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1Dalton Cardiovascular Research Center and 2Department of Biomedical Science, College of Veterinary Medicine, University of Missouri, Columbia, Missouri; and 3Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada
Submitted 3 August 2007; accepted in final form 2 December 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). Respiratory rhythm is continually modulated by a number of inputs including chemosensory drive from central and peripheral chemoreceptors, dynamic feedback from pulmonary and somatosensory afferents as well as local release of neuropeptides such as substance P (SP). Considerable attention has focused on the effect of SP on breathing in brain stem regions that predominately contain inspiratory neurons (Gray et al. 1999, 2001; Morgado-Valle and Feldman 2004; Ptak et al. 2000a). Neurons in pre-Bötzinger Complex (preBötC) of the ventral respiratory group (VRG) express high levels of the SP receptor, neurokinin-1 (NK1-R) (Gray et al. 1999; Guyenet et al. 2002; Nakaya et al. 1994; Wang et al. 2001). Ablation of NK1-R expressing neurons in preBötC, the putative kernel for respiratory rhythmogenesis (Smith et al. 1991), severely disrupts breathing and markedly depresses the ventilatory response to inspired CO2 (Gray et al. 2001; McKay et al. 2005; Wenninger et al. 2004). Earlier studies have shown that exogenous SP excites respiratory activity by increasing breathing frequency and tidal volume (Chen et al. 1990a; Gray et al. 1999; Hedner et al. 1984; Johnson et al. 1996; Monteau et al. 1996; Morgado-Valle and Feldman 2004; Ptak et al. 2000a). However, in a recent study, we showed that NK1-R activation could excite both inspiratory and expiratory activity depending on the VRG neuronal subtype activated (Fong and Potts 2006). Despite the central effect of SP on breathing, NK1-R do not appear to be essential for the generation of respiratory rhythm because genetic deletion of NK1-R or SP synthesizing neurons failed to alter basal breathing (Cao et al. 1998; De Felipe et al. 1998; Ptak et al. 1999; Telgkamp et al. 2002). These findings suggest that SP may be involved in modulating respiratory rhythm rather than in the generation of respiratory rhythm.
To date, no study has directly examined the role of NK1-R on expiratory (E) neurons in the VRG. Given that NK1-R are expressed in regions containing E neurons (Guyenet et al. 2002; Wang et al. 2001), it is reasonable to propose that SP evoked excitation of E neurons may provide a mechanism to modulate breathing. The present study was designed to test this prediction and to demonstrate that SP-induced excitation of E neurons in the VRG selectively modulates respiratory rhythm in a reflex pathway-dependent manner. Because reflex activation of E neurons contributes to changes in respiratory rhythm (Ezure et al. 2002; Fong and Potts 2006; Hayashi et al. 1996; Kubin et al. 2006; Potts et al. 2005) and respiratory rhythm can be coupled to locomotor activity via somatosensory-evoked excitation of expiratory neurons (Funk et al. 1992; Iscoe 1981; Potts et al. 2005), we hypothesized that locomotor-respiratory coupling (LRC) would require endogenous SP release in the VRG.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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In situ working heart-brain stem preparation
Experiments were performed on 39 juvenile male Wistar rats (55–120 g, Harlan) using the in situ arterial-perfused juvenile rat preparation. The surgical procedures and the extracorporeal circuit for this preparation have been described in detail previously (Fong and Potts 2006; Paton 1996; Potts et al. 2000). In brief, rats were deeply anesthetized with isoflurane via spontaneous inhalation, and the depth of anesthesia was gauged by the absence of limb withdrawal to noxious pinch and the lack of corneal reflex. The rat was transected subdiaphragmatically and the upper torso immediately submerged into ice-cold Ringer solution bubbled with carbogen gas (95% O2-5% CO2) and decerebrated precollicularly, and the cerebellum was removed to expose the dorsal surface of the brain stem. The preparation was skinned, and a portion of the posterior thoracic wall was removed to expose the heart and lungs. The descending aorta and the left phrenic nerve (PN) were blunt dissected, isolated, and cut. After the surgery, the preparation was transferred to an acrylic chamber and the thoracic aorta cannulated with a double lumen catheter (16 and 18 gauge; Braintree Scientific) and secured. Peristaltic pump perfusion of Ringer solution bubbled with 95% O2-5% CO2 was started immediately, and the perfusate was warmed to 32–33°C using an in-line heat exchanger, pumped through two in-line bubble traps and a filter (polypropylene mesh; pore size: 40 µm, Millipore). Perfusion pressure was measured via one of the lumens of the double-lumen catheter using a pressure transducer (model PT300, Grass Instruments), connected to an amplifier (Model No. 13-6615-50, Gould). Pump flow rate was calibrated at the beginning of each experiment and flow rates between 25 and 36 ml/min were used in this study. Whole nerve activity was recorded from the PN via suction electrodes (tip diameter, 0.2–0.3 mm), amplified (20,000–50,000) and filtered (100 Hz to 3 kHz, Model No. P511, Grass Astro-Med), and sampled at 2.5 kHz. The electrocardiogram (ECG) was measured via silver wires placed directly on the heart, amplified (10,000–20,000), filtered (100 Hz to 3 kHz, Model No. P511; Grass Astro-Med), displayed on an oscilloscope and a discriminator circuit (Model N-750, Mentor) was used to generate transistor-transistor logic (TTL) pulses triggered from the upstroke of the R wave. Instantaneous heart rate was derived from measuring the interbeat interval and displayed as beats per minute (bpm).
