Brain Research, 566 (1991) 290-294 (~) 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/911503.50
2qO BRES 17237
Microinjection of glycine into the nucleus ambiguus elicits tachycardia in spinal rats V.C. C h i t r a v a n s h i , S.K. A g a r w a l a n d E R . C a l a r e s u Department of Physiology, University of Western Ontario, London, Ont. (Canada) (Accepted 23 July 1991)
Key words: Cardiovascular regulation; Glycine; Microinjection; Nucleus ambiguus
in 30 male Wistar spinal (CI) rats, anaesthetized with urethane and artificially ventilated, experiments were done to study the effect on heart rate (HR) and arterial pressure (AP) of microinjection of the inhibitory amino acid glycine (Gly) into the nucleus ambiguus (NA). L-Glutamate (Glu; 1.5 nmol) was microinjected into the region of the right NA to search for sites from which decreases in AP and HR could be elicited. The decreases in HR were found to be 73.1 - 7.0 bpm (n - 30). No changes in AP were observed. Microinjection of Gly (1 M; 2-20 nmoi in 2-20 ni; n - 12) elicited a dose dependent increase in HR with no changes in AP. Microinjection of Gly 1-2 rain before microinjection of Glu in 7 sites reduced significantly (P < 0.05) the decrease in HR elicited by Glu from 87.0 ± 27.3 bpm to 17.7 ± 7.2 bpm. Increases in HR elicited by Gly in the right NA of another 12 rats were not affected significantly by prior microinjection of the Gly antagonist strychnine hydrochloride (30-90 pmol in 10-30 nl in one group of animals, n - 6; and 2.5 nmol in 50 nl in another group, n - 6). In addition, to determine whether the effects of Gly were caused by actions on N-methyI-D-aspartate (NMDA) receptors, kynurenic acid (KYN; 4.5 nmol in 30 hi) was microinjected into the right NA of 6 rats prior to microinjection of Gly. KYN failed to block the response to Gly microinjection and instead potentiated the HR increase elicited by Gly. These results suggest that Gly acts as an inhibitory amino acid transmitter influencing vagal neurons in the right NA controlling HR and that the effects of Gly are mediated probably through strychnine-insensitive non-NMDA receptors.
INTRODUCTION
It is well established that vagal inhibitory fibers to the heart arise from two nuclei in the medulla oblongata, the dorsal vagal nucleus (DVN), and the ventrolateral component of the nucleus ambiguus (NA) ~'l~, but the detailed pathways and the putative neurotransmitters between baroreceptor afferents and these vagal moto. neurons have begun to be studied only recently t's'7. At least two putative neurotransmitters have been proposed to be involved in inhibitory control of cardiac vagal motoneurons (CVM). The inhibitory amino acids glycine (Gly) and ~, aminobutyric acid (GABA) have both been shown to depress the activity of CVM when applied iontophoretically 7,t°. In addition, the specific antagonists strychnine and bicuculline have been shown to block the effects of Gly and GABA7't° Furthermore, as the microiontophoresis of bicuculline often evoked an increase in CVM discharge it has been suggested that there may be a tonic release of GABA regulating the activity of CVM and hence heart rate 7,t° (HR). Additional information about glycinergic mechanisms in medullary cardiovascular control has been obtained by
the demonstration that microinjection of Gly into the rat DVN elicits increases in arterial pressure (AP) and HR tT, whereas a decrease in AP and HR is elicited by Gly injection into the adjacent nucleus tractus solitarius t~ (NTS), Finally, GABA and Oly, the two major inhibitory neurotransmitters in the mammalian central nervous system, may also contribute to cardiovascular control by their actions on structures in the medulla other than the vagal nuclei. For instance, it has been shown that microinjection of Gly into the caudal ventrolateral medulla of the rabbit causes an increase in AP4, In the present study Gly was microinjected into the right NA to study its effects on HR and AP in spinal rats. The spinal transection was done at the C1 level to eliminate the descending bulbar pathways that control the sympathetic outflow to the heart thereby limiting the effects of medullary microinjection to influences on vagal nerve fibers to the heart. The objectives of our experiments were to determine: (I) the cardiovascular effects of microinjection of Gly at selected sites in the NA from which decreases in HR could be obtained by glutamate (Glu) microinjection, and (2) the mechanisms of
Correspondence: V.C, Chitravanshi, Department of Physiology, The University of Western Ontario, London, Ont. N6A 5CI, Canada. Fax: (1) (519) 661 3827.
