Muscimol inhibition of medullary raphé neurons decreases the CO2 response and alters sleep in newborn piglets

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Muscimol inhibition of medullary raphe´ neurons decreases the CO2 response and alters sleep in newborn piglets Michelle L. Messier *, Aihua Li, Eugene E. Nattie Department of Physiology, Dartmouth Medical School, Borwell Building, 1 Medical Center Drive, Lebanon, NH 03756-0001, USA Accepted 20 August 2002

Abstract Medullary raphe´ neurons are chemosensitive in vitro (Wang et al., J. Physiol. Lond. 511 (1998)), are involved in the ventilatory response to CO2 in vivo (Dreshaj et al., Respir. Physiol. 111 (1998); Nattie and Li, J. Appl. Physiol. 90 (2001)), and are abnormal in many Sudden Infant Death Syndrome (SIDS) victims (Panigrahy et al., J. Neuropathol. Exp. Neurol. 59 (2000)). In this study we determine whether the ventilatory response to CO2 is altered when medullary raphe´ neuronal function is focally and reversibly inhibited in chronically instrumented newborn piglets. Ventilation was measured by whole body plethysmography in room air and in 5% CO2 before and during microdialysis of muscimol, a g-amino butyric acid (GABAA) receptor agonist, into the medullary raphe´. Muscimol (10 mM in the dialysate), had no effect on eupneic ventilation, but reduced significantly the CO2 response by 17% during wakefulness. Sleep cycling was also disrupted, as characterized by a significant increase in the percentage of time spent awake and a significant decrease in the percentage of time spent in NREM sleep. Disturbances of medullary raphe´ function can alter central chemoreception and normal sleep architecture, which may contribute to the pathogenesis of SIDS. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Brainstem, medullary raphe´; Carbon dioxide, central chemosensitivity; Chemosensitivity, central, CO2; Control of breathing, CO2 sensitivity; Disease, SIDS; Mammals, piglet; Pharmacological agents, muscimol

1. Introduction Medullary raphe´ neurons are situated in the midline and parapyramidal regions extending from the pontomedullary border to the brainstem
/ spinal cord junction. This neuronal population is heterogeneous, containing both serotonergic and

* Corresponding author. Tel.: /1-603-650-7726; fax: /1603-650-6130 E-mail address: [email protected] (M.L. Messier).

non-serotonergic neurons that fire differently across the sleep/wake cycle (Sheu et al., 1974; Heym et al., 1982). Medullary raphe´ neurons project extensively throughout the brainstem and spinal cord and play a role in the modulation of a variety of homeostatic processes, including: sensory and motor output (Jacobs and Fornal, 1991, 1997), blood pressure regulation (Rathner and McAllen, 1999) and blood flow (Blessing and Nalivaiko, 2000), respiration (Bernard et al., 1996; Wang et al., 1998, 2001; Dreshaj et al., 1998, Nattie and Li, 2001), thermoregulation

1569-9048/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 9 - 9 0 4 8 ( 0 2 ) 0 0 1 6 8 - 4

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(Berner et al., 1999; Morrison et al., 1999), nociception (Leung and Mason, 1999), and sleep and arousal (Portas et al., 2000). The medullary raphe´ contains chemosensitive neurons that increase ventilation in response to elevations in brain CO2 and/or H  levels (Bernard et al., 1996; Dreshaj et al., 1998; Wang et al., 1998, 2001). The firing rate of medullary raphe´ neurons in culture increases in response to increased CO2 (Wang et al., 1998, 2001). Excitation of the raphe´ by either microinjection of acetazolamide (1 nl, 5 /106 M) (Bernard et al., 1996) or glutamate (10 nl, 10
/1000 mM) (Bernard, 1998) results in an increase in phrenic nerve activity in the anesthetized rat. Inhibition of the raphe´ by successive microinjections (300 nl each) of either ibotenic acid (5 mg/ml) or lidocaine (2%) along the rostrocaudal extent of the raphe´ significantly reduces phrenic and hypoglossal nerve responses to CO2 in decorticate or anesthetized piglets (Dreshaj et al., 1998). These studies demonstrate that the raphe´ is chemosensitive and is involved in the normal ventilatory response to CO2. Recent brainstem research in Sudden Infant Death Syndrome (SIDS) victims has uncovered an abnormality in the medullary serotonergic system (Kinney et al., 2001), including the medullary raphe´ (Panigrahy et al., 2000), supporting the hypothesis that SIDS victims are not normal prior to death, although they appear to be in good health (Filiano and Kinney, 1994). In one data set, made up of 84 cases collected at The Boston Children’s Hospital over a 10-year period (1985
/ 1995), mean serotonergic binding was analyzed in 52 SIDS cases (cause of death undetermined), 15 acute controls (cause of death established at autopsy), and 17 chronic controls (deaths attributed to known oxygenation disorders) (Panigrahy et al., 2000). Nineteen brainstem nuclei, including the medullary raphe´, were analyzed for [3H]lysergic acid diethylamide ([3H]-LSD) binding, a serotonin receptor agonist binding to 5HT1A-D and 5HT2 receptors. Results showed reduced mean serotonergic receptor binding in the raphe´ (289/3 fmol/mg tissue) compared to either the acute controls (669/6 fmol/mg tissue) or chronic controls (599/1 fmol/mg tissue). This 50
/60% decrease in mean serotonergic receptor binding suggests an