The elapsed time from induction of anesthesia to the start of systemic perfusion was generally 10–15 min. Neuromuscular paralysis was produced by addition of vecuronium bromide (50 µg) directly to the perfusate. Only preparations that exhibited ramping phrenic discharge with regular burst intervals, an index of adequate brain stem perfusion (Paton 1996), were included in this study.
Extracellular recording of VRG neurons
Multibarrel glass micropipettes containing three to five barrels (1.2 mm OD, 0.68 mm ID, World Precision Instruments) were pulled and broken back to yield a total tip diameter of <10 µm for single-unit recording and picoejection or <30 µm for multiunit recording and microinjection. One barrel was filled with 3 M NaCl, and a silver wire was introduced into the NaCl solution for recording neuronal activity (electrode tip impedance: 12–28 M
). Extracellular recordings were amplified using an AC amplifier (5,000–10,000x; NeuroLog NL104, Digitimer, Welwyn Garden City, UK) and filtered (300 Hz to 3 kHz, Neurolog NL126, Digitimer). Action potentials (AP) were continuously monitored using a window discriminator (Neurolog NL201, Digitimer) using dual threshold windowing to capture both the trajectory and amplitude of each unit and displayed on a storage oscilloscope (20 mHz, Kikisui). For picoejection studies, only neurons the APs of which could be clearly discriminated were studied. Single-unit activity was digitized using 12 bit A/D conversion (micro 1401, Cambridge Electronics Design, Cambridge, UK) at 25 kHz and recorded using Spike2 software (Cambridge Electronics Design). The system recorded and displayed discriminated unit activity in real time. Wave-shape template matching was used off-line to confirm the waveshape of all recorded units as single units.
Drug preparation for picoejection or microinjection
The remaining drug barrels of the combined recording/picoejection microelectrode array were back-filled with one of the following solutions: 1 mM of the selective NK1-R agonist, [Sar9-Met(O2)11]-substance P (SSP); 10 mM of the selective nonpeptide NK1-R antagonist, CP99,994; vehicle (Ringer solution). In experiments designed to characterize the role of NK1-R in LRC, each barrel of a triple-barrel pipette was back-filled with 3 M NaCl (extracellular recording), CP99,994 (NK1-R antagonist), and D-L-homocysteic acid (DLH). DLH was used to functionally identify the BötC region as previously described (Fong and Potts 2006; Monnier et al. 2003; Wang et al. 2002). All drugs were dissolved in the Ringer solution and pH adjusted to 7.4. The ends of the microinjection barrels were sealed with silicone tubing connected to a pneumatic pressure injection system (Picospritzer II, General Valve) via polyethylene tubing.