291 interaction between G l y a n d Glu microinjected into the NA. MATERIALS AND METHODS
General procedure Experiments were done in 30 adult male Wistar rats (250-350 g, Charles River, Montreal, Canada), anaesthetized with urethane (Sigma, St. Louis, M e , 1,4 g/kg, i,p. initially and 0.25 g/kg supplements as required), Cannulation of the trachea, femoral artery and vein was done and the animal was artificially ventilated with room air using a small animal ventilator (Harvard Apparatus, model 683), The arterial cannula was connected to a pressure transducer (Century Technology, Inglewood, CA, model CP 01) that was connected to a Grass polygraph (model 79C) for continuous recording of AE HR was monitored by a Grass tachograph (7 P44B) triggered by the AP pulse, The animal was placed in a stereotaxic apparatus, with the bite bar 20 mm below the interaural line, The dorsal neck muscles were retracted and the medulla was exposed by incising the atlanto-occipitai membrane and removing part of the occipital bone and the dura. Transection of the spinal cord was done at the C1 level. Phenylephrine (PE; Sigma) in physiological saline (2 mg/ml) was infused into the venous cannula, initially at a rate of I ml/h for 3-5 min and then at 0.25 ml/h to maintain AP within the physiological range (mean arterial pressure between 90 and 100 mmHg). Animals that did not have a steady level of AP over a period of several hours were not included in the results. Rectal temperature was maintained at 37.5 --. 0.50 C with a thermostatically controlled heating blanket. Pressure microinjection L-Glutamate (Olu; Sigma, 0.15 M), glycine (Gly; Sigma, 1 M), strychnine hydrochloride (Sigma, 3 raM) and kynurenic acid (KYN; Sigma, 0,15 M) were dissolved in phosphate-buffered saline (PBS, pH 7.4) and pressure microinjeeted through ntulfibarrelled glass micropipettes pulled from glass capillary tubing (Socorex 851-5, Temehem Laboratories, Mississauga, Ont., Canada). Long shanks were drawn to minimize tissue distortion and the tips were broken to an external diameter of ~50 ~m, The pipettes were inclined 200 from the vertical in the sagittal plane with the tip pointing rostrally. The stereotaxic coordinates used for the placement of the angled tip in the NA were 0.2-0.6 mm caudal to the obex, 1.6-2.0 mm lateral to the midline and 2.0-2.4 mm below the dorsal surface of the medulla, Volumes of injections were measured directly by monitoring the movement of the fluid meniscus in the micropipette through a 40x microscope fitted with an ocular scale that allowed a resolution of 1 nl.
Histology Microinjection sites were marked for histological verification with India ink usi,,g a method previously described 2. The animals were perfused with 50 ml of PBS followed by 50 ml of a 10% formalin solution in PBS. After fixation in formalin for 3-4 days, frozen transverse sections (50 ~m) were cut and stained with thionin. Injection ~itc~ were mapped on drawings of transverse sections of the ,'at brain from an atlas '5. Statistics Mean changes in AP and HR from control values were compared by Student's t-tests. For dose-response relationships, percentage changes in HR vs doses were plotted. The probability level taken to indicate a significant difference was P < 0.05 for all statistical tests. All data in text and figures are expressed as means -- S.E.M.
RESULTS Glu (1.5 nmol in 10 nl) was microinjected into the right N A to identify the location of cell bodies from which decreases in H R could be elicited. Microinjection of Glu elicited decreases in H R of 73.1 -- 7.0 bpm from a baseline of 440.0 ± 4.1 b p m (n = 30 sites). The Glu response had an onset latency of 1.5 ± 0.6 s, reached a p e a k in 7.4 ± 1.0 s and lasted 77.8 ± 7.4 s. There were no decreases in AP. A characteristic response to Glu is shown in Fig. 1A. Microinjection of PBS (30 nl in 12 rats) into the same sites h a d no effect on A P or H R (Fig. 1B). T h e locations of the Glu injection sites are shown in Fig. 2.
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Fig. 1. Tracings of heart rate (HR) and arterial pressure (AP) after microinjection of (A) L-glutamate (Glu: 1.5 nmol in 10 nl), (B) phosphate-buffered saline (PBS; 30 nl), and (C) glycine (Gly; 10 nmol in 10 nl) into the right nucleus ambiguus of a spinal rat. All records are from the same rat. Arrowheads indicate beginning of microinjections.
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Fig. 2. Diagrammatic transverse sections of the medulla (modified from Paxinos and Watson 's) showing locations of sites in the right NA where responses to Glu microinjection were obtained. Numbers indicate distances in mm caudal to the interaural line. IO, inferior olive; LRN, lateral reticular nucleus: NA, nucleus ambiguus: py, pyramid; RO, raphe obscurus.
292 HR
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Fig. 3. Microinjcction of Gly into the right nucleus ambiguus elicited dose dependent increases in HR in 12 rats: the doses ranged between 2 and 20 nmol and the points represent mean values (S.E.M.) of % increases in HR.
Microin/ection of Gly into the NA In 12 rats, glycine microinjected at the doses of 2, 6, 10 and 20 nmol (2-20 nl) into the right NA at sites where microinjection of Glu elicited bradycardia, elicited dose dependent increases in HR (Fig, 3), The threshold dose was 2 nmol, The maximum dose of 20 nmol ~aused an increase in HR of 17,5 ± 2,4 bpm (n -- 12), An example of a response to Gly injection is shown in Fig, IC, Oly injection did not change AP, The average onset la. tency of the HR response was 10,5 ± 1,8 s, the peak response was reached in 17,3 ± 5,2 s and HR gradually returned to preinjection values in 171,3 ± 34.1 s,
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Fig. 5. Effect of kynurenic acid (KYN) microinjection into the fight NA on decrease and increase in HR elicited by microinjection of Glu and Gly, respectively. A: microinjection of Glu (1.5 nmol in 10 nl). B: microinjection of Gly (20 nmol in 20 nl) into the fight NA. C: microinjection of KYN (4.5 nmol in 30 nl). D: microinjection of Glu (1.5 nmol in 10 nl) 3-5 min after KYN microinjection. Note that Glu response was attenuated. E: HR response to microinjection of Gly (20 nmol in 20 nl) is potentiated after KYN and F: HR response to Glu (1.5 nmol) into the right NA has recovered after 40-50 min. All records are from the same rat.
