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Baroreceptor effects on medullary respiratory neurones of the cat
168
Brah~ Research, 86 (1975) 168-171 :~5)Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Baroreceptor effects on medullary respiratory neurones of the cat
D. W. RICHTER* AND H. SELLER* Physiologisches Institut, Universitdt Miinchen, 8 Munich 2 (G.F.R.)
(Accepted November 27th, 1974)
A rise in pressure in the carotid sinus or in the aortic arch produces a reflex inhibition of respiration 4,5,7. This respiratory inhibition by baroreceptor activation takes place most likely at the level of the respiratory neurones which have been localized within the lower brain stem. Extracellular recordings from respiratory single units revealed a decrease in discharge frequency of inspiratory neurones when the baroreceptors were activated 3. The burst discharge of expiratory neurones was prolonged and the mean discharge frequency remained constant or decreased slightly 3,6. These results lead to the question of the nature of synaptic connections between baroreceptors and medullary respiratory neurones. Therefore the effect of baroreceptor activation was studied in intracellular recordings from medullary respiratory neurones. The experiments were performed on cats (2-4 kg body wt.), which were anaesthetized with chloralose (50-60 mg/kg, i.v.) and artificially ventilated after paralysis with Pancuronium-bromid (0.5-0.7 mg/kg, i.v.). A bilateral pneumothorax was applied to abolish respiratory movements of the brain tissue. The arterial blood pressure was maintained at 120-150 m m Hg by i.v. infusion of adrenaline (2-5 #g/kg/min) to reduce changes in pressure following baroreceptor activation. Body temperature was kept constant at 37-38 °C by a ventral heating pad. Carotid sinus nerves and aortic nerves were exposed and identified by recording. All vessels around the carotid bifurcation were ligated except the common and external carotid arteries, taking care not to damage the carotid sinus nerves. The c o m m o n and external carotid arteries were surrounded by silicone-cuffs and a small catheter filled with a heparin-Ringer solution was inserted into the lingual artery. Baroreceptors were stimulated by clamping both common and external carotid arteries and by increasing the intracarotid pressure via the lingual artery by means of a syringe. Both vagal and aortic nerves were sectioned and the central end of the right aortic nerve was stimulated by bipolar platinum electrodes (trains of 5 square pulses of 0.1 msec duration, 3-4 V intensity, and 30-40 c/sec frequency). Arterial and intracarotid pressure were measured by a strain gauge, and end-tidal CO2 by a rapid infra-red analyzer. Renal nerve and phrenic nerve activity were recorded by bipolar platinum electrodes. Respiratory neurones were recorded intracellularly by glass microelectrodes with a tip diameter of less than 1/zm. The electrodes were filled with 2 M potassium citrate and their * Present address: Physiologisches lnstitut, Universit~it Heidelberg, 69 Heidelberg, G.F.R.
169 B
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0.5s Fig. l. Changes in discharge and membrane potential of a medullary inspiratory neurone after bilateral carotid sinus distension during inspiration (A), during the transition period of inspiration and expiration (B) and during expiration (C). Traces show from top to bottom: phrenic nerve activity, membrane potential, renal nerve activity and pressure within carotid sinus. Spikes partly retouched.
DC resistance ranged between 40-50 M ~ in saline. Intracellular potentials were recorded by a voltage follower which was provided with a circuit for capacitance compensation. Constant current was injected through the recording electrode and the voltage drop Over the electrode resistance was balanced by an appropriate bridge circuit. Respiratory neurones were recorded within the retro-ambigual region and were identified by their periodic burst discharge or the spontaneous changes of their membrane potential synchronous with the phrenic nerve activity (Fig. 2A). The only neurones selected were those whose membrane potential reached a level of at least 50 mV during the neurone's 'silent period'. The activation of baroreceptor afferents by either intracarotid pressure increase or aortic nerve stimulation was verified by reflex inhibition of renal sympathetic and phrenic nerve activity. Measurements were rejected whenever inhibition of sympathetic activity failed or activation of phrenic nerve activity was observed. The discharge frequency of inspiratory neurones decreased after both kinds of baroreceptor activation. During this decrease in firing rate the membrane potential was A 0.1mV ]
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Fig. 2. Changes in membrane potential of a medullary expiratory neurone after the spontaneous discharge was blocked by intracellular injection of a hyperpolarizing DC current (A) and ipsilateral aortic nerve stimulation (B). Traces show from top to bottom: phrenic nerve activity, membrane potential, renal nerve activity. Lowest trace in B: aortic nerve stimulation.