Experimental procedure
The micropipette was secured to a pipette holder attached to a piezoelectric stepper motor (Inch Worm, model IW-711-01; Burleigh Instruments) driven by a low-noise controller (model 6200ULN-1-1; Burleigh Instruments) mounted on a stereotaxic frame. The tip of the micropipette was placed at calamus scriptorius (CS) using a stereomicroscope (total magnification = 66X, Stemi SV11, Carl Zeiss), and this landmark was used as relative zero for the rostrocaudal and lateral displacement of the micropipette array. The cranium was tilted nose down at an angle of 
30° from the horizontal such that the brain stem was horizontal. The pipette was positioned into the VRG using the following coordinates: AP: +0.3–1.8 mm, ML: ±1.4–2.0 mm from CS, DV: 1,800–2,700 µm below the dorsal surface of the medulla. Extracellular unit activity was continuously monitored as the micropipette was advanced ventrally into the medulla and the locations of respiratory neurons were noted. Once a respiratory neuron was located, the neuron was classified as decrementing-expiratory (EDEC), augmenting expiratory (EAUG), or tonic expiratory (ETONIC) based on its discharge pattern relative to phrenic nerve discharge (PND) (Ezure 1990). Following characterization of bursting profile of the expiratory neuron, the chosen protocol was used as described in the following text. The final stereotaxic location of recorded neurons was documented and subsequently plotted on a reconstruction of a representative rat brain stem of the weight range used in this study.
Protocol 1: effect of SSP picoejection on expiratory neurons
The first series of experiments determined the effect of SSP on excitability of single unit expiratory neurons in the VRG. To facilitate recording of single units during drug delivery to the recorded cell, picoejection pressure was maintained as low as possible (pressures range between 10 and 25 psi). Ejection pressure was slowly adjusted to the drug barrel until a minimum pressure that produced 10% change in AP amplitude was achieved. In the majority of cases, this resulted in a decrease in AP amplitude. However, in two cases, AP amplitude increased during pressure application. The drugs were pressure-ejected over a 20- to 30-s period, which typically coincided with six to seven respiratory cycles while the amplitude of the recorded unit was constantly monitored. The ejection volumes could not be determined as the ejected volume was below the visual resolution of the microscope (3 nl).
To verify that the SSP-evoked response was, in fact, due to activation of NK1-R, the picoejection was repeated following delivery of CP99,994 (CP) from an adjacent barrel. Pressure transfer between picoejection barrels was facilitated by a series of four-way stopcocks that were connected in-series with the external pressure source. In preliminary studies, we determined that a minimum period of 1 min was required between repeat applications of SSP to prevent receptor desensitization and for the response to SSP to be reproducible (see Fig. 1A). Therefore to ensure sufficient time for recovery, 
1 min was given between sequential SSP applications. In a subset of E neurons excited by SSP (n = 5), the nonpeptide NK1-R antagonist CP was picoejected onto the same neuron. Immediately following CP, SSP picoejection was repeated. Finally, SSP was picoejected serially over the next 10 min to examine recovery of the SSP response.
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Protocol 2: effect of NK1-R antagonism on LRC
Somatic afferents innervating the forelimb were activated by electrical stimulation of the forelimb flexors as previously described (Potts et al. 2000, 2005). The ipsilateral scapula was stabilized using a vertebral clamp and the distal end of the forelimb attached to a force displacement transducer (FT-100, CB Science) with developed tension measured using a recorder amplifier (Neurolog NL107, Digitimer, Welwyn Garden City, UK). Stimuli were delivered using two fine silver wires (0.005-in OD, A-M Systems, Carlsborg, WA) introduced into the biceps brachii and coracobrachialis muscle groups parallel to the humerus. Pulse-train stimuli (0.4–1.3 Hz, 50- to 75-ms train duration, 1- to 3-ms pulse duration, 250 µA to 1 mA) were generated by a stimulator (Model S88, Astro-Med Grass) and a stimulation isolation unit (Model PSIU6, Astro-Med Grass). This stimulus activated somatic afferents as evidenced by tachypnea and pressor/tachycardic responses (Potts et al. 2000, 2005).
Once the forelimb was stabilized, the multibarrel microelectrode (3 barrels, <30 µm total OD) was introduced into the VRG ipsilateral to the stimulated forelimb (CS: AP +1.6–1.8 mm, ML ±1.6–2.0 mm, depth from surface: 1,800–2,600 µm). Multiunit neuronal activity was recorded as the electrode was advanced ventrally. Once a region containing expiratory activity was located, the electrode was left in place for 10 min prior to microinjection of DLH (6 pmol in 6 nl) to functionally confirm that the electrode tip was in the BötC region as evidenced by an evoked bradypnea represented by an increase in expiratory duration of >1.5-fold of baseline for over five respiratory cycles (Fong and Potts 2006; Monnier et al. 2003; Wang et al. 2002). Following functional identification of the bradypneic region, the effect of somatic afferent stimulation on LRC was determined before and after blockade of NK1-R using CP99,994 (500 pmol in 50 nl).