Interactions between Glu and Gly in the NA Microinjection of Giu (1.5 nmol in 10 nl) into the right NA (7 sites) decreased HR 87.0 ± 27.3 bpm from a baseline of 437,6 ± 7,9 bpm. Microinjection of the same volume of Olu 1-2 min after injection of Oly (10 nmol in 10 nl) into the same sites elicited decreases in HR of only 17,7 • 7.2 bpm from a baseline of 433.1 ± 7.3 bpm. The attenuation of the Glu response following Oly injection was significant (P < 0.05). An example of a response to Glu before (Fig. 4A) and after (Fig. 4B) Gly injection and the recovery of the response (Fig. 4C) is shown in Fig. 4. Microinjection of 30 nl of PBS (n ffi 7) at the same sites had no effect on AP and HR (Fig. 4D).
Microinjection of strychnine into the NA AP
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Fig. 4. Heart rate (HR) and arterial pressure (AP) tracings after microinjection of Glu before and after Gly. A: bradycardia elicited by microinjection of Glu (1.5 nmol in 10 nl). B: response to the injection of Glu (1.5 nmol in 10 nl) after Gly injection at the same site was markedly attenuated when compared with ~he response before glycine (A). C: ten minutes after the injection of Gly the response to Glu had recovered. D: phosphate buffered saline (PBS, ni) did not elicit any changes in either AP or HR. No changes in AP were observed with either Glu or Gly. All records are from the same rat.
To determine whether the cardiovascular responses to Oly were mediated through specific receptors, the Oly receptor antagonist strychnine hydrochloride was microinjected (30-90 pmol in 10-30 hi; n - 6) into the right NA. Strychnine did not block the increases in H R elicited by Gly injection from an adjacent barrel of a multibarrel pipette. In an additional series of experiments higher doses of strychnine (2.5 nmol in 50 nl; n ffi 6) also failed to block the effects of Gly.
Microin]ection of Glu and Gly before and after KYN into the NA To determine whether the Gly responses were medi-
293 ated by actions on N-methyl-D-aspartate (NMDA) receptors, Glu and Gly were microinjected before and after the microinjection of the Glu receptor antagonist KYN into the right NA. Microinjecttion of Glu (1.5 nmol in 10 nl) and Gly (20 nmol in 20 nl) into the NA produced decreases (74.2 -- 9.3 bpm) and increases (14.0 - 3.4 bpm) in HR respectively (Fig. 5A,B). Microinjection of KYN (4.5 nmol in 30 nl; n = 6) decreased HR by 14.2 - 4.5 bpm from a baseline of 377.5 -+ 7.4 bpm. The onset latency was 14.0 +- 3.7 s and the peak response was reached in 30.8 +- 11.3 s (Fig. 5C). Glu microinjection 3-5 min after KYN microinjection elicited a significantly (P < 0.001) attenuated decrease in HR from 74.2 - 9.3 bpm to 8.5 +- 2.3 bpm (n = 6). An example is shown in Fig. 5D. The responses to Glu microinjection recovered 40-50 min after microinjection of KYN (Fig. 5F). In contrast, Gly responses were significantly potentiated (n = 6; P < 0.05) 6-10 min after KYN microinjection. There was a change in the original Gly response from 14.0 +- 3.4 bpm to 25.7 - 3.1 bpm after KYN injection (Fig. 5E). Responses to Gly microinjection returned to baseline values 40-45 rain after KYN injection. DISCUSSION This study has shown that microinjection of Gly into the NA elicits dose dependent increases in HR which are mediated by inhibition of vagal neurons to the heart. These novel results are in agreement with an earlier report that microinjection of Oly into the rat DVN, another medullary nucleus containing cardioinhibitory neurons 17, elicits dose dependent increases in HR that are not seen with injections into adjacent areas. Gly had no effect on AP in our study whereas microinjection of Oly into the DVN has been previously shown to increase Ap 17. Gly is a putative inhibitory neurotransmitter in the central nervous system of mammals and may have an important role in the control of AP and HR 3 probably mediated through structures in the caudal ventrolateral medulla 4. In addition, Gly has been found in high concentrations in the DVN n'x4't6, further supporting a role in cardiovascular control. REFERENCES 1 Agarwal, S.K. and Calaresu, ER., Enkephalins, substance P and acetylcholinemicroinjectedinto the nucleusambiguuselicit vagal bradycardia in rats, Brain Research, (1991)., in press. 2 Agarwal, S.K., Oelsema, A.J. and Calaresu, F.R., Neurons in the rostral VLM are inhibited by chemical stimulation of caudal VLM in rats, Am. Y. Physiol., 257 (1989) R265-R270. 3 Antonaccio, M. and Taylor, D.G., Involvement of central
As microinjection of up to 30 nl of PBS into the NA did not elicit changes in HR, the changes after microinjection of Gly and Glu were likely not the result of nonspecific vehicle effects, such as local distortion of neural tissue caused by the micropipettes or by the volume of the injected fluid. Furthermore, the responses to Glu and Gly were site dependent since they could be eliminated by moving the tip of the micropipettes as little as 150 ~m. Finally, if distortion of nervous tissue were responsible for the observed chtuiges in HR it would be expected that microinjection of either Gly or Glu would elicit similar increases or decreases in HR. Microinjection of 67 pmol of strychnine has been reported to block the effects of a 15 nmol dose of Gly in the NTS of the rat TM. In our studies, however, microinjection of strychnine (30-90 pmol and 2.5 nmol) into the NA did not block the increases in HR elicited by Gly suggesting that this amino acid was acting through strychnine-insensitive Gly receptors. This possibility is supported by the previous observation that strychnine also failed to block the effects elicited by Gly on the NMDA receptor complex in cultured mouse brain neurons9. The interpretation of our results is further supported by the finding that (1) KYN microinjected into the NA could inhibit strychnine-insensitive [3H]GIy binding sites~'xl probably by an action at the Gly modulatory site on the NMDA receptor complex 19 and (2) in this study microinjection of KYN potentiated the HR responses to Gly instead of blocking them. Taken together, these results indicate that HR responses to Gly were mediated through strychnine-insensitive non-NMDA receptors on NA neurons controlling HR. In summary, these results suggest a role for Oly as an inhibitor of the vagal neurons in the NA controlling heart rate. As the increases in HR elicited by Gly were not blocked by strychnine or KYN, it may be concluded that Gly acts through strychnine-insensitive non-NMDA receptors on the cell bodies of the NA.
