while the NTS was systematically explored for evoked field potentials. The electrode was inserted dorsoventrally at 200-/~m steps to a maximum depth of 2.0-2.2 mm, and the activity was recorded and amplified with a pre-amplifier (band pass 160 H z - 3 kHz). At each step, evoked field potentials elicited by 50 repetitive stimuli applied to the CSN were averaged by a conventional signal averager (NTS response). The NTS response was recorded under 3 different conditions: (1) normal condition without polarizing current; (2) condition of polarizing anodal current; (3) condition of polarizing cathodal current. In order to avoid the record o f the NTS response being interfered with by an electrical artefact associated with the switching of the pulse on and off, the polarizing pulse (3 /~A, 300 ms) was started 200 ms prior to the NTS response. In addition, the effect of electrical stimulation of the site of the NTS response on blood pressure, heart rate and respiration was examined by a train (6 s) of square-wave pulses (1 ms, 5 ~tA, 50 Hz). To test the effect of a drug on the NTS re-
181
sponse, the microelectrode was placed at the site of the maximal NTS response in the microelectrode track, and a drug was injected through the barrel by passing a DC anodal current of 5 ffA from a constant current supply (Nihon Kohden) in 30-50 repetitive cycles of 0.3 s on 0.2 s off (45-75 flA. s). At the end of the experiment, the position of the microelectrode was marked by depositing H R P (4 ffA, 30 s, electrode-positive pulse).
Results
The effect of polarizing current on the N T S response As detailed in the previous paper from our laboratory [19], the NTS response is a field potential which is evoked by electrical stimulation of the afferent A6 fibers of the ipsilateral carotid sinus nerve (CSN), and is recorded in confined areas within the NTS. Fig. 1 shows the typical NTS response recorded at different depths and under different conditions in a microelectrode track which penetrated the area lateral to the solitary tract in the rostral regions of the NTS. At depths of 1.6 m m and 1.7 mm, the amplitude of the negative potential of the NTS response reached the maximal level. It was augmented under anodal current, which we called anodal facilita-
Histologo, Each animal was perfused transcardially by a conventional method [29]. The brain was removed, fixed, frozen and sectioned serially at 50 ffm. The sections were then processed for histochemical demonstration of H R P deposition.
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Fig. 1. A map of the NTS response, surface-negative field potential evoked by electrical stimulation of the carotid sinus nerve (CSN) in the solitary tract nucleus (NTS). Records are averaged responses to 50 CSN stimuli at different depths in the rostral regions of the cat NTS and under different conditions consisting of normal, polarizing anodal current and polarizing cathodal current. A: photograph of the dorsal medulla oblongata and a microelectrode track in the NTS. Attached panels show ESB effects on blood pressure (BP, mm Hg), heart rate (HR, bpm) and tidal volume (VT, ml) at a depth of 1.7 mm. B: the NTS response recorded at 0.2or 0.1-mm steps from a depth of 2.3-1.3 mm from the surface. Note that the NTS response reached the maximal level at 1.6 and 1.7 mm. Time and voltage calibration, 200/~V and 5 ms. Ic, nucleus intercalatus; insp, inspiration; Ts, tractus solitarius; X, nucleus nervi vagi dorsalis motorius; XII, nucleus nervi hypoglossi.
182 tion. On the contrary, it was depressed under cathodal current, which we called cathodal inhibition. Electrical stimulation of the site of the maximal NTS response always produced marked effects on circulation and respiration. The insert of Fig. 1A shows the marked effects of the NTS stimulation which were a set of pressor-depressor, cardiac slowing and apneustic responses. Since both anodal facilitation and cathodal inhibition remained unchanged when the 0.9% NaC1 solution of the recording barrel was replaced with 1 M choline chloride solution, it cannot have been due to the effect of sodium ions. Such effects of polarizing current on the NTS response were marked in the pressor and apneustic (or inspiratory) response zone covering the lateral area of the rostral and intermediate regions, but far less in the depressor and apneic (or hypopneic) response zone covering the medial area of the intermediate regions and almost absent in the commissure regions. Since the effect of polarizing current on the NTS response was none or small at depths some distance from the site of the maximal response, the effect of polarizing current may be due to the mechanism working in the site of the maximal response. We suggest that at the site of the maximal response anodal current may suppress the background inhibitory influence on the NTS response and eventually augment the size of the NTS response, while cathodal current may intensify the background inhibitory influence on the NTS response and eventually depress the size of the NTS response.