Protocol 3: effect of NK1-R antagonism on somatic afferent stimulation evoked excitation of expiratory neurons
A multibarrel microelectrode for single-unit recording and picoejection of SSP and CP as described in protocol 1 was positioned into the VRG. Additionally, the forelimb was stabilized and stimulation electrodes were placed in the forelimb flexors as described for protocol 2. E neurons were examined for responsiveness to both picoejection of SSP and somatic afferent stimulation. Only neurons that were excited by both exogenous SSP and somatic afferent stimulation were used in this series of experiments. Once an E neuron that fitted the above profile was localized, CP was picoejected onto the neuron and the effect of somatic afferent stimulation was immediately repeated.
Data and statistical analyses
All data were collected on-line using commercially available data-acquisition hardware and software (micro1401 A/D converter, Spike 2 software, version 5.16; Cambridge Electronics Design). Wave-shape template matching algorithms (Spike2; Cambridge Electronic Design) was used off-line to confirm the shape of action potentials belonging to a single unit or to discriminate different units prior to analysis; all other analyses were performed off-line using custom-written scripts.
The following variables were measured from the phrenic neurogram: inspiratory duration (TI), determined as duration of PND; expiratory duration (TE) determined as the duration from the end of one PND to the onset of the following burst; respiratory cycle length (TTot)was calculated by summing TI and the subsequent TE; respiratory frequency was calculated from TTot and expressed in hertz. The latency between somatic afferent stimulation and the following PND was measured using a event correlation algorithm (Spike 2, V 5.16) over a series of somatic afferent stimulation events (generally 9–19 events).
For extracellular unit activity, the duration of each burst and the number of action potentials per burst was recorded and used to calculate the mean discharge frequency. In addition, the peak discharge frequency (determined from instantaneous firing frequency) was also recorded. Changes in neuronal activity in response to SSP or CP picoejection were expressed as percent of baseline. Baseline was determined by averaging the variables measured for 10–15 respiratory cycles immediately before drug ejection. Effect of the drug on extracellular activity was determined by averaging over all the respiratory cycles during picoejection (generally 6–7 respiratory cycles). Following drug ejection, the variables were measured over 10–15 respiratory cycles to determine recovery of neuronal activity toward baseline levels. This time frame was chosen based on preliminary experiments showing that this was sufficient for extracellular neuronal activity to recover to control values. Drug application was determined to have elicited an effect on the recorded neuron if the parameters measured during picoejection increased 15% above baseline.
All graphs were plotted using the software package SigmaPlot (version 9). All data are presented as means ± SE; n is the number of neurons, unless otherwise indicated. All statistical analysis was performed on raw data using the software program SAS (version 9.1, SAS Institute, Cary, NC). Data were tested for normality using the Kolmogorov-Smirnov test. Data that were normally distributed was test for statistical significance on the effect of drug on neuronal activity or PND parameters using one-way repeated-measures ANOVA followed by Student Newman-Keul (SNK) post hoc test. Data that were not normally distributed were evaluated using repeated-measures ANOVA on ranks followed by SNK post hoc test. The effect of somatic afferent stimulation on E neurons in the absence and presence of NK1-R blockade was determined using paired t-test. The effect of NK1-R blockade on respiratory rhythm entrainment was determined using Student's t-test. P < 0.05 was considered significant in all cases.