Acknowledgements. The critical comments and suggestionsprovided by D.J. McKitrick and K. Hayes are gratefully acknowledged. This study was supported by a grant from the Medical Research Council of Canada. S.K.A. is a Fellow of the Canadian Heart and Stroke Foundation.
GABA receptors in the regulation of blood pressure and heart rate of anaesthetized cats, Eur. J. Pharrnacol., 46 (1977) 283287. 4 Blessing, W.W. and Reis, DJ., Evidence that GABA and glycine-like inputs inhibits vasodepressor neurons in the caudal ventrolateral medullaof the rabbit, Neuroscience, 37 (1983) 5762. 5 Caverson, M.M. and Zhang, T.X., Substance P (SP) innervation of the vagal cardiomotor neurons (VCN) in the nucleus
294 ambiguus, Soc. Neurosci. Abstr., 15 (1989) 966. 6 Chiamulera, C., Costa, S. and Reggiani, A., Effect of NMDAand strychnine insensitive glycine site antagonists on NMDAmediated convulsions and learning, Psychopharmacology, 102 (1990) 551-552. 7 Gilbey, M.P., Spyer, K.M. and Wood, L.M., The inhibitory actions of GABA on cardiac vagal motoneurons in the cat, J. Physiol., 361 (1985) 49P. 8 Hopkins, D.A. and Holstege, G., Amygdaloid projections to the mesencephalon, ports and medulla oblongata in the cat, Exp. Brain Res., 32 (1978) 529-547. 9 Johnson, LW. and Ascher. P., Giycine potent.lares the NMDA response in cultured mouse brain neurons, Nature, 325 (1987) 529-531. 10 Jordan, D., Gilbey, M.P., Richter, W.D., Spyer, K.M. and Wood, L.M., Respiratory-vagal interactions in the nucleus ambiguus of the cat. In A.L. Bianchi and M. Denavit Saubie (Eds.), Neurogenesis of Central Respiratory Rhythm, MTP, Lancaster, 1985, pp. 370-378. 11 Kessler, M., Lynch, G., Terramani, T. and Bandry, M., A glycine site associated with N.methyl-D-aspartic receptors: characterization and identification of a new class of antagonists, J. Sreurochem., 52 (1989) 1319-1328. 12 Kubo, T. and Kihara, M., Evidence for presence of GABAergic and glycine like systems responsible for cardiovascular control in the nucleus tractus solitarius of rat, Neurosci. Lett., 74
(1987) 331-336. 13 Loewy, A.D. and Spyer, K.M., Vagal preganglionic neurons. In A.D. Loewy and K.M. Spyer (Eds.), Central Regulation of Autonomic Function, Oxford University Press, U.K., 1990, pp. 68-87. 14 Meeley, M.P., Underwood, M.D., Talman, W.T. and Reis, D.J., Content and in vitro release of endogenous aminoacids in the area of nucleus tractus solitarius (NTS), NeuroscL Absir., 9 (1983) 262. 15 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986. 16 Perrone, M.H., Biochemical evidence that L-glutamate is a neurotransmitter of primary vagal afferent nerve fibers, Brain Research, 230 (1981) 283-293. 17 Talman, W.T., Glycine microinjected in the rat dorsal vagal nucleus increases arterial pressure, Hypertension, 11 (1988) 664667. 18 Talman, W.T. and Robertson, S.C., Glycine, like glutamate, microinjected into the nucleus tractus solitarii of rat decreases arterial pressure and heart rate, Brain Research, 447 (1989) 7-13. 19 Watson, G.B., Hood, W.F., Monahan, J.B. and Lanthorn, T.H., Kynurenate antagonizes actions of N-methyl-D-aspartate through a glycine-sensitive receptor, Neurosci. Res. Commun., 2 (1988) 169-174.
