Respiratory Sinus Arrhythmia

Integrative Physiology 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 ␣4␤2-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 neurochemical 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.) Downloaded from http://ahajournals.org by on May 29, 2020 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 gradient for oxygen in the lungs.1–3 RSA has been observed in neonatal4 and adult5,6 humans, baboons,7 dogs,1 rabbits,8 and seals9 but has not been well characterized in rats. Heart rate is controlled by the activity of premotor cardioinhibitory parasympathetic neurons (CPNs) in the brainstem, 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 –13 CPNs 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 GABAand/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 GABAA antagonist bicuculline or the glycine antagonist strychnine.2 ACh has been shown to inhibit CPN activity,15 and 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 parasympathetic and reduces sympathetic control of the heart rate17 and 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 [email protected] © 2003 American Heart Association, Inc. Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000090361.45027.5B 565 566 Circulation Research September 19, 2003 experience a sustained bradycardia, which is preceded or accompanied by a life-threatening apnea.19,20 These lifethreatening 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 respiratory 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 Recordings Downloaded from http://ahajournals.org by on May 29, 2020 Adult female Sprague-Dawley rats were anesthetized with a combination 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 unanesthetized, freely moving rats were placed in a whole-body plethysmographic 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 anesthetized 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, CaCl2 2, 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 800 ␮m) 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, CaCl2 2, NaHCO3 26, glucose 5, and HEPES 5) equilibrated with 95% O2/5% CO2, pH 7.4. hypoglossal motor neurons, which generate inspiratory-phase motor discharge in hypoglossal cranial nerves.13 As 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.21 Spontaneous respiratory-related activity was recorded by monitoring the motor neuron population activity from hypoglossal 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.22 Patch 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, MgCl2 4, 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 recordings 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 120 ␮m downstream from the drug pipette and considerably less behind the drug pipette.23 GABAergic neurotransmission was isolated by focal application of D-2-amino-5-phosphonovalerate (AP-5, 50 ␮mol/L), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50␮mol/L), and strychnine (1 mmol/L) to block N-methyl-Daspartate (NMDA), non-NMDA, and glycinergic receptors, respectively. Glycinergic neurotransmission was isolated by focal application of AP-5, CNQX, and gabazine (25 ␮mol/L) to block NMDA, non-NMDA, and GABAA receptors, respectively. Nicotinic receptors were blocked with dihydro-␤-erythroidine (DH␤E, 100 ␮mol/L) or curare (10 ␮mol/L). The role of different receptor subtypes was tested by applying DH␤E at concentration selective for the ␣4␤2 nicotinic receptor (3 ␮mol/L),24 and ␣7 nicotinic receptor subtypes were tested with the ␣7 nicotinic 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.25 Pumps 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 Recording Respiratory Network Activity Plethysmographic/Blood Pressure Experiments The thick medullary slice preparation contains the pre-Botzinger complex, local circuits for motor output generation, and respiratory Heart rate intervals were measured during inspiratory and subsequent expiratory periods in 6 animals using Acqknowledge (version 3.7.3, Neff et al Mechanism of Respiratory Sinus Arrhythmia 567 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 expiration 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). 