Solutions and drugs
The Ringer solution, containing (in mM) 125 NaCl, 24 NaHCO3, 5 KCl, 2.5 CaCl2, 1.25 MgSO4, 1.25 KH2PO4, and 1 D-glucose, was made fresh for each experiment. A high-molecular-weight compound (1.25%, Ficoll, type 70, 70 kDa, Sigma) was added to the Ringer solution as an oncotic agent with the resulting oncotic pressure of the perfusate of 310 mosM/Kg H2O. The neuromuscular blocking agent, vecuronium bromide (0.04 µg/ml, Sicor Pharmaceuticals), was also applied to the perfusate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Thirty-five E neurons located within the confines of the VRG were successfully recorded and included in the study. Picoejection of SSP excited 57% (20/35 cells) of E neurons, while the remaining 43% failed to alter their firing pattern. Table 1 summarizes the classification of E cells based on neuronal discharge subtype and summarizes their response to exogenous SSP. ETONIC neurons were the most commonly recorded and SSP excited 71% of these neurons. Fifty-five percent of EAUG neurons and 50% of EDEC neurons were also excited by SSP. One neuron whose activity spanned from late expiration to early inspiration was also excited by SSP. Exogenous application of SSP significantly increased the total number of APs per burst (15/20 cells), burst duration (8/20 cells), as well as the mean (9/20 cells) and peak (11/20 cells) firing frequency (see Table 2). We also evaluated the effect of SSP on respiratory rhythm and pattern and found that picoejection of SSP had no significant effect on inspiratory duration (TI), expiratory duration (TE), total respiratory cycle time (Ttot), or phrenic burst frequency (Table 3). These results suggest that the delivery of exogenous SSP was highly localized and predominately affected the recorded neuron while having little to no effect on other neighboring respiratory neurons.
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To determine whether phasic coupling between locomotor and respiratory rhythms depended on SPergic signaling in the VRG, we unilaterally blocked NK1-R by microinjecting CP into a region that contained expiratory neurons, verified by extracellular recording (Fig. 5 A) and functionally identified as the BötC region by DLH microinjection evoked bradypnea (Fig. 5B). Prior to blockade of NK1-Rs, somatic stimulation increased the PND frequency and phase-locked PND with the onset of muscle contraction (Fig. 6 A), indicated by constant latency between contraction and subsequent phrenic burst (Fig. 6C). Unilateral microinjection of CP, ipsilateral to somatic afferent stimulation, did not affect basal respiratory rhythm but eliminated the phasic coupling of locomotor and respiratory rhythms (Fig. 6, B and C). However, it should be noted that somatic stimulation increased overall PND frequency to the same extent as during the control condition (Fig. 6, A and B), which was in agreement with previous work (Potts et al. 2005). A peristimulus-timed histogram (PSTH) between the onset of somatic stimulation (i.e., forelimb tension development) and PND demonstrated that phase-coupling was completely eliminated following NK1-R blockade (Fig. 6C). In addition, CP significantly increased the average latency and its variability between the onset of locomotor and respiratory activity (Fig. 6D).
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Based on the preceding finding that blockade of NK1-R abolished LRC and that activation of somatic afferents selectively augments the discharge frequency of EAUG neurons (Potts et al. 2005), we hypothesized that somatic-evoked excitation of EAUG neurons require local SP release. In recorded EAUG neurons that were excited by exogenous SSP (n = 4), somatic stimulation increased mean and peak instantaneous firing frequency (Fig. 7 A). This was accompanied by a shortening of expiratory duration and a premature onset of phrenic nerve discharge. Picoejection of CP completely abolished somatic-evoked excitation of EAUG neurons (Fig. 7B). However, CP alone failed to alter the cell's overall discharge profile (Fig. 7B). Group results found that NK1-R blockade significantly prevented somatic-evoked excitation of EAUG (Fig. 7C).
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), we tested whether blockade of NK1-Rs would alter this inhibition. As we have previously shown, somatic afferent stimulation reduced the burst duration of EDEC neurons, accompanied by a reduction in expiratory duration (Fig. 8A). NK1-R antagonism had no effect on somatic evoked reduction in either EDEC burst duration or expiratory duration (Fig. 8B). Table 5 contains group data showing that blockade of NK1-R had no effect on somatic evoked inhibition of EDEC burst duration, the number of action potentials per burst, or the reduction in expiratory duration (n = 7). In addition, the latency for inhibition of EDEC neurons was not altered by NK1-R blockade (89.7 ± 17.8 vs. 109 ± 30.8 ms, control vs. NK1-R blockade, P = 0.372, paired t-test, n = 7).