2qO BRES 17237
Microinjection of glycine into the nucleus ambiguus elicits tachycardia in spinal rats V.C. C h i t r a v a n s h i , S.K. A g a r w a l a n d E R . C a l a r e s u Department of Physiology, University of Western Ontario, London, Ont. (Canada) (Accepted 23 July 1991)
Key words: Cardiovascular regulation; Glycine; Microinjection; Nucleus ambiguus
in 30 male Wistar spinal (CI) rats, anaesthetized with urethane and artificially ventilated, experiments were done to study the effect on heart rate (HR) and arterial pressure (AP) of microinjection of the inhibitory amino acid glycine (Gly) into the nucleus ambiguus (NA). L-Glutamate (Glu; 1.5 nmol) was microinjected into the region of the right NA to search for sites from which decreases in AP and HR could be elicited. The decreases in HR were found to be 73.1 - 7.0 bpm (n - 30). No changes in AP were observed. Microinjection of Gly (1 M; 2-20 nmoi in 2-20 ni; n - 12) elicited a dose dependent increase in HR with no changes in AP. Microinjection of Gly 1-2 rain before microinjection of Glu in 7 sites reduced significantly (P < 0.05) the decrease in HR elicited by Glu from 87.0 ± 27.3 bpm to 17.7 ± 7.2 bpm. Increases in HR elicited by Gly in the right NA of another 12 rats were not affected significantly by prior microinjection of the Gly antagonist strychnine hydrochloride (30-90 pmol in 10-30 nl in one group of animals, n - 6; and 2.5 nmol in 50 nl in another group, n - 6). In addition, to determine whether the effects of Gly were caused by actions on N-methyI-D-aspartate (NMDA) receptors, kynurenic acid (KYN; 4.5 nmol in 30 hi) was microinjected into the right NA of 6 rats prior to microinjection of Gly. KYN failed to block the response to Gly microinjection and instead potentiated the HR increase elicited by Gly. These results suggest that Gly acts as an inhibitory amino acid transmitter influencing vagal neurons in the right NA controlling HR and that the effects of Gly are mediated probably through strychnine-insensitive non-NMDA receptors.
INTRODUCTION
It is well established that vagal inhibitory fibers to the heart arise from two nuclei in the medulla oblongata, the dorsal vagal nucleus (DVN), and the ventrolateral component of the nucleus ambiguus (NA) ~'l~, but the detailed pathways and the putative neurotransmitters between baroreceptor afferents and these vagal moto. neurons have begun to be studied only recently t's'7. At least two putative neurotransmitters have been proposed to be involved in inhibitory control of cardiac vagal motoneurons (CVM). The inhibitory amino acids glycine (Gly) and ~, aminobutyric acid (GABA) have both been shown to depress the activity of CVM when applied iontophoretically 7,t°. In addition, the specific antagonists strychnine and bicuculline have been shown to block the effects of Gly and GABA7't° Furthermore, as the microiontophoresis of bicuculline often evoked an increase in CVM discharge it has been suggested that there may be a tonic release of GABA regulating the activity of CVM and hence heart rate 7,t° (HR). Additional information about glycinergic mechanisms in medullary cardiovascular control has been obtained by
the demonstration that microinjection of Gly into the rat DVN elicits increases in arterial pressure (AP) and HR tT, whereas a decrease in AP and HR is elicited by Gly injection into the adjacent nucleus tractus solitarius t~ (NTS), Finally, GABA and Oly, the two major inhibitory neurotransmitters in the mammalian central nervous system, may also contribute to cardiovascular control by their actions on structures in the medulla other than the vagal nuclei. For instance, it has been shown that microinjection of Gly into the caudal ventrolateral medulla of the rabbit causes an increase in AP4, In the present study Gly was microinjected into the right NA to study its effects on HR and AP in spinal rats. The spinal transection was done at the C1 level to eliminate the descending bulbar pathways that control the sympathetic outflow to the heart thereby limiting the effects of medullary microinjection to influences on vagal nerve fibers to the heart. The objectives of our experiments were to determine: (I) the cardiovascular effects of microinjection of Gly at selected sites in the NA from which decreases in HR could be obtained by glutamate (Glu) microinjection, and (2) the mechanisms of
Correspondence: V.C, Chitravanshi, Department of Physiology, The University of Western Ontario, London, Ont. N6A 5CI, Canada. Fax: (1) (519) 661 3827.