170 hyperpolarized (Fig. 1A). The amplitude of these hyperpolarizing potentials varied within the respiratory cycle (Fig. 1A-C). The amplitude was largest during the transition periods of the respiratory phases and lowest during the period of spontaneous membrane hyperpolarization (Fig. 1C). Expiratory neurones failed to exhibit a significant change of their mean discharge frequency when short-lasting baroreceptive stimuli were applied. However, the membrane potential shifted in depolarizing direction when identical stimuli were applied during the period of spontaneous membrane hyperpolarization. This reflex effect also varied in amplitude. The potential changes were smaller during the early period of inspiration than during the later period of inspiration (Fig. 2B). The reflex changes of membrane potential completely failed to appear during the period of expiration (Fig. 2B). In the same region of the lower brain stem and close to the respiratory neurones, other neurones were recorded, whose spontaneous discharge was not in phase with the phrenic nerve activity. Some of these neurones responded with a decrease in discharge frequency and hyperpolarizing potentials when baroreceptor afferents were activated (Fig. 3). The result that baroreceptor activation depolarizes expiratory neurones only during the period of spontaneous membrane hyperpolarization indicates that baroreceptor afferents have no direct synaptic connections with these neurones. The spontaneous cyclic membrane hyperpolarization of expiratory neurones results from an inhibition by inspiratory neurones 9-11. The depolarizing shifts of the potential therefore can be explained by a disinhibition of expiratory neurones when the inspiratory neurones are inhibited. The increase in amplitude of those depolarizing potentials during the progress of an inspiratory period corresponds well with the result that inspiratory neurones were most inhibited during the later period of inspiration. Disinhibition of expiratory neurones also explains the observation that the conspicuous effect of a long-lasting baroreceptor activation is a prolongation of the burst discharge of expiratory neurones without major changes in the mean discharge frequency3, 6. The hyperpolarizing potentials in inspiratory neurones which were observed during the whole period of the respiratory cycle may indicate the existence ot O.lmV],
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Fig. 3. Changes in discharge and membrane potential of a medullary non-respiratory neurone after bilateral carotid sinus distension. Traces show from top to bottom: phrenic nerve activity,membrane potential, renal nerve activity and pressure within carotid sinus. Spikes retouched.
171 a direct inhibitory connection o f baroreceptor afferents to inspiratory neurones. The reduction in amplitude o f these potentials during the period o f expiration can be explained by the simultaneous m e m b r a n e hyperpolarization which is produced by the reciprocal inhibition by expiratory neurones 1°,11. Baroreceptor afferents are also known to depress the neuronal activity o f reticular formation s and cerebral cortex 1. This is in agreement with our finding that even non-respiratory medullary neurones exhibit hyperpolarizing postsynaptic potentials in response to baroreceptor stimulation. These non-respiratory neurones may belong to the reticular activating system which is t h o u g h t to exert a tonic activation o n medullary respiratory neurones2,12. Thus, the hyperpolarizing shift o f the m e m b r a n e potential o f inspiratory neurones after baroreceptor stimulation m a y be the result o f a decrease o f this tonic activation. Further experiments are planned to answer the question o f whether the effect o f baroreceptor afferents on the activity o f medullary inspiratory neurones is p r o d u c e d by inhibition or by disfacilitation. This w o r k was supported by the Deutsche Forschungsgemeinschaft.
