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Department of Pharmacology and Physiology, The George Washington University, Washington, DC
Submitted 20 April 2007; accepted in final form 13 August 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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), and the activity of CVNs is intricately linked to respiratory function. For example, respiratory sinus arrhythmia, in which heart rate increases during each inspiration to optimize blood flow to the lungs, is mediated by increases in inhibitory GABAergic and glycinergic neurotransmission to CVNs during inspiration (Neff et al. 2003). The increase in GABAergic but not glycinergic neurotransmission to CVNs during inspiratory activity is dependent on the activation of nicotinic receptors (Neff et al. 2003).
Hypoxia elicits a parasympathetically mediated bradycardia, which decreases metabolic demands on the heart and increases survival (Guntheroth and Kawabori 1975). The biphasic respiratory and heart rate response to hypoxia is paralleled by a biphasic change in inhibitory neurotransmission to CVNs (Neff et al. 2004). However, exposure to prenatal nicotine can alter the nicotinic modulation of CVN activity and significantly alter the responses of the cardiorespiratory network to physiological challenges such as hypoxia and/or hypercapnia (Evans et al. 2005; Huang et al. 2005; Neff et al. 2004).
The purine nucleotide adenosine 5'-triphosphate (ATP) is a clearly identified neurotransmitter within the CNS. Purinergic receptors are present in key central cardiovascular and respiratory control centers such as the nucleus of the solitary tract (NTS), rostroventrolateral medulla, ventral respiratory group, and hypoglossal nucleus (Collo et al. 1996; Gourine and Spyer 2003). ATP is released synaptically to mediate both pre- and postsynaptic effects at ionotropic P2X and/or metabotropic P2Y cell surface receptors (Jo and Schlichter 1999; Ralevic and Burnstock 1998; Robertson and Edwards 1998; Zhang et al. 2000). Purinergic signaling is important in respiratory network interactions; ATP release in the ventral medulla during hypoxia and hypercapnia facilitates central chemoreception and respiratory network plasticity (Gourine et al. 2005a, b).
Although purinergic receptors are present within the medulla and contribute to respiratory control, very little is known regarding the purinergic contribution to respiratory modulation of parasympathetic cardiac neurons or their role in the responses to hypoxia/hypercapnia in brain stem parasympathetic cardiac neurons. Recent work also indicates P2X and nicotinic acetylcholine receptors (nicotinic receptor) exhibit mutual occlusion and suggests P2X and nicotinic receptors form heterooligomers within the plasma membrane, effecting cross-inhibition when one or both receptors are activated (Khakh et al. 2000, 2005). In this study, we examined the cardiorespiratory network responses in an in vitro brain stem preparation to a mimicked apneic event by decreasing oxygen and increasing carbon dioxide levels in the perfusate and tested whether P2X and nicotinic receptors play an opposing role in the recruitment of excitatory synaptic neurotransmission to cardiac vagal neurons in response to hypoxia/hypercapnia and whether prenatal nicotine exposure alters this competition.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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CVNs were identified in a thick medullary slice that generates rhythmic respiratory-related motor discharge in hypoglossal cranial nerves by injecting rhodamine (XRITC, Invitrogen) into the fat pads at the base of the heart 1–5 days prior to sacrifice as described previously (Bouairi et al. 2006; Evans et al. 2005; Huang et al. 2005, 2006; Neff et al. 2003, 2004; Wang et al. 2003a). This slice was placed in a recording chamber that allowed perfusion (4 ml/min) with artificial cerebrospinal fluid (containing, in mM: 125 NaCl, 3 KCl, 2 CaCl2, 26 NaHCO3, 5 glucose, and 5 HEPES, equilibrated with 95% O2-5% CO2, pH 7.35–7.4). Slices were maintained at 21°C. Spontaneous respiratory-related activity was recorded by monitoring motoneuron population activity from hypoglossal nerve rootlets using a suction electrode. Hypoglossal rootlet activity was amplified 50,000 times and filtered (10- to 300-Hz band-pass; CWE, Ardmore, PA) and electronically integrated (
= 50 ms; CWE). 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 Guide for the Care and Use of Laboratory Animals.