291 interaction between G l y a n d Glu microinjected into the NA. MATERIALS AND METHODS
General procedure Experiments were done in 30 adult male Wistar rats (250-350 g, Charles River, Montreal, Canada), anaesthetized with urethane (Sigma, St. Louis, M e , 1,4 g/kg, i,p. initially and 0.25 g/kg supplements as required), Cannulation of the trachea, femoral artery and vein was done and the animal was artificially ventilated with room air using a small animal ventilator (Harvard Apparatus, model 683), The arterial cannula was connected to a pressure transducer (Century Technology, Inglewood, CA, model CP 01) that was connected to a Grass polygraph (model 79C) for continuous recording of AE HR was monitored by a Grass tachograph (7 P44B) triggered by the AP pulse, The animal was placed in a stereotaxic apparatus, with the bite bar 20 mm below the interaural line, The dorsal neck muscles were retracted and the medulla was exposed by incising the atlanto-occipitai membrane and removing part of the occipital bone and the dura. Transection of the spinal cord was done at the C1 level. Phenylephrine (PE; Sigma) in physiological saline (2 mg/ml) was infused into the venous cannula, initially at a rate of I ml/h for 3-5 min and then at 0.25 ml/h to maintain AP within the physiological range (mean arterial pressure between 90 and 100 mmHg). Animals that did not have a steady level of AP over a period of several hours were not included in the results. Rectal temperature was maintained at 37.5 --. 0.50 C with a thermostatically controlled heating blanket. Pressure microinjection L-Glutamate (Olu; Sigma, 0.15 M), glycine (Gly; Sigma, 1 M), strychnine hydrochloride (Sigma, 3 raM) and kynurenic acid (KYN; Sigma, 0,15 M) were dissolved in phosphate-buffered saline (PBS, pH 7.4) and pressure microinjeeted through ntulfibarrelled glass micropipettes pulled from glass capillary tubing (Socorex 851-5, Temehem Laboratories, Mississauga, Ont., Canada). Long shanks were drawn to minimize tissue distortion and the tips were broken to an external diameter of ~50 ~m, The pipettes were inclined 200 from the vertical in the sagittal plane with the tip pointing rostrally. The stereotaxic coordinates used for the placement of the angled tip in the NA were 0.2-0.6 mm caudal to the obex, 1.6-2.0 mm lateral to the midline and 2.0-2.4 mm below the dorsal surface of the medulla, Volumes of injections were measured directly by monitoring the movement of the fluid meniscus in the micropipette through a 40x microscope fitted with an ocular scale that allowed a resolution of 1 nl.
Histology Microinjection sites were marked for histological verification with India ink usi,,g a method previously described 2. The animals were perfused with 50 ml of PBS followed by 50 ml of a 10% formalin solution in PBS. After fixation in formalin for 3-4 days, frozen transverse sections (50 ~m) were cut and stained with thionin. Injection ~itc~ were mapped on drawings of transverse sections of the ,'at brain from an atlas '5. Statistics Mean changes in AP and HR from control values were compared by Student's t-tests. For dose-response relationships, percentage changes in HR vs doses were plotted. The probability level taken to indicate a significant difference was P < 0.05 for all statistical tests. All data in text and figures are expressed as means -- S.E.M.
RESULTS Glu (1.5 nmol in 10 nl) was microinjected into the right N A to identify the location of cell bodies from which decreases in H R could be elicited. Microinjection of Glu elicited decreases in H R of 73.1 -- 7.0 bpm from a baseline of 440.0 ± 4.1 b p m (n = 30 sites). The Glu response had an onset latency of 1.5 ± 0.6 s, reached a p e a k in 7.4 ± 1.0 s and lasted 77.8 ± 7.4 s. There were no decreases in AP. A characteristic response to Glu is shown in Fig. 1A. Microinjection of PBS (30 nl in 12 rats) into the same sites h a d no effect on A P or H R (Fig. 1B). T h e locations of the Glu injection sites are shown in Fig. 2.
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Fig. 1. Tracings of heart rate (HR) and arterial pressure (AP) after microinjection of (A) L-glutamate (Glu: 1.5 nmol in 10 nl), (B) phosphate-buffered saline (PBS; 30 nl), and (C) glycine (Gly; 10 nmol in 10 nl) into the right nucleus ambiguus of a spinal rat. All records are from the same rat. Arrowheads indicate beginning of microinjections.
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Fig. 2. Diagrammatic transverse sections of the medulla (modified from Paxinos and Watson 's) showing locations of sites in the right NA where responses to Glu microinjection were obtained. Numbers indicate distances in mm caudal to the interaural line. IO, inferior olive; LRN, lateral reticular nucleus: NA, nucleus ambiguus: py, pyramid; RO, raphe obscurus.
292 HR
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Fig. 3. Microinjcction of Gly into the right nucleus ambiguus elicited dose dependent increases in HR in 12 rats: the doses ranged between 2 and 20 nmol and the points represent mean values (S.E.M.) of % increases in HR.
Microin/ection of Gly into the NA In 12 rats, glycine microinjected at the doses of 2, 6, 10 and 20 nmol (2-20 nl) into the right NA at sites where microinjection of Glu elicited bradycardia, elicited dose dependent increases in HR (Fig, 3), The threshold dose was 2 nmol, The maximum dose of 20 nmol ~aused an increase in HR of 17,5 ± 2,4 bpm (n -- 12), An example of a response to Gly injection is shown in Fig, IC, Oly injection did not change AP, The average onset la. tency of the HR response was 10,5 ± 1,8 s, the peak response was reached in 17,3 ± 5,2 s and HR gradually returned to preinjection values in 171,3 ± 34.1 s,
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Fig. 5. Effect of kynurenic acid (KYN) microinjection into the fight NA on decrease and increase in HR elicited by microinjection of Glu and Gly, respectively. A: microinjection of Glu (1.5 nmol in 10 nl). B: microinjection of Gly (20 nmol in 20 nl) into the fight NA. C: microinjection of KYN (4.5 nmol in 30 nl). D: microinjection of Glu (1.5 nmol in 10 nl) 3-5 min after KYN microinjection. Note that Glu response was attenuated. E: HR response to microinjection of Gly (20 nmol in 20 nl) is potentiated after KYN and F: HR response to Glu (1.5 nmol) into the right NA has recovered after 40-50 min. All records are from the same rat.
