Respiratory Sinus Arrhythmia
Endogenous Activation of Nicotinic Receptors Mediates Respiratory Modulation of Brainstem Cardioinhibitory Parasympathetic Neurons
Robert A. Neff, Jijiang Wang, Sunit Baxi, Cory Evans, David Mendelowitz
Abstract—The heart rate increases during inspiration and decreases during expiration. This respiratory sinus arrhythmia (RSA) occurs by modulation of premotor cardioinhibitory parasympathetic neuron (CPN) activity. However, RSA has not been fully characterized in rats, and despite the critical role of CPNs in the generation of RSA, little is known about the mechanisms that mediate this cardiorespiratory interaction. This study demonstrates that RSA in conscious rats is similar to that in other species. The mechanism of RSA was then examined in vitro. Rhythmic inspiratory-related activity was recorded from the hypoglossal rootlet of 700- to 800-m medullary sections. CPNs were identified by retrograde fluorescent labeling, and neurotransmission to CPNs was examined using patch-clamp electrophysiological techniques. During inspiratory bursts, the frequency of both spontaneous ␥-aminobutyric acidergic (GABAergic) and spontaneous glycinergic synaptic events in CPNs was significantly increased. Focal application of the nicotinic antagonist dihydro--erythroidine in an ␣42-selective concentration (3 mol/L) abolished the respiratory-evoked increase in GABAergic frequency. In contrast, the increase in glycinergic frequency during inspiration was not altered by nicotinic antagonists. Prenatal nicotine exposure exaggerated the increase in GABAergic frequency during inspiration and enhanced GABAergic synaptic amplitude both between and during inspiratory events. Glycinergic synaptic frequency and amplitude were unchanged by prenatal nicotine exposure. This study establishes a neurochem- ical link between neurons essential for respiration and CPNs, reveals a functional role for endogenous acetylcholine release and the activation of nicotinic receptors in the generation of RSA, and demonstrates that this cardiorespiratory interaction is exaggerated in rats prenatally exposed to nicotine. (Circ Res. 2003;93:565-572.)
Key Words: nucleus ambiguus 䡲 vagal activity 䡲 respiratory sinus arrhythmia 䡲 prenatal nicotine 䡲 sudden infant death syndrome
T
he heart rate increases during inspiration and decreases during the post-inspiration/expiration period. This respiratory-related change in heart rate, respiratory sinus arrhythmia (RSA), helps to match pulmonary blood flow to lung inflation and to maintain an appropriate diffusion gra- dient for oxygen in the lungs.1–3RSA has been observed in neonatal4and adult5,6humans, baboons,7dogs,1rabbits,8and seals9but has not been well characterized in rats.Heart rate is controlled by the activity of premotor car- dioinhibitory parasympathetic neurons (CPNs) in the brain- stem, and RSA is mediated in part by central respiratory modulation of CPN activity. CPNs are primarily located in the nucleus ambiguus (NA), in proximity to neurons thought to be essential for respiratory rhythmogenesis.2,3,10 –13CPNs in the NA are intrinsically silent and therefore rely on synaptic inputs to dictate their activity.14
Although the pathways and transmitters responsible for respiratory modulation of CPNs are unknown,
␥-aminobutyric acid (GABA), glycine, and acetylcholine
(ACh) are all neurotransmitters that have been implicated in the central generation of RSA. CPNs are inhibited during inspiration, and this inhibition has been reversed by the intracellular injection of Cl⫺.15 This suggests that GABA- and/or glycine-mediated chloride channels may be involved in respiratory modulation of CPNs. Paradoxically, however, one author of the same study described in a later review that the inhibition of CPNs during inspiration could not be inhibited by the GABAAantagonist bicuculline or the glycine antagonist strychnine.2
ACh has been shown to inhibit CPN activity,15and recent work has shown that endogenous ACh activates presynaptic nicotinic acetylcholine receptors (nAChRs), which enhance both GABAergic and glycinergic inputs to CPNs.16 The possible involvement of nicotinic receptors in mediating RSA is interesting because prenatal nicotine augments parasympa- thetic and reduces sympathetic control of the heart rate17and is among the highest risk factors for sudden infant death syndrome (SIDS).18,19 Infants that succumb to SIDS often
Original received April 1, 2003; revision received July 29, 2003; accepted July 30, 2003.
