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2-receptor inhibition of the Ih current or presynaptic glutamate release
Department of Physiology, Faculty of Medicine and Health Science, University of Auckland, Auckland, New Zealand
Submitted 7 September 2004 ; accepted in final form 30 November 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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2-adrenoceptor-mediated, presynaptic inhibition of glutamatergic inspiratory drive (Selvaratnam SR, Parkis MA, and Funk GD. Brain Res 805: 104–115, 1998) and postsynaptic inhibition of the hyperpolarization-activated inward current (Ih) (Parkis MA and Berger AJ. Brain Res 769: 108–118, 1997) modulate XII inspiratory activity. Nerve and whole cell recordings were applied to rhythmic medullary slice preparations from neonatal rats (postnatal days 0–4) to monitor XII inspiratory burst amplitude and motoneuron properties. Application of an 2-receptor agonist (clonidine, 1 mM) to the XII nucleus reduced inspiratory burst amplitude to 71 ± 3% of control but had no effect on inspiratory synaptic currents. It also reduced the Ih current by 
40%, but an Ih current blocker (ZD7288), at concentrations that blocked 80% of Ih, had no effect on inspiratory burst amplitude. The clonidine inhibition was unaffected by the GABAA antagonist (+)bicuculline but attenuated by the 2-antagonist rauwolscine and the imidazoline 1 (I1) antagonist efaroxan. The I1 agonist rilmenidine, but not the 2-agonist UK14304, inhibited XII output. Clonidine also reduced action potential amplitude or impaired repetitive firing. Although a contribution from 2, and in particular I1, receptors remains possible, results demonstrate that 1) noradrenergic modulation of XII inspiratory activity is unlikely to involve 2-receptor-mediated presynaptic inhibition of glutamate release or modulation of Ih; 2) inhibition of repetitive firing is a major factor underlying the inhibition of XII output by clonidine; and 3) Ih is present in neonatal XII motoneurons but does not contribute to shaping their inspiratory activity.
airway control; 2-adrenoceptor; hyperpolarization-activated inward current; whole cell recording; rat; clonidine
Noradrenergic signaling cascades, for example, exert powerful and diverse modulatory effects on inspiratory activity of XII motoneurons (20, 49, 51, 58). The predominant effect in mice is an 1-receptor-mediated potentiation of inspiratory output that increases approximately ninefold between postnatal day 0 (P0) and postnatal day 14 (P14). This excitatory effect is complemented by a small, 
-receptor-mediated component and attenuated by a putative 2-receptor-mediated inhibition of inspiratory output (58). The 2-receptor-mediated inhibition of inspiratory output is proposed to involve 2C-receptors because these, but not 2A-receptors, are expressed in the XII nucleus (54, 63). However, the underlying mechanisms are not known. One hypothesis is that postsynaptic 2-receptors hyperpolarize and inhibit XII motoneurons by shifting the voltage dependence of activation of the Ih current (hyperpolarization-activated inward current) toward more negative potentials (50). An alternative hypothesis is that the inhibition is due to 2-receptor-mediated presynaptic inhibition of glutamatergic synaptic drive to XII motoneurons. Indeed, inspiratory drive to XII motoneurons is glutamatergic (21), and 2-receptor-mediated, presynaptic inhibition of glutamatergic transmission is well documented in the nervous system (6, 7, 30, 42, 46).
Thus the primary goal of this study was to determine, using rhythmically active medullary slice preparations from neonatal rats, whether the noradrenergic modulation of XII inspiratory activity involves 2-receptor-mediated presynaptic inhibition of glutamate release or postsynaptic modulation of the Ih current. A secondary objective was to determine the role of the Ih current in shaping the inspiratory activity of XII motoneurons. In most rhythmic motor behaviors, block of Ih attenuates both frequency and output magnitude (24, 37, 39). However, the effects on respiratory-related behavior recorded from rhythmically active medullary slices of blocking Ih are controversial. At low concentrations the Ih blocker ZD7288 has no effect on rhythm or motor output (41), whereas at higher concentrations it potentiates both frequency and burst amplitude (64). Because ZD7288 was applied to the bath in these experiments where it would affect both rhythm generating and drive transmission elements, it remains to be determined whether block of Ih solely within the motoneuron pool has any impact on inspiratory output.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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For studies examining changes in XII nerve inspiratory output, slices were pinned caudal surface up in a recording chamber (10-ml volume) and superfused with aCSF (27–29°C, chamber pH between 7.50 and 7.56) (36) at a flow rate of 20 ml/min. In whole cell recording experiments, slices were perfused at 2–3 ml/min in a 0.5-ml chamber. The pH of the solution in the recording chamber was between 7.50 and 7.56 and within the range necessary for maintaining relative alkalinity. This is a particularly important factor in these experiments because, in addition to the typical concern regarding maintaining the charge of proteins within the slice, clonidine contains an imidazoline ring. Thus its chemical activity might be altered if the pH of the aCSF deviated significantly from that required to maintain relative alkalinity. Further supporting that the efficacy of clonidine persists under in vitro conditions are the observations that application of clonidine to the ventrolateral medulla causes a reduction in frequency whether applied in vivo (Ref. 12; anesthetized rats) or in vitro (Ref. 27; rhythmically active medullary slices).