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 unanesthetized, freely moving rats (n⫽6 animals). The heartbeat-toheartbeat interval was significantly decreased from 171⫾4 to 167⫾4 ms during Insp (P⬍0.05). Biopac Systems). Heart rate was recorded from at least 20 respiratory 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. Downloaded from http://ahajournals.org by on May 29, 2020 Electrophysiology Synaptic events were detected using MiniAnalysis (version 5.6.12, Synaptosoft). The frequency and amplitude of inhibitory postsynaptic currents (IPSCs) that occurred in CPNs during inspiratory-related 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 neurotransmission 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 2. Endogenous activation of nicotinic receptors mediates GABAergic inhibition of premotor CPNs during inspiration. Inspiratory-related bursting activity was recorded from the hypoglossal rootlet (XII), rectified, and adjacent-averaged (XII with bar above). Fluorescently identified 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 (50 ␮mol/L), CNQX (50 ␮mol/L), and strychnine (1 mmol/L), respectively. a, During inspiratory activity, the frequency of GABAergic IPSCs in CPNs was significantly increased (P⬍0.05). b, GABAA antagonist 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. 568 Circulation Research September 19, 2003 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 Downloaded from http://ahajournals.org by on May 29, 2020 To determine the specific nicotinic ACh receptor (nAChR) subtype mediating the respiratory-related increase in GABAergic 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 DH␤E, in a concentration selective for the ␣4␤2 receptor subtype (3 ␮mol/L), abolished the increase in GABAergic frequency during inspiration (control basal 8.5⫾1.7 Hz, control inspiration 13.9⫾2.7 Hz, DH␤E control 6.4⫾1.2 Hz, DH␤E 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 (control 8.5⫾1.7 Hz, DH␤E 6.4⫾1.2 Hz, P⬎0.05, n⫽6 cells from 3 preparations). GABAergic synaptic amplitude was not significantly altered by ␣BTX (control 49.7⫾8.2 pA, ␣BTX 40.5⫾8.9 pA, P⬎0.05, n⫽4 cells) or DH␤E (control 57.2⫾11.4 pA, DH␤E 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 GABAergic 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 DH␤E in a high concentration that blocks all nicotinic receptors (100 ␮mol/L, Figures 4b, 4c, and 4f) did not significantly alter the inspiratoryrelated increase in glycinergic frequency (control basal 13.4⫾3.8 Hz, control inspiration 22.3⫾6.3 Hz, DH␤E 13.4⫾4.0 to 22.0⫾6.1 Hz, P⬎0.05, n⫽7 cells from 5 preparations). DH␤E 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 Figure 3. ␣4␤2 nicotinic 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 inspiratoryrelated increase in GABA synaptic frequency was abolished by focal application of the nicotinic receptor antagonist DH␤E at a concentration selective for the ␣4␤2 receptor subtype (3 ␮mol/L). DH␤E did not significantly change GABA synaptic frequency between bursts. c and d, Gabazine (25 ␮mol/L) reversibly blocked all IPSCs under these recording conditions. Representative 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. 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 application of DH␤E (control 52.0⫾5.1 pA, DH␤E 44.9⫾7.7 pA, P⬎0.05, n⫽7 cells). Application of 100 ␮mol/L DH␤E 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 Neff et al Mechanism of Respiratory Sinus Arrhythmia 569 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 GABAA receptor 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 significantly altered by focal application of a high concentration (100 ␮mol/L) of the nicotinic receptor antagonist DH␤E. d and e, Glycine antagonist strychnine (Strx) reversibly blocked all IPSCs under these recording conditions. Representative traces in panel a and panels b through e are from 2 CPNs. f, Values are mean⫾SEM from 7 cells. *P⬍0.05. Downloaded from http://ahajournals.org