Patch-clamp techniques
CVNs in the external formation of the nucleus ambiguus were identified by the presence of the fluorescent tracer and studied using patch-clamp techniques as described in detail previously (Bouairi et al. 2006). Pipettes were filled with a solution containing (in mM) 135 K-gluconic acid, 10 HEPES, 10 EGTA, 1 CaCl2, and 1 MgCl2, at a pH of 7.35–7.4. All synaptic activity in CVNs was recorded at –80 mV. Only one experiment was performed per preparation.
Focal drug application was performed using a pneumatic picopump pressure-delivery system (WPI, Sarasota, FL). Drugs were continuously ejected from a patch pipette positioned within 30 µm of the patched CVN. The maximum range of drug application has been previously determined to be 100–120 µm downstream from the drug pipette and considerably less behind the drug pipette. Glutamatergic neurotransmission was isolated by focal application of strychnine hydrochloride (1 µM) and gabazine (25 µM) to block glycine and GABA receptors, respectively. In some experiments, 
-bungarotoxin (100 nM), dihydro-beta-erythroidine (DH
E) 3 or 100 µM were included to block 7 nicotinic receptors, 42 nicotinic receptors, or all nicotinic receptors, respectively (Alkondon and Albuquerque 1993). In some experiments, the broad P2 purinergic receptor antagonist suramin (100 µM) or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 100 µM) were included in the drug pipette. All drugs were obtained from Sigma.
ATP application
To examine miniature EPSCs, gabazine (25 µM) and strychnine (1 µM) were included in the perfusate to block GABA and glycine receptors. In addition, TTX (1 µM) was added to the perfusate to block voltage-gated sodium channels. ATP-Na+ salt (1 mM) was dissolved in ACSF and focally applied to whole cell patch-clamped CVNs. Nicotine (10 µM) was subsequently added to the perfusate.
Hypoxia/hypercapnia
Rhythmic inspiratory-related activity glutamatergic EPSCs in a single CVN were recorded simultaneously for four minutes in control ACSF (containing, in mM: 125 NaCl, 3 KCl, 2 CaCl2, 26 NaHCO3, 5 glucose, and 5 HEPES, equilibrated with 95% O2-5% CO2, pH 7.35–7.4). Hypoxia/hypercapnia was induced by changing the control perfusate to an identical solution bubbled with 9% CO2-6% O2-85% N2, and readjusted back to 7.35–7.4 immediately prior to use. Slices were exposed to hypoxia/hypercapnia for 10 min and then returned to the original perfusate for 30 min. At the end of each experiment, glutamatergic synaptic activity was reversibly inhibited using D-2-amino-5-phosphonovalerate (AP5, 50 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 µM) to block N-methyl-D-Aspartate (NMDA) and non-NMDA receptors.
Prenatal nicotine exposure
Adult female rats were anesthetized with ketamine-xylazine (87/13 mg/kg ip; Phoenix Pharmaceuticals, St. Joseph, MO) on the third day of gestation and implanted subcutaneously with Alzet osmotic minipumps (Durect, Cupertine, CA) 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 (Slotkin 1998). Pumps delivered nicotine at 6 mg·kg–1·day–1 to produce a blood nicotine concentration approximately equivalent to those that occur in moderate to heavy smokers (i.e., 30–40 ng/ml) for 28 days (Benowitz et al. 1982; Isaac and Rand 1972; Slotkin 1998).