abnormality in the medullary raphe´ of SIDS victims in this data set (Panigrahy et al., 2000). Since the raphe´ is involved in the modulation of a variety of homeostatic processes, including respiratory control, abnormalities in this brainstem region could prevent a vulnerable infant from responding to a life-threatening challenge, such as hypercapnia, during sleep. The purpose of this study was to determine the effects of medullary raphe´ inhibition on the CO2 response in the unanesthetized newborn piglet across the sleep/wake cycle. Using the microdialysis technique, we focally and reversibly inhibited medullary raphe´ neurons via the GABAA receptor agonist muscimol. We chose this agent since GABAA receptors colocalize with both serotonergic and non-serotonergic (GABAergic) medullary raphe´ neurons (Gao et al., 1993). Ventilation, measured by whole body plethysmography, was studied in room air and in 5% CO2 before and during 10 mM muscimol microdialysis into the raphe´. We hypothesized that muscimol would inhibit both serotonergic and non-serotonergic medullary raphe´ neurons expressing the GABAA receptor, resulting in a decreased ventilatory response to CO2.

2. Methods Experiments were performed on 21 newborn Yorkshire or Duroc piglets of either sex, ages 4
/12 days old (mean age9/S.E.M.; 7.49/0.55) and weighing between 1.6 to 3.6 kg (2.39/0.12). Piglets were housed pre- and post-operatively in a farrowing crate with sow in a controlled light (12-h light
/ dark cycle) and temperature (229/1 8C) environment in the Animal Research Facility. The Institutional Animal Care and Use Committee of Dartmouth College approved all surgical procedures and experimental protocols. Experiments were performed within the first 4 days after surgery. In some instances piglets were used for an additional experimental protocol with a different drug treatment after testing with muscimol was complete.

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2.1. Chronic instrumentation The surgical preparation is similar to previously described methods (Curran et al., 2001; Darnall et al., 2001). Briefly, piglets were anesthetized (2% isofluorane in O2) and artificially ventilated for the duration of surgery. They were kept hyperoxic and ventilated at a level to maintain a PCO2 between 35 and 40 mmHg. A heating pad was used to maintain body temperature between 37 and 39 8C. A dual-lumen umbilical catheter (3.5 French) was inserted 7.5 cm into a branch of the femoral artery. This catheter served the dual purposes of monitoring blood pressure, while also allowing for the sampling of arterial blood for blood gas measurements. Both lumens were flushed daily and the dead space was filled with heparin (1000 U/ml) to prevent the lines from clotting. A thermister was implanted subcutaneously into the abdomen, approximately 1
/2 cm lateral to the midline. Both the arterial catheter and thermister were tunneled subcutaneously and exited out the back. Piglets were mounted in a Kopf stereotaxic frame and the skull was exposed by a midline incision. Stereotaxic measurements that included bregma, lambda, and a fiducial mark on the right ear bar were taken and the midline was predicted using a regression equation. A burr hole was then drilled at the predicted coordinate and a guidetube with stylette (Bioanalytical Systems, Inc.) was inserted into the medullary raphe´ with the tip located approximately 1 mm above the ventral surface of the brainstem. Electroencephalogram (EEG) electrodes were screwed into the left frontal and right parietal bones. A common electrode was screwed into the right frontal bone. Electrooculogram (EOG) electrodes were positioned lateral to the left eye and superior to the orbit of the right eye. A bipolar electrode was used to record neck electromyogram (EMG) activity. A ground electrode was implanted subcutaneously in the neck. Brass connectors attached to the end of the electrodes were plugged into two plastic pedestals and the pedestals and guidetube were cemented to the skull with cranioplastic cement. Cefazolin (20 mg/kg, i.v.) was given prior to surgery and dexamethasone (1
/2 mg/kg, i.v.) was