by on May 29, 2020 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 GABAergic 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 DH␤E at a concentration (3 ␮mol/L) selective for ␣4␤2 nicotinic receptors significantly inhibited the inspiratory-related increase in GABAergic frequency in animals prenatally exposed to nicotine (control basal 6.2⫾1.2 Hz, control inspiration 14.8⫾2.2 Hz, DH␤E basal 2.8⫾0.8 Hz, DH␤E inspiration 4.6⫾1.3 Hz, P⬍0.05, n⫽6 cells from 5 preparations). In addition, DH␤E 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 between (unexposed 43.7⫾4.5 pA [n⫽22 cells], prenatal nicotine 61.0⫾5.5 pA [n⫽18 cells], P⬍0.05) and during (unexposed 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 significantly altered by prenatal nicotine exposure between (unexposed 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 ␣4␤2 nicotinic ACh receptors. (4) Prenatal nicotine exposure 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 neonatal4 and 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 cardiorespiratory 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.31 However, there are several factors that make the results from that study difficult to interpret. These include a desynchronization of central respiratory activity 570 Circulation Research September 19, 2003 Figure 5. Prenatal nicotine exposure exaggerates 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 frequency of GABAergic IPSCs increased 260⫾90% during inspiration. In animals prenatally exposed to nicotine, the frequency of GABAergic IPSCs increased 460⫾40% during inspiration, which is a significant augmentation (P⬍0.05) of this response. Representative 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 inspiratory 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 significantly exaggerates the inspiratory-related increase in GABA frequency. In contrast, the inspiratory-related increase in glycinergic frequency is not significantly altered by prenatal nicotine exposure. Downloaded from http://ahajournals.org by on May 29, 2020 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 –9,26 The 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 activation of nicotinic receptors. Further investigation revealed that this increase in GABAergic frequency was unaffected by ␣BTX, demonstrating that it was not mediated by the activation of the ␣7 nAChR subtype. However, an ␣4␤2-selective concentration of DH␤E abolished the GABAergic inhibition of CPNs during inspiration, demonstrating that activation of ␣4␤2 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 ␣4␤2 receptors located in the presynaptic terminals of GABAergic neurons.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 DH␤E abolished this increase. In contrast, greater (100 ␮mol/L) concentrations of DH␤E did not significantly alter the respiratory-related increase in glycinergic synaptic frequency in CPNs. Interestingly, previous studies have shown that spontaneous glycinergic inputs to CPNs are also enhanced by the activation of ␣4␤2 nicotinic receptors in glycinergic presynaptic terminals.16 This suggests that although glycinergic inputs to CPNs possess presynaptic nicotinic receptors, the respiratory-evoked increase 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 inspiratoryrelated increase in GABAergic synaptic frequency was nearly Neff et al Downloaded from http://ahajournals.org by on May 29, 2020 twice that of unexposed animals, whereas the inspiratorydependent increase in glycinergic frequency was not significantly different in control and nicotine-exposed animals. This is consistent with the results that nicotinic antagonists abolished the inspiratory-related increase in GABAergic frequency 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 ␣4␤2 nAChR subtype is significantly upregulated in rat brains chronically exposed to nicotine34,35 and that ␣4␤2 receptors chronically exposed to nicotine exhibit enhanced responses to ACh and are less sensitive to desensitization.36 A greater number of, enhanced responses to, and reduced