Interactions between Glu and Gly in the NA Microinjection of Giu (1.5 nmol in 10 nl) into the right NA (7 sites) decreased HR 87.0 ± 27.3 bpm from a baseline of 437,6 ± 7,9 bpm. Microinjection of the same volume of Olu 1-2 min after injection of Oly (10 nmol in 10 nl) into the same sites elicited decreases in HR of only 17,7 • 7.2 bpm from a baseline of 433.1 ± 7.3 bpm. The attenuation of the Glu response following Oly injection was significant (P < 0.05). An example of a response to Glu before (Fig. 4A) and after (Fig. 4B) Gly injection and the recovery of the response (Fig. 4C) is shown in Fig. 4. Microinjection of 30 nl of PBS (n ffi 7) at the same sites had no effect on AP and HR (Fig. 4D).
Microinjection of strychnine into the NA AP
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Fig. 4. Heart rate (HR) and arterial pressure (AP) tracings after microinjection of Glu before and after Gly. A: bradycardia elicited by microinjection of Glu (1.5 nmol in 10 nl). B: response to the injection of Glu (1.5 nmol in 10 nl) after Gly injection at the same site was markedly attenuated when compared with ~he response before glycine (A). C: ten minutes after the injection of Gly the response to Glu had recovered. D: phosphate buffered saline (PBS, ni) did not elicit any changes in either AP or HR. No changes in AP were observed with either Glu or Gly. All records are from the same rat.
To determine whether the cardiovascular responses to Oly were mediated through specific receptors, the Oly receptor antagonist strychnine hydrochloride was microinjected (30-90 pmol in 10-30 hi; n - 6) into the right NA. Strychnine did not block the increases in H R elicited by Gly injection from an adjacent barrel of a multibarrel pipette. In an additional series of experiments higher doses of strychnine (2.5 nmol in 50 nl; n ffi 6) also failed to block the effects of Gly.
Microin]ection of Glu and Gly before and after KYN into the NA To determine whether the Gly responses were medi-
293 ated by actions on N-methyl-D-aspartate (NMDA) receptors, Glu and Gly were microinjected before and after the microinjection of the Glu receptor antagonist KYN into the right NA. Microinjecttion of Glu (1.5 nmol in 10 nl) and Gly (20 nmol in 20 nl) into the NA produced decreases (74.2 -- 9.3 bpm) and increases (14.0 - 3.4 bpm) in HR respectively (Fig. 5A,B). Microinjection of KYN (4.5 nmol in 30 nl; n = 6) decreased HR by 14.2 - 4.5 bpm from a baseline of 377.5 -+ 7.4 bpm. The onset latency was 14.0 +- 3.7 s and the peak response was reached in 30.8 +- 11.3 s (Fig. 5C). Glu microinjection 3-5 min after KYN microinjection elicited a significantly (P < 0.001) attenuated decrease in HR from 74.2 - 9.3 bpm to 8.5 +- 2.3 bpm (n = 6). An example is shown in Fig. 5D. The responses to Glu microinjection recovered 40-50 min after microinjection of KYN (Fig. 5F). In contrast, Gly responses were significantly potentiated (n = 6; P < 0.05) 6-10 min after KYN microinjection. There was a change in the original Gly response from 14.0 +- 3.4 bpm to 25.7 - 3.1 bpm after KYN injection (Fig. 5E). Responses to Gly microinjection returned to baseline values 40-45 rain after KYN injection. DISCUSSION This study has shown that microinjection of Gly into the NA elicits dose dependent increases in HR which are mediated by inhibition of vagal neurons to the heart. These novel results are in agreement with an earlier report that microinjection of Oly into the rat DVN, another medullary nucleus containing cardioinhibitory neurons 17, elicits dose dependent increases in HR that are not seen with injections into adjacent areas. Gly had no effect on AP in our study whereas microinjection of Oly into the DVN has been previously shown to increase Ap 17. Gly is a putative inhibitory neurotransmitter in the central nervous system of mammals and may have an important role in the control of AP and HR 3 probably mediated through structures in the caudal ventrolateral medulla 4. In addition, Gly has been found in high concentrations in the DVN n'x4't6, further supporting a role in cardiovascular control. REFERENCES 1 Agarwal, S.K. and Calaresu, ER., Enkephalins, substance P and acetylcholinemicroinjectedinto the nucleusambiguuselicit vagal bradycardia in rats, Brain Research, (1991)., in press. 2 Agarwal, S.K., Oelsema, A.J. and Calaresu, F.R., Neurons in the rostral VLM are inhibited by chemical stimulation of caudal VLM in rats, Am. Y. Physiol., 257 (1989) R265-R270. 3 Antonaccio, M. and Taylor, D.G., Involvement of central
As microinjection of up to 30 nl of PBS into the NA did not elicit changes in HR, the changes after microinjection of Gly and Glu were likely not the result of nonspecific vehicle effects, such as local distortion of neural tissue caused by the micropipettes or by the volume of the injected fluid. Furthermore, the responses to Glu and Gly were site dependent since they could be eliminated by moving the tip of the micropipettes as little as 150 ~m. Finally, if distortion of nervous tissue were responsible for the observed chtuiges in HR it would be expected that microinjection of either Gly or Glu would elicit similar increases or decreases in HR. Microinjection of 67 pmol of strychnine has been reported to block the effects of a 15 nmol dose of Gly in the NTS of the rat TM. In our studies, however, microinjection of strychnine (30-90 pmol and 2.5 nmol) into the NA did not block the increases in HR elicited by Gly suggesting that this amino acid was acting through strychnine-insensitive Gly receptors. This possibility is supported by the previous observation that strychnine also failed to block the effects elicited by Gly on the NMDA receptor complex in cultured mouse brain neurons9. The interpretation of our results is further supported by the finding that (1) KYN microinjected into the NA could inhibit strychnine-insensitive [3H]GIy binding sites~'xl probably by an action at the Gly modulatory site on the NMDA receptor complex 19 and (2) in this study microinjection of KYN potentiated the HR responses to Gly instead of blocking them. Taken together, these results indicate that HR responses to Gly were mediated through strychnine-insensitive non-NMDA receptors on NA neurons controlling HR. In summary, these results suggest a role for Oly as an inhibitor of the vagal neurons in the NA controlling heart rate. As the increases in HR elicited by Gly were not blocked by strychnine or KYN, it may be concluded that Gly acts through strychnine-insensitive non-NMDA receptors on the cell bodies of the NA.