Thirty minutes before the start of data collection, extracellular K+ concentration was raised from 3 to 9 mM. Elevated K+ was not required to activate rhythmic network activity but to maintain long-term respiratory network activity (21). Although the confounding influence of elevated K+ must always be considered, the possibility that it affected our conclusions is remote. Our main objectives were to determine whether clonidine presynaptically inhibited glutamatergic inspiratory activity and whether its modulation of Ih influenced inspiratory output. Elevated K+ depolarizes XII motoneurons by 10 mV (68). This will cause a mild reduction in the driving force for the K+ component of the glutamatergic current [predominantly a DL--amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA)-mediated Na+ and K+ current (21)]. However, the reversal potential of the glutamate current will remain significantly above resting potential. Thus glutamate will still produce a depolarizing input and any attenuation of glutamate release by clonidine will appear as a reduction in XII burst amplitude. In the context of Ih, extracellular K+ has minimal effect on its reversal potential and increases the slope of its current-voltage relationship (47). This will enhance Ih magnitude and should in fact enhance our ability to detect any effect of blocking Ih on inspiratory activity. All experiments and procedures were approved by the local animal ethics committees and performed in accordance with New Zealand and Canadian guidelines for the care, handling, and treatment of experimental animals.
Drugs and drug application.
Drugs used included the 2-receptor agonists clonidine (Sigma, Oakville, Ontario, Canada; 0.1–1 mM) and UK14304 (Tocris, Northpoint, Bristol, UK; 0.5 mM), the 2-receptor antagonists rauwolscine (RBI, Natick, MA; 10 µM) and idazoxan (RBI; 10 µM), the organic Ih blocker ZD7288 [N-ethyl-1,6-dihydro-1,2-dimethyl-6-(methylimino)-N-phenyl-4-pyrimidinamine; Tocris; 0.1–0.5 mM], the imidazoline 1 (I1) receptor agonist rilmenidine (RBI; 1 mM), the I1 antagonist efaroxan (RBI; 10 µM), and the GABAA antagonist (+)bicuculline (Sigma; 10 µM). Most of drugs were made up as concentrated stock in aCSF solution, then diluted with aCSF containing 9 mM K+, aliquoted into 1-ml Eppendorf tubes, and frozen for future use. UK14304 was made up as 100 mM stock in DMSO. (+)Bicuculline was dissolved in chloroform to make 20 mM stock solution. Effects of UK14304 and (+)bicuculline were compared with effects of vehicle. Rauwolscine and (+)bicuculline were made fresh before each experiment. The concentration of K+ in injected solutions always matched the concentration of K+ in the extracellular solution.
Clonidine, ZD7288, UK14304, and rilmenidine were pressure injected via triple-barreled pipettes (6–7 µm per barrel, outside-tip diameter) pulled from borosilicate glass capillaries (Clark Electromedical Instruments, Pangbourne, UK). Drugs were delivered at 10 psi, and injections were controlled via a programmable stimulator (Master-8, A.M.P.I., Jerusalem, Israel) linked to a solenoid. Injection pipettes were positioned over the ventromedial aspect of the XII nucleus by use of a motorized microdrive (PCM100, Newport, Irvine, CA) attached to a three-axis manual micromanipulator (M443 series, Newport).
Note that the concentrations of drugs used in the present study should not be directly compared with those in experiments in which similar agents are bath applied or applied to isolated cells. First, the concentration of drug decreases exponentially with distance from the pipette tip (43). Second, previous experiments with this preparation indicate that drug concentration must be at least 10-fold greater than the bath-applied concentration to produce similar effects (34).
Recording of XII nerve activity.
XII nerve inspiratory activity was recorded bilaterally by using suction electrodes with internal tip diameters ranging from 70 to 100 µm. Signals were amplified (50,000 times), band-pass filtered (0.1–3 kHz), full-wave rectified, and integrated (
= 50 ms). The output of the integrator was displayed on an oscilloscope (5111 Storage Oscilloscope, Tektronix) and chart recorder (2400S, Gould Instruments) and stored via pulse code modulation on VCR cassette (Vetter 400A).
