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Meeting Abstracts  |   September 2001
Opioid Action on Respiratory Neuron Activity of the Isolated Respiratory Network in Newborn Rats
Author Affiliations & Notes
  • Shinhiro Takeda, M.D., Ph.D.
    *
  • Lars I. Eriksson, M.D., Ph.D.
  • Yuji Yamamoto, Ph.D.
  • Henning Joensen, M.D.
    §
  • Hiroshi Onimaru, Ph.D.
    ‖‖
  • Sten G. E. Lindahl, M.D., Ph.D.
    #
  • * Research Fellow, Department of Anesthesiology and Intensive Care, Karolinska Institute and Hospital. Current position: Assistant Professor, Department of Anesthesiology and Intensive Care, Nippon Medical School, Tokyo, Japan. † Associate Professor, § Assistant Professor, # Professor and Chairman, Department of Anesthesiology and Intensive Care, ‡ Assistant Professor, Department of Woman and Child Health, Karolinska Institute and Hospital. ‖‖ Assistant Professor, Department of Physiology, Showa University School of Medicine, Tokyo, Japan.
  • Received from the Department of Anesthesiology and Intensive Care, Karolinska Institute and Hospital, Stockholm, Sweden.
Article Information
Meeting Abstracts   |   September 2001
Opioid Action on Respiratory Neuron Activity of the Isolated Respiratory Network in Newborn Rats
Anesthesiology 9 2001, Vol.95, 740-749. doi:
Anesthesiology 9 2001, Vol.95, 740-749. doi:
RESPIRATORY depression is the most important acute side effect of morphine and other opioids in the adult and pediatric population. Opioid-induced respiratory depression is caused by μ-, κ-, and δ-opioid receptor activation within the brainstem. 1–6 
In the brainstem, the rostral ventrolateral medulla (RVLM) is thought to be an important area for respiratory rhythm generation 7–9 and may be the target area for opioid-induced respiratory depression. Respiratory neurons in the brainstem–spinal cord preparation fall into three major classes according to the pattern of membrane potential (Em) oscillations and spike discharges: inspiratory, preinspiratory, and expiratory neurons. 8,10 The patterns of burst activity and postsynaptic potentials of these three major respiratory neurons suggest a complex pattern of mutual synaptic connections. 11 However, the effects of opioids on membrane properties of medullary neurons of the ventral respiratory group in the RVLM have not been fully established.
Previous data 5,12 suggest a change in Emafter application of opioid receptor agonists and that the response to opioids might differ between subtypes of respiratory neurons. We therefore hypothesized that the depression of respiratory neuronal activity by opioids is dependent on both presynaptic and postsynaptic interference within the respiratory neuronal network. The purpose of the current study was to examine opioid (μ, κ, and δ) effects on medullary respiratory neurons and to evaluate how opioid receptor agonists exert inhibitory effects on respiration in the brainstem–spinal cord preparation from newborn rats.
Materials and Methods
Preparation
The study was performed in accordance with the protocol approved by the institutional Animal Care and Use Committee of the Karolinska Institute, Stockholm, Sweden. Brainstem–spinal cord preparations from 0- to 4-day-old Sprague-Dawley rats (163 preparations) were used. 8,9,11 Brainstem and spinal cord were isolated with deep ether anesthesia. 3 The brainstem was rostrally decerebrated between the VIth cranial nerve roots and the lower border of the trapezoid body, so that most of the pons was removed. The preparation with the ventral surface up was then placed in a perfusion chamber (2 ml) continuously superfused at a rate of 3.0–3.5 ml/min with artificial cerebrospinal fluid: 124 mm NaCl, 5.0 mm KCl, 1.2 mm KH2PO4, 2.4 mm CaCl2, 1.3 mm MgSO4, 26 mm NaHCO3, and 30 mm glucose, equilibrated with 95% O2and 5% CO2, and kept at 26–27°C and a pH of 7.4.
Intracellular Recording
Intracellular whole cell recordings were made from three major classes of respiratory neurons using the blind patch clamp technique. 10,13–15 Electrodes were pulled in a single stage from thin-wall borosilicate glass (GC100TF-10, OD = 1.0 mm, with a filament, Clark Electromedical, Reading, United Kingdom) on a vertical puller. The electrodes had an inner tip diameter of 1.2–2.0 μm, a DC resistance of 3–8 MΩ, and the electrode solution consisted of 130 mm potassium gluconate, 10 mm EGTA, 10 mm HEPES, 2 mm Na2–adenosine triphosphate, 1 mm CaCl2, and 1 mm MgCl2, with a pH of 7.2–7.3 adjusted by KOH. The electrodes were placed in the RVLM, including the ventral respiratory group, by insertion through a small region of the ventral surface of the RVLM where the pia mater was removed with a thin glass needle. The Em(millivolts) was recorded with a single-electrode voltage clamp amplifier (CEZ-300, Nihon Kohden, Tokyo, Japan) after compensation of the series resistance (20–60 MΩ) and capacitance. For comparison with previous studies, 13,14 the membrane potential values were not corrected for junction potentials (< −10 mV). Input membrane resistance (Rm, megaohms) was determined from the voltage change in response to intracellular injection of DC current pulses (500 ms, −20 and −40 pA) during the silent phase of the respiratory cycle or during negative holding potentials (−50 or −55 mV) of expiratory neurons. Discharges of respiratory motor activity were recorded extracellularly with suction electrodes applied to the proximal ends of cut ventral roots of spinal (C4 or C5) nerves and through a high-pass filter with a 0.3-s time constant. During the experiments, neuronal activity was displayed on a chart recorder, monitored on oscilloscope, digitized (Digidata 1200B, Axon Instruments, Foster, CA), and stored on a DAT tape (RD-120TE, TEAC, Tokyo, Japan) or a hard disk for offline analysis with a personal computer and data acquisition software (Axoscope, Axon Instruments).
Definition of Respiratory Neurons of the Rostral Ventrolateral Medulla
Membrane potential was recorded from three major classes of respiratory neurons in the RVLM (fig. 1). Preinspiratory neuron action potential discharges are observed during the preinspiratory and postinspiratory phases. 13,16 The expiratory neurons are characterized by tonic action potential discharges during the expiratory phase and by inhibitory postsynaptic potentials (IPSPs) that cause hyperpolarization and inhibited spontaneous firing during the inspiratory phase. 14,17 Preinspiratory and expiratory neurons receive inhibitory postsynaptic inputs from inspiratory neurons. 10,11 Inspiratory neurons were classified into three subtypes. 13,14,18 Type I inspiratory neurons show excitatory postsynaptic potentials (EPSPs) before onset and after termination of inspiratory nerve bursts. In type II neurons, EPSPs are only revealed during the inspiratory phase, whereas type III neurons are hyperpolarized by synchronized IPSPs during the preinspiratory and postinspiratory phases. 8,13 Types I and III inspiratory neurons receive excitatory and inhibitory postsynaptic inputs, respectively, from preinspiratory neurons. 10,11 
Fig. 1. (A  ) Membrane potential traces from preinspiratory, inspiratory (types I, II, and III), and expiratory neurons in the brainstem–spinal cord preparation along with inspiratory motor nerve activity from the C4 ventral root. (B  ) Ventral aspect of the preparation and recording sites. The area examined in the current study (shaded area) seems to be located more rostrally, but partly overlapped, to pre-Bötzinger complex.