1 BONVALLET,M., DELL, P., ET HIEBEL,G., Tonus sympathique et activit6 61ectrique corticale, Electroenceph. clin. Neurophysiol., 6 (1954) 119-144. 2 COHEN,M. I., How respiratory rhythm originates: evidence from discharge patterns of brainstem respiratory neurons. In R. PORTER (Ed.), Breathing: Hering-Breuer Centenary Symposium. A Ciba Foundation Symposium, Churchill, London, 1970, pp. 125-150. 3 GABRIEL,M., ANDSELLER,H., Excitation of expiratory neurones adjacent to the nucleus ambiguus by carotid sinus baroreceptor and trigeminal afferents, Pfliigers Arch. ges. Physiol., 313 (1969) 1-10. 4 HEYMANS,J. F., ET HEYMANS, C., Sur les modifications directes et sur la r6gulation r6fiexe de l'activit6 du centre respiratoire de la t6te isol6e du chien, Arch. int. Pharmacodyn., 33 (1927) 273-372. 5 HERING,H. E., Die Karotissinusreflexe aufHerz und Gefiisse, Steinkopff, Leipzig, 1927. 6 KLf)SSENDORF,D., ANDKOEPCHEN,H. P., Automatic analysis of discharge patterns of respiratory neurons of dog during respiratory reflexes from baroreceptor, chemoreceptor and pulmonary afferents, Pfliigers Arch. ges. Physiol., 313 (1969) R58. 7 KOCH, E. B., Die reflektorisehe Selbststeuerung des Kreislaufs, Steinkopff, Dresden, 1931. 8 KOEPCHEN, H.P., LANGHORST,P., SELLER, H., POLSTER, J., UND WAGNER, P.H., Neuronale Aktivit/it im unteren Hirnstamm mit Beziehung zum Kreislauf, Pfl@ers Arch. ges. Physiol., 294 (1967) 4(Y-64. 9 MITCHELL, R.A., AND HERBERT, D.A., Synchronized high frequency synaptic potentials in medullary respiratory neurones, Brain Research, 75 (1974) 350-355. 10 RICHTER,D. W., AND HEYDE,F., Reciprocal innervation of medullary inspiratory and expiratory neurons, Pfliigers Arch. ges. Physiol., Suppl. 347 (1974) R39. 11 RICHTER,D. W., HEYDE, F., AND GABRIEL,M., Intracellular potentials recorded from different types of medullary respiratory neurones of the cat, J. NeurophysioL, submitted for publication. 12 SALMOIRAGHI,G. C., AND BURNS,B. D., Notes on mechanism of rhythmic respiration, J. Neurophysiol., 23 (1960) 14-26.
Brah~ Research, 86 (1975) 168-171 :~5)Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Baroreceptor effects on medullary respiratory neurones of the cat
D. W. RICHTER* AND H. SELLER* Physiologisches Institut, Universitdt Miinchen, 8 Munich 2 (G.F.R.)
(Accepted November 27th, 1974)
A rise in pressure in the carotid sinus or in the aortic arch produces a reflex inhibition of respiration 4,5,7. This respiratory inhibition by baroreceptor activation takes place most likely at the level of the respiratory neurones which have been localized within the lower brain stem. Extracellular recordings from respiratory single units revealed a decrease in discharge frequency of inspiratory neurones when the baroreceptors were activated 3. The burst discharge of expiratory neurones was prolonged and the mean discharge frequency remained constant or decreased slightly 3,6. These results lead to the question of the nature of synaptic connections between baroreceptors and medullary respiratory neurones. Therefore the effect of baroreceptor activation was studied in intracellular recordings from medullary respiratory neurones. The experiments were performed on cats (2-4 kg body wt.), which were anaesthetized with chloralose (50-60 mg/kg, i.v.) and artificially ventilated after paralysis with Pancuronium-bromid (0.5-0.7 mg/kg, i.v.). A bilateral pneumothorax was applied to abolish respiratory movements of the brain tissue. The arterial blood pressure was maintained at 120-150 m m Hg by i.v. infusion of adrenaline (2-5 #g/kg/min) to reduce changes in pressure following baroreceptor activation. Body temperature was kept constant at 37-38 °C by a ventral heating pad. Carotid sinus nerves and aortic nerves were exposed and identified by recording. All vessels around the carotid bifurcation were ligated except the common and external carotid arteries, taking care not to damage the carotid sinus nerves. The c o m m o n and external carotid arteries were surrounded by silicone-cuffs and a small catheter filled with a heparin-Ringer solution was inserted into the lingual artery. Baroreceptors were stimulated by clamping both common and external carotid arteries and by increasing the intracarotid pressure via the lingual artery by means of a syringe. Both vagal and aortic nerves were sectioned and the central end of the right aortic nerve was stimulated by bipolar platinum electrodes (trains of 5 square pulses of 0.1 msec duration, 3-4 V intensity, and 30-40 c/sec frequency). Arterial and intracarotid pressure were measured by a strain gauge, and end-tidal CO2 by a rapid infra-red analyzer. Renal nerve and phrenic nerve activity were recorded by bipolar platinum electrodes. Respiratory neurones were recorded intracellularly by glass microelectrodes with a tip diameter of less than 1/zm. The electrodes were filled with 2 M potassium citrate and their * Present address: Physiologisches lnstitut, Universit~it Heidelberg, 69 Heidelberg, G.F.R.
169 B
IAIIIIIIUUII LUUU IU"ULII UUUlL t aUtt[l mmHg
200
0J
C
/,,
~__
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0.5s Fig. l. Changes in discharge and membrane potential of a medullary inspiratory neurone after bilateral carotid sinus distension during inspiration (A), during the transition period of inspiration and expiration (B) and during expiration (C). Traces show from top to bottom: phrenic nerve activity, membrane potential, renal nerve activity and pressure within carotid sinus. Spikes partly retouched.
DC resistance ranged between 40-50 M ~ in saline. Intracellular potentials were recorded by a voltage follower which was provided with a circuit for capacitance compensation. Constant current was injected through the recording electrode and the voltage drop Over the electrode resistance was balanced by an appropriate bridge circuit. Respiratory neurones were recorded within the retro-ambigual region and were identified by their periodic burst discharge or the spontaneous changes of their membrane potential synchronous with the phrenic nerve activity (Fig. 2A). The only neurones selected were those whose membrane potential reached a level of at least 50 mV during the neurone's 'silent period'. The activation of baroreceptor afferents by either intracarotid pressure increase or aortic nerve stimulation was verified by reflex inhibition of renal sympathetic and phrenic nerve activity. Measurements were rejected whenever inhibition of sympathetic activity failed or activation of phrenic nerve activity was observed. The discharge frequency of inspiratory neurones decreased after both kinds of baroreceptor activation. During this decrease in firing rate the membrane potential was A 0.1mV ]
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_ - - . . ~ #w~'~'%'~%-:~'':-~-L~-~'~'-'~'-
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Fig. 2. Changes in membrane potential of a medullary expiratory neurone after the spontaneous discharge was blocked by intracellular injection of a hyperpolarizing DC current (A) and ipsilateral aortic nerve stimulation (B). Traces show from top to bottom: phrenic nerve activity, membrane potential, renal nerve activity. Lowest trace in B: aortic nerve stimulation.
170 hyperpolarized (Fig. 1A). The amplitude of these hyperpolarizing potentials varied within the respiratory cycle (Fig. 1A-C). The amplitude was largest during the transition periods of the respiratory phases and lowest during the period of spontaneous membrane hyperpolarization (Fig. 1C). Expiratory neurones failed to exhibit a significant change of their mean discharge frequency when short-lasting baroreceptive stimuli were applied. However, the membrane potential shifted in depolarizing direction when identical stimuli were applied during the period of spontaneous membrane hyperpolarization. This reflex effect also varied in amplitude. The potential changes were smaller during the early period of inspiration than during the later period of inspiration (Fig. 2B). The reflex changes of membrane potential completely failed to appear during the period of expiration (Fig. 2B). In the same region of the lower brain stem and close to the respiratory neurones, other neurones were recorded, whose spontaneous discharge was not in phase with the phrenic nerve activity. Some of these neurones responded with a decrease in discharge frequency and hyperpolarizing potentials when baroreceptor afferents were activated (Fig. 3). The result that baroreceptor activation depolarizes expiratory neurones only during the period of spontaneous membrane hyperpolarization indicates that baroreceptor afferents have no direct synaptic connections with these neurones. The spontaneous cyclic membrane hyperpolarization of expiratory neurones results from an inhibition by inspiratory neurones 9-11. The depolarizing shifts of the potential therefore can be explained by a disinhibition of expiratory neurones when the inspiratory neurones are inhibited. The increase in amplitude of those depolarizing potentials during the progress of an inspiratory period corresponds well with the result that inspiratory neurones were most inhibited during the later period of inspiration. Disinhibition of expiratory neurones also explains the observation that the conspicuous effect of a long-lasting baroreceptor activation is a prolongation of the burst discharge of expiratory neurones without major changes in the mean discharge frequency3, 6. The hyperpolarizing potentials in inspiratory neurones which were observed during the whole period of the respiratory cycle may indicate the existence ot O.lmV],
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Fig. 3. Changes in discharge and membrane potential of a medullary non-respiratory neurone after bilateral carotid sinus distension. Traces show from top to bottom: phrenic nerve activity,membrane potential, renal nerve activity and pressure within carotid sinus. Spikes retouched.