From the Department of Pharmacology, The George Washington University, Washington, DC.
Correspondence to Robert A. Neff, Department of Pharmacology, The George Washington University, 2300 Eye St NW, Washington, DC 20037.
E-mail phmdxm@gwumc.edu
© 2003 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000090361.45027.5B 565
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experience a sustained bradycardia, which is preceded or accompanied by a life-threatening apnea.19,20 These life- threatening events in SIDS victims are thought to be caused by exaggerated central cardiorespiratory interactions.17,19
The aim of the present study was to test whether rats have an RSA pattern similar to that of other species and to elucidate the cellular mechanisms responsible for the respi- ratory modulation of CPNs. Specifically, we tested the hypothesis that heart rate increases during inspiration in rats.
In addition, we tested whether CPNs receive increased GABAergic and glycinergic synaptic inputs during inspiratory-related activity. Furthermore, we examined whether the respiratory modulation of CPNs is dependent on endogenous activation of nicotinic receptors. Because our work demonstrates that endogenous activation of nicotinic receptors is responsible for RSA, we tested whether prenatal nicotine exposure alters these cardiorespiratory interactions.
Materials and Methods
Plethysmographic/Blood Pressure RecordingsAdult female Sprague-Dawley rats were anesthetized with a combi- nation of ketamine (40 mg/kg) and xylazine (5 mg/kg IP, Phoenix Pharmaceuticals). The femoral artery was exposed and catheterized with Micro-Renthane tubing (Braintree Scientific) that had been soaked overnight in heparinized bacteriostatic saline. The animals were placed in a Covance infusion harness (Instech Labs, Inc) and allowed to recover for 24 to 48 hours. After recovery, the unanes- thetized, freely moving rats were placed in a whole-body plethys- mographic chamber, which allowed simultaneous measurement of blood pressure, heart rate, and respiratory airflow using Biosystem XA software (Buxco Electronics, Inc). Only measurements recorded during periods in which the animals were awake and sedentary were analyzed.
Fluorescent Labeling of CPNs and Medullary Slice Preparations
Neonatal Sprague-Dawley rats (P1-P5, Hilltop, Scottdale, Pa) were initially exposed to isoflurane (Abbott Laboratories) until anesthe- tized and cooled to⬇4°C. A right thoracotomy was performed, and the retrograde fluorescent tracer X-rhodamine-5- (and -6)- isothiocyanate (Molecular Probes) was injected into the fat pads at the base of the heart. After 24 hours of recovery, each animal was anesthetized with isoflurane and decapitated, and the head was placed in a 4°C physiological saline solution (mmol/L: NaCl 140, KCl 5, CaCl22, glucose 5, and HEPES 10) bubbled with 100% O2, pH 7.4. All animal procedures were performed with the approval of the Animal Care and Use Committee of The George Washington University in accordance with the recommendations of the panel on euthanasia of the American Veterinary Medical Association and the National Institutes of Health publication Guide for the Care and Use of Laboratory Animals. The medulla was removed with care to preserve the hypoglossal cranial nerve rootlet. The medulla was mounted on a cutting block and placed into a vibrating blade microtome (Leica). Serial transverse sections were sliced in a rostrocaudal progression until the inferior olives and the NA could be visualized on the rostral surface of the tissue. A single thick (700 to 800m) section that included CPNs, the hypoglossal nerve rootlet, the pre-Botzinger complex, and the rostral portion of the hypoglossal nucleus was cut, transferred to a recording chamber and perfused (4 mL/min) with room temperature artificial cerebrospinal fluid (mmol/L: NaCl 125, KCl 3, CaCl22, NaHCO326, glucose 5, and HEPES 5) equilibrated with 95% O2/5% CO2, pH 7.4.
Recording Respiratory Network Activity
The thick medullary slice preparation contains the pre-Botzinger complex, local circuits for motor output generation, and respiratory
hypoglossal motor neurons, which generate inspiratory-phase motor discharge in hypoglossal cranial nerves.13As seen in other similar medullary respiratory slice preparations, the frequency of respiratory-related hypoglossal discharge is significantly lower than that in in vivo preparations; this is likely due to the reduced temperature of the preparation and the absence of sensory input to the medulla.21Spontaneous respiratory-related activity was recorded by monitoring the motor neuron population activity from hypoglos- sal nerve rootlets using a suction electrode. Hypoglossal rootlet activity was amplified (50 000 times), filtered (10- to 300-Hz bandpass, CWE Inc), and adjacent-averaged (50-ms windows).