Fig. 1. (A 
	) Membrane potential traces from preinspiratory, inspiratory (types I, II, and III), and expiratory neurons in the brainstem–spinal cord preparation along with inspiratory motor nerve activity from the C4 ventral root. (B 
	) Ventral aspect of the preparation and recording sites. The area examined in the current study (shaded area) seems to be located more rostrally, but partly overlapped, to pre-Bötzinger complex.
Fig. 1. (A  ) Membrane potential traces from preinspiratory, inspiratory (types I, II, and III), and expiratory neurons in the brainstem–spinal cord preparation along with inspiratory motor nerve activity from the C4 ventral root. (B  ) Ventral aspect of the preparation and recording sites. The area examined in the current study (shaded area) seems to be located more rostrally, but partly overlapped, to pre-Bötzinger complex.
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Contralateral Medulla Stimulation and Simultaneous Intracellular and Extracellular Recordings
Neuronal projections and evoked EPSPs were studied using whole cell recordings of inspiratory neurons (n = 17). Contralateral stimulation of the RVLM (0.1 ms duration single square wave impulses of 15 V) was delivered by tungsten electrodes with 2 MΩ resistance and 30-μm tip diameters. Simultaneous recordings of intracellular and extracellular Emof inspiratory–preinspiratory neuronal pairs (in the contralateral medulla) were made in 10 preparations to elucidate whether opioid receptor agonists interfered with the correlation between these neuronal bursts. Extracellular unit activity of respiratory neurons in the RVLM was recorded by using glass microelectrodes filled with 0.5 m sodium acetate (5–20 MΩ).
Application of Pharmacologic Agents
Actions of four opioid receptor agonists were examined:μ-opioid receptor agonists by the use of morphine hydrochloride (Pharmacia, Stockholm, Sweden) and DAGO (Try-D-Ala- Gly-[N MePhe]-Gly-ol; Sigma, St. Louis, MO), κ-opioid receptor agonist by U50488 (trans-(±)-3,4-Dichloro-N  -methyl-N  -[2-(1-pyrrolidiny)-cyclohexyl]-nzeneacetamide methanesulfonate; Research Biochemicals International, Natick, MA), and the δ-opioid receptor agonist by DPDPE ([D-pen2,5]-enkephalin; Sigma). Drug concentration and application time were determined according to results from previous pharmacologic studies. 4,6 Artificial cerebrospinal fluid containing 1 μm DAGO and 10 μm morphine were applied for 10 min, while artificial cerebrospinal fluid with 10 μm DPDPE and 10 μm U50488 were applied for 20 min. This was followed by the application of opioid receptor antagonist naloxone hydrochloride (Sigma; 1 μm) for 10 min. In 68 preparations (47 inspiratory, 11 preinspiratory, and 10 expiratory neurons), the opioid receptor agonists were applied after blockade of ongoing synaptic activity with 0.5 μm tetrodotoxin (Sigma). The whole brainstem–spinal cord preparation was exposed via  bath application of pharmacologic agents. All solutions were adjusted to a pH of 7.4 and were applied to the recording chamber through the perfusing system.
Statistical Analysis
Repeated-measurement analysis of variance and Bonferroni tests were performed to distinguish within-group differences over time. The one-way analysis of variance and Bonferroni tests were performed to evaluate differences among the respiratory neurons and the test drugs. All statistical analyses were conducted using the SPSS version 8.0J (SPSS Inc., Chicago, IL). All values are reported as the mean ± SD, and all P  values < 0.05 were considered statistically significant.
Results
Control Conditions and Summary Table
Control membrane properties of 163 respiratory neurons are presented in table 1. The values were comparable to those of previous studies. 13,14 Table 2summarizes effects of opioids on the various neuron groups in standard artificial cerebrospinal fluid and tetrodotoxin-containing solution.
Table 1. Resting Membrane Potentials, Amplitude of Action Potentials, and Input Membrane Resistance after Establishing Whole Cell Configuration
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Table 1. Resting Membrane Potentials, Amplitude of Action Potentials, and Input Membrane Resistance after Establishing Whole Cell Configuration
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Table 2. Responses of Respiratory Neurons after Application of Opioid Receptor Agonists in Standard Artificial Cerebrospinal Fluid (aCSF) or in the Presence of Tetrodotoxin (TTX)
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Table 2. Responses of Respiratory Neurons after Application of Opioid Receptor Agonists in Standard Artificial Cerebrospinal Fluid (aCSF) or in the Presence of Tetrodotoxin (TTX)
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Inspiratory Neurons
Application of morphine, DAGO, and U50488 resulted in a synchronous decrease in the burst rates of all classes of inspiratory neuron and of the C4 activity (table 3, figs. 2 and 3). The depressant effects by these three opioid receptor agonists were antagonized by 1 μm naloxone. However, inspiratory neuron intraburst firing frequency did not significantly change during opioid-induced respiratory depression (17.0 ± 6.8 to 16.8 ± 6.8 Hz).
Table 3. Effects of Opioid Receptor Agonists on Burst Rate of Inspiratory Neurons (bursts/min)
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Table 3. Effects of Opioid Receptor Agonists on Burst Rate of Inspiratory Neurons (bursts/min)
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Fig. 2. Type II inspiratory neuron and effects of opioid receptor agonists on membrane potential. (A  ) Continuous recording of membrane potential. Current pulses with different intensity were injected into the neuron to examine the current–voltage relation at the three time intervals denoted by bars above the neuron tracing. (B  ) Representation of recordings in (A  ) on extended time scale. (C  ) Reversal potential was −70 mV according to the current–voltage relation.
Fig. 2. Type II inspiratory neuron and effects of opioid receptor agonists on membrane potential. (A 
	) Continuous recording of membrane potential. Current pulses with different intensity were injected into the neuron to examine the current–voltage relation at the three time intervals denoted by bars above the neuron tracing. (B 
	) Representation of recordings in (A 
	) on extended time scale. (C 
	) Reversal potential was −70 mV according to the current–voltage relation.
Fig. 2. Type II inspiratory neuron and effects of opioid receptor agonists on membrane potential. (A  ) Continuous recording of membrane potential. Current pulses with different intensity were injected into the neuron to examine the current–voltage relation at the three time intervals denoted by bars above the neuron tracing. (B  ) Representation of recordings in (A  ) on extended time scale. (C  ) Reversal potential was −70 mV according to the current–voltage relation.
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Fig. 3. Effects of opioid receptor agonists on membrane potential of types I and III inspiratory neurons. (A  ) Type I inspiratory neuron. Excitatory postsynaptic potentials, the source of which are presumably preinspiratory neurons, were still observed during opioid-induced respiratory depression. Arrows indicate excitatory postsynaptic potentials. Reversal potential was −83 mV according to the current–voltage relation. (B  ) Type III inspiratory neuron. Inhibitory postsynaptic potentials, the source of which are presumably preinspiratory neurons, were still observed during opioid-induced respiratory depression. Arrows indicate inhibitory postsynaptic potentials. Reverse potential was −75 mV according to the current–voltage relation.
Fig. 3. Effects of opioid receptor agonists on membrane potential of types I and III inspiratory neurons. (A 
	) Type I inspiratory neuron. Excitatory postsynaptic potentials, the source of which are presumably preinspiratory neurons, were still observed during opioid-induced respiratory depression. Arrows indicate excitatory postsynaptic potentials. Reversal potential was −83 mV according to the current–voltage relation. (B 
	) Type III inspiratory neuron. Inhibitory postsynaptic potentials, the source of which are presumably preinspiratory neurons, were still observed during opioid-induced respiratory depression. Arrows indicate inhibitory postsynaptic potentials. Reverse potential was −75 mV according to the current–voltage relation.