Whole cell recording.
Whole cell patch-clamp recordings from XII motoneurons were made under visualization by using infrared differential interference contrast microscopy. Patch electrodes (resistance 3.0–4.0 M
) were pulled on a horizontal puller (Sutter model P-97) from 1.2-mm outer diameter filamented borosilicate glass (Clark/WPI) and filled with potassium gluconate solution containing (in mM) 122.5 potassium gluconate, 17.5 KCl, 9 NaCl, 1 MgCl2, 10 HEPES, 0.2 EGTA, 3 ATP-Mg, 0.3 GTP-Tris. Intracellular solution pH was adjusted to 7.2–7.3 with 5 M KOH. Intracellular signals were amplified and filtered (2–5 kHz low-pass Bessel filter) with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), acquired at 8–20 kHz by using a Digidata 1200B analog-to-digital board and pClamp 6.0 software, and stored via pulse code modulation for offline analysis. Series resistance and whole cell capacitance were estimated under voltage-clamp conditions by using short voltage pulses (100 Hz, –10 mV, 3.0 ms). Series resistance averaged 15 ± 5 M, was compensated by 75%, and was monitored throughout the experiments. Data were discarded if it changed by >5% between control and test conditions. Depolarizing ramps from –90 to –30 mV were used to obtain current-voltage (I-V) curves. Input resistance (RN) was calculated from the inverse of the slope of a least squares regression line fitted to the I-V curves.
Data analysis. Effects of drugs on XII nerve inspiratory burst amplitude were assessed with custom-written LabVIEW acquisition and analysis protocols and Excel software. The maximum inhibition induced by clonidine alone or in the presence of various antagonists was determined from a moving average of XII inspiratory burst amplitude (on the basis of 5 consecutive inspiratory events) during the 2-min control period immediately before drug onset, the 2-min period of drug application, and 5 min of drug washout. The maximum inhibition was then calculated by comparing the smallest value of the moving average obtained during drug application to the smallest value of the moving average obtained during control period. To determine whether a given effect was significant, the maximum inhibition induced by a given drug was compared with the inhibition produced by injection of vehicle into the same location.
To address the possibility that clonidine was acting on premotoneuron pools outside the XII nucleus, we verified that injections in the lateral tegmental field within 300 µm of the lateral margin of the ventral half of the XII nucleus (n = 3), and in the dorsal portion of the XII nucleus (n = 5), were without effect on inspiratory output. Moreover, clonidine-mediated decreases in frequency, indicative of clonidine diffusion to rhythm-generating regions in the ventrolateral medulla (14, 15) were never observed.
Whole cell data were analyzed offline by using AxoGraph 4.4, Clampfit 8.0, and Excel software. Data are presented as means ± SE. All statistical analyses were performed on raw data, and differences in means were tested with Student's or paired t-tests. Values of P < 0.05 were assumed significant. Because of the slow onset of the clonidine response and evidence in the literature that the effects are slow to reverse (50), most slices or motoneurons were exposed to clonidine only once. Thus the effects of rauwolscine, efaroxan, and rilmenidine were not explored with paired protocols, but by comparing the effects of clonidine in a control group with its effects in a different group. In only two sets of experiments were paired protocols used. The first compared the actions of clonidine with those of UK14304 and the order of treatment was varied. The second compared the actions of clonidine first in the presence and then the absence of (+) bicuculline.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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2-receptors was established by demonstrating that norepinephrine (NE), when applied in the presence of 1- and -adrenoceptor antagonists, produces an inhibition of XII nerve inspiratory burst amplitude that is similar to the clonidine effect (58). Our first objective in this study was to establish that this same inhibitory mechanism operates in rats. Clonidine was used at a high concentration (1 mM) to ensure maximal activation of 2-receptors throughout the XII nucleus, so that in subsequent experiments we could exclude the possibility that a negative effect on glutamatergic inputs or Ih reflected insufficient receptor activation. As shown for a single preparation in Fig. 1A, local application of clonidine (120 s) over the XII nucleus produced a significant inhibition of inspiratory burst amplitude that reached a nadir during the second minute of application before returning to control 7.5 ± 0.5 min (n = 7) after clonidine application was stopped. Burst amplitude was reduced on average to 71 ± 3% (n = 10) of control. Injections of vehicle did not affect burst amplitude (Fig. 1B).
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Modulation of Ih by clonidine does not contribute to the inhibition of XII inspiratory output. To test the hypothesis that inhibition of Ih contributes to the clonidine-mediated inhibition of XII nerve inspiratory output, we examined the effects on XII burst amplitude and Ih amplitude of locally applying the Ih channel blocker, ZD7288, to the XII nucleus. At 0.1 mM, ZD7288 blocked 80 ± 8% of Ih, reducing it from –377 ± 70 pA (n = 13) under control conditions to –40 ± 33 pA (Fig. 4, B and C). In spite of this, 0.1 mM ZD7288 had no effect on XII burst amplitude (Fig. 4, A and C). Inhibition of XII burst amplitude was only observed when the concentration of ZD7288 was increased to 0.5 mM, at which point Ih was no longer detectable in individual XII motoneurons (n = 2) and XII burst amplitude fell significantly to 85 ± 5% of control (n = 5). This inhibition of XII burst amplitude peaked within 2–3 min, and recovery occurred gradually over 8–10 min.
Reports in supraoptic neurons in superfused explants of rat hypothalamus suggest that high concentrations (30–60 µM in the bath) of ZD7288 caused a significant decrease in spike amplitude, and this effect was readily reversed with a wash period lasting 20–40 min, suggesting that ZD7288 has nonspecific inhibitory actions on voltage-gated Na+ channels (23). We therefore examined the effects of ZD7288 on the repetitive firing properties of inspiratory XII motoneurons to help determine whether the inhibitory actions of ZD7288 at 0.5 mM were due to indirect actions or inhibition of the Ih current. Under current clamp conditions, motoneurons were held at –60 mV and a series of 5 incrementing, depolarizing current pulses (600 ms, 20 pA increments) were delivered starting with an initial step of +200 pA. This protocol was repeated before, during, and after local application of ZD7288 to establish its effects on action potential amplitude, and the relationship between firing frequency and current (the f/I relationship). From the voltage responses to the same single current step before, during, and after ZD7288 shown in Fig. 4D, action potential amplitude and the f/I relationship were unaffected at the lower dose of 0.1 mM ZD7288 where control and ZD7288 curves overlap completely. However, at the higher dose of 0.5 mM, where effects on XII burst amplitude were apparent, ZD7288 significantly, and reversibly, reduced action potential amplitude. The effect on repetitive firing was variable. In some cells, the f/I relationship was minimally affected (Fig. 4D). However, in others the ability to fire continuously in response to a 600-ms depolarizing pulse was compromised. In these motoneurons, firing could not be sustained throughout the pulse while consecutive action potentials became progressively smaller in amplitude and longer in duration (data not shown). In summary, concentrations of ZD7228 (0.1 mM) that blocked 80 ± 8% of Ih were without effect on XII burst amplitude. Effects on XII burst amplitude were only observed at 0.5 mM where action potential amplitude or repetitive firing was inhibited.
Are the inhibitory effects of clonidine mediated by 2-receptors?
Involvement of an 2-receptor mechanism was established previously by the observation in mice that NE inhibits XII nerve inspiratory burst amplitude in the presence of propranolol (-adrenoceptor antagonist) and prazosin (1-adrenoceptor antagonist). We tested the involvement of 2-receptor signaling in the inhibition in rat by determining whether the inhibition was sensitive to 2-receptor antagonism. The 2-receptor antagonists, rauwolscine or idazoxan (10 µM), were applied to the bath 30 min before the local application of clonidine (1 mM) to ensure sufficient time for diffusion into the tissue. Rauwolscine (10 µM), which unlike idazoxan does not bind to I1 receptors (16), blocked the inhibitory actions of clonidine (n = 6; Fig. 5A). Idazoxan (10 µM) did not completely block the clonidine-mediated inhibition of inspiratory output, but reduced it to 83 ± 7% (n = 5) from 71 ± 3% (n = 10; Fig. 5B) in control.
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2-antagonists also reduced XII inspiratory burst amplitude. Rauwolscine reduced XII inspiratory burst amplitude to 83 ± 4% of control (Fig. 5C). The effects of idazoxan alone were variable. It reduced burst amplitude in some but not all preparations, such that average values (90 ± 6% of control) were not different from control. Yohimbine, another 2-antagonist, completely and irreversibly blocked XII nerve output (data not shown). A consequence of the fact that the 2-antagonists alone had an effect on inspiratory output was that the effects of clonidine tested in the absence and presence of antagonist are not directly comparable because they were performed on different baselines of respiratory activity.
Therefore, to further test whether 2-receptor activation inhibits XII output in rats as seen in mice (58), we compared in eight preparations the effects on XII output of clonidine and a saturating concentration of the 2-agonist, UK14304 (0.5 mM) (2, 48). Responses of a single rhythmic slice to local application of both agonists are shown in Fig. 6A. UK14304 had no effect when applied to the same site where clonidine potently inhibited XII output. This was the case whether UK14304 preceded or followed clonidine. Pooled data similarly indicate that burst amplitude in UK14304 (94 ± 5% of control, n = 8) was no different than in control (Fig. 6B).