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given prior to implantation of the guidetube into the brainstem. Buprenorphine (0.1 mg/kg i.m.) was given immediately post-surgery for analgesia. Piglets received the antibiotic trimethaprim sulfa (120 mg) mixed in with piglet formula each day post surgery until euthanasia. Bacitracin antibiotic ointment was applied topically to all incisions daily. The animals tolerated surgery well and were healthy, as evidenced by active ambulation and increased weight gain postoperatively. 2.2. /V˙ E measurement Whole body plethysmography (Drorbaugh and Fenn, 1955; Bartlett and Tenney, 1970) was performed as modified by Pappenheimer (1977), Jacky (1978) and Curran et al. (2001). A pressure transducer (Validyne) was used to measure respiratory related pressure deflections that result from inspiration and expiration. Large pressure fluctuations were avoided by creating a leak between the plethysmograph and a reference chamber. Analog signals from the pressure transducer were digitized, sampled at 1 kHz by a computer data acquisition system (PowerLab, ADInstruments), and stored for off-line analysis. A flowmeter (Hastings) was used to maintain a constant and balanced flow of inlet and outlet gas through the chamber via high resistance inlet and outlet ports. A high flow rate through the plethysmograph (/8 L/min) prevented animals from rebreathing CO2 and maintained high inspired O2 levels. Inlet gas was warmed to 38 8C and humidified. An O2 analyzer (Applied Electrochemistry) was used to sample both inlet and outlet [O2]. These measurements were used in conjunction with gas flow rate to calculate oxygen consumption. A CO2 analyzer (CWI) was used to measure inspired [CO2]. CO2 challenges were performed by using a gas mixer (Bird Corp.) to bleed 40% CO2 into the plethysmograph inflow until the inspired [CO2] reached 5%. It took approximately 3 minutes for the plethysmograph to equilibrate to 5% CO2. Neither baseline plethysmograph pressure nor flow rate were affected by the CO2 challenge. Prior to the start of each experiment the plethysmograph was calibrated with triplicate injections of 1, 2, 3 and 5 ml of air.

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2.3. Experimental protocol Piglets were placed in the prone position in a sling that was suspended from a metal frame inside the plethysmograph. Respiration, blood pressure (MAP), body temperature (Tb), and sleep cycling were continuously recorded (YSI). Piglets were videotaped during the experiment for later analysis of behavioral sleep state. On the day of an experiment, the stylette was replaced with a microdialysis probe (1 mm tip length, 250 mm diameter) that was continuously dialyzed with artificial cerebrospinal fluid (aCSF) at a flow rate of either 8.5 ml/min (n/14) or 4.0 ml/min (n/7). These different flow rates had no discernible effect so the results were pooled. The plethysmograph, with the piglet in it, was equilibrated over a 40-min period, during which time the inspired [CO2] and [O2], humidity, and plethysmograph air temperature were allowed to stabilize, and the piglet was allowed to acclimate to the box. Approximately 20 min of baseline measurements were taken with the piglet breathing room air (baseline I). A 5% CO2 challenge was then performed, in which inspired [CO2] was raised to 5% for approximately 10 min (pre-drug CO2 challenge). The box was then flushed of the CO2, and after an equilibration period, another 20 min of baseline measurements were taken with the piglet breathing room air (baseline II). At this point, aCSF microdialysis was stopped and 10 mM muscimol microdialysis was begun. Drug dialysis lasted an average of 20 min, 10 min of which were at room air breathing, followed by a 10 min 5% CO2 challenge. The experiment was then ended. 2.4. Neuroanatomy At the end of an experiment 10 mM fluorescein, a fluorescent dye with a similar molecular weight to muscimol, was microdialyzed at the probe placement site for the same length of time that muscimol was dialyzed. The brainstem was then quickly removed, frozen on dry ice, and sectioned at 50 mm in a Reichert-Jung cryostat. Sections were viewed under a fluorescence microscope (Olympus), allowed to dry, and then stained with