Respiratory activity was also electronically integrated (⫽50 ms, CWE Inc) during experiments examining glycinergic synaptic inputs to CPNs.
Patch-Clamp Techniques
CPNs in the NA were identified by the presence of the fluorescent tracer.22Patch pipettes (2.5 to 3.5 M⍀) were visually guided to the surface of individual CPNs using differential interference optics and infrared illumination (Zeiss). Patch pipettes contained (mmol/L) KCl 150, MgCl24, EGTA 2, Na-ATP 2, and HEPES 10, pH 7.4. This pipette solution causes the Cl⫺current induced by the activation of GABA or glycine receptors to be recorded as an inward current (calculated reversal potential of Cl⫺4 mV). Voltage-clamp record- ings were made with an Axopatch 200B and pClamp 8 software (Axon Instruments). All synaptic activity in CPNs was recorded at
⫺80 mV. Only preparations in which synaptic activity increased in CPNs during inspiration (in 95 of 117 [81%] of the preparations) were used for further experimentation and analysis. Only one cell was recorded per nucleus for an experiment. In 12 slices, an additional cell was recorded from the same slice in the contralateral NA.
Focal Drug Application
Focal drug application was performed using a pneumatic Picopump pressure delivery system (WPI). Drugs were ejected from a patch pipette positioned within 30 m from the patched CPN. The maximum range of drug application has been previously determined to 100 to 120m downstream from the drug pipette and consider- ably less behind the drug pipette.23GABAergic neurotransmission was isolated by focal application ofD-2-amino-5-phosphonovalerate (AP-5, 50mol/L), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50mol/L), and strychnine (1 mmol/L) to block N-methyl-D- aspartate (NMDA), non-NMDA, and glycinergic receptors, respec- tively. Glycinergic neurotransmission was isolated by focal applica- tion of AP-5, CNQX, and gabazine (25mol/L) to block NMDA, non-NMDA, and GABAA receptors, respectively. Nicotinic recep- tors were blocked with dihydro--erythroidine (DHE, 100 mol/L) or curare (10mol/L). The role of different receptor subtypes was tested by applying DHE at concentration selective for the ␣42
nicotinic receptor (3mol/L),24and␣7nicotinic receptor subtypes were tested with the␣7nicotinic receptor antagonist␣-bungarotoxin (␣BTX, 100 nmol/L). All drugs were obtained from Sigma.
Prenatal Nicotine Exposure
Adult female rats were anesthetized with ketamine (40 mg/kg)/
xylazine (5 mg/kg IP, Phoenix Pharmaceuticals) on the third day of gestation and implanted with Alzet osmotic minipumps (Durect) containing (⫺)-nicotine (56.1 mg/mL bacteriostatic saline, Sigma).
Osmotic minipumps were chosen to avoid the high plasma nicotine concentrations and subsequent episodic fetal hypoxia/ischemia that can be produced by nicotine injections.25Pumps delivered 2.1 mg nicotine per day, a level approximately equivalent to levels that occur in moderate to heavy smokers, for 28 days.17
Data Analysis
Plethysmographic/Blood Pressure Experiments
Heart rate intervals were measured during inspiratory and subsequent expiratory periods in 6 animals using Acqknowledge (version 3.7.3,
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Biopac Systems). Heart rate was recorded from at least 20 respira- tory cycles while the animal was awake and sedentary. Data are presented as mean⫾SEM. Statistical comparisons were made using paired Student t tests. A value of P⬍0.05 indicates significant differences.
Electrophysiology
Synaptic events were detected using MiniAnalysis (version 5.6.12, Synaptosoft). The frequency and amplitude of inhibitory postsynap- tic currents (IPSCs) that occurred in CPNs during inspiratory-related
hypoglossal activity were averaged to determine inspiratory values.