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2-receptors were hypothesized to act in the XII nucleus, and that the UK14304 data directly question a role for 2-receptors in the clonidine-mediated inhibition in rats, the mechanism(s) underlying the inhibition remains unknown. We tested three additional possibilities.
Imidazoline receptors.
UK14303 does not inhibit XII output and is selective for 2-receptors, whereas clonidine inhibits XII output and activates both 2-receptors and imidazoline (I1) receptors. We therefore tested the effects of the I1 receptor antagonist, efaroxan (10 µM), on the clonidine inhibition. Efaroxan did not completely block the clonidine-mediated inhibition, but significantly reduced its magnitude from 71 ± 3% to 91 ± 5% of control (n = 6; Fig. 7). Local application of the I1 agonist rilmenidine over the XII nucleus also produced a significant inhibition of XII inspiratory burst amplitude (85 ± 5%) that was similar in time course but smaller in amplitude relative to that evoked by clonidine.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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1-receptor-mediated excitation (20, 49, 51, 58). However, considerable evidence supports 2-receptor-mediated inhibition of motoneuron output. For example, 2-receptor mechanisms contribute to the long-term depression of phrenic and XII output induced by episodic hypercapnia (3) and suppress the responses of trigeminal motoneurons to oral stimulation (29). In addition, clonidine inhibits EMG activity and the flexor responses of spinal rats to afferent nerve volleys (65). Finally, the inspiratory output of XII motoneurons is inhibited by clonidine alone and by NE when coapplied with 1- and -adrenoceptor antagonists (58). Whether these inhibitory actions on motoneuron output are mediated presynaptically via inhibition of glutamate release or postsynaptically via modulation of Ih (50) is not known and was investigated at the level of the XII premotor-to-XII motoneuron synapse.
Noradrenergic modulation of XII inspiratory activity does not involve 2-receptor-mediated presynaptic inhibition of glutamatergic drive.
Presynaptic, 2-receptor-mediated inhibition of glutamatergic transmission is well documented at multiple levels of the nervous system (6, 7, 30, 42, 46). For example, glutamatergic excitatory postsynaptic potentials (EPSCs) in dorsal motor vagal neurons (6), sympathetic preganglionic neurons (42), dorsal horn neurons (46), and cultured hippocampal neurons are inhibited by activation of presynaptic 2-receptors. Clonidine also reduces the amount of glutamate that is released spontaneously from spinal synaptosomes (28) and from slices in response to capsaicin (66).
In spite of this database, our observation that clonidine, at concentrations that should completely saturate 2-receptors, has no effect on glutamatergic inspiratory currents in XII motoneurons strongly suggests that a presynaptic 2-receptor-mediated inhibition of glutamate drive does not operate at the XII preinspiratory motoneuron-to-XII motoneuron synapse. Because the inspiratory synaptic current is composed of multiple events distributed over the somatodendritic tree, we cannot exclude the possibility that an 2 inhibitory effect was masked by a postsynaptic effect of clonidine which, through increases in motoneuron RN, effectively "boosted" synaptic currents arriving on unclamped regions of the dendritic tree and counterbalanced an 2 inhibition. We consider this possibility unlikely. Individual synapses contributing to the inspiratory current will be widely distributed over the tree, with distal synapses less effectively "space-clamped" than proximal synapses. Thus the potential boosting effect will vary between individual synapses as a function of their distance from the soma. We consider it a remote possibility that this variable amplification of distal inputs would exactly match a presynaptic inhibition of glutamatergic drive.
Analysis of miniature EPSC (mEPSC) amplitude and frequency distributions would not resolve this problem. Like the inspiratory current, these distributions are constructed from analysis of widely distributed inputs. Direct stimulation of synaptic inputs to the XII motoneurons and comparison of paired-pulse responses in control and clonidine would address the space-clamp problem. However, the disadvantage of this approach, which is common to the analysis of mEPSCs, is that it is impossible to determine whether the synapses assessed with these methods are inspiratory in nature. The purpose of this study was to test whether an 2-receptor mechanism presynaptically inhibits glutamatergic inspiratory inputs. This should not imply that mEPSC analysis and evoked potentials are not of enormous value. For example, in the context of the inspiratory synapse examined here, if 2-receptor activation decreased mEPSC frequency without affecting glutamate inspiratory drive, it would suggest the differential modulation of inspiratory and noninspiratory glutamatergic inputs. Evoked potentials are also of considerable value in that, whereas the functional relevance of an evoked input (from the lateral tegmental field, for example) may be unknown, the input will be relatively stable and involve a reasonably constant pool of synapses. Evoked potential are also important in developmental studies because of the difficulty in older animals of maintaining spontaneously active networks in vitro. In summary, our analysis does not exclude the possibility that a presynaptic 2-receptor mechanism operates at noninspiratory glutamatergic synapses. It does, however, suggest that this mechanism does not modulate inspiratory inputs to XII motoneurons.