Fig. 3. Effects of opioid receptor agonists on membrane potential of types I and III inspiratory neurons. (A  ) Type I inspiratory neuron. Excitatory postsynaptic potentials, the source of which are presumably preinspiratory neurons, were still observed during opioid-induced respiratory depression. Arrows indicate excitatory postsynaptic potentials. Reversal potential was −83 mV according to the current–voltage relation. (B  ) Type III inspiratory neuron. Inhibitory postsynaptic potentials, the source of which are presumably preinspiratory neurons, were still observed during opioid-induced respiratory depression. Arrows indicate inhibitory postsynaptic potentials. Reverse potential was −75 mV according to the current–voltage relation.
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In four (27%) of 15 inspiratory neurons, the decrease in burst rate was associated with a hyperpolarizing shift in resting Emby, on average, 5.5 ± 2.9 mV (range, 3–8 mV) and a reduction in Rmby, on average, 77.5 ± 39.7 MΩ (i.e.  , 24%; range, 36–121 MΩ) after application of morphine (fig. 2). Resting Emand Rmin the remaining 11 inspiratory neurons did not change despite a decrease in C4 and inspiratory neuron burst rates. To further study the membrane properties of the hyperpolarized neurons, we analyzed the current–voltage relations of steady state Emresponses to injection of DC pulses. In the example shown in fig. 2C, the control current–voltage relation and that obtained during morphine application intersected at −70 mV. This Emvalue represents the reversal potential (Erev) of the opioid-induced hyperpolarization, which averaged −75.1 ± 18.6 mV (n = 4).
DAGO resulted in a hyperpolarization by, on average, 5.0 ± 2.5 mV (range, 2–9 mV) and a decrease in Rmby, on average, 78.3 ± 33.2 MΩ (i.e.  , 20%; range, 25–130 MΩ), whereas Erevaveraged −76.2 ± 12.1 mV in 11 (50%) of 22 inspiratory neurons (fig. 3A). The remaining 11 neurons were unchanged for resting Emand Rm.
Within 15 min after application of U50488, steady state hyperpolarization of down to 5.7 ± 1.7 mV (range, 2–7 mV) occurred, and Rmdecreased by, on average, 65.2 ± 32.2 MΩ (i.e.  , 19%; range, 21–125 MΩ) in nine (45%) of 20 inspiratory neurons (fig. 3B). Erevaveraged −83.0 ± 15.5 mV (n = 9). The remaining 11 inspiratory neurons did not change resting Emand Rm.
In all three classes of inspiratory neurons, the δ-opioid receptor agonist DPDPE did not cause any effects on burst rate, Em, and Rm(n = 9).
As shown in fig. 3, the type I and III inspiratory neurons showed a high probability of EPSPs and IPSPs, respectively, during the preinspiratory and postinspiratory phases. EPSPs and IPSPs, as indicated by arrows in figure 3, were still observed periodically despite a decrease in the burst rates of C4 and inspiratory neuron during opioid-induced respiratory depression. Summarizing the effects of all three agonists (morphine, DAGO, and U50488), approximately 42% inspiratory neurons responded with membrane hyperpolarization and Rmdecrease during application of these agonists (table 2).
Preinspiratory Neurons
Opioid receptor agonists did not affect Emand Rmin preinspiratory neurons (n = 15: 5 DAGO, 4 morphine, 4 U50488, 2 DPDPE). As shown in figure 4, the preinspiratory neurons were discharging at unchanged rate while the C4 respiratory rate decreased markedly after application of DAGO (table 4). Similar effects were also caused by morphine or U50488 (table 4). Intraburst firing frequency of preinspiratory neurons did not significantly change during opioid-induced respiratory depression (12.4 ± 3.3 to 12.1 ± 2.9 Hz).
Fig. 4. Effects of opioid receptor agonists on membrane potential of preinspiratory neuron. (A  ) Continuous recording of membrane potential. Current pulses with different intensity were injected into the neuron to examine current-voltage relation at the three time intervals denoted by bars above the neuron tracing. (B  ) Representation of recordings in (A  ) on extended time scale. During opioid-induced respiratory depression, preinspiratory neuron activity, followed by absence of inhibitory postsynaptic potentials from inspiratory neurons, indicated “throughout” burst pattern. When C4 inspiratory burst occurred, preinspiratory neurons showed long preinspiratory but short postinspiratory activity.
Fig. 4. Effects of opioid receptor agonists on membrane potential of preinspiratory neuron. (A 
	) Continuous recording of membrane potential. Current pulses with different intensity were injected into the neuron to examine current-voltage relation at the three time intervals denoted by bars above the neuron tracing. (B 
	) Representation of recordings in (A 
	) on extended time scale. During opioid-induced respiratory depression, preinspiratory neuron activity, followed by absence of inhibitory postsynaptic potentials from inspiratory neurons, indicated “throughout” burst pattern. When C4 inspiratory burst occurred, preinspiratory neurons showed long preinspiratory but short postinspiratory activity.
Fig. 4. Effects of opioid receptor agonists on membrane potential of preinspiratory neuron. (A  ) Continuous recording of membrane potential. Current pulses with different intensity were injected into the neuron to examine current-voltage relation at the three time intervals denoted by bars above the neuron tracing. (B  ) Representation of recordings in (A  ) on extended time scale. During opioid-induced respiratory depression, preinspiratory neuron activity, followed by absence of inhibitory postsynaptic potentials from inspiratory neurons, indicated “throughout” burst pattern. When C4 inspiratory burst occurred, preinspiratory neurons showed long preinspiratory but short postinspiratory activity.
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Table 4. Effects of Opioid Receptor Agonists on C4 Respiratory Frequency (bursts/min) and Preinspiratory (Pre-I) Neuron Burst Rate (bursts/min)
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Table 4. Effects of Opioid Receptor Agonists on C4 Respiratory Frequency (bursts/min) and Preinspiratory (Pre-I) Neuron Burst Rate (bursts/min)
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Expiratory Neurons
In the 12 expiratory neurons (4 DAGO, 3 morphine, 3 U50488, 2 DPDPE), opioid receptor agonists did not affect action potentials, Em, and Rm(fig. 5A). Intraburst firing frequency of these expiratory neurons did not significantly change during opioid-induced respiratory depression (8.3 ± 4.0 to 8.5 ± 3.4 Hz). However, we observed that U50488 hyperpolarized the membrane by 3–5 mV and decreased intraburst firing frequency from 5.3 ± 1.1 to 1.3 ± 1.1 Hz in a naloxone-reversible manner in two (40%) of five expiratory neurons (fig. 5Band table 2).
Fig. 5. Effects of opioid receptor agonists on membrane potential of expiratory neuron. (A  ) After U50488 application, C4 inspiratory discharges were decreased, but discharges of an expiratory neuron were not affected. (B  ) Expiratory neuron discharges were depressed with hyperpolarization produced by U50488.
Fig. 5. Effects of opioid receptor agonists on membrane potential of expiratory neuron. (A 
	) After U50488 application, C4 inspiratory discharges were decreased, but discharges of an expiratory neuron were not affected. (B 
	) Expiratory neuron discharges were depressed with hyperpolarization produced by U50488.