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The stereotaxic coordinates where E neurons were recorded in the VRG (n = 35) is illustrated in Fig. 9. Recorded neurons were distributed in a longitudinal column ventral of nucleus ambiguus extending rostrocaudally from the caudal pole of the facial nucleus to calamus scriptorius (CS). These neurons were concentrated in two regions, 300–600 µm caudal to the caudal pole of the facial nucleus and 300–600 µm rostral of CS corresponding to the BötC and rVRG, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Substance P modulates the excitability of expiratory neurons
Substance P is a potent stimulator of respiration and can excite inspiration and prolong expiration by selectively activating separate populations of respiratory neurons in the VRG (Chen et al. 1990a,b; Fong and Potts 2006; Gray et al. 1999; Hedner et al. 1984; Morgado-Valle and Feldman 2004; Ptak et al. 2000a). The role of SP and NK1-R has been the focus of much research as it is involved in mediating a number of respiratory reflex responses, including hypoxic ventilatory response and central chemosensitivity (Chen et al. 1990a; Gray et al. 2001; Mazzone et al. 1997; Nattie and Li 2002; Ptak et al. 2002; Wickstrom et al. 2004). Although NK1-R are localized throughout the VRG (Gray et al. 1999; Guyenet et al. 2002; Nakaya et al. 1994; Wang et al. 2001) with expression confirmed on preinspiratory neurons (Guyenet and Wang 2001), the effect of direct activation of NK1-R expressing expiratory neurons on respiratory rhythm has not been thoroughly explored. Although substance P can decrease respiratory frequency by prolonging expiratory duration (Chen et al. 1990b; Fong and Potts 2006; Makeham et al. 2005), the functional expression of NK1-R on individual expiratory neurons has not been demonstrated. We now provide evidence that exogenous SP modulates the excitability of a subpopulation of E neurons suggesting a possible functional role for NK1-Rs in an expiratory region of the VRG. The ability of the selective NK1-R antagonist, CP99,994, to block the effects of exogenous SSP further strengthens the proposal that excitation of E neurons by SSP was mediated by selective activation of NK1-Rs.
The increase in excitability of E neurons by SSP agrees with previous data demonstrating that exogenous SP can increase the discharge rate of several neuronal populations including, phrenic motor neurons (Ptak et al. 2000b), C1 bulbospinal neurons in the rostroventral lateral medulla (Li and Guyenet 1997), midbrain periaqueductal gray neurons (Drew et al. 2005), and NTS neurons (Boscan et al. 2002). Although the mechanism by which NK1-R activation leads to an increase in neuronal excitability remains to be determined, this effect may be mediated through a reduction in leak K+ conductance that would result in raising resting membrane potential, possibly via inhibition of TASK channels (Boscan et al. 2002; Li and Guyenet 1997; Ptak et al. 2000b; Washburn et al. 2003). As TASK channels are modulated by receptors coupled to G
q proteins, such as NK1-R, and these channels are expressed on majority of NK1-R expressing neurons throughout the VRG (Washburn et al. 2003), this is a likely possibility. Alternatively, NK1-R activation could increase the excitability of E neurons through phosphorylation of G proteins leading to turnover of intracellular inositol triphosphate (IP3) to raise intracellular calcium (Harrison and Geppetti 2001; Washburn et al. 2003). Finally, NK1-R may increase neuronal excitability by modulating R-type Ca2+ currents (Meza et al. 2007). Thus future work is needed to identify the cellular mechanism(s) of NK1-R activation on membrane excitability of E neurons.
Reflex activation of NK1-R on expiratory neurons modulates respiratory rhythm
The lack of effect on the basal activity of E neurons following blockade of NK1-R (Table 4) suggests there is no tonic release of endogenous SP onto expiratory neurons of the VRG. Thus this raises the following question: under what condition(s) is endogenous SP released? Data from the present study found that a heterogeneous population of E neurons, including EDEC and EAUG neurons, was excited by exogenous SP. This suggests that endogenous SP and NK1-R may be involved in modulating the excitability of EDEC and EAUG neurons, and these neurons are involved in modulating respiratory rhythm in response to activation of a variety of afferents, including arterial chemoreceptors (Sun and Reis 1996), pulmonary stretch receptors (Hayashi et al. 1996), and somatic afferents (Potts et al. 2005). It has previously been shown that activation of the slowly adapting pulmonary stretch receptors (SARs) by the Hering-Breuer reflex (HB-reflex) excites EDEC neurons in the VRG (Hayashi et al. 1996). Recently we have demonstrated that extension of expiratory duration by SARs of the Hering-Breuer reflex requires the activation of NK1-R in the BötC (Fong and Potts 2006). We therefore posited that activation of SARs evokes endogenous release of SP in the BötC, which, in turn, activates NK1-R and excites a subpopulation of EDEC neurons. This excitation leads to prolonged inhibition of EAUG neurons, resulting in extension of expiration, as proposed by current models of respiratory rhythm generation (Fong and Potts 2006; Richter et al. 1986; Rybak et al. 2004) (see also Fig. 10). We now show that NK1-Rs are functionally expressed on a subpopulation of propriobulbar EDEC neurons that support our earlier proposal.