Acknowledgements. The critical comments and suggestionsprovided by D.J. McKitrick and K. Hayes are gratefully acknowledged. This study was supported by a grant from the Medical Research Council of Canada. S.K.A. is a Fellow of the Canadian Heart and Stroke Foundation.
GABA receptors in the regulation of blood pressure and heart rate of anaesthetized cats, Eur. J. Pharrnacol., 46 (1977) 283287. 4 Blessing, W.W. and Reis, DJ., Evidence that GABA and glycine-like inputs inhibits vasodepressor neurons in the caudal ventrolateral medullaof the rabbit, Neuroscience, 37 (1983) 5762. 5 Caverson, M.M. and Zhang, T.X., Substance P (SP) innervation of the vagal cardiomotor neurons (VCN) in the nucleus
294 ambiguus, Soc. Neurosci. Abstr., 15 (1989) 966. 6 Chiamulera, C., Costa, S. and Reggiani, A., Effect of NMDAand strychnine insensitive glycine site antagonists on NMDAmediated convulsions and learning, Psychopharmacology, 102 (1990) 551-552. 7 Gilbey, M.P., Spyer, K.M. and Wood, L.M., The inhibitory actions of GABA on cardiac vagal motoneurons in the cat, J. Physiol., 361 (1985) 49P. 8 Hopkins, D.A. and Holstege, G., Amygdaloid projections to the mesencephalon, ports and medulla oblongata in the cat, Exp. Brain Res., 32 (1978) 529-547. 9 Johnson, LW. and Ascher. P., Giycine potent.lares the NMDA response in cultured mouse brain neurons, Nature, 325 (1987) 529-531. 10 Jordan, D., Gilbey, M.P., Richter, W.D., Spyer, K.M. and Wood, L.M., Respiratory-vagal interactions in the nucleus ambiguus of the cat. In A.L. Bianchi and M. Denavit Saubie (Eds.), Neurogenesis of Central Respiratory Rhythm, MTP, Lancaster, 1985, pp. 370-378. 11 Kessler, M., Lynch, G., Terramani, T. and Bandry, M., A glycine site associated with N.methyl-D-aspartic receptors: characterization and identification of a new class of antagonists, J. Sreurochem., 52 (1989) 1319-1328. 12 Kubo, T. and Kihara, M., Evidence for presence of GABAergic and glycine like systems responsible for cardiovascular control in the nucleus tractus solitarius of rat, Neurosci. Lett., 74
(1987) 331-336. 13 Loewy, A.D. and Spyer, K.M., Vagal preganglionic neurons. In A.D. Loewy and K.M. Spyer (Eds.), Central Regulation of Autonomic Function, Oxford University Press, U.K., 1990, pp. 68-87. 14 Meeley, M.P., Underwood, M.D., Talman, W.T. and Reis, D.J., Content and in vitro release of endogenous aminoacids in the area of nucleus tractus solitarius (NTS), NeuroscL Absir., 9 (1983) 262. 15 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986. 16 Perrone, M.H., Biochemical evidence that L-glutamate is a neurotransmitter of primary vagal afferent nerve fibers, Brain Research, 230 (1981) 283-293. 17 Talman, W.T., Glycine microinjected in the rat dorsal vagal nucleus increases arterial pressure, Hypertension, 11 (1988) 664667. 18 Talman, W.T. and Robertson, S.C., Glycine, like glutamate, microinjected into the nucleus tractus solitarii of rat decreases arterial pressure and heart rate, Brain Research, 447 (1989) 7-13. 19 Watson, G.B., Hood, W.F., Monahan, J.B. and Lanthorn, T.H., Kynurenate antagonizes actions of N-methyl-D-aspartate through a glycine-sensitive receptor, Neurosci. Res. Commun., 2 (1988) 169-174.