Control values were determined by averaging IPSCs that occurred in a 5- to 10-second window ending 1 second before inspiratory burst onset and beginning at least 3 seconds after the end of any previous inspiratory activity. All data are presented as mean⫾SEM. Statistical comparisons were made using paired or unpaired Student t tests, as appropriate. A value of P⬍0.05 indicates significant differences.
Results
Sprague-Dawley Rats Exhibit RSA Pattern Similar to That of Other Species
Respiratory airflow (Figure 1, top) and blood pressure (Figure 1, bottom) were simultaneously recorded in 6 conscious, freely moving rats. During inspiration, the average heartbeat interval decreased significantly from 171⫾4 ms during expi- ration to 168⫾4 ms during inspiration (P⬍0.05). This change in heart period corresponds to an average inspiratory-related increase in heart rate of 6.9⫾1.9 bpm (P⬍0.05).
CPNs Are Inhibited During Inspiration by Endogenous Nicotinic ACh Receptor–Mediated Increases in GABAergic Activity
To determine the cellular basis of RSA, the synaptic activity of CPNs was measured in vitro. GABAergic neurotransmis- sion was isolated by focal application of the glutamatergic and glycinergic antagonists AP-5, CNQX, and strychnine in 22 cells (from 17 preparations). The focal application of these antagonists did not significantly alter the frequency (P⬎0.05) or duration (P⬎0.05) of the respiratory activity.
During inspiration, the frequency of GABAergic synaptic inputs to CPNs was significantly increased (basal 5.2⫾0.7 Hz, inspiration 10.5⫾1.3 Hz, P⬍0.01, n⫽22 cells; Figure 2a). All IPSCs under these recording conditions were blocked Figure 1. Heart rate increases during inspiration (Insp) and
decreases during expiration (Exp) in the rat. Blood pressure (BP) and respiratory airflow were recorded simultaneously in unanes- thetized, freely moving rats (n⫽6 animals). The heartbeat-to- heartbeat interval was significantly decreased from 171⫾4 to 167⫾4 ms during Insp (P⬍0.05).
Figure 2. Endogenous activation of nico- tinic receptors mediates GABAergic inhi- bition of premotor CPNs during inspira- tion. Inspiratory-related bursting activity was recorded from the hypoglossal root- let (XII), rectified, and adjacent-averaged (XII with bar above). Fluorescently identi- fied CPNs were patch-clamped in the whole-cell configuration, and GABAergic IPSCs were isolated by focal application of the NMDA, non-NMDA, and glycine receptor antagonists AP-5 (50mol/L), CNQX (50mol/L), and strychnine (1 mmol/L), respectively. a, During inspiratory activity, the frequency of GABAergic IPSCs in CPNs was signifi- cantly increased (P⬍0.05). b, GABAAan- tagonist gabazine blocked all IPSCs. c, Nicotinic receptor antagonist curare (10
mol/L) significantly inhibited (P⬍0.05) the inspiratory-related increase in GABAergic synaptic frequency in CPNs.
Representative traces in panels a, b, and c are from 3 CPNs.
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by focal application of the GABAA antagonist gabazine (Figure 2b). Focal application of the nicotinic antagonist curare significantly reduced the inspiratory-related increase in GABAergic synaptic frequency (control basal 3.5⫾0.6 Hz, control inspiration 7.6⫾1.3 Hz, curare basal 2.6⫾0.4 Hz, curare inspiration 4.4⫾0.6 Hz, P⬍0.05, n⫽12 cells from 10 preparations; Figure 2c) but did not significantly affect the basal frequency of IPSCs between bursts (P⬍0.05, n⫽12 cells). GABAergic synaptic amplitude was not significantly altered by inspiratory activity or by the application of curare (P⬎0.05, n⫽12 cells).
Determination of the nAChR Subtype Mediating Inspiratory-Related Increase in GABAergic Input to CPNs
To determine the specific nicotinic ACh receptor (nAChR) subtype mediating the respiratory-related increase in GABAer- gic frequency, subtype-selective nicotinic antagonists were used.