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; Potts et al. 2005; Rybak et al. 2004). However, because EAUG neurons are excited by somatic afferent stimulation (Potts et al. 2005), their excitation may require endogenous SP. Substance P has been demonstrated to participate in the somatic afferent reflex at the level of the spinal cord (Wilson et al. 1993a,b) and the nucleus tractus solitarius (NTS) (Potts et al. 1999). Because spinal dorsal horn neurons project predominately ipsilaterally throughout the VRG (Potts et al. 2002), we hypothesized that excitation of EAUG neurons and LRC evoked by somatic afferent stimulation requires NK1-R activation in the ipsilateral VRG. We determined that NK1-R blockade in the ipsilateral BötC by microinjection of CP eliminated the phasic coupling of respiratory and skeletal muscle activity. Additionally, we determined this phasic coupling was mediated by activation of NK1-Rs on EAUG neurons, as the contraction evoked excitation of EAUG neurons was abolished following NK1-R blockade. Together these findings demonstrate that endogenous SP is released in the VRG during somatic afferent stimulation to excite BötC EAUG neurons that participate in the coupling of respiratory and locomotor rhythms. However, it is interesting to note that although LRC was blocked by CP, the overall excitation of respiratory activity (i.e., PND frequency) was unaltered. This finding was similar to the effect we observed following pharmacological blockade of synaptic transmission in the lateral parabrachial nucleus (LPBN) (Potts et al. 2005), suggesting that SPergic mechanisms in the BötC contribute to phase-transitions in respiratory rhythm.
Interestingly, both the HB-reflex (Fong and Potts 2006) and LRC require SPergic signaling in the BötC. Activation of these two reflex pathways result in opposing effects on respiratory rhythm: the HB-reflex extends expiration and slows respiratory rhythm; whereas LRC excites respiration by reducing expiratory duration. These two reflex pathways also have different synaptic targets: the HB-reflex excites EDEC neurons while somatic afferent stimulation excites EAUG neurons (although both of these E populations are excited by exogenous SSP). This finding suggests that multiple afferent pathways innervating different E populations utilize SP as a neuromodulator.
In the present study, we did not directly determine whether the neurons we recorded were bulbospinal neurons or vagal motorneurons. However, our data support the idea that these neurons were likely propriobulbar neurons based on the finding that a subpopulation of NK1-R expressing EDEC neurons that inhibit EAUG neurons in the BötC following HB-reflex activation (Fong and Potts 2006). Additionally, earlier work from our lab found that EAUG neurons increase respiratory frequency by inhibiting EDEC neurons in the BötC (Potts et al. 2005). Thus our present data are consistent with the notion that NK1-Rs are expressed on a subpopulation of propriobulbar EAUG neurons. However, we cannot rule out the possibility that NK1-Rs may also be expressed on expiratory premotor neurons as has been reported on a subpopulation of inspiratory premotor neurons (Guyenet et al. 2002).
Previously, we hypothesized that a possible source of SP for the excitation of EDEC neurons by SARs may be pump neurons in the NTS (Fong and Potts 2006) (see Fig. 10). We now propose that LPBN may be an additional source of SP to the BötC that specifically targets EAUG neurons. This is based on of the following observations: 1) inhibition of LPBN (Potts et al. 2005) as well as blockade of NK1-R in the BötC, result in a similar loss of phase coupling of respiration to muscle contraction without a loss in overall excitation of respiration; 2) SPergic neurons are localized in the LPBN (Block and Hoffman 1987; Douglas et al. 1982; Leger et al. 1983; Potts et al. 2005); and 3) BötC receives direct projections from respiratory neurons in the dorsolateral pons region, including the LPBN (Ezure and Tanaka 2006). We propose that ascending somatosensory input from the spinal dorsal horn transmitting sensory information from contracting muscles excites SPergic neurons in the LPBN that in turn project to and excite EAUG neurons in the VRG through release of SP (Fig. 10), although a direct SPergic projection from the LPBN to the BötC remains to be confirmed. Taking these current findings together with the existing knowledge that NK1-R are vital to the hypoxic ventilatory response and central chemosensitivity (Chen et al. 1990a; Ezure and Tanaka 2006; Gray et al. 2001; Mazzone et al. 1997; Nattie and Li 2002; Ptak et al. 2002; Wickstrom et al. 2004) further establishes a fundamental role for the endogenous release of SP in modulating E neuron excitability and demonstrate that the modulation and formation of respiratory rhythm depends on reflex specific inputs to BötC.