Fig. 5. Effects of opioid receptor agonists on membrane potential of expiratory neuron. (A  ) After U50488 application, C4 inspiratory discharges were decreased, but discharges of an expiratory neuron were not affected. (B  ) Expiratory neuron discharges were depressed with hyperpolarization produced by U50488.
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Contralateral Medulla Stimulation
The stimulation induced orthodromic responses in the 15 of 17 neurons, whereas two neurons were antidromically activated (6 DAGO, 4 morphine, 4 U50488, 3 DPDPE). The latencies of orthodromic EPSPs in the individual neurons were determined from responses to three trials, and the SD indicates variance of the latencies less than 0.6 ms. The average latencies of the first EPSPs (9.2 ± 0.9 ms, n = 15) closely fitted with antidromic latencies (9.4 ± 0.2 ms, n = 2).
After application of opioid receptor agonists, latencies of EPSP responses did not significantly change (table 5), whether neurons examined were hyperpolarized or not. In contrast, the slope in the rising phase of the first EPSPs, as indicated by arrowheads in figure 6, was reduced during opioid-induced respiratory depression (n = 12: 4 DAGO, 4 morphine, 4 U50488). Therefore, latencies of first action potential responses were significantly prolonged after application of μ- and κ-opioid receptors (table 5). Seven (58%) of these 12 inspiratory neurons did not change the resting Emand Rm(fig. 6A). The other five neurons were hyperpolarized by 4–7 mV, and Rmdecreased by 36–130 MΩ during opioid-induced respiratory depression (fig. 6B).
Table 5. Latencies of Excitatory Postsynaptic Potentials (EPSPs) and of First Action Potential in Inspiratory Neurons’ Induced Orthodromic Response by Contralateral Medulla Stimulation
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Table 5. Latencies of Excitatory Postsynaptic Potentials (EPSPs) and of First Action Potential in Inspiratory Neurons’ Induced Orthodromic Response by Contralateral Medulla Stimulation
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Fig. 6. Orthodromic responses evoked in inspiratory neurons by stimulation of the rostral ventrolateral medulla on the contralateral side after application of opioid receptor agonists. Arrows indicate time of stimulation. Arrowheads indicate slopes of excitatory postsynaptic potentials (  EPSPs). (A  ) Resting membrane potential did not change. Latencies of EPSPs were 8.6 ms in control and 10.3 ms after treatment with DAGO. Latencies of first action potential were prolonged from 11.3 ms in control to 18.6 ms after treatment with DAGO because the slope of EPSPs reduced. (B  ) Resting membrane potential shifted by −4 mV. Latencies of EPSPs were 9.3 ms in control and 8.7 ms after treatment with DAGO. Latencies of first action potential were also prolonged from 12.3 ms in control to 15.3 ms after treatment with DAGO because the slope of EPSPs reduced and resting membrane potential hyperpolarized.
Fig. 6. Orthodromic responses evoked in inspiratory neurons by stimulation of the rostral ventrolateral medulla on the contralateral side after application of opioid receptor agonists. Arrows indicate time of stimulation. Arrowheads indicate slopes of excitatory postsynaptic potentials ( 
	EPSPs). (A 
	) Resting membrane potential did not change. Latencies of EPSPs were 8.6 ms in control and 10.3 ms after treatment with DAGO. Latencies of first action potential were prolonged from 11.3 ms in control to 18.6 ms after treatment with DAGO because the slope of EPSPs reduced. (B 
	) Resting membrane potential shifted by −4 mV. Latencies of EPSPs were 9.3 ms in control and 8.7 ms after treatment with DAGO. Latencies of first action potential were also prolonged from 12.3 ms in control to 15.3 ms after treatment with DAGO because the slope of EPSPs reduced and resting membrane potential hyperpolarized.
Fig. 6. Orthodromic responses evoked in inspiratory neurons by stimulation of the rostral ventrolateral medulla on the contralateral side after application of opioid receptor agonists. Arrows indicate time of stimulation. Arrowheads indicate slopes of excitatory postsynaptic potentials (  EPSPs). (A  ) Resting membrane potential did not change. Latencies of EPSPs were 8.6 ms in control and 10.3 ms after treatment with DAGO. Latencies of first action potential were prolonged from 11.3 ms in control to 18.6 ms after treatment with DAGO because the slope of EPSPs reduced. (B  ) Resting membrane potential shifted by −4 mV. Latencies of EPSPs were 9.3 ms in control and 8.7 ms after treatment with DAGO. Latencies of first action potential were also prolonged from 12.3 ms in control to 15.3 ms after treatment with DAGO because the slope of EPSPs reduced and resting membrane potential hyperpolarized.
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Simultaneous Intracellular and Extracellular Recordings
After treatment with DAGO (n = 4), morphine (n = 3), and U50488 (n = 3), the inspiratory neuron burst rate and the C4 respiratory rate decreased, while the preinspiratory neuron was still discharging at the same rate as in the control state (fig. 7). Furthermore, we confirmed that EPSPs and IPSPs, which corresponded to the period of extracellular preinspiratory neuron burst, were still observed in type I and III inspiratory neurons during opioid-induced respiratory depression (fig. 7).
Fig. 7. Responses to opioid receptor agonists of inspiratory neuron recorded intracellularly (upper traces  ), accompanied by extracellular preinspiratory neuron activity (middle traces  ) in contralateral rostral ventrolateral medulla. The type III inspiratory neuron was hyperpolarized during preinspiratory and postinspiratory phases corresponding to the activity of preinspiratory neuron in the control state and after treatment with naloxone. After application of DAGO, the C4 and inspiratory neuron burst rates decreased, while the preinspiratory neuron was still discharging at the same rate as in the control state. Furthermore, inhibitory postsynaptic potentials, which corresponded to the period of preinspiratory neuron, were still observed. Arrows indicate inhibitory postsynaptic potentials.
Fig. 7. Responses to opioid receptor agonists of inspiratory neuron recorded intracellularly (upper traces 
	), accompanied by extracellular preinspiratory neuron activity (middle traces 
	) in contralateral rostral ventrolateral medulla. The type III inspiratory neuron was hyperpolarized during preinspiratory and postinspiratory phases corresponding to the activity of preinspiratory neuron in the control state and after treatment with naloxone. After application of DAGO, the C4 and inspiratory neuron burst rates decreased, while the preinspiratory neuron was still discharging at the same rate as in the control state. Furthermore, inhibitory postsynaptic potentials, which corresponded to the period of preinspiratory neuron, were still observed. Arrows indicate inhibitory postsynaptic potentials.
Fig. 7. Responses to opioid receptor agonists of inspiratory neuron recorded intracellularly (upper traces  ), accompanied by extracellular preinspiratory neuron activity (middle traces  ) in contralateral rostral ventrolateral medulla. The type III inspiratory neuron was hyperpolarized during preinspiratory and postinspiratory phases corresponding to the activity of preinspiratory neuron in the control state and after treatment with naloxone. After application of DAGO, the C4 and inspiratory neuron burst rates decreased, while the preinspiratory neuron was still discharging at the same rate as in the control state. Furthermore, inhibitory postsynaptic potentials, which corresponded to the period of preinspiratory neuron, were still observed. Arrows indicate inhibitory postsynaptic potentials.