Data analysis
Analysis of spontaneous synaptic currents was performed using MiniAnalysis (version 5.6.12, Synaptosoft) with minimal acceptable amplitude set at the amplitude at which AP5 and CNQX blocked all synaptic events. The frequency of EPSCs that occurred in CVNs was grouped into 1-s bins and cross-correlated with the onset of inspiratory–related hypoglossal activity. Data were analyzed from all bursts during the last 2 min of the control period, the last 2 min of hypoxia/hypercapnia, and from 10 to 12 min during the recovery. These periods were chosen for analysis because during these periods, synaptic activities were consistent, and any hypoxia/hypercapnia-evoked changes reached a steady state. Burst duration, frequency, and amplitude were measured using pClamp 7 software (Axon Instruments, Union City, CA) and from the filtered (10- to 300-Hz band-pass) and electronically integrated hypoglossal rootlet activity. mEPSC frequency was analyzed for 10 s before and after ATP application and for 10 s 1 min after ATP application. Results are presented as means ± SE. Statistical comparisons were performed using ANOVA with repeated measures to examine the responses throughout the time course of the experiments and two-way ANOVA when comparing the results from different series of experiments such as between control animals and animals that were exposed to nicotine prenatally. Significant difference was set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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We employed an in vitro brain stem slice that retains spontaneous respiratory-related rhythms and allows simultaneous whole cell recordings of CVNs, the dominant control of heart rate (Blitz and Ramirez 2002; Feldman and Gray 2000; Smith et al. 1991). As previously reported (Huang et al. 2005), hypoxia/hypercapnia evoked a biphasic change in central respiratory activity. Hypoxia/hypercapnia elicited a transient increase, followed by a decrease in respiratory frequency, see Fig. 1. In addition, hypoxia/hypercapnia significantly depressed (P < 0.01) respiratory burst duration. Hypoxia and hypercapnia induced a small but significant increase in the amplitude of respiratory bursts, (P < 0.01). The responses in animals exposed to prenatal nicotine closely mimicked and were not significantly different from the responses in unexposed animals, see Fig. 1.
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CVNs exhibit no respiratory modulation of excitatory neurotransmission under control conditions (Fig. 2; n = 7; P > 0.05) or during hypoxia/hypercapnia (P > 0.05). However, on recovery from hypoxia/hypercapnia, there is a significant respiratory-related increase in excitatory neurotransmission (Fig. 2; P < 0.01). These responses persisted for 
48 min post hypoxia-hypercapnia.
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, 2003). To determine if nicotinic acetylcholine receptors also mediate excitatory synaptic neurotransmission to CVNs after hypoxia/hypercapnia, nicotinic antagonists were focally applied to CVNs throughout the entire experiment. Application of -bungarotoxin (100 nM) had no effect on excitatory inputs to CVNs during hypoxia/hypercapnia or during the recovery period, see Fig. 3 (P > 0.05; n = 7). Similarly, 3 µM DHE, a concentration selective for 42* nicotinic receptors, failed to alter glutamatergic neurotransmission in CVNs throughout hypoxia/hypercapnia or during recovery (n = 9; P > 0.05), see Fig. 3D. Inhibition of all nicotinic receptors with 100 µM DHE (n = 11) likewise had no effect on excitatory inputs to CVNs during hypoxia/hypercapnia administration or during recovery (Fig. 3E; P > 0.05).
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Because P2 receptors facilitate central cardiorespiratory network responses to hypoxia, we hypothesized that respiratory-related glutamatergic neurotransmission to CVNs on recovery from hypoxia/hypercapnia is mediated by ATP receptors. To test this hypothesis, we focally applied the broad purinergic antagonist, suramin (25 or 100 µM) throughout the experiments, or in the case of PPADS (100 µM) after a delay in the hypoxia/hypercapnia period. Suramin (25 µM) significantly depressed the respiratory-related glutamatergic inputs to CVNs during recovery from hypoxia, see Fig. 4B (n = 8, P < 0.05, from a peak of 8.9 ± 0.4 to 7.1 ± 0.3 Hz, a 19.8% decrease from vehicle control). Suramin (100 µM) further reduced respiratory-related EPSCs in CVNs during recovery from hypoxia/hypercapnia (Fig. 4C; n = 7; P < 0.05, from a peak of 8.9 ± 0.4 to 6.2 ± 0.3 Hz, a 30.4% decrease from vehicle control).