cresyl violet. Fluorescence and cresyl violet images were captured and overlaid in the computer program ImagePro. These anatomical techniques allowed for an approximation of drug spread and determination of probe location within the brainstem. 2.5. Data analysis Data were analyzed as previously described by Curran et al. (2001) and Darnall et al. (2001). Briefly, mean arterial blood pressure (MAP) and heart rate (HR) were derived from arterial blood pressure recordings (MATLAB). Tidal volume (VT), respiratory rate (fR), and minute ventilation (/V˙ E ) were computed from plethysmograph respiratory pressure fluctuations. All signals were resampled at 100 Hz. EEG and EOG signals were filtered with a bandpass of 0.3
/30 Hz. The EEG record was divided into five-second epochs and power spectral analysis was performed (delta, 0.5
/ 4.0 Hz; theta, 5.0
/9.0 Hz; sigma, 10.0
/14.0 Hz). The EMG signal was filtered with a band pass of 10
/100 Hz (Grass). Animals were videotaped for behavioral sleep analysis. Each five-second epoch of the videotape was scored for eye openings and body movements. Wakefulness was defined as a period of low to moderate EEG amplitude and delta power, eyes open or closed, moderate EMG activity, and occasional body movements. Non rapid eye movement (NREM) sleep was defined as a period of high EEG amplitude and delta power, with eyes closed, reduced EMG tone and infrequent body movements. Rapid eye movement (REM) sleep was defined as a period of low EEG amplitude and delta power, decreased V˙ E and MAP, absent EMG tone, rapid eye movements, eyes closed, and limb twitches. Not all criteria were always available to determine sleep state. Breaths were selected from representative sleep states (quiet wakefulness, NREM sleep, REM sleep). Breaths from baseline I and baseline II were averaged together since breathing was not significantly different between baselines. Periods of active wakefulness were omitted from ventilatory analysis. During quite wakefulness, each period selected consisted of an average of 39/0.8

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min (mean9/S.E.M.) of data, made up of an average of 1429/46 breaths (mean9/S.E.M.). Periods were averaged together (e.g. all periods of wakefulness during baseline), for each animal. Data from all piglets were then averaged together for each experimental condition. To be included in respiratory analysis, piglets had to have an increase in V˙ E (e.g. an increase in both VT and fR) at the pre-drug CO2 challenge. For inclusion in sleep analysis, each piglet had to cycle through NREM sleep at least once during both of the pre-drug room air measurements; the first one occurring prior to the pre-drug CO2 challenge (baseline I) and the second one occurring subsequent to the CO2 challenge, but prior to the start of muscimol dialysis (baseline II). 2.6. Statistics The results for V˙ E ; VT, fR, V˙ O2 ; MAP, HR and Tb measured during quiet wakefulness were compared before and during muscimol dialysis using a student’s paired t-test. Ventilation, measured during NREM sleep, was compared with an unpaired t-test. Sleep disruption was analyzed by one way repeated measures ANOVA with the appropriate post hoc test performed when statistical significance was reached. In some instances, the Friedman repeated measures ANOVA on ranks was necessary. A two way repeated measures ANOVA was used to examine the interaction between muscimol and CO2. All data is in the form of mean9/S.E.M.

3. Results 3.1. Neuroanatomy Piglets were grouped according to anatomical localization of probe placement, as verified by fluorescein spread and cresyl violet staining. Probe placement was considered to be in the medullary raphe´ if the fluorescein spread was found either in the midline or parapyramidal region, lying between the pontomedullary border to the brainstem
/spinal cord junction. However, the majority of the probes were located at the level

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of the facial nucleus, an area that contains serotonergic neurons in the newborn piglet brainstem (Kinney et al., Personal communication). We did not differentiate between the three raphe´ subdivisions (magnus, pallidus, obscurus) in this paper. Of the 21 piglets studied, five were excluded from data analysis. One animal was excluded because of an undetermined probe location, two because fