30
Brain Research, 511 (1990) 30-40 Elsevier
BRES 15250
Excitation of rostral medullary pacemaker neurons with putative sympathoexcitatory function by cyclic AMP and/3-adrenoceptor agonists 'in vitro' Miao-Kun Sun and Patrice G. Guyenet Department of Pharmacology, University of Virginia, School of Medicine, Charlottesville, VA 22908 (U.S.A.) (Accepted 1 August 1989)
Key words: Adenosine 3",5"-cyclic-monophosphate; fl-Adrenoceptor; Catecholamine; Medullary pacemaker; Sympathoexcitation
This study explores the mechanism of action of catecholamines on rostral medullary pacemaker neurons with putative sympathoexcitatory function, in tissue slices. The firing rate of the pacemaker neurons of nucleus reticularis rostroventrolateralis (RVL pacemakers) was reversibly increased by agents which elevate intracellular levels of cAMP (forskolin and 8-br-cAMP). Forskolin dideoxy, an analog without action on adenylate cyclase, was ineffective and adenosine, a potential degradation product of 8-br cAMP produced inhibition exclusively and only in high doses (0.1-1 mM). The firing rate of these cells was uniformly increased by epinephrine and isoproterenol (10 ktM) but unaffected by both phenylephrine (100/tM) and clonidine (up to 1 aM). These effects were abolished by pretreatment with the fl-adrenoceptor antagonist propranolol (10 gM) but they were unaffected by the a-antagonist phentolamine (100 #M). The indirectly-acting sympathomimetic amine tyramine (0.1-1 raM) activated all the ceils tested. The effect of tyramine was antagonized by the fl-blocker pindolol and was absent 7 days after microinjection of the neurotoxin 6-hydroxydopamine into the lateral aspect of the RVL. IntraceUular recordings indicated that both isoproterenol and tyramine enhanced the rate of depolarization of the pacemaker neurons during the interspike interval and produced a decrease in input resistance. After tetrodotoxin (TI'X) pretreatment, isoproterenol produced a depolarization also associated with a reduction in input resistance. Three conclusions are proposed. First, RVL pacemakers have functional fl-adrenergic receptors whose activation increases their discharge rate via the intracellular production of cAMP. The effect of cAMP is due at least in part to the activation of an inward current which may be carried by a cation. Secondly, RVL neurons are in close proximity to a releasable pool of catecholamines which is susceptible to destruction by the cytotoxic agent 6-hydroxydopamine (6-OHDA). Finally it is tentatively suggested that the reduction in sympathetic tone produced by centrally acting/5-blockers could be due, at least in part, to an action of these agents on RVL pacemaker cells. INTRODUCTION T h e nucleus reticularis rostroventrolateralis ( R V L , Ross et al. 21) contains the cell bodies of reticulospinal neurons with direct projections to the intermediolateral cell column, some of which m a k e monosynaptic connections with sympathetic preganglionic neurons x4. Many such neurons receive inhibitory polysynaptic inputs from b a r o r e c e p t o r s and are tonically active 'in vivo '27'28. Their tonic activity is currently viewed as providing an essential excitatory drive to v a s o m o t o r preganglionic neurons, thus sustaining arterial b l o o d pressure at least under anesthesia (for review see G u y e n e t et al.5). This reticulospinal efferent p a t h w a y consists of adrenergic and n o n - a d r e n e r g i c neurons whose relative proportions are still u n d e r d e b a t e 35. T h e non-adrenergic ceils display intrinsic p a c e m a k e r activity 'in vitro '29 which can be also o b s e r v e d 'in vivo '2s. Since the R V L has been described as a p r o b a b l e site
of action of several types of hypotensive agents which interfere with catecholaminergic transmission (clonidine 19, fl-blockersa,X8), the p u r p o s e of the present study was to investigate the effect of catecholamines on the activity of p a c e m a k e r neurons with putative sympathoexcitatory function. This study was carried out in tissue slices in o r d e r to study the neuronal effects of drugs i n d e p e n d e n t l y of their actions on the local vasculature and also to obtain intracellular information. MATERIALS AND METHODS
Slice preparation Slice containing the nucleus reticularis rostroventrolateralis (RVL) were prepared exactly as previously described2s'29. Briefly, brains obtained from ether-anesthetized male Sprague-Dawley rats (80-140 g) were cut with a Vibratome. A 500-#m coronal section of the brainstem containing the RVL was obtained and incubated in a chamber continuously perfused with a saline medium at a rate of 2.0 ml per rain. The saline medium, equilibrated with 95% 02-5% CO2 (pH 7.35-7.4) was composed of (in mM) NaC! 124; KC! 4.9;
Correspondence." P.G. Guyenet, Department of Pharmacology, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
31
A
0.5 mM 8Br-cAMP
10
B
10 p.M Forskolin
15'--
C
D 10 tiM Forskolin dideoxv
1 mM Ado
Fig. 1. Effects of 8-br-cAMP, forskolin, forskolin dideoxy and adenosine on the firing rate of medullary pacemaker neurons recorded extracellularly in brain slices. A: 8-Br-cAMP (500/~M at bar above histogram) caused a reversible increase in spontaneous activity of the cell. B: forskolin (10/~M) had a similar action. C: forskolin dideoxy (10/~M) did not alter the firing rate of the cell. D: adenosine (1 mM) inhibited spontaneous firing. In this and subsequent figures, extracellularly recorded firing rate is presented in the form of integrated rate histograms (pen reset every 10 s). Calibrations: vertical, 100 spikes/10 s; horizontal bars below histograms, 5 min.
KH2PO 4 1.2; MgSO 4 1.3; CaCI2 3.1: NaHCO 3 25.6; glucose 10. The temperature of the slice chamber was maintained at 30-31 °C.
A 10 ~M ISO
Electrical recordings Extracellular potentials were recorded with glass electrodes filled with 2 M NaCl (6-10 M~). The rostral ventrolateral medulla was visually identified as an area 1.5-2.0 mm lateral to the midline, within 0.5 mm of the ventral surface, and ventromedial to the rostral compact portion of the nucleus ambiguus 28'29"a°. Neurons used for extracellular studies had the following characteristics: a triphasic waveform spike, regular non-bursting activity, and spontaneous firing rates between 3 and 12 spikes per second 28'29. Intracellular recording was conducted using an Axoclamp -2 amplifier. Glass microelectrodes (100-130 MQ) filled with 3 M potassium acetate or 2 M lithium chloride were used. All successful impalements were obtained by passing 19.9 nA positive current of 25 ms duration through the electrode tip. Bridge balance was readjusted after cell impalement by observing the shape of the capacitative current following a small hyperpolarizing step (20 ms, 20 mV). The time constant of the membrane voltage response to such hyperpolarizing steps averaged 8.2 ms in 5 neurons (_+ S.E.M. 0.9 ms). The second criterion for bridge balance was that currents of opposite polarities produced steady-state voltage deflections of approx, equal but opposite amplitude. In the case of spontaneouslyactive cells, the last criterium was not always very useful because the cells have no resting membrane potential during a depolarizing step. In TTX-blocked preparations, stricter criteria for bridge balance could be met. In any event, input resistance measurements were made for the purpose of evaluating drug-induced changes in conductance in a qualitative manner. Although the accuracy of the input resistance measurement cannot be guaranteed, conclusions regarding the effects of fl-agonists should not be affected.
Microinjections Unilateral microinjections of 6-hydroxydopamine (6-OHDA) on
I B
10 pM ISO 10 pM PRO
C 10 pM ISO
Fig. 2. fl-adrenoccptor excitation of medullary pacemaker neurons. Excitation of the pacemaker neurons by isoproterenol (A) was abolished by propranolol (B). The excitation elicited by isoproterenol was recovered after 2 h of washing with saline medium (C). Calibration: vertical, 100 spikes/10 s; horizontal, 5 rain.
32 Histology
the right side of the rats were performed using a 1-,ul Hamilton syringe with the needle glued to a glass micropipette pulled to a 10-20 ~m tip (o.d.). Prior to lowering the injection pipettes, the coordinates of the medullary pacemaker neurons were determined in each rat. This was done by mapping the contour of the posterior extent of the facial motornucleus with antidromic field potentials as described before 28. 6-OHDA (bromide, 4 mg/ml) was dissolved in artificial CSF bubbled with 5% CO 2, 95% N2 (same composition as saline used for brain slice work, see above) and kept under oil to prevent oxidation. Less than 1 rain later, the 6-OHDA injection (2 #g, 0.5/~1) was initiated (duration 3-5 rain) 300 #m posterior to the caudal edge of the facial motornucleus and 2 mm lateral to the midline. Histological verification of the injection site was performed in each case (for technical details, see below). All injection sites were found 200-300 gm lateral to the compact portion of the Cj adrenergic cell group in RVL.
A
At the end of the experiments on slices harvested from 6OHDA-treated rats, the tissue was processed for the immunocytochemical localization of tyrosine-hydroxylase, a specific marker enzyme for all types of catecholamine-synthesizing neurons. The experimental protocol, identical to that described previously3°, is based on the use of a commercially available antiserum (Eugene Tech International) and a Texas red-tagged secondary antibody. Staining was carried out in 32-#m-thick cryostat sections cut from the aldehyde-fixed slices.
Materials Forskolin and forskolin dideoxy were obtained from Calbiochem; 8-Br-cAMP, tetrodotoxin (TTX) and tyramine, from Sigma; isoproterenol HCt, from Abbott; DL-propranolol (Inderal), from Ayerst; phentolamine from Ciba-Geigy; chlorpromazine HCI, from Smith,
B
C
0 o
10 pM ISO \
> E
J
10 ~M ISO
:~
>/!!'!I
!!~!
Control
-6040 ms
2ms
=-02 ] 0.2S
o
! !
o-| ~ I I"" "'i "'i "i, '" ii II >/ ! i I I I ' ' i '
-60
E
~
i
'
i
'
'
'
:
:
H nA
°]
Brain Research, 511 (1990) 30-40 Elsevier
BRES 15250
Excitation of rostral medullary pacemaker neurons with putative sympathoexcitatory function by cyclic AMP and/3-adrenoceptor agonists 'in vitro' Miao-Kun Sun and Patrice G. Guyenet Department of Pharmacology, University of Virginia, School of Medicine, Charlottesville, VA 22908 (U.S.A.) (Accepted 1 August 1989)
Key words: Adenosine 3",5"-cyclic-monophosphate; fl-Adrenoceptor; Catecholamine; Medullary pacemaker; Sympathoexcitation
This study explores the mechanism of action of catecholamines on rostral medullary pacemaker neurons with putative sympathoexcitatory function, in tissue slices. The firing rate of the pacemaker neurons of nucleus reticularis rostroventrolateralis (RVL pacemakers) was reversibly increased by agents which elevate intracellular levels of cAMP (forskolin and 8-br-cAMP). Forskolin dideoxy, an analog without action on adenylate cyclase, was ineffective and adenosine, a potential degradation product of 8-br cAMP produced inhibition exclusively and only in high doses (0.1-1 mM). The firing rate of these cells was uniformly increased by epinephrine and isoproterenol (10 ktM) but unaffected by both phenylephrine (100/tM) and clonidine (up to 1 aM). These effects were abolished by pretreatment with the fl-adrenoceptor antagonist propranolol (10 gM) but they were unaffected by the a-antagonist phentolamine (100 #M). The indirectly-acting sympathomimetic amine tyramine (0.1-1 raM) activated all the ceils tested. The effect of tyramine was antagonized by the fl-blocker pindolol and was absent 7 days after microinjection of the neurotoxin 6-hydroxydopamine into the lateral aspect of the RVL. IntraceUular recordings indicated that both isoproterenol and tyramine enhanced the rate of depolarization of the pacemaker neurons during the interspike interval and produced a decrease in input resistance. After tetrodotoxin (TI'X) pretreatment, isoproterenol produced a depolarization also associated with a reduction in input resistance. Three conclusions are proposed. First, RVL pacemakers have functional fl-adrenergic receptors whose activation increases their discharge rate via the intracellular production of cAMP. The effect of cAMP is due at least in part to the activation of an inward current which may be carried by a cation. Secondly, RVL neurons are in close proximity to a releasable pool of catecholamines which is susceptible to destruction by the cytotoxic agent 6-hydroxydopamine (6-OHDA). Finally it is tentatively suggested that the reduction in sympathetic tone produced by centrally acting/5-blockers could be due, at least in part, to an action of these agents on RVL pacemaker cells. INTRODUCTION T h e nucleus reticularis rostroventrolateralis ( R V L , Ross et al. 21) contains the cell bodies of reticulospinal neurons with direct projections to the intermediolateral cell column, some of which m a k e monosynaptic connections with sympathetic preganglionic neurons x4. Many such neurons receive inhibitory polysynaptic inputs from b a r o r e c e p t o r s and are tonically active 'in vivo '27'28. Their tonic activity is currently viewed as providing an essential excitatory drive to v a s o m o t o r preganglionic neurons, thus sustaining arterial b l o o d pressure at least under anesthesia (for review see G u y e n e t et al.5). This reticulospinal efferent p a t h w a y consists of adrenergic and n o n - a d r e n e r g i c neurons whose relative proportions are still u n d e r d e b a t e 35. T h e non-adrenergic ceils display intrinsic p a c e m a k e r activity 'in vitro '29 which can be also o b s e r v e d 'in vivo '2s. Since the R V L has been described as a p r o b a b l e site
of action of several types of hypotensive agents which interfere with catecholaminergic transmission (clonidine 19, fl-blockersa,X8), the p u r p o s e of the present study was to investigate the effect of catecholamines on the activity of p a c e m a k e r neurons with putative sympathoexcitatory function. This study was carried out in tissue slices in o r d e r to study the neuronal effects of drugs i n d e p e n d e n t l y of their actions on the local vasculature and also to obtain intracellular information. MATERIALS AND METHODS
Slice preparation Slice containing the nucleus reticularis rostroventrolateralis (RVL) were prepared exactly as previously described2s'29. Briefly, brains obtained from ether-anesthetized male Sprague-Dawley rats (80-140 g) were cut with a Vibratome. A 500-#m coronal section of the brainstem containing the RVL was obtained and incubated in a chamber continuously perfused with a saline medium at a rate of 2.0 ml per rain. The saline medium, equilibrated with 95% 02-5% CO2 (pH 7.35-7.4) was composed of (in mM) NaC! 124; KC! 4.9;
Correspondence." P.G. Guyenet, Department of Pharmacology, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
31
A
0.5 mM 8Br-cAMP
10
B
10 p.M Forskolin
15'--
C
D 10 tiM Forskolin dideoxv
1 mM Ado
Fig. 1. Effects of 8-br-cAMP, forskolin, forskolin dideoxy and adenosine on the firing rate of medullary pacemaker neurons recorded extracellularly in brain slices. A: 8-Br-cAMP (500/~M at bar above histogram) caused a reversible increase in spontaneous activity of the cell. B: forskolin (10/~M) had a similar action. C: forskolin dideoxy (10/~M) did not alter the firing rate of the cell. D: adenosine (1 mM) inhibited spontaneous firing. In this and subsequent figures, extracellularly recorded firing rate is presented in the form of integrated rate histograms (pen reset every 10 s). Calibrations: vertical, 100 spikes/10 s; horizontal bars below histograms, 5 min.
KH2PO 4 1.2; MgSO 4 1.3; CaCI2 3.1: NaHCO 3 25.6; glucose 10. The temperature of the slice chamber was maintained at 30-31 °C.
A 10 ~M ISO
Electrical recordings Extracellular potentials were recorded with glass electrodes filled with 2 M NaCl (6-10 M~). The rostral ventrolateral medulla was visually identified as an area 1.5-2.0 mm lateral to the midline, within 0.5 mm of the ventral surface, and ventromedial to the rostral compact portion of the nucleus ambiguus 28'29"a°. Neurons used for extracellular studies had the following characteristics: a triphasic waveform spike, regular non-bursting activity, and spontaneous firing rates between 3 and 12 spikes per second 28'29. Intracellular recording was conducted using an Axoclamp -2 amplifier. Glass microelectrodes (100-130 MQ) filled with 3 M potassium acetate or 2 M lithium chloride were used. All successful impalements were obtained by passing 19.9 nA positive current of 25 ms duration through the electrode tip. Bridge balance was readjusted after cell impalement by observing the shape of the capacitative current following a small hyperpolarizing step (20 ms, 20 mV). The time constant of the membrane voltage response to such hyperpolarizing steps averaged 8.2 ms in 5 neurons (_+ S.E.M. 0.9 ms). The second criterion for bridge balance was that currents of opposite polarities produced steady-state voltage deflections of approx, equal but opposite amplitude. In the case of spontaneouslyactive cells, the last criterium was not always very useful because the cells have no resting membrane potential during a depolarizing step. In TTX-blocked preparations, stricter criteria for bridge balance could be met. In any event, input resistance measurements were made for the purpose of evaluating drug-induced changes in conductance in a qualitative manner. Although the accuracy of the input resistance measurement cannot be guaranteed, conclusions regarding the effects of fl-agonists should not be affected.
Microinjections Unilateral microinjections of 6-hydroxydopamine (6-OHDA) on
I B
10 pM ISO 10 pM PRO
C 10 pM ISO
Fig. 2. fl-adrenoccptor excitation of medullary pacemaker neurons. Excitation of the pacemaker neurons by isoproterenol (A) was abolished by propranolol (B). The excitation elicited by isoproterenol was recovered after 2 h of washing with saline medium (C). Calibration: vertical, 100 spikes/10 s; horizontal, 5 rain.
32 Histology
the right side of the rats were performed using a 1-,ul Hamilton syringe with the needle glued to a glass micropipette pulled to a 10-20 ~m tip (o.d.). Prior to lowering the injection pipettes, the coordinates of the medullary pacemaker neurons were determined in each rat. This was done by mapping the contour of the posterior extent of the facial motornucleus with antidromic field potentials as described before 28. 6-OHDA (bromide, 4 mg/ml) was dissolved in artificial CSF bubbled with 5% CO 2, 95% N2 (same composition as saline used for brain slice work, see above) and kept under oil to prevent oxidation. Less than 1 rain later, the 6-OHDA injection (2 #g, 0.5/~1) was initiated (duration 3-5 rain) 300 #m posterior to the caudal edge of the facial motornucleus and 2 mm lateral to the midline. Histological verification of the injection site was performed in each case (for technical details, see below). All injection sites were found 200-300 gm lateral to the compact portion of the Cj adrenergic cell group in RVL.
A
At the end of the experiments on slices harvested from 6OHDA-treated rats, the tissue was processed for the immunocytochemical localization of tyrosine-hydroxylase, a specific marker enzyme for all types of catecholamine-synthesizing neurons. The experimental protocol, identical to that described previously3°, is based on the use of a commercially available antiserum (Eugene Tech International) and a Texas red-tagged secondary antibody. Staining was carried out in 32-#m-thick cryostat sections cut from the aldehyde-fixed slices.
Materials Forskolin and forskolin dideoxy were obtained from Calbiochem; 8-Br-cAMP, tetrodotoxin (TTX) and tyramine, from Sigma; isoproterenol HCt, from Abbott; DL-propranolol (Inderal), from Ayerst; phentolamine from Ciba-Geigy; chlorpromazine HCI, from Smith,
B
C
0 o
10 pM ISO \
> E
J
10 ~M ISO
:~
>/!!'!I
!!~!
Control
-6040 ms
2ms
=-02 ] 0.2S
o
! !
o-| ~ I I"" "'i "'i "i, '" ii II >/ ! i I I I ' ' i '
-60
E
~
i
'
i
'
'
'
:
:
H nA
°]