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Nicotinic and purinergic receptors can exhibit cross-inhibition (Khakh et al. 2000). To test whether nicotinic receptor activation blocks P2 receptor facilitation of glutamatergic neurotransmission, miniature EPSCs (mEPSCs) were isolated by inclusion of gabazine (25 µM), strychnine (1 µM), and TTX (1 µM) in the perfusate. Focal application of ATP (1 mM) significantly increased mEPSC frequency in CVNs (Fig. 6; n = 9; P < 0.05). However, in the presence of nicotine (10 µM), ATP failed to significantly alter mEPSC frequency (P > 0.05). In animals exposed to prenatal nicotine, ATP also significantly increased mEPSC frequency (n = 7; P < 0.05), which was abolished in the presence of nicotine (10 µM; P > 0.05).
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In contrast to unexposed animals, in prenatal nicotine-exposed animals, CVNs receive a respiratory-related glutamatergic neurotransmission during hypoxia/hypercapnia but not on recovery from hypoxia/hypercapnia, see Fig. 7A. To test whether this transformation results from nicotinic receptor activation, we focally applied nicotinic receptor antagonists. Application of 3 µM DHE to block 42* receptors (n = 8) did not alter respiratory-evoked excitatory inputs to CVNs during or on recovery from hypoxia/hypercapnia (Fig. 7B; P > 0.05). Focal application of the 7 nicotinic receptor antagonist -bungarotoxin (100 nM) partially depressed respiratory-related excitatory inputs evoked during hypoxia/hypercapnia (n = 11) without altering glutamatergic neurotransmission during recovery (Fig. 7C). Application of 100 µM DHE to block all nicotinic receptors (n = 11) ablated respiratory-related excitatory neurotransmission during hypoxia/hypercapnia (Fig. 8, A and B) and restored a respiratory-evoked glutamatergic neurotransmission during recovery as in unexposed animals.
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E (100 µM) and suramin (100 µM) in preparations from prenatal nicotine-exposed pups (n = 7). Suramin blocked the restored glutamatergic neurotransmission to CVNs during recovery from hypoxia/hypercapnia (Fig. 8C).
Nearly identical results were obtained with continuous and focal application of the P2 receptor antagonist PPADS (100 µM). As shown in Fig. 9, in prenatal nicotine-exposed animals, continuous application of DHE (100 µM) ablated respiratory-related excitatory neurotransmission during hypoxia/hypercapnia (Fig. 9, A and B) and restored a respiratory-evoked glutamatergic neurotransmission during recovery as in unexposed animals. This respiratory-evoked glutamatergic neurotransmission during recovery was abolished by focal application of the P2 receptor antagonist PPADS (100 µM, n = 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Similar to studies both in vivo and in vitro, our data indicate hypoxia/hypercapnia evokes a biphasic response in the central respiratory network; respiratory bursts exhibited a transient increase followed by a secondary decrease in frequency and acquired a gasp-like quality characterized by high-amplitude bursts of short-duration. Prenatal nicotine exposure elicited no significant changes in the central respiratory responses to hypoxia/hypercapnia. However, some other studies have reported significant changes in cardiorespiratory integrity following prenatal nicotine exposure. Maternal cigarette smoking has been reported to have no effect on ventilatory responses to hypoxia or hypercapnia (Lewis and Bosque 1995), whereas in other work, prenatal nicotine exposure diminished the hypoxic ventilatory responses and respiratory drive (Ueda et al. 1999). Similarly it is reported that prenatal nicotine exposure both does not alter (Bamford et al. 1996; Schuen et al. 1997; Slotkin et al. 1997) and diminishes the ventilatory response to hypoxia or hypercapnia (Hafstrom et al. 2002a,b; Simakajornboon et al. 2004). The results from this study indicate that prenatal nicotine exposure does not alter central respiratory responses to hypercapnia and suggests any potential changes in the ventilatory responses to prenatal nicotine in vivo may depend on changes in the activity of peripheral chemoreceptors rather than within the medulla (Bamford et al. 1999; Holgert et al. 1995; Simakajornboon et al. 2004).