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Responses after Blockade of Ongoing Synaptic Activity by Tetrodotoxin
After the application of DAGO, 7 (39%) of 18 inspiratory neurons hyperpolarized by, on average, 3.4 ± 1.6 mV (range, 2–6 mV) and decreased Rmby, on average, 68.0 ± 46.9 MΩ (i.e.  , 27%; range, 24–151 MΩ). Erevaveraged −65.5 ± 4.6 mV (n = 7;fig. 8). Application of U50488 resulted in a hyperpolarization by, on average, 5.6 ± 2.8 mV (range, 2–11 mV), and Rmdecreased by, on average, 46.6 ± 27.5 MΩ (i.e.  , 20%; range, 22–109 MΩ), while Erevaveraged −83.2 ± 10.8 mV in 9 (56%) of 16 inspiratory neurons. Morphine also hyperpolarized by, on average, 5.3 ± 3.9 mV (range, 3–11 mV) and decreased Rmby, on average, 51.0 ± 34.9 MΩ (i.e.  , 18%; range, 17–84 MΩ), while Erevaveraged −82.6 ± 21.1 mV in four (40%) of 10 inspiratory neurons. This means that a membrane hyperpolarization and reduction of Rmwere recorded in 20 (45%) of 44 inspiratory neurons during application of these three agonists. In inspiratory neurons, DPDPE did not cause any effects on Emand Rm. The opioid receptor agonists with tetrodotoxin did not affect Emand Rmin preinspiratory and expiratory neurons.
Fig. 8. Responses to opioid receptor agonists of inspiratory neuron after blockade of ongoing synaptic activity by tetrodotoxin (TTX). The application of 0.5 μm tetrodotoxin abolished C4 and inspiratory neuron discharges. Resting membrane potential shifted by −4 mV. After the application of 1 μm DAGO with 0.5 μm tetrodotoxin, membrane potential hyperpolarized farther by 6 mV, and input membrane resistance decreased 48% (315 to 164 MΩ) in a naloxone-reversible manner. (A  ) Continuous recording of membrane potential with injecting hyperpolarizing current pulse (40 pA, 0.2 Hz, 500 ms). Current pulses with different intensity were injected into the neuron to examine current–voltage relation at the three time intervals denoted by bars above the neuron tracing. (B  ) The injected hyperpolarizing and depolarizing current pulses and the generated electronic potentials. (C  ) Reversal potential was −71 mV according to the current–voltage relation.
Fig. 8. Responses to opioid receptor agonists of inspiratory neuron after blockade of ongoing synaptic activity by tetrodotoxin (TTX). The application of 0.5 μm tetrodotoxin abolished C4 and inspiratory neuron discharges. Resting membrane potential shifted by −4 mV. After the application of 1 μm DAGO with 0.5 μm tetrodotoxin, membrane potential hyperpolarized farther by 6 mV, and input membrane resistance decreased 48% (315 to 164 MΩ) in a naloxone-reversible manner. (A 
	) Continuous recording of membrane potential with injecting hyperpolarizing current pulse (40 pA, 0.2 Hz, 500 ms). Current pulses with different intensity were injected into the neuron to examine current–voltage relation at the three time intervals denoted by bars above the neuron tracing. (B 
	) The injected hyperpolarizing and depolarizing current pulses and the generated electronic potentials. (C 
	) Reversal potential was −71 mV according to the current–voltage relation.
Fig. 8. Responses to opioid receptor agonists of inspiratory neuron after blockade of ongoing synaptic activity by tetrodotoxin (TTX). The application of 0.5 μm tetrodotoxin abolished C4 and inspiratory neuron discharges. Resting membrane potential shifted by −4 mV. After the application of 1 μm DAGO with 0.5 μm tetrodotoxin, membrane potential hyperpolarized farther by 6 mV, and input membrane resistance decreased 48% (315 to 164 MΩ) in a naloxone-reversible manner. (A  ) Continuous recording of membrane potential with injecting hyperpolarizing current pulse (40 pA, 0.2 Hz, 500 ms). Current pulses with different intensity were injected into the neuron to examine current–voltage relation at the three time intervals denoted by bars above the neuron tracing. (B  ) The injected hyperpolarizing and depolarizing current pulses and the generated electronic potentials. (C  ) Reversal potential was −71 mV according to the current–voltage relation.
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Discussion
The major findings in this investigation were that both μ- and κ-opioid receptor agonists inhibited inspiratory neuron bursts, whereas these agonists were less effective on the preinspiratory and expiratory neurons, as well as on the synaptic transmission from preinspiratory neurons to inspiratory neurons. Furthermore, this inhibition of inspiratory neurons seems to be caused by both a presynaptic and postsynaptic inhibition. Thus, opioids in used concentration caused reduction of final motor outputs by mainly inhibiting medullary inspiratory neuron network, exerting only moderate effects on the central respiratory rhythm produced by preinspiratory neurons. However, it should be carefully considered which neuron activity is a good indicator of the central rhythm generator. The current results are consistent with a hypothesis that the preinspiratory neuron network is a primary rhythm generator for respiration in the neonatal in vitro  preparation. Therefore, measurement of central burst frequency could be practically performed by measuring preinspiratory burst rate in the current study.
In the current study, central mechanisms of respiratory depression induced by opioid receptor agonists were investigated using a brainstem–spinal cord preparation from newborn rats. Although this reduced preparation is advantageous for the purpose, extrapolation of the current results to the mature and intact animal should be made with a high degree of caution.
Reduction of C4 Respiratory Frequency
The current results showing an inhibitory effect of opioid receptor agonists on the C4 respiratory frequency were consistent with those of previous studies. 6,19 In detail, μ- and κ-opioid receptors were involved in the reduction of C4 and inspiratory neuron burst frequency, whereas δ-opioid receptor did not participate in respiratory depression. Although drugs were applied to the isolated brainstem preparation in a perfusion chamber, concomitant inhibition of inspiratory neuron discharge in the medulla and C4 inspiratory activity indicates that such inhibitory effects on respiratory outputs originated from the effects of drugs in the medulla. This is further supported by the lack of effects on preinspiratory burst rate. In rats, μ- and κ-opioid receptors are present at birth and have a similar distribution in the brainstem, whereas δ-opioid receptors appear during the postnatal period. 20–22 These results from previous histologic studies may explain pharmacologic findings that activation of δ-opioid receptor in the neonatal preparations did not always produce consistent inhibitory effects. 4–6 It is possible that δ-opioid receptors in the medulla oblongata may participate in respiratory depression produced by opioids in older rats. 23,24 
Using the neonatal rat brainstem–spinal cord preparation, Greer et al.  4 reported that DAGO (0.2–1.0 μm) decreased in the C4 respiratory frequency, whereas the respiratory frequency was unaffected by U50488 (0.1–4.0 μm) and DPDPE (0.1–2.0 μm). However, they measured respiratory frequency only 5 min after drug application. It was also shown that inhibitory effects via  κ-opioid receptor could be observed with a slower time course than those mediated via  μ-opioid receptor. 6 In the current study, we showed that the effects of U50488 usually appeared within 15 min after the application. Several in vivo  studies have shown that κ-opioid receptors do not participate in the opioid-induced respiratory depression. 4,24,25 This discrepancy between the results of the current in vitro  study and these in vivo  studies suggests that the suprabulbar structures may modify the medullary κ-opioid receptor-mediated respiratory depression.