171 a direct inhibitory connection o f baroreceptor afferents to inspiratory neurones. The reduction in amplitude o f these potentials during the period o f expiration can be explained by the simultaneous m e m b r a n e hyperpolarization which is produced by the reciprocal inhibition by expiratory neurones 1°,11. Baroreceptor afferents are also known to depress the neuronal activity o f reticular formation s and cerebral cortex 1. This is in agreement with our finding that even non-respiratory medullary neurones exhibit hyperpolarizing postsynaptic potentials in response to baroreceptor stimulation. These non-respiratory neurones may belong to the reticular activating system which is t h o u g h t to exert a tonic activation o n medullary respiratory neurones2,12. Thus, the hyperpolarizing shift o f the m e m b r a n e potential o f inspiratory neurones after baroreceptor stimulation m a y be the result o f a decrease o f this tonic activation. Further experiments are planned to answer the question o f whether the effect o f baroreceptor afferents on the activity o f medullary inspiratory neurones is p r o d u c e d by inhibition or by disfacilitation. This w o r k was supported by the Deutsche Forschungsgemeinschaft.
1 BONVALLET,M., DELL, P., ET HIEBEL,G., Tonus sympathique et activit6 61ectrique corticale, Electroenceph. clin. Neurophysiol., 6 (1954) 119-144. 2 COHEN,M. I., How respiratory rhythm originates: evidence from discharge patterns of brainstem respiratory neurons. In R. PORTER (Ed.), Breathing: Hering-Breuer Centenary Symposium. A Ciba Foundation Symposium, Churchill, London, 1970, pp. 125-150. 3 GABRIEL,M., ANDSELLER,H., Excitation of expiratory neurones adjacent to the nucleus ambiguus by carotid sinus baroreceptor and trigeminal afferents, Pfliigers Arch. ges. Physiol., 313 (1969) 1-10. 4 HEYMANS,J. F., ET HEYMANS, C., Sur les modifications directes et sur la r6gulation r6fiexe de l'activit6 du centre respiratoire de la t6te isol6e du chien, Arch. int. Pharmacodyn., 33 (1927) 273-372. 5 HERING,H. E., Die Karotissinusreflexe aufHerz und Gefiisse, Steinkopff, Leipzig, 1927. 6 KLf)SSENDORF,D., ANDKOEPCHEN,H. P., Automatic analysis of discharge patterns of respiratory neurons of dog during respiratory reflexes from baroreceptor, chemoreceptor and pulmonary afferents, Pfliigers Arch. ges. Physiol., 313 (1969) R58. 7 KOCH, E. B., Die reflektorisehe Selbststeuerung des Kreislaufs, Steinkopff, Dresden, 1931. 8 KOEPCHEN, H.P., LANGHORST,P., SELLER, H., POLSTER, J., UND WAGNER, P.H., Neuronale Aktivit/it im unteren Hirnstamm mit Beziehung zum Kreislauf, Pfl@ers Arch. ges. Physiol., 294 (1967) 4(Y-64. 9 MITCHELL, R.A., AND HERBERT, D.A., Synchronized high frequency synaptic potentials in medullary respiratory neurones, Brain Research, 75 (1974) 350-355. 10 RICHTER,D. W., AND HEYDE,F., Reciprocal innervation of medullary inspiratory and expiratory neurons, Pfliigers Arch. ges. Physiol., Suppl. 347 (1974) R39. 11 RICHTER,D. W., HEYDE, F., AND GABRIEL,M., Intracellular potentials recorded from different types of medullary respiratory neurones of the cat, J. NeurophysioL, submitted for publication. 12 SALMOIRAGHI,G. C., AND BURNS,B. D., Notes on mechanism of rhythmic respiration, J. Neurophysiol., 23 (1960) 14-26.