Technical considerations
The picoejection method employed in the current study has been used by a number of other investigators to examine the effect of discrete drug application onto recorded neurons (Boscan et al. 2002, 2005; Brandes et al. 2006). This method allows for highly localized application of drugs onto functionally identified neurons using drug solutions at physiological pH 7.4, compared with iontophoresis, which typically requires the solutions to carry an ionic charge that alters solution pH. The ability to use solutions at physiological pH is particularly important as neurons in the ventrolateral medulla, including the VRG neurons, which have been shown to express TASK channels, can be inhibited by changing extracellular pH (Washburn et al. 2003). Due to this characteristic of neurons, we felt that the use of drug solutions departing from neutral pH may make it difficult to differentiate between the direct effects of the drug versus the indirect effect of pH, thus making the results difficult to interpret.
Although we were unable to directly measure the volume of drug ejected, we are confident that each ejection was localized to a finite volume as there was no effect on network output, including no change in the frequency or amplitude of the phrenic motor output (Table 3). An earlier study by Monnier and colleagues (2003) showed that microinjection volumes as small as 3 nl into the VRG can affect network output. Similarly, we recently showed in the same preparation that microinjection volumes as small as 4–6 nl of SSP at a similar concentration to that used in the current study could produce profound effects on respiratory frequency (Fong and Potts 2006). Together with the slow ejection rate, the short-duration of ejection time (30 s) and the rapid decrease in concentration of the drug relative to the distance from the pipette tip (Stone 1985), we propose that the sphere of influence for the picoejected drug was highly localized and mainly affected the recorded neuron. While we cannot exclude the possibility that the drug may have produced presynaptic effects, NK1-R labeling is predominantly localized to the soma and dendrites of VRG neurons (Liu et al. 2004), suggesting that the likely site of action was postsynaptic.
Summary
Data from the present study identify a subpopulation of E neurons that express NK1-R the activation of which increases neuronal excitability. Following blockade of NK1-R, the basal activity of E neurons remained unchanged, suggesting that SP does not significantly contribute to the basal level of excitability of these neurons nor to the generation of respiratory rhythm during eupnea. However, its endogenous release plays an important role in modulating respiratory rhythm during reflex activation. Indeed, LRC evoked by somatic afferent stimulation requires functional NK1-R expression on EAUG neurons in the BötC. Together with existing data, the present findings further establish the functional importance of SPergic mechanisms in modulating respiratory rhythm by increasing the excitability of EDEC and EAUG neurons in a reflex pathway-specific manner.
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Present address and address for reprint requests and other correspondence: J. T. Potts, Dept. of Integrative Physiology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 (E-mail: jepotts{at}hsc.unt.edu)
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PND). B: waveform analysis of action potential trajectory before (baseline), during (SSP), and after (recovery) SSP picoejection from A1. These data confirm that this recording was from a single unit and that shape of the action potential was not affected by picoejection of SSP. The number of action potential shown in each overlay is indicated. C: quantification of the effect of picoejection of SSP alone or immediately after NK1-R antagonist, CP99,994. SSP alone resulted in an increase in the number of action potentials per burst (# APs), mean, and peak frequency. The effect of SSP was completely blocked in the presence CP99,994. * = P < 0.05, One-way repeated-measures ANOVA, post hoc Student Newman-Keul (SNK), n = 5. Burst Dur, Burst duration; Mean freq, Mean frequency, Peak freq, Peak frequency.
) there was a tight correlation between the onset of muscle tension and PND (average latency = 126 ms). This relationship was lost following NK1-R blockade with CP99,994 (
) in the BötC (average latency = 606 ms). D: box-whisker plot of the latency between contraction onset and PND during control (