Concerning action of endogenous opioids, we did not test the sole administration of naloxone in the current study. Previous studies have shown that naloxone has no effect on medullary respiratory control during similar experimental conditions as in the current study, and that endogenous opioids do not participate in basal respiratory control in the medulla oblongata of newborn rats. 4,6 
Inspiratory Neurons
Forty-two percent of the inspiratory neurons were hyperpolarized and decreased in Rmduring opioid-induced respiratory depression. In addition, a similar percentage of inspiratory neurons were also hyperpolarized and decreased in Rmduring application of μ- and κ-opioid receptor agonists after blockade of ongoing synaptic activity by tetrodotoxin. This type of neuron is thought to be inhibited by postsynaptic action of opioids. Results from the current–voltage relation of the neuron and the value of the Erevare consistent with a suggestion that opioid-induced hyperpolarization is induced by an increase in the K+conductance. These results are consistent with those of rhythmically active slices from mice in which a subpopulation of inspiratory neurons was hyperpolarized via  direct postsynaptic action accompanying an increase in K+conductance during application of μ-opioid receptor agonist. 12 Interestingly, half of inspiratory neurons inhibited with μ- and κ-opioid receptor agonists remained unchanged for resting Emand Rm. These inhibitory actions on neuron burst discharges may be induced by a decrease in excitatory drives from presynaptic neurons. Opioids in presynaptic sites inhibit Ca2+-dependent neurotransmitter release along with K+channel activation and inhibitory actions on voltage-dependent Ca2+channels. 26,27 It has been suggested that the main action of opioids is a presynaptic inhibition of the excitatory synaptic input. 28 Intermediate- and high-voltage–activated Ca2+channels may contribute to the potentiation of burst activity in respiratory neurons. 10 In addition to K+conductance increase, therefore, we presume that the insufficient activation of Ca2+channels at the presynaptic and postsynaptic membrane may be involved in the inhibition of inspiratory neuron bursts by opioids.
The slope of the first EPSPs, evoked by stimulation to the contralateral medulla, was reduced in inspiratory neurons during opioid-induced respiratory depression. Fifty-eight percent (7 of 12) of these inspiratory neurons did not show any significant change in the resting Emand Rm. We hypothesized that the first EPSPs were induced monosynaptically because of the steady latency, which corresponds well to antidromic latency and significant small variance of latencies of EPSPs. Based on this assumption, the results indicate that opioids exert inhibitory effects at presynaptic sites on these inspiratory neurons, as suggested in other neuronal systems. 26,27 Even in hyperpolarized neurons (42%), the slope of the EPSPs was reduced, which suggests the presence of neurons that simultaneously show both presynaptic and postsynaptic inhibitory effects. The reduction of slope of EPSPs causes delay of initiation of action potentials and, consequently, may elicit failure of synaptic transmission at polysynaptic pathways in the inspiratory neuron network. In addition to the postsynaptic inhibition, the presynaptic inhibition caused by opioids may effectively depress inspiratory burst generation by mutual excitatory couplings between inspiratory neurons. 29 
Ballanyi et al.  5 reported that opioid receptor agonists blocked respiration-related Emfluctuations in a cyclic adenosine monophosphate–dependent manner. Their data also indicated a possibility that the neuronal responses to opioid receptor agonists differ depending on subtypes of respiratory neurons. Type I and III inspiratory neurons receive excitatory and inhibitory synaptic inputs, respectively, from preinspiratory neurons. 10,11 These synaptic potentials, which correlated to preinspiratory neuron burst, remained during opioid-induced respiratory depression, as confirmed by simultaneous recordings from inspiratory and preinspiratory neurons. Thus, we conclude that μ- and κ-opioid receptor agonists inhibited inspiratory neuron burst via  direct or indirect synaptic mechanism. Synaptic inputs from preinspiratory neurons in the neonatal brainstem–spinal cord preparation were, however, unchanged, indicating a lack of effect of these agonists on central respiratory rhythm generation.
Preinspiratory Neurons
We observed that the burst activities of preinspiratory neurons were still present even with the disappearance of inspiratory neuron bursts and C4 respiratory discharges. In addition, Emand Rmdid not change during administration of opioid receptor agonists with or without tetrodotoxin perfusion. Such resistance of preinspiratory neurons to opioids does not mean that inspiratory neuron burst and C4 inspiratory activity occur out of phase with the preinspiratory neuron burst during opioid-induced respiratory depression. Rather, we noted that during opioid-induced respiratory depression, the firing of a preinspiratory neuron always preceded the initiation even of slow inspiratory neuron bursts.
In the previous studies using the brainstem–spinal cord preparation from neonatal rats, it was suggested that bath-applied neuromodulators induced change in respiratory rhythm through primarily altering preinspiratory neuron activity. 8,11,30 In contrast, inhibition of respiratory rhythm (i.e.  , reduction of frequency of the C4 motoneuron outputs) by opioids was caused by inhibition of the inspiratory neuron network but not attributable to effects on the preinspiratory neuron network in the medulla. Hence, inspiratory neurons (i.e.  , output neurons) are affected either directly or via  drive reduction without any significant effects on the central rhythm generated by preinspiratory neurons.
Expiratory Neurons
Most expiratory neurons showed unchanged discharging action potentials, Em, and Rmduring application of opioid receptor agonists. However, the κ-opioid receptor agonist U50488 with standard artificial cerebrospinal fluid induced a naloxone-reversible hyperpolarization and decrease of spike firing frequency in two expiratory neurons. This response may be a result of an activation of K+conductance via  opioid receptors present on expiratory neurons. Expiratory neurons in the RVLM of neonatal rats express three different K+channels. One of these, which may be expressed specifically in the expiratory neuron, is voltage-dependent and determines the neuronal level of depolarization. 31 Although most expiratory neurons are thought to be basically insensitive to opioids, some expiratory neurons have opioid receptors and may be influenced by opioids. 5 
In conclusion, respiratory depression caused by μ- and κ-opioid receptor agonists in the brainstem–spinal cord preparation is caused by a combined and reversible presynaptic and postsynaptic inhibition of inspiratory neurons, while membrane properties of preinspiratory and most expiratory neurons are left unaffected. Thus, opioids exerted the inhibitory effects mainly on the inspiratory neuron network in the medulla and only minor effects on the central respiratory rhythm by preinspiratory neurons.
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Fig. 1. (A  ) Membrane potential traces from preinspiratory, inspiratory (types I, II, and III), and expiratory neurons in the brainstem–spinal cord preparation along with inspiratory motor nerve activity from the C4 ventral root. (B  ) Ventral aspect of the preparation and recording sites. The area examined in the current study (shaded area) seems to be located more rostrally, but partly overlapped, to pre-Bötzinger complex.
Fig. 1. (A 
	) Membrane potential traces from preinspiratory, inspiratory (types I, II, and III), and expiratory neurons in the brainstem–spinal cord preparation along with inspiratory motor nerve activity from the C4 ventral root. (B 
	) Ventral aspect of the preparation and recording sites. The area examined in the current study (shaded area) seems to be located more rostrally, but partly overlapped, to pre-Bötzinger complex.
Fig. 1. (A  ) Membrane potential traces from preinspiratory, inspiratory (types I, II, and III), and expiratory neurons in the brainstem–spinal cord preparation along with inspiratory motor nerve activity from the C4 ventral root. (B  ) Ventral aspect of the preparation and recording sites. The area examined in the current study (shaded area) seems to be located more rostrally, but partly overlapped, to pre-Bötzinger complex.
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Fig. 2. Type II inspiratory neuron and effects of opioid receptor agonists on membrane potential. (A  ) Continuous recording of membrane potential. Current pulses with different intensity were injected into the neuron to examine the current–voltage relation at the three time intervals denoted by bars above the neuron tracing. (B  ) Representation of recordings in (A  ) on extended time scale. (C  ) Reversal potential was −70 mV according to the current–voltage relation.