Focal application of ␣BTX (␣7-selective nicotinic antagonist) had no significant effect on the frequency of GABAergic synaptic inputs to CPNs between inspiratory bursts (control 4.9⫾1.2 Hz,␣BTX 5.5⫾1.2 Hz, P⬎0.05, n⫽4 cells) and did not alter the inspiratory-related increase in GABAergic synaptic frequency (control basal 5.0⫾1.2 Hz, control inspiration 15.2⫾2.7 Hz, ␣BTX basal 5.7⫾1.1 Hz, ␣BTX inspiration 11.4⫾2.4, P⬎0.05, n⫽4 cells from 4 preparations).
However, focal application of the nicotinic antagonist DHE, in a concentration selective for the ␣42 receptor subtype (3
mol/L), abolished the increase in GABAergic frequency dur- ing inspiration (control basal 8.5⫾1.7 Hz, control inspiration 13.9⫾2.7 Hz, DHE control 6.4⫾1.2 Hz, DHE inspiration 8.2⫾1.8 Hz, P⬍0.05, n⫽6 cells; Figures 3a, 3b, and 3e) but did not affect GABAergic synaptic frequency between bursts (con- trol 8.5⫾1.7 Hz, DHE 6.4⫾1.2 Hz, P⬎0.05, n⫽6 cells from 3 preparations). GABAergic synaptic amplitude was not signifi- cantly altered by␣BTX (control 49.7⫾8.2 pA, ␣BTX 40.5⫾8.9 pA, P⬎0.05, n⫽4 cells) or DHE (control 57.2⫾11.4 pA, DHE 52.9⫾9.2 pA, P⬎0.05, n⫽6 cells). Focal application of gabazine reversibly blocked all GABAergic synaptic events (Figures 3c through 3e).
Glycinergic Respiratory Inputs to CPNs
In an additional 13 cells (from 10 preparations), glycinergic activity was isolated by focal application of the glutamatergic and GABAer- gic antagonists AP-5, CNQX, and gabazine. The application of these antagonists did not significantly alter the frequency (P⬎0.05) or duration (P⬎0.05) of the respiratory activity. During inspiration, glycinergic synaptic frequency was also significantly increased (basal 11.0⫾2.2 Hz, inspiration 19.0⫾3.5 Hz, P⬍0.01, n⫽13 cells;
Figure 4a). Focal application of the nicotinic antagonist DHE in a high concentration that blocks all nicotinic receptors (100mol/L, Figures 4b, 4c, and 4f) did not significantly alter the inspiratory- related increase in glycinergic frequency (control basal 13.4⫾3.8 Hz, control inspiration 22.3⫾6.3 Hz, DHE 13.4⫾4.0 to 22.0⫾6.1 Hz, P⬎0.05, n⫽7 cells from 5 preparations). DHE did not significantly alter the frequency of glycinergic synaptic events between inspiratory events (Figure 4f, n⫽7 cells, P⬎0.05). All IPSCs under these recording conditions were reversibly blocked by focal application of strychnine (Figures 4d through 4f). Glycinergic
amplitude was not altered by respiratory activity (basal 52.0⫾5.1 pA, inspiration 59.6⫾9.1 pA, P⬎0.05, n⫽13 cells) or by applica- tion of DHE (control 52.0⫾5.1 pA, DHE 44.9⫾7.7 pA, P⬎0.05, n⫽7 cells). Application of 100 mol/L DHE did not significantly alter the frequency (P⬎0.05) or duration (P⬎0.05) of the respiratory activity.
Effect of Prenatal Nicotine Exposure on GABAergic and Glycinergic Synaptic Inputs to CPNs
GABA
The frequency of GABAergic synaptic events increased 460⫾90% in animals prenatally exposed to nicotine (n⫽18 cells from 17 preparations), a significant exaggeration of the Figure 3.␣42nicotinic receptors mediate the inspiratory-related GABAergic inhibition of CPNs. GABAergic IPSCs were isolated by focal application of the NMDA, non-NMDA, and glycine receptor antagonists AP-5, CNQX, and strychnine, respectively.
a, Inspiratory activity evoked a significant increase (P⬍0.01) in the frequency of GABAergic IPSCs in CPNs. b, This inspiratory- related increase in GABA synaptic frequency was abolished by focal application of the nicotinic receptor antagonist DHE at a concentration selective for the␣42receptor subtype (3mol/L).