It is somewhat surprising that nicotinic receptors do not mediate respiratory-related excitatory neurotransmission to CVNs in unexposed animals. CVNs receive tonic endogenously active cholinergic input that mediates both excitatory and inhibitory neurotransmission (Neff et al. 1998; Wang et al. 2003b). Furthermore, 42* nicotinic receptors mediate inspiratory-evoked GABAergic neurotransmission to CVNs responsible for respiratory sinus arrhythmia (Neff et al. 2003). However, excitatory neurotransmission to CVNs was not altered by nicotinic antagonists under control conditions, hypoxia/ hypercapnia, nor during recovery. Although activation of nicotinic receptors is one mechanism by which glutamatergic neurotransmission is facilitated to CVNs, this input does not mediate the respiratory-related enhancement of excitatory neurotransmission during recovery from hypoxia/hypercapnia.
Whereas nicotinic receptor antagonists did not alter excitatory neurotransmission to CVNs, both of the P2 receptor antagonists suramin and PPADS inhibited respiratory-related increases in glutamatergic neurotransmission during recovery from hypoxia/hypercapnia. Because suramin and PPADS were focally applied to CVNs, ATP is acting on local P2 receptors to enhance the release of glutamate. Recent work has shown P2X but not P2Y receptor agonists can facilitate glutamatergic neurotransmission to cardiac vagal neurons (Griffioen et al. 2007). Focal application of the selective P2X agonist ,-methylene ATP but not the P2Y agonists UTP and adenosine 5'-0-(Z-thiodiphosphate) facilitates glutamatergic EPSCs in CVNs, demonstrating P2X but not P2Y receptors are localized on glutamatergic synaptic terminals on CVNs and can enhance excitatory neurotransmission to CVNs (Griffioen et al. 2007).
However, whether P2X, as well as P2Y receptor activation at distant sites also contributes to glutamatergic neurotransmission to CVNs is unknown. Purinergic neurotransmission plays a role in respiratory neuron activity during hypoxia and hypercapnia (Gourine et al. 2005b), hypoglossal motoneuron output and NTS activation (Funk et al. 1997; Shigetomi and Kato 2004). Therefore in addition to local P2 receptor facilitation of glutamatergic neurotransmission, P2 receptor activation in other cardiorespiratory control sites outside of the influence of the applied drug in this study may also contribute to the enhancement of excitatory neurotransmission to CVNs.
ATP facilitated the presynaptic release of glutamate in both unexposed and prenatal nicotine-exposed animals. However, this facilitation is prevented in the presence of nicotine. Recent work has suggested cross-talk occurs between nicotinic and purinergic receptors leading to occlusion of purinergic receptor function. As a result of physical receptor coupling, 42* nicotinic receptors and P2X2 receptors physically interact and effect mutual cross-inhibition when coactivated (Khakh et al. 2000, 2005). In the dorsal motor nucleus of the vagus, ATP and nicotinic receptors colocalize (Nabekura et al. 1995). In myenteric neurons, P2X and nicotinic receptors are mutually inhibitory (Zhou and Galligan 1998). Further, in sympathetic neurons of the celiac ganglia, nicotine occludes ATP currents (Searl et al. 1998). The mechanisms for this cross-inhibition are unknown. In addition to physical receptor coupling another possibility is that the inability of ATP to increase mEPSC frequency after nicotine application is due to nicotine-mediated depletion of vesicles. It is possible the prior exposure of nicotine released all or most vesicles in the presynaptic terminal leaving subsequent application of ATP ineffective due to depletion of the vesicle pool.
In addition to physical receptor coupling or vesicle depletion, purinergic and nicotinic receptors could exhibit mutual occlusion by competitively increasing calcium in the presynaptic terminal (Boehm 1999). P2X receptors are nonselective cation channels with equal permeability to potassium and sodium and a significant permeability to calcium (Evans et al. 1996). Activation of P2X receptors is reported to facilitate neurotransmitter release by direct calcium entry through P2X receptors (Khakh and Henderson 1998) or through activation of voltage gated calcium channels (Gu and MacDermott 1997). Therefore P2X receptors may facilitate glutamate release by directly mediating presynaptic calcium entry or alternatively, through depolarizing the presynaptic terminal to open voltage-gated calcium channels that then elicit glutamate release. Similarly, nicotinic receptor activation facilitates glutamate release onto cardiac vagal neurons, and this increase in glutamatergic neurotransmission occurs via activation of voltage-dependent calcium channels, especially the calcium channels sensitive to agatoxin IVA (Neff et al. 1998; Wang et al. 2001). The data in this study, which shows an increase in glutamatergic release with nicotine prevents any further increase with ATP, in both unexposed and nicotine-treated animals, suggest that, as in other systems, nicotinic and purinergic receptors present within the nucleus ambiguus functionally compete and that nicotinic receptor activation occludes purinergic signaling at the presynaptic glutamatergic synaptic terminal. Further work will be necessary to elucidate the mechanisms, such as direct receptor interactions or competition for calcium channel activation for this cross-inhibition of nicotinic and purinergic receptors.
In unexposed animals, there is a burst of GABAergic neurotransmission to CVNs during inspiratory activity, and this respiratory related increase in GABAergic neurotransmission is dependent on activation of 42* nicotinic receptors (Neff et al. 2004). This respiratory-related GABAergic neurotransmission to CVNs is exaggerated by prenatal nicotine exposure (Neff et al. 2004). However, instead of exaggerating an already existing neurotransmission, prenatal nicotine exposure generates a novel excitatory synaptic input not present in unexposed animals. Prenatal nicotine exposure recruits an excitatory neurotransmission to CVNs during hypoxia/hypercapnia and prevents respiratory-related increases in glutamatergic neurotransmission on recovery from hypoxia/hypercapnia (Huang et al. 2005). Further, nicotinic receptor antagonists restore prenatal nicotine responses to that of unexposed animals. Therefore prenatal nicotine exposure evokes a dual modification of cardiorespiratory responses to hypoxia/hypercapnia and causes cardiorespiratory responses not normally under nicotinic control to rely on nicotinic receptor activation.
The changes in glutamatergic neurotransmission evoked by prenatal nicotine exposure likely results from alterations in nicotinic receptor expression and/or activation. The glutamatergic neurotransmission evoked during hypoxia/hypercapnia is mediated by multiple nicotinic receptors, as -bungarotoxin blunted and 100 µM DHE completely blocked this input. Previous work has shown that prenatal nicotine exposure alters the types and location of nicotinic receptors mediating excitatory neurotransmission to CVNs (Huang et al. 2004). The results from this study indicate glutamatergic neurotransmission evoked during hypoxia/hypercapnia is mediated in part by 7 but not 42* nicotinic receptors. This is consistent with previous work that shows that 42* nicotinic receptors are not involved in glutamatergic neurotransmission in CVNs and that 7 partially mediates responses to nicotine (Huang et al. 2004). Further, although in unexposed animals 7 nicotinic receptors are located presynaptically and non-42* receptors are located postsynaptically on CVNs, prenatal nicotine exposure causes the additional expression of 7 nicotinic receptors postsynaptically (Huang et al. 2004).
Whereas during hypoxia/hypercapnia CVNs receive enhanced respiratory-related glutamatergic neurotransmission, prenatal nicotine depresses inspiratory-evoked excitatory neurotransmission during recovery from hypoxia/hypercapnia. Furthermore, application of nicotinic antagonists restored the respiratory-evoked increase in glutamatergic neurotransmission during recovery, thereby converting a nicotine-exposed response to one that resembles an unexposed response. Neither 7 nor 42* nicotinic receptors mediate prenatal nicotine-evoked modifications during recovery from hypoxia/hypercapnia; control responses were only restored by 100 µM DHE. These responses are likely evoked by the conversion of excitatory neurotransmission control by 32*/6X/34 to solely 34 nicotinic receptors (Kamendi et al. 2006).
The alterations in cardiorespiratory responses to hypoxia/hypercapnia after prenatal nicotine exposure may be of clinical significance. Smoking during pregnancy increases the risk of SIDS two to four times (Bulterys et al. 1990; Haglund and Cnattingius 1990; Malloy et al. 1988; Schoendorf and Kiely 1992), and nicotine has been proposed to be the link between maternal smoking and SIDS (Bamford and Carroll 1999; Nachmanoff et al. 1998; Slotkin et al. 1997; St-John and Leiter 1999). Some have suggested that SIDS may result from a direct alteration of the development of brain stem sites responsible for cardiorespiratory control and arousal due to prenatal nicotine exposure (Meny et al. 1994; Nachmanoff et al. 1998; Slotkin et al. 1997; St-John and Leiter 1999). During hypercapnia in vivo, the firing rate of CVNs increases during inspiration (Yen et al. 2000), and infants at risk for SIDS exhibit a more pronounced bradycardia during hypercapnia than healthy infants (Edner et al. 2002; Meny et al. 1994; Poets et al. 1999). The recruitment of a glutamatergic neurotransmission to CVNs during hypoxia/hypercapnia observed in the present study would result in a significant reduction in heart rate and provides a possible mechanism by which severe bradycardia is evoked by hypoxia/hypercapnia in SIDS.
Purinergic neurotransmission is an important component of central cardiovascular and respiratory responses to hypoxia and/or hypercapnia (Gourine 2005; Gourine et al. 2005a, b). Our data suggest novel nicotinic receptor activity after prenatal nicotine exposure prevents purinergic modulation of central cardiorespiratory interactions. When nicotinic activity is blocked, purinergic-mediated cardiorespiratory responses are restored. To our knowledge, this is the first report linking prenatal nicotine exposure to altered purinergic neurotransmission. This hypothesis may provide a cellular mechanism for in vivo studies that report increased parasympathetic outflow after apnea in control infants, but absent parasympathetic increases after apnea in future SIDS victims (Franco et al. 2003). In summary, our study suggests purinergic neurotransmission is a key component of central cardiorespiratory interactions and nicotinic receptor activity modification by prenatal nicotine exposure reversibly precludes purinergic control of cardiorespiratory function.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. Mendelowitz, Dept. of Pharmacology and Physiology, The George Washington University, 2300 Eye St. NW, Washington, DC 20037 (E-mail: dmendel{at}gwu.edu)
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REFERENCES |
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) and prenatal nicotine-exposed (
) animals. C: hypoxia/hypercapnia significantly depressed burst duration in both unexposed and prenatal nicotine-exposed animals. D: hypoxia/hypercapnia induced a small but significant increase in burst amplitude in both unexposed and prenatal nicotine-exposed animals. There were no statistically significant differences in the respiratory responses between unexposed and prenatal nicotine-exposed animals. +/*P < 0.05, ++/**P < 0.01. In this and all subsequent figures, error bars are SE.
, n = 9), during recovery from H/H, CVNs received significant increases in glutamatergic synaptic inputs during respiratory bursts. In the presence of suramin at concentrations of 25 µM (B; n = 8) and 100 µM (C; n = 7, and top representative experiment), the excitatory input to CVNs was significantly decreased by 19.8 and 30.4%, respectively, in unexposed animals.