Fig. 2. Type II inspiratory neuron and effects of opioid receptor agonists on membrane potential. (A 
	) Continuous recording of membrane potential. Current pulses with different intensity were injected into the neuron to examine the current–voltage relation at the three time intervals denoted by bars above the neuron tracing. (B 
	) Representation of recordings in (A 
	) on extended time scale. (C 
	) Reversal potential was −70 mV according to the current–voltage relation.
Fig. 2. Type II inspiratory neuron and effects of opioid receptor agonists on membrane potential. (A  ) Continuous recording of membrane potential. Current pulses with different intensity were injected into the neuron to examine the current–voltage relation at the three time intervals denoted by bars above the neuron tracing. (B  ) Representation of recordings in (A  ) on extended time scale. (C  ) Reversal potential was −70 mV according to the current–voltage relation.
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Fig. 3. Effects of opioid receptor agonists on membrane potential of types I and III inspiratory neurons. (A  ) Type I inspiratory neuron. Excitatory postsynaptic potentials, the source of which are presumably preinspiratory neurons, were still observed during opioid-induced respiratory depression. Arrows indicate excitatory postsynaptic potentials. Reversal potential was −83 mV according to the current–voltage relation. (B  ) Type III inspiratory neuron. Inhibitory postsynaptic potentials, the source of which are presumably preinspiratory neurons, were still observed during opioid-induced respiratory depression. Arrows indicate inhibitory postsynaptic potentials. Reverse potential was −75 mV according to the current–voltage relation.
Fig. 3. Effects of opioid receptor agonists on membrane potential of types I and III inspiratory neurons. (A 
	) Type I inspiratory neuron. Excitatory postsynaptic potentials, the source of which are presumably preinspiratory neurons, were still observed during opioid-induced respiratory depression. Arrows indicate excitatory postsynaptic potentials. Reversal potential was −83 mV according to the current–voltage relation. (B 
	) Type III inspiratory neuron. Inhibitory postsynaptic potentials, the source of which are presumably preinspiratory neurons, were still observed during opioid-induced respiratory depression. Arrows indicate inhibitory postsynaptic potentials. Reverse potential was −75 mV according to the current–voltage relation.
Fig. 3. Effects of opioid receptor agonists on membrane potential of types I and III inspiratory neurons. (A  ) Type I inspiratory neuron. Excitatory postsynaptic potentials, the source of which are presumably preinspiratory neurons, were still observed during opioid-induced respiratory depression. Arrows indicate excitatory postsynaptic potentials. Reversal potential was −83 mV according to the current–voltage relation. (B  ) Type III inspiratory neuron. Inhibitory postsynaptic potentials, the source of which are presumably preinspiratory neurons, were still observed during opioid-induced respiratory depression. Arrows indicate inhibitory postsynaptic potentials. Reverse potential was −75 mV according to the current–voltage relation.
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Fig. 4. Effects of opioid receptor agonists on membrane potential of preinspiratory neuron. (A  ) Continuous recording of membrane potential. Current pulses with different intensity were injected into the neuron to examine current-voltage relation at the three time intervals denoted by bars above the neuron tracing. (B  ) Representation of recordings in (A  ) on extended time scale. During opioid-induced respiratory depression, preinspiratory neuron activity, followed by absence of inhibitory postsynaptic potentials from inspiratory neurons, indicated “throughout” burst pattern. When C4 inspiratory burst occurred, preinspiratory neurons showed long preinspiratory but short postinspiratory activity.
Fig. 4. Effects of opioid receptor agonists on membrane potential of preinspiratory neuron. (A 
	) Continuous recording of membrane potential. Current pulses with different intensity were injected into the neuron to examine current-voltage relation at the three time intervals denoted by bars above the neuron tracing. (B 
	) Representation of recordings in (A 
	) on extended time scale. During opioid-induced respiratory depression, preinspiratory neuron activity, followed by absence of inhibitory postsynaptic potentials from inspiratory neurons, indicated “throughout” burst pattern. When C4 inspiratory burst occurred, preinspiratory neurons showed long preinspiratory but short postinspiratory activity.
Fig. 4. Effects of opioid receptor agonists on membrane potential of preinspiratory neuron. (A  ) Continuous recording of membrane potential. Current pulses with different intensity were injected into the neuron to examine current-voltage relation at the three time intervals denoted by bars above the neuron tracing. (B  ) Representation of recordings in (A  ) on extended time scale. During opioid-induced respiratory depression, preinspiratory neuron activity, followed by absence of inhibitory postsynaptic potentials from inspiratory neurons, indicated “throughout” burst pattern. When C4 inspiratory burst occurred, preinspiratory neurons showed long preinspiratory but short postinspiratory activity.
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Fig. 5. Effects of opioid receptor agonists on membrane potential of expiratory neuron. (A  ) After U50488 application, C4 inspiratory discharges were decreased, but discharges of an expiratory neuron were not affected. (B  ) Expiratory neuron discharges were depressed with hyperpolarization produced by U50488.
Fig. 5. Effects of opioid receptor agonists on membrane potential of expiratory neuron. (A 
	) After U50488 application, C4 inspiratory discharges were decreased, but discharges of an expiratory neuron were not affected. (B 
	) Expiratory neuron discharges were depressed with hyperpolarization produced by U50488.
Fig. 5. Effects of opioid receptor agonists on membrane potential of expiratory neuron. (A  ) After U50488 application, C4 inspiratory discharges were decreased, but discharges of an expiratory neuron were not affected. (B  ) Expiratory neuron discharges were depressed with hyperpolarization produced by U50488.
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Fig. 6. Orthodromic responses evoked in inspiratory neurons by stimulation of the rostral ventrolateral medulla on the contralateral side after application of opioid receptor agonists. Arrows indicate time of stimulation. Arrowheads indicate slopes of excitatory postsynaptic potentials (  EPSPs). (A  ) Resting membrane potential did not change. Latencies of EPSPs were 8.6 ms in control and 10.3 ms after treatment with DAGO. Latencies of first action potential were prolonged from 11.3 ms in control to 18.6 ms after treatment with DAGO because the slope of EPSPs reduced. (B  ) Resting membrane potential shifted by −4 mV. Latencies of EPSPs were 9.3 ms in control and 8.7 ms after treatment with DAGO. Latencies of first action potential were also prolonged from 12.3 ms in control to 15.3 ms after treatment with DAGO because the slope of EPSPs reduced and resting membrane potential hyperpolarized.
Fig. 6. Orthodromic responses evoked in inspiratory neurons by stimulation of the rostral ventrolateral medulla on the contralateral side after application of opioid receptor agonists. Arrows indicate time of stimulation. Arrowheads indicate slopes of excitatory postsynaptic potentials ( 
	EPSPs). (A 
	) Resting membrane potential did not change. Latencies of EPSPs were 8.6 ms in control and 10.3 ms after treatment with DAGO. Latencies of first action potential were prolonged from 11.3 ms in control to 18.6 ms after treatment with DAGO because the slope of EPSPs reduced. (B 
	) Resting membrane potential shifted by −4 mV. Latencies of EPSPs were 9.3 ms in control and 8.7 ms after treatment with DAGO. Latencies of first action potential were also prolonged from 12.3 ms in control to 15.3 ms after treatment with DAGO because the slope of EPSPs reduced and resting membrane potential hyperpolarized.
Fig. 6. Orthodromic responses evoked in inspiratory neurons by stimulation of the rostral ventrolateral medulla on the contralateral side after application of opioid receptor agonists. Arrows indicate time of stimulation. Arrowheads indicate slopes of excitatory postsynaptic potentials (  EPSPs). (A  ) Resting membrane potential did not change. Latencies of EPSPs were 8.6 ms in control and 10.3 ms after treatment with DAGO. Latencies of first action potential were prolonged from 11.3 ms in control to 18.6 ms after treatment with DAGO because the slope of EPSPs reduced. (B  ) Resting membrane potential shifted by −4 mV. Latencies of EPSPs were 9.3 ms in control and 8.7 ms after treatment with DAGO. Latencies of first action potential were also prolonged from 12.3 ms in control to 15.3 ms after treatment with DAGO because the slope of EPSPs reduced and resting membrane potential hyperpolarized.
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Fig. 7. Responses to opioid receptor agonists of inspiratory neuron recorded intracellularly (upper traces  ), accompanied by extracellular preinspiratory neuron activity (middle traces  ) in contralateral rostral ventrolateral medulla. The type III inspiratory neuron was hyperpolarized during preinspiratory and postinspiratory phases corresponding to the activity of preinspiratory neuron in the control state and after treatment with naloxone. After application of DAGO, the C4 and inspiratory neuron burst rates decreased, while the preinspiratory neuron was still discharging at the same rate as in the control state. Furthermore, inhibitory postsynaptic potentials, which corresponded to the period of preinspiratory neuron, were still observed. Arrows indicate inhibitory postsynaptic potentials.
Fig. 7. Responses to opioid receptor agonists of inspiratory neuron recorded intracellularly (upper traces 
	), accompanied by extracellular preinspiratory neuron activity (middle traces 
	) in contralateral rostral ventrolateral medulla. The type III inspiratory neuron was hyperpolarized during preinspiratory and postinspiratory phases corresponding to the activity of preinspiratory neuron in the control state and after treatment with naloxone. After application of DAGO, the C4 and inspiratory neuron burst rates decreased, while the preinspiratory neuron was still discharging at the same rate as in the control state. Furthermore, inhibitory postsynaptic potentials, which corresponded to the period of preinspiratory neuron, were still observed. Arrows indicate inhibitory postsynaptic potentials.
Fig. 7. Responses to opioid receptor agonists of inspiratory neuron recorded intracellularly (upper traces  ), accompanied by extracellular preinspiratory neuron activity (middle traces  ) in contralateral rostral ventrolateral medulla. The type III inspiratory neuron was hyperpolarized during preinspiratory and postinspiratory phases corresponding to the activity of preinspiratory neuron in the control state and after treatment with naloxone. After application of DAGO, the C4 and inspiratory neuron burst rates decreased, while the preinspiratory neuron was still discharging at the same rate as in the control state. Furthermore, inhibitory postsynaptic potentials, which corresponded to the period of preinspiratory neuron, were still observed. Arrows indicate inhibitory postsynaptic potentials.
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Fig. 8. Responses to opioid receptor agonists of inspiratory neuron after blockade of ongoing synaptic activity by tetrodotoxin (TTX). The application of 0.5 μm tetrodotoxin abolished C4 and inspiratory neuron discharges. Resting membrane potential shifted by −4 mV. After the application of 1 μm DAGO with 0.5 μm tetrodotoxin, membrane potential hyperpolarized farther by 6 mV, and input membrane resistance decreased 48% (315 to 164 MΩ) in a naloxone-reversible manner. (A  ) Continuous recording of membrane potential with injecting hyperpolarizing current pulse (40 pA, 0.2 Hz, 500 ms). Current pulses with different intensity were injected into the neuron to examine current–voltage relation at the three time intervals denoted by bars above the neuron tracing. (B  ) The injected hyperpolarizing and depolarizing current pulses and the generated electronic potentials. (C  ) Reversal potential was −71 mV according to the current–voltage relation.
Fig. 8. Responses to opioid receptor agonists of inspiratory neuron after blockade of ongoing synaptic activity by tetrodotoxin (TTX). The application of 0.5 μm tetrodotoxin abolished C4 and inspiratory neuron discharges. Resting membrane potential shifted by −4 mV. After the application of 1 μm DAGO with 0.5 μm tetrodotoxin, membrane potential hyperpolarized farther by 6 mV, and input membrane resistance decreased 48% (315 to 164 MΩ) in a naloxone-reversible manner. (A 
	) Continuous recording of membrane potential with injecting hyperpolarizing current pulse (40 pA, 0.2 Hz, 500 ms). Current pulses with different intensity were injected into the neuron to examine current–voltage relation at the three time intervals denoted by bars above the neuron tracing. (B 
	) The injected hyperpolarizing and depolarizing current pulses and the generated electronic potentials. (C 
	) Reversal potential was −71 mV according to the current–voltage relation.
Fig. 8. Responses to opioid receptor agonists of inspiratory neuron after blockade of ongoing synaptic activity by tetrodotoxin (TTX). The application of 0.5 μm tetrodotoxin abolished C4 and inspiratory neuron discharges. Resting membrane potential shifted by −4 mV. After the application of 1 μm DAGO with 0.5 μm tetrodotoxin, membrane potential hyperpolarized farther by 6 mV, and input membrane resistance decreased 48% (315 to 164 MΩ) in a naloxone-reversible manner. (A  ) Continuous recording of membrane potential with injecting hyperpolarizing current pulse (40 pA, 0.2 Hz, 500 ms). Current pulses with different intensity were injected into the neuron to examine current–voltage relation at the three time intervals denoted by bars above the neuron tracing. (B  ) The injected hyperpolarizing and depolarizing current pulses and the generated electronic potentials. (C  ) Reversal potential was −71 mV according to the current–voltage relation.
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Table 1. Resting Membrane Potentials, Amplitude of Action Potentials, and Input Membrane Resistance after Establishing Whole Cell Configuration
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Table 1. Resting Membrane Potentials, Amplitude of Action Potentials, and Input Membrane Resistance after Establishing Whole Cell Configuration
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Table 2. Responses of Respiratory Neurons after Application of Opioid Receptor Agonists in Standard Artificial Cerebrospinal Fluid (aCSF) or in the Presence of Tetrodotoxin (TTX)
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Table 2. Responses of Respiratory Neurons after Application of Opioid Receptor Agonists in Standard Artificial Cerebrospinal Fluid (aCSF) or in the Presence of Tetrodotoxin (TTX)
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Table 3. Effects of Opioid Receptor Agonists on Burst Rate of Inspiratory Neurons (bursts/min)
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Table 3. Effects of Opioid Receptor Agonists on Burst Rate of Inspiratory Neurons (bursts/min)
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Table 4. Effects of Opioid Receptor Agonists on C4 Respiratory Frequency (bursts/min) and Preinspiratory (Pre-I) Neuron Burst Rate (bursts/min)
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Table 4. Effects of Opioid Receptor Agonists on C4 Respiratory Frequency (bursts/min) and Preinspiratory (Pre-I) Neuron Burst Rate (bursts/min)
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Table 5. Latencies of Excitatory Postsynaptic Potentials (EPSPs) and of First Action Potential in Inspiratory Neurons’ Induced Orthodromic Response by Contralateral Medulla Stimulation
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Table 5. Latencies of Excitatory Postsynaptic Potentials (EPSPs) and of First Action Potential in Inspiratory Neurons’ Induced Orthodromic Response by Contralateral Medulla Stimulation
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