Noradrenergic modulation of XII inspiratory activity does not involve 2-receptor-mediated inhibition of the Ih current.
Inwardly rectifying currents such as Ih have been characterized in diverse groups of cells (47). The unique property of Ih to increase its activation on hyperpolarization is of particular interest in rhythm-generating systems for its ability to produce pacemaker-like depolarizations between consecutive oscillations. However, the depolarizing sag that follows membrane hyperpolarization and results from activation of Ih and the postinhibitory rebound that occurs with removal of the hyperpolarizing influence are important determinants of the integrative and firing properties of many nonpacemaking neurons, including motoneurons (47, 51). In XII motoneurons, Ih expression increases developmentally (5). However, a significant current is present in neonatal motoneurons through which 2-receptors, like many other receptor systems (51), could act to modulate inspiratory activity. Indeed, the observations in juvenile XII motoneurons (49) that clonidine modulates Ih in an idazoxan-sensitive manner was the basis of the hypothesis tested here, that 2-receptor-mediated inhibition of Ih in XII motoneurons inhibits inspiratory-related activity. In juveniles, clonidine has multiple effects on Ih, reducing the peak amplitude, shifting the voltage dependence of activation in the hyperpolarizing direction, and slowing the time course of activation. Clonidine also inhibited Ih in neonatal XII motoneurons. Surprisingly, however, concentrations of clonidine or ZD7288 that inhibited Ih in individual XII motoneurons were without effect on XII burst amplitude, indicating that 2-receptor-induced inhibition of Ih does not contribute to the modulation of XII inspiratory output.
Whether the effects of clonidine on Ih in neonates are similar to those described in juveniles remains unknown. It was not examined in this study because our objective was to determine whether modulation of Ih by clonidine affected inspiratory activity. With the observation that complete block of Ih did not influence inspiratory output, defining the mechanisms underlying the 40% attenuation of Ih by clonidine was not a priority.
Modulation of Ih itself does not contribute to the baseline activity of neonatal XII motoneurons. The role of Ih in modulating the inspiratory activity of XII motoneurons is controversial. As outlined by Bayliss et al. (5), Ih is normally active at rest and therefore contributes to setting the resting membrane potential (5, 67). They also proposed that Ih contributes to the subthreshold and repetitive firing behavior of motoneurons, as well as the integration of synaptic inputs. Because resting membrane potential sits on the lower portion of the Ih activation curve, Ih will be activated with only minor membrane hyperpolarization. In contrast, depolarizations will have a smaller influence on Ih magnitude. In other words, Ih may prevent prolonged hyperpolarizations in response to inhibitory inputs without compromising depolarizing responses to excitatory inputs (5). On the basis of this hypothesis, one would not predict a major influence of Ih or its modulation on excitatory inspiratory motoneuron activity in neonates in vitro. Only two studies have examined the role of Ih in modulating rhythmic inspiratory activity (41, 64). However, these studies yielded conflicting results. Moreover, they were not designed to focus specifically on Ih modulation of XII inspiratory motoneuron activity. The Ih antagonist ZD7288 was applied to the solution bathing the entire rhythmically active medullary slice preparation. Thus it was not possible to determine whether the effect on XII output was due to an action at the XII premotor-to-inspiratory motoneuron synapse or further upstream at the level of the rhythm generator or premotor network.
Nevertheless, our data in which ZD7288 was focally applied at 100 µM to the XII nucleus (approximately equivalent to 10 µM bath application) are consistent with data from late neonatal mice (P6–P11), indicating that bath application of 1–10 µM ZD7288 is without effect on rhythm or motor output (41). These data, however, are inconsistent with data from slightly older mice (P7–P22) in which bath application of 100 µM ZD7288 increased frequency and potentiated burst amplitude (64). Strain or developmental differences may account for some of the discrepancy between these two studies. However, it is also possible that the alteration of respiratory activity in the latter study reflects the higher concentration of ZD7288 and actions via mechanisms other than inhibition of Ih. For example, it is now apparent that ZD7288 also inhibits voltage-gated Na+ channels, reduces AMPA and N-methyl-D-aspartate glutamate receptor currents (9), reduces T-type calcium currents in expression systems (17), and shortens the interspike interval in neurons (38), possibly by modulating calcium-dependent K+ channels.