desensitization of ␣4␤2 nicotinic receptors may be responsible for the prenatal nicotineinduced exaggeration of the ␣4␤2-mediated increase in GABAergic synaptic frequency in CPNs during inspiration. The ␣4␤2 receptor antagonist DH␤E (3 ␮mol/L) significantly inhibited the inspiratory-related increase in GABAergic frequency in animals prenatally exposed to nicotine, indicating that activation of ␣4␤2 nicotinic receptors by endogenous ACh also mediates this cardiorespiratory interaction in animals prenatally exposed to nicotine. In addition, ␣4␤2 block significantly reduced the frequency of spontaneous, 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 control.17,19,37,38 Infants that subsequently succumb to SIDS have heart rates higher than those in other infants.39 The exaggerated amplitude of all GABAergic inputs to CPNs and enhanced 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 facilitation 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 frequency is mediated by the activation of ␣4␤2 nicotinic receptors and is significantly exaggerated by prenatal exposure 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 endogenous ACh release and the activation of nicotinic receptors in the generation of RSA. Mechanism of Respiratory Sinus Arrhythmia 571 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 Fellowship from the American Heart Association, Mid-Atlantic affiliate. References 1. Hayano J, Yasuma F, Okada A, Mukai S, Fujinami T. Respiratory sinus arrhythmia: a phenomenon improving pulmonary gas exchange and circulatory efficiency. Circulation. 1996;94:842– 847. 2. Loewy AD, Spyer KM, eds. Central Regulation of Autonomic Functions. New York, NY: Oxford University Press; 1990. 3. Taylor EW, Jordan D, Coote JH. Central control of the cardiovascular and respiratory systems and their interactions in vertebrates. Physiol Rev. 1999;79:855–916. 4. Hathorn MK. Respiratory sinus arrhythmia in new-born infants. J Physiol. 1987;385:1–12. 5. Eckberg DL. Human sinus arrhythmia as an index of vagal cardiac outflow. J Appl Physiol. 1983;54:961–966. 6. Hirsch JA, Bishop B. Respiratory sinus arrhythmia in humans: how breathing pattern modulates heart rate. Am J Physiol. 1981;241: H620 –H629. 7. Myers MM, Fifer W, Haiken J, Stark RI. Relationships between breathing activity and heart rate in fetal baboons. Am J Physiol. 1990;258: R1479 –R1485. 8. Jordan D, Khalid ME, Schneiderman N, Spyer KM. The location and properties of preganglionic vagal cardiomotor neurones in the rabbit. Pflugers Arch. 1982;395:244 –250. 9. Castellini MA, Rea LD, Sanders JL, Castellini JM, Zenteno-Savin T. Developmental changes in cardiorespiratory patterns of sleep-associated apnea in northern elephant seals. Am J Physiol. 1994;267:R1294 –R1301. 10. Machado BH, Brody MJ. Role of the nucleus ambiguus in the regulation of heart rate and arterial pressure. Hypertension. 1988;11:602– 607. 11. McAllen RM, Spyer KM. The location of cardiac vagal preganglionic motoneurones in the medulla of the cat. J Physiol. 1976;258:187–204. 12. Mendelowitz D. Advances in parasympathetic control of heart rate and cardiac function. News Physiol Sci. 1999;14:155–161. 13. Smith JC, Ellenberger HH, Ballanyi K, Richter DW, Feldman JL. PreBotzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science. 1991;254:726 –729. 14. Mendelowitz D. Firing properties of identified parasympathetic cardiac neurons in nucleus ambiguus. Am J Physiol. 1996;271:H2609 –H2614. 15. Gilbey MP, Jordan D, Richter DW, Spyer KM. Synaptic mechanisms involved in the inspiratory modulation of vagal cardio-inhibitory neurones in the cat. J Physiol. 1984;356:65–78. 16. Wang J, Wang X, Irnaten M, Venkatesan P, Evans C, Baxi S, Mendelowitz D. Endogenous acetylcholine and nicotine activation enhances GABAergic and glycinergic inputs to cardiac vagal neurons. J Neurophysiol. 2003;89:2473–2481. 17. Slotkin TA, Saleh JL, McCook EC, Seidler FJ. Impaired cardiac function during postnatal hypoxia in rats exposed to nicotine prenatally: implications for perinatal morbidity and mortality, and for sudden infant death syndrome. Teratology. 