the maximal NTS response, we determined whether it was decreased in size a n d / o r reversed in polarity. In 11 experiments, it was shown that both SP antagonist and naloxone were effective on the same maximal NTS response recorded at 28 sites. Fig. 2 shows the distribution of 28 sites, consisting of 12 in the rostral regions, I0 in the intermediate regions and 6 in the commissure regions. The effect of an antagonist was strong on the NTS response recorded in the pressor-apneustic response zone of the rostral and intermediate regions, while weak on those in the depressorapneic response zone of the intermediate and commissure regions. Fig. 3 shows the data determining the site of the maximal NTS response and the effect of SP antagonist and naloxone on the maximal NTS response in the rostral regions. The microelectrode track ran through just lateral to the solitary tract. At depths of 1.4-2.0 mm, the effects of the NTS
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The effect of antagonist of the possible excitatory transmitter If the NTS response is mediated through the possible transmitter released from primary afferent terminals of the CSN, an antagonist of the possible transmitter should alter the NTS response. So far, m a n y possible transmitters have been thought to be effective transmitters which mediate the NTS response. In this study, we tested the effect of reliable antagonists like SP antagonist ([D-Arg 1, D-Pro 2, D-Trp7'9, LeuU]-SP) and naloxone on the maximal NTS response. After the injection of the selective antagonist into the site of
commissure region
Fig. 2. Distribution of sites where substance P antagonist as well as naloxone blocked the NTS response. Twenty-eight positive sites were superimposed on 3 representative frontal sections of the lower medulla oblongata. Rostrat, intermediate and commissure regions represent levels 0.8-2.5 ram, 0-0.8 mm ahead of the obex and 0-2 mm behind the obex.
183
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stimulation were a set of pressor, cardiac slowing and apneustic responses. At a depth of 1.8 m m both the NTS response and the stimulation effect reached the maximal level. Immediately after injection of SP antagonist and naloxone (75 ~ A . s) into the site of the maximal NTS response, the negative potential was decreased in size and reversed in polarity. This phenomenon suggests that SP antagonist as well as naloxone may influence the site of the maximal NTS response. The reversal of polarity of the potentials may be due to a shift of the sink from the site of the maximal NTS response to the site of the CSN afferent in which the conduction of a nerve impulse is not interrupted [18]. The NTS response, however, recovered completely from the effect of the antagonist with 10 min. Fig. 4A and B shows the site of the NTS response in the intermediate regions and the effect of SP antagonist and naloxone on the NTS response. The microelectrode track ran through the area just medial to the solitary tract. At depths of 1.0 1.2 ram, the effects of the NTS stimulation were a set of depressor, cardiac slowing and apneic responses, and the amplitude of the NTS response
was low. Fig. 4C shows that SP antagonist as well as naloxone altered the NTS response at a depth of 1.1 mm, but this recovered within 10 min. Although no example is shown, a similar effect of SP antagonist and naloxone on the NTS response was recognized in the commissure regions.
The effect of antagonist of the possible inhibito O, transmitter The effect of polarizing current on the NTS response, i.e. anodal facilitation and cathodal inhibition, suggested that the NTS response may be influenced by the background inhibitory input. Therefore, we attempted to obtain an electrophysiological evidence that the possible inhibitory transmitter influences on the NTS response. Immunohistochemical studies of the NTS suggest that GABA, ~-adrenergic agonist and opioid peptides are candidates as inhibitory transmitters. If the selective antagonists of these possible inhibitory transmitters, like bicuculline, prazosin and naloxone, block the background inhibitory input to the site of the NTS response, the amplitude of the NTS response should be enhanced by removal of the background inhibitory input. In order to
184
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Fig. 4. Effects of substance P antagonist and naloxone on the NTS response in the intermediate regions. Style of figure is the same as that of Fig. 3. Note that the NTS response and effects of the NTS stimulation reached the maximal level at 1.1 mm. Time and Voltage calibration, 50/~V and 5 ms.
v e r i f y this h y p o t h e s i s , w e i n j e c t e d s e l e c t i v e a n t a g o n i s t i n t o t h e site o f the m a x i m a l N T S res p o n s e a n d d e t e r m i n e d w h e t h e r o r n o t the N T S r e s p o n s e w a s i n c r e a s e d in size. Fig. 5 s h o w s s u c h a d i s i n h i b i t o r y e f f e c t of bicuculline and prazosin on the NTS response. T h e sites o f t h e d i s i n h i b i t o r y effect, c o n s i s t i n g of
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11 for b i c u c u l l i n e a n d 16 for p r a z o s i n , w e r e dist r i b u t e d a r o u n d t h e s o l i t a r y t r a c t in t h e r o s t r a l a n d i n t e r m e d i a t e r e g i o n s (Fig. 5A). In o r d e r to a c c e n t u a t e the d i s i n h i b i t o r y effect, t h e N T S res p o n s e was e x a m i n e d u n d e r c a t h o d a l c u r r e n t . A n e x a m p l e o f the e f f e c t of b i c u c u U i n e was s h o w n in t h e N T S r e s p o n s e r e c o r d e d at the site o f p r e s s o r a n d a p n e u s t i c r e s p o n s e s in t h e v e n t r o l a t e r a l a r e a o f t h e r o s t r a l r e g i o n s ( t i n Fig. 5A).
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185
Five minutes after injection, the amplitude of the negative potential began to increase. Twenty minutes after injection, it attained a maximal size which was similar to that of the NTS response recorded under anodal current. The disinhibitory effect of bicuculline lasted for at least 200 rain. An example of the effect of prazosin was shown in the NTS response recorded at the site of pressor and apneustic responses in the area just lateral to the intermediate regions (. in Fig. 5A). Five minutes after injection, the amplitude of the negative potential began to increase. Thirty minutes after injection, it attained a maximal size which was similar to that of the NTS response recorded under anodal current. The disinhibitory effect of prazosin lasted for at least 150 rain. Generally, bicuculline and prazosin had a long-term disinhibitory effect on the NTS response in the pressor and apneustic response zone of the lateral area. Since the recovery from the disinhibitory effect was incomplete, we could not test both drugs on the same NTS response. Thus, it is uncertain whether or not both drugs have a disinhibitory effect on the same NTS response. The disinhibitory effect of naloxone on the
A
B Naloxone effect
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C
NTS response was observed only in the depressor and apneic response zone in the medial area of the intermediate regions. Fig. 6 shows the representative example (o in Fig. 6A). Immediately after the application of naloxone, the negative potentials between 5.5 and 9 ms disappeared and the positive potential between 9 and 12.5 ms was reversed in polarity, but the change in these potentials recovered within 8 min. Such a fast onset of, and fast recovery from the effect of naloxone seem very different in quality from the effect of bicuculline as well as that of prazosin.
Discussion
Several types of substances have been proposed as transmitters working at the site between primary afferent terminals of the CSN and the secondary neurons in the NTS: (1) substance P (SP) [6,9,12,15]; (2) glutamate [8,30]; and (3) opioid peptide [3,15]. In addition to these possible transmitters in the afferent fibers, y-aminobutyric acid (GABA) [8,25], noradrenaline [1,7], adrenaline [1,11] and opioid peptide [15] are represented in
under c(]thodal onodol normal current current
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Fig. 6. Effect of naloxone on the NTS response. Style of figure is the same as that of Fig. 5. A: distribution of sites of the maximal NTS response where positive effects of naloxone were obtained (C)), superimposed on a frontal section at the level of intermediate regions of the NTS. A representative example of the effect of naloxone was derived from a site in the medial area of the intermediate regions (e). B: stimulation effects at the site of the maximal NTS response. C: the NTS response recorded under different conditions (before) and effects of naloxone recorded under cathodal current in order to accentuate the disinhibitory effect. Time and voltage calibration, 50 /LV and 5 ms.
186
the NTS neurons, and regarded as transmitters. Since antagonists of SP, opioid peptide, G A B A and a-adrenergic agonist have been well established, these substances were used for testing. It is generally agreed that opioid peptides act as an inhibitory transmitter on most of the central neurons. However, Okada and Miura [23] discovered that small oval-type neurons of the cultured rat brainstem are excited by the application of opioid peptide and blocked by the concomitant application of naloxone. Since the cat NTS contains many oval-type neurons [27], we attempted to determine whether opioid peptide acts as a transmitter in the cat N T K and found that circulatory reflex response as well as evoked field potentials elicited by stimulation of the ipsilateral CSN were inhibited by the application of naloxone (250 mM, 300/~A. s) [18]. This finding suggested that opioid peptide might be an excitatory transmitter of the CSN fibers mediating circulatory reflex. However, in this previous experiment, the NTS response was not explored in relation to functional subdivisions of the NTS. After we had succeeded in subdividing the NTS in relation to the functional properties of the CSN reflex [19], we showed that SP antagonist as well as naloxone had an inhibitory effect on the same NTS response. The effect was strong in the pressor-apneustic response zone of the rostral regions and mild in the depressor-apneic response zone of both intermediate and commissure regions of the NTS [20,21]. This phenomenon has been confirmed in the present study, and suggests that SP as well as opioid peptide may act as an excitatory transmitter at the site of the CSN baroreceptor and chemoreceptor inputs. Recently, it has been shown that SP-immunoreactive terminals and SP binding sites are distributed throughout the whole extent of the rat NTS [14,16] and that SP exhibits a potent excitatory action on the cat NTS neuron [22]. These findings also support the notion that SP is a possible transmitter in the NTS. Bicuculline and prazosin augmented the NTS response which had been depressed by the application of cathodal current. This phenomenon was conspicuous in the pressor-apneustic response zone of the lateral NTS. Since cathodal inhibition of the NTS response may be due to activation of the
background inhibitory influence on the NTS response~ it is possible that the application of an antagonist of possible inhibitory transmitter may augment the NTS response. Recently, GABAergic neurons were detected immunocytochemically in the rat NTS [5,13,17,26]. They were observed in all subdivisions of the NTS with an increasing concentration from the caudal to rostral regions [26], and mostly detected around the solitary tract in the rostral and intermediate regions [17]. Such a characteristic distribution of the NTS GABAergic neurons is consistent with the site of the NTS response on which bicuculline had a disinhibitory effect. On the other hand, a-adrenergic neurons were also detected immunocytochemically in the rat NTS [1,2], and it has been demonstrated that adrenergic neurons are predominant in the C2 group of H~Skfelt [11], and noradrenergic neurons in the A2 group of DahlstrOm and Fuxe [7]. It has been reported in a study of cultured neurons from the rat lower brainstem [24] that GABA always acts as an inhibitory transmitter, while c~-adrenergic agonist acts as an inhibitory transmitter to a majority of the neurons and as an excitatory transmitter to a minority. Since the NTS neurons in the rostral regions are innervated not only by nearby A2 and C2 group neurons [11,28] but by distant A1 group neurons [4], it is possible that the NTS neurons may receive the inhibitory input from A1, A2 and C1 group neurons. In the medial NTS we showed that under cathodal current the early part of the NTS response was inhibited and the late part was disinhibited by the application of naloxone. This finding suggests that opioid peptides act as inhibitory as well as excitatory transmitters. Such a dual transmitter activity of opioid peptides has been observed in cultured neurons from the rat lower brainstem [23]. Recently, enkephalinergic neurons were detected immunocytochemically in the rat NTS [15], being concentrated in the medial area of the intermediate regions. Such a characteristic distribution of enkephalinergic neurons is consistent with the site of the NTS response on which naloxone had a disinhibitory effect. In conclusion, while we have provided an evidence that opioid peptide, substance P, GABA, a-adrenergic agonist may be transmitters working
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in the NTS, it is important to point out that our study is limited because we have used the field potentials instead of unit potentials as an indicator and we have not tried all the transmitter candidates. Future studies will have to address the problem concerning all the transmitter candidates acting in the NTS in relation to the carotid sinus baro- and chemoreceptor reflex.
Acknowledgement
This work was supported by a Grant-in-Aid for Scientific Research B from the Japanese Ministry of Education, Science and Culture (no. 59480102).
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