DHE did not significantly change GABA synaptic frequency between bursts. c and d, Gabazine (25mol/L) reversibly blocked all IPSCs under these recording conditions. Represen- tative traces in panels a through d are from the same CPN. e, Values are mean⫾SEM from 6 cells. *P⬍0.05 and **P⬍0.01.
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260⫾40% increase observed in unexposed animals (n⫽22 cells, P⬍0.01; Figures 5a through 5c and 5e). Prenatal nicotine did not significantly alter the frequency of GABAer- gic synaptic events between inspiratory bursts (unexposed 5.2⫾0.7 Hz [n⫽22 cells], prenatal nicotine 7.7⫾0.7 [n⫽18 cells], P⬎0.05). Focal application of DHE at a concentra- tion (3mol/L) selective for ␣42nicotinic receptors signif- icantly inhibited the inspiratory-related increase in GABAer- gic frequency in animals prenatally exposed to nicotine (control basal 6.2⫾1.2 Hz, control inspiration 14.8⫾2.2 Hz, DHE basal 2.8⫾0.8 Hz, DHE inspiration 4.6⫾1.3 Hz, P⬍0.05, n⫽6 cells from 5 preparations). In addition, DHE significantly reduced basal GABAergic synaptic frequency (P⬎0.05, n⫽6 cells).
Prenatal nicotine also significantly increased GABAergic synaptic amplitude relative to unexposed animals both be- tween (unexposed 43.7⫾4.5 pA [n⫽22 cells], prenatal nico- tine 61.0⫾5.5 pA [n⫽18 cells], P⬍0.05) and during (unex- posed 45.3⫾5.3 pA [n⫽22 cells], prenatal nicotine 66.1⫾6.2 pA [n⫽18 cells], P⬍0.05) inspiratory bursts (Figures 5d and 5e). All GABAergic synaptic events were reversibly blocked by focal application of gabazine.
Glycine
The inspiratory-related increase in glycinergic frequency was not significantly altered in animals prenatally exposed to nicotine (179⫾17%, n⫽11 cells from 8 animals) compared with unexposed animals (191⫾15%, n⫽13 cells, P⬎0.05;
Figure 5e). Glycinergic synaptic amplitude was not signifi- cantly altered by prenatal nicotine exposure between (unex- posed 52.0⫾5.0 pA [n⫽13 cells], prenatal nicotine 43.3⫾5.5 pA [n⫽11 cells], P⬎0.05) or during inspiratory bursts (un-
exposed 59.6⫾9.1 pA [n⫽13 cells], prenatal nicotine 45.2⫾7.9 pA [n⫽11 cells], P⬍0.05). All glycinergic IPSCs were reversibly blocked by focal application of strychnine.
Discussion
There are four major findings from the present study: (1) Heart rate increases during inspiration and decreases during expiration in conscious, unrestrained rats. (2) CPNs in the brainstem are inhibited during inspiration by an increase in both GABAergic and glycinergic synaptic inputs. (3) The respiratory-related increase in GABAergic activity, but not glycinergic activity, is mediated by the endogenous activation of␣42nicotinic ACh receptors. (4) Prenatal nicotine expo- sure significantly exaggerates the GABA-mediated, but not glycine-mediated, inhibition of CPNs during inspiration.
RSA Pattern in Rats
It is well established in many species (including neonatal4and adult5,6 humans, baboons,7 dogs,26 seals,9 and rabbits8) that the heart rate increases during inspiration and decreases during expiration. This RSA improves the efficiency of pulmonary gas exchange by better matching ventilation and pulmonary blood flow.1,2,9 This inspiratory-related tachycardia is predominantly mediated by a reduction in cardiac vagal activity due to the decreased activity of CPNs in the NA.2,3,8,15,27–30 However, data describing cardiorespira- tory interactions in the rat are contradictory. A recent in vivo study has paradoxically shown that in contrast to all other studied species, the activity of CPNs is enhanced during inspiration in rats.31However, there are several factors that make the results from that study difficult to interpret. These include a desynchronization of central respiratory activity
Figure 4. CPNs are inhibited during inspiration by an increased frequency of glycinergic IPSCs, which is not mediated by the activation of nicotinic receptors.
Glycinergic IPSCs were isolated by focal application of the NMDA, non-NMDA, and GABAAreceptor antagonists AP-5, CNQX, and gabazine, respectively. a, Inspiratory activity evoked a significant increase (P⬍0.05) in glycinergic IPSCs in CPNs. b and c, This inspiratory-related increase in IPSC frequency was not sig- nificantly altered by focal application of a high concentration (100mol/L) of the nicotinic receptor antagonist DHE. d and e, Glycine antagonist strychnine (Strx) reversibly blocked all IPSCs under these recording conditions. Representa- tive traces in panel a and panels b through e are from 2 CPNs. f, Values are mean⫾SEM from 7 cells. *P⬍0.05.
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from lung inflation and using anesthetics, which in general reduce or eliminate parasympathetic cardiac activity. In contrast, a recent study using the rat working heart brainstem preparation32(a preparation without anesthetics) demonstrates that heart rate increases during inspiration, consistent with studies using other species.4 –9,26The results from the present study unequivocally demonstrate that similar to the RSA that occurs in other species, heart rate increases during inspiration in conscious, freely moving rats.
Inhibitory Respiratory-Related Synaptic Inputs to CPNs
Previous in vivo work has shown that CPNs are inhibited during inspiration via a chloride-mediated current.15 The present study demonstrates that CPNs receive an increased frequency of both GABAergic and glycinergic IPSCs during inspiration. This respiratory-dependent inhibition of CPN activity provides a cellular mechanism for the tachycardia that occurs during the inspiratory phase of the respiratory cycle.
Involvement of Nicotinic Receptors in RSA
The GABA-mediated inhibition of CPNs during inspiration was significantly inhibited by curare, indicating that the increase in GABAergic frequency is mediated by the activa- tion of nicotinic receptors. Further investigation revealed that this increase in GABAergic frequency was unaffected by
␣BTX, demonstrating that it was not mediated by the activa- tion of the ␣7nAChR subtype. However, an ␣42-selective concentration of DHE abolished the GABAergic inhibition
of CPNs during inspiration, demonstrating that activation of
␣42 nAChRs by endogenous ACh is essential for the GABAergic component of this cardiorespiratory interaction.
The facilitation of GABAergic inputs to CPNs by activation of nicotinic receptors is consistent with previous work, which has shown that spontaneous GABAergic synaptic inputs to CPNs are enhanced by the activation of ␣42 receptors located in the presynaptic terminals of GABAergic neu- rons.16,33 The present study also indicates that the nicotinic receptors responsible for the increased GABAergic activity during inspiration are in proximity to the CPNs, inasmuch as the focal application of the nicotinic antagonist DHE abol- ished this increase.
In contrast, greater (100mol/L) concentrations of DHE did not significantly alter the respiratory-related increase in glycinergic synaptic frequency in CPNs. Interestingly, previ- ous studies have shown that spontaneous glycinergic inputs to CPNs are also enhanced by the activation of␣42nicotinic receptors in glycinergic presynaptic terminals.16 This sug- gests that although glycinergic inputs to CPNs possess presynaptic nicotinic receptors, the respiratory-evoked in- crease in glycinergic synaptic input to CPNs is not mediated by the activation of nicotinic receptors. Alternatively, there may be nicotinic modulation of glycinergic activity that occurs at a location distant from the CPNs and out of the range of the focal application of drugs in the present study.
Prenatal Nicotine Exposure and
Respiratory-Related Synaptic Inputs to CPNs In animals prenatally exposed to nicotine, the inspiratory- related increase in GABAergic synaptic frequency was nearly Figure 5. Prenatal nicotine exposure exag- gerates the inspiratory-related increase in GABAergic, but not glycinergic, synaptic inputs to CPNs. a, In animals prenatally exposed to nicotine, CPNs received increased GABAergic synaptic input during inspiration. b, In unexposed animals, the fre- quency of GABAergic IPSCs increased 260⫾90% during inspiration. In animals pre- natally exposed to nicotine, the frequency of GABAergic IPSCs increased 460⫾40% dur- ing inspiration, which is a significant aug- mentation (P⬍0.05) of this response. Repre- sentative traces in panels a and b are from 3 CPNs. c, Values are mean⫾SEM from 22 control and 18 prenatal nicotine experiments.
**P⬍0.01. d, GABAergic IPSC amplitude is significantly increased by prenatal nicotine exposure both between and during inspira- tory bursts. Unexp indicates the group not exposed to nicotine; Pre Nic, the group exposed to prenatal nicotine. Values are mean⫾SEM from 22 control and 18 prenatal nicotine experiments. *P⬍0.05. e, Values are mean⫾SEM percent increase of the inspiratory-related increase in GABAergic (n⫽22 Unexp cells, n⫽18 Pre Nic cells) and glycinergic (n⫽13 Unexp cells, n⫽11 Pre Nic cells) IPSCs in CPNs during inspiration.
*P⬍0.05. Prenatal nicotine exposure signifi- cantly exaggerates the inspiratory-related increase in GABA frequency. In contrast, the inspiratory-related increase in glycinergic frequency is not significantly altered by pre- natal nicotine exposure.
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twice that of unexposed animals, whereas the inspiratory- dependent increase in glycinergic frequency was not signifi- cantly different in control and nicotine-exposed animals. This is consistent with the results that nicotinic antagonists abol- ished the inspiratory-related increase in GABAergic fre- quency to CPNs but did not alter the respiratory modulation of glycinergic IPSCs to CPNs. In addition to enhancing the increase in frequency of GABAergic synaptic inputs to CPNs during inspiration, prenatal nicotine exposure also caused a significant increase in the amplitude of both spontaneous and inspiratory-evoked GABAergic synaptic inputs to CPNs.
Other studies have shown that the␣42nAChR subtype is significantly upregulated in rat brains chronically exposed to nicotine34,35 and that ␣42 receptors chronically exposed to nicotine exhibit enhanced responses to ACh and are less sensitive to desensitization.36A greater number of, enhanced responses to, and reduced desensitization of ␣42 nicotinic receptors may be responsible for the prenatal nicotine- induced exaggeration of the ␣42-mediated increase in GABAergic synaptic frequency in CPNs during inspiration.
The ␣42 receptor antagonist DHE (3 mol/L) signifi- cantly inhibited the inspiratory-related increase in GABAer- gic frequency in animals prenatally exposed to nicotine, indicating that activation of ␣42 nicotinic receptors by endogenous ACh also mediates this cardiorespiratory inter- action in animals prenatally exposed to nicotine. In addition,
␣42block significantly reduced the frequency of spontane- ous, non–inspiratory-related GABAergic synaptic inputs to CPNs in animals prenatally exposed to nicotine but not in unexposed animals. This suggests that prenatal nicotine augments endogenous cholinergic control of GABAergic inputs to CPNs in animals prenatally exposed to nicotine.
These alterations in cardiorespiratory control with prenatal nicotine exposure may be clinically important. Maternal cigarette smoking is highly correlated with SIDS, and it has been suggested that SIDS is caused by an alteration of brainstem sites responsible for cardiorespiratory con- trol.17,19,37,38Infants that subsequently succumb to SIDS have heart rates higher than those in other infants.39The exagger- ated amplitude of all GABAergic inputs to CPNs and en- hanced increase in GABAergic synaptic frequency during inspiration observed the present study would be expected to evoke an elevated basal heart rate and a greater than normal tachycardia during inspiration. This nicotine-mediated facil- itation of GABAergic neurotransmission to CPNs provides possible mechanisms for the elevated heart rate in SIDS victims and the elevated heart rate prevalent in smokers.
In summary, rats possess normal RSA, and CPNs are inhibited during inspiration by an increase in the frequency of both GABAergic and glycinergic synaptic inputs. The inspiratory-related increase in GABAergic synaptic fre- quency is mediated by the activation of ␣42 nicotinic receptors and is significantly exaggerated by prenatal expo- sure to nicotine. These data show a neurochemical link between the neurons essential for respiration and neurons that control heart rate and reveal a physiological role of endoge- nous ACh release and the activation of nicotinic receptors in the generation of RSA.
Acknowledgments
This study was supported by NIH National Heart, Lung, and Blood Institute grants HL-72006 and HL-59895 to Dr Mendelowitz. Dr Neff was supported by the Jocelyn Beard Moran Memorial Fellow- ship from the American Heart Association, Mid-Atlantic affiliate.
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