Regardless of the reasons underlying the differences between these earlier studies in mice, our data demonstrate at the level of the XII nucleus that modulation of Ih does not contribute to inspiratory burst pattern formation in the neonatal rat. XII burst amplitude was unaffected by concentrations of ZD7288 that almost completely blocked Ih. Effects on inspiratory output were only observed when the concentration of ZD7288 was elevated to the point that it inhibited action potential amplitude and repetitive firing.
Whether a role for Ih in controlling inspiratory activity of XII motoneurons develops postnatally is not known. Certainly, the density of the Ih current increases developmentally (5). Developmental changes in the voltage dependence of activation would impact how Ih influences XII inspiratory activity, but this property does not change postnatally (5). Our measurements for the voltage dependence of Ih activation in neonatal rats appear somewhat more hyperpolarized than previously reported for juveniles. Some of this may reflect an artifact of whole cell recording. However, the majority of it likely reflects that our voltage protocol stepped from –60 to –75 to –90 mV. In many motoneurons, a slowly developing inward current was apparent with the step to –75 mV. Thus Ih began to activate between –60 and –75 mV, consistent with the value of approximately –65 mV reported previously (5).
Independent of changes in Ih itself, developmental changes in membrane, synaptic, and network properties, particularly the maturation of inhibitory systems and the hyperpolarization of the Cl– reversal potential (60), mean it is possible that respiratory-related oscillations in membrane potential will fall into more hyperpolarized ranges and Ih will play a more conspicuous role in controlling inspiratory motor output. Alternatively, Ih may contribute to nonrespiratory behaviors of the XII motoneurons, such as coughing or swallowing (53), in which stronger inhibitory inputs may lower membrane potential into the relevant window.
2-Receptor-mediated modulation of XII motoneuron excitability?
The objective of this study was to determine whether the previously demonstrated 2-receptor-mediated inhibition of XII inspiratory output (58) was due to presynaptic inhibition of glutamate release or modulation of Ih (50). Because we used high concentrations of clonidine and clonidine affected Ih, we are confident that adequate drug was delivered to the target area and therefore that neither of these two proposed mechanisms contribute to the noradrenergic modulation of XII excitability. Dialysis of intracellular components critical in signal transduction is always a concern with whole cell recording that can account for false negative data. However, dialysis was not a factor in our conclusions regarding the potential involvement of these two mechanisms. First, a presynaptic 2-receptor-mediated inhibitory mechanism would not have been altered by dialysis of the postsynaptic cell. Second, in the context of Ih, clonidine inhibited Ih under whole cell conditions, indicating that the necessary signaling cascades were still, at least partially, intact. The possibility that a clonidine-mediated potentiation of tonic or phasic inspiratory GABAergic inhibitory inputs to XII motoneurons (8, 56) contributed to the inhibition was also eliminated by the observation that the clonidine inhibition was similar in (+)bicuculline and control. However, this leaves the question as to the mechanism underlying the clonidine-mediated inhibition.
In neurons of the dorsal motor nucleus of the vagus (19), substantia gelatinosa (44), and sympathetic preganglionic neurons (26), clonidine, via 2-receptors, induces a hyperpolarization that is accompanied by decreased RN and has a reversal potential near equilibrium potential for K+ (EK+) that shifts with changing extracellular K+ as predicted by the Nernst equation for a K+ conductance. Although such a mechanism might contribute in XII motoneurons, we found no evidence for a clonidine-induced outward current that reduces input resistance and reverses near EK+.
As mentioned previously, within motoneurons there is considerable evidence for 2-receptor-mediated inhibition of excitability (3), including inhibition of XII inspiratory activity in neonatal mice (58). However, a similar 2-mediated inhibition of inspiratory output in rat is difficult to reconcile with several observations. First, inhibition is only produced when high concentrations of clonidine are used. Moreover, UK14304 has no effect even when applied to the same regions where clonidine produces an inhibition. The attenuation of the clonidine inhibition by rauwolscine supports an 2 mechanism because it, unlike idazoxan, is selective for 2- and not I1 receptors (16). However, the rauwolscine data are compromised by the fact that rauwolscine itself inhibits XII output, which means that the actions of clonidine in control and antagonist are not directly comparable. The rauwolscine-mediated inhibition of XII output is also surprising for two reasons. First, most sources of endogenous noradrenergic inputs to XII motoneurons are not included in the rhythmic slice (1). Thus an effect of the antagonist alone is unexpected. Second, clonidine inhibits XII inspiratory output. If this effect is due to activation of 2-receptors, inhibition of an endogenous 2-receptor-mediated effect should manifest as an increase in burst amplitude. Thus either there is an endogenous 2-receptor-mediated potentiation of inspiratory activity that accounts for the rauwolscine-mediated inhibition or there is an unidentified action of these antagonists on inspiratory burst amplitude that is not mediated through 2-receptors. The former possibility is unlikely because 2-agonists do not increase amplitude. Nonspecific actions are supported by the fact that the three 2-antagonists used here, yohimbine, rauwolscine, and idazoxan, inhibited XII inspiratory output to different degrees. The inhibition may reflect that all three compounds can act as agonists at 5HT1A receptors (11, 35, 40). In turn the greater efficacy of yohimbine in inhibiting XII inspiratory output may reflect that it, unlike rauwolscine and idazoxan, can also act as an agonist at 5HT1B receptors (40), which act presynaptically to inhibit glutamatergic inputs to XII motoneurons (59) and glutamatergic inspiratory inputs to phrenic motoneurons (33).
Additional evidence suggesting that 2-receptors are not involved in the inhibition is that at high concentration, clonidine has non-2-receptor-mediated effects. As described previously in rabbit (13, 22), we demonstrated in every XII motoneuron examined that clonidine inhibited either action potential amplitude or repetitive firing. This inhibition, combined with the fact that the time course of the effect on repetitive firing was similar to the inhibitory actions of clonidine on XII inspiratory burst amplitude, suggests that a major mechanism underlying the inhibitory actions of clonidine on XII burst amplitude in neonatal rat is via nonspecific inhibition of Na+ channels.
Imidazoline receptor involvement must be considered because clonidine can activate I1 receptors (16). Indeed, imidazoline receptors, as defined by [3H]rilmenidine binding, are present in the XII nucleus (31). Pharmacological data are most consistent with the activation of I1 receptors by clonidine. First, the effects of clonidine were attenuated by the I1 antagonist efaroxan. Second, the I1 agonist, rilmenidine, inhibited XII inspiratory output. Note, however, that both rilmenidine and efaroxan can also act on 2-receptors (16, 25). Inhibition of clonidine effects by rauwolscine, an 2-antagonist that does not affect I1 receptors (16), also questions I1 receptor involvement, but the inhibition may have resulted from the nonspecific actions of rauwolscine. Thus, while pharmacological data more strongly support clonidine activation of I1 receptors, data must be interpreted cautiously. It is also possible that inhibition of voltage-gated calcium currents by 2-receptor activation, as seen in mitral cells of Xenopus and in mammalian sympathetic and raphe neurons (10, 32, 57), contributes to the inhibition of XII burst amplitude.
Thus multiple mechanisms are likely to contribute to the clonidine-mediated inhibition of XII inspiratory burst amplitude. A contribution from 2- and I1 receptor cascades and inhibition of voltage-gated calcium channels is possible. However, a major factor contributing to the inspiratory inhibition appears to be the disruption of repetitive firing.
In summary, these data reinforce the view that the dominant effect of NE within the XII nucleus is mediated through an 1-receptor mechanism that increases motoneuron excitability and potentiates inspiratory output. Both mechanisms through which 2-receptors were hypothesized to act within the XII nucleus were shown not to influence inspiratory activity. First, there is no 2-receptor mechanism acting presynaptically to inhibit glutamate release from the inspiratory premotor-to-XII-motoneuron synapse. Second, inhibition of Ih does not impact on the inspiratory behavior of XII motoneurons. What then is the role of 2-receptors in the XII nucleus (54, 63)? First, the role of Ih and its modulation may change developmentally owing to increases in Ih current density (5), as well as the maturation of membrane and network properties. 2-Receptors may also control nonrespiratory activity of XII motoneurons or the activity of other neuromodulatory systems. Certainly, a role for 2-receptor mechanisms in the presynaptic regulation of NE release is well established (69). It is also possible, as suggested by the observation that repeated hypercapnia leads to an 2-receptor-dependent long-term depression of inspiratory activity in both XII and phrenic nerves (3), that an 2-receptor-mediated effect exists but is only expressed under specific conditions or patterns of input.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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, current-voltage (I-V) ramp or series resistance check.
) and instantaneous current jump measured immediately after the capacitive transient at the beginning of the pulse (
). C: average I-V relationship for Ih under control conditions and in the presence of 0.1 mM clonidine (n = 6). D: histogram showing the magnitude of the Ih current evoked in 0.1 (n = 6) and 1.0 mM (n = 4) clonidine by a step from –60 to –120 mV, relative to that evoked by the same step in control. Note that different neurons were exposed to 0.1 or 1.0 mM clonidine *Significantly different from control, P < 0.05.
, relative to control, n = 5) and the magnitude of the Ih current (
; as measured in 
significant difference from clonidine; P < 0.05.