1997;55:177–184. 18. Taylor JA, Sanderson M. A reexamination of the risk factors for the sudden infant death syndrome. J Pediatr. 1995;126:887– 891. 19. Meny RG, Carroll JL, Carbone MT, Kelly DH. Cardiorespiratory recordings from infants dying suddenly and unexpectedly at home. Pediatrics. 1994;93:44 – 49. 20. Cote A, Hum C, Brouillette RT, Themens M. Frequency and timing of recurrent events in infants using home cardiorespiratory monitors. J Pediatr. 1998;132:783–789. 21. Rekling JC, Feldman JL. PreBotzinger complex and pacemaker neurons: hypothesized site and kernel for respiratory rhythm generation. Annu Rev Physiol. 1998;60:385– 405. 22. Mendelowitz D, Kunze DL. Identification and dissociation of cardiovascular neurons from the medulla for patch clamp analysis. Neurosci Lett. 1991;132:217–221. 23. Wang J, Irnaten M, Venkatesan P, Evans C, Baxi S, Mendelowitz D. Synaptic activation of hypoglossal respiratory motorneurons during inspiration in rats. Neurosci Lett. 2002;332:195–199. 24. Alkondon M, Albuquerque EX. Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons, I: pharmacological and functional evidence for distinct structural subtypes. J Pharmacol Exp Ther. 1993; 265:1455–1473. 572 Circulation Research September 19, 2003 25. Slotkin TA. Fetal nicotine or cocaine exposure: which one is worse? J Pharmacol Exp Ther. 1998;285:931–945. 26. Warner MR, deTarnowsky JM, Whitson CC, Loeb JM. Beat-by-beat modulation of AV conduction, II: autonomic neural mechanisms. Am J Physiol. 1986;251:H1134 –H1142. 27. Kollai M, Koizumi K. Reciprocal and non-reciprocal action of the vagal and sympathetic nerves innervating the heart. J Auton Nerv Syst. 1979; 1:33–52. 28. Kunze DL. Reflex discharge patterns of cardiac vagal efferent fibres. J Physiol. 1972;222:1–15. 29. Anrep G, Pascual F, Rossler R. Respiratory variations of the heart rate, I: the reflex mechanism of the respiratory sinus arrhythmia. Proc R Soc Lond B Biol Sci. 1936;119:191–217. 30. Anrep G, Pascual F, Rossler R. Respiratory variations of the heart rate, II: the central mechanism of the respiratory sinus arrhythmia and the interrelations between the central and the reflex mechanisms. Proc R Soc Lond B Biol Sci. 1936;119:218 –232. 31. Rentero N, Cividjian A, Trevaks D, Pequignot JM, Quintin L, McAllen RM. Activity patterns of cardiac vagal motoneurons in rat nucleus ambiguus. Am J Physiol. 2002;283:R1327–R1334. 32. Pickering AE, Waki H, Headley PM, Paton JF. Investigation of systemic bupivacaine toxicity using the in situ perfused working heart-brainstem preparation of the rat. Anesthesiology. 2002;97:1550 –1556. 33. Wang J, Irnaten M, Mendelowitz D. Characteristics of spontaneous and evoked GABAergic synaptic currents in cardiac vagal neurons in rats. Brain Res. 2001;889:78 – 83. 34. Peng X, Gerzanich V, Anand R, Whiting PJ, Lindstrom J. Nicotineinduced increase in neuronal nicotinic receptors results from a decrease in the rate of receptor turnover. Mol Pharmacol. 1994;46:523–530. 35. Flores CM, Rogers SW, Pabreza LA, Wolfe BB, Kellar KJ. A subtype of nicotinic cholinergic receptor in rat brain is composed of ␣4 and ␤2 subunits and is up-regulated by chronic nicotine treatment. Mol Pharmacol. 1992;41:31–37. 36. Buisson B, Bertrand D. Chronic exposure to nicotine upregulates the human ␣4␤2 nicotinic acetylcholine receptor function. J Neurosci. 2001; 21:1819 –1829. 37. Nachmanoff DB, Panigrahy A, Filiano JJ, Mandell F, Sleeper LA, Valdes-Dapena M, Krous HF, White WF, Kinney HC. Brainstem 3 H-nicotine receptor binding in the sudden infant death syndrome. J Neuropathol Exp Neurol. 1998;57:1018 –1025. 38. St-John WM, Leiter JC. Maternal nicotine depresses eupneic ventilation of neonatal rats. Neurosci Lett. 1999;267:206 –208. 39. Schechtman VL, Raetz SL, Harper RK, Garfinkel A, Wilson AJ, Southall DP, Harper RM. Dynamic analysis of cardiac R-R intervals in normal infants and in infants who subsequently succumbed to the sudden infant death syndrome. Pediatr Res. 1992;31:606 – 612. Downloaded from http://ahajournals.org by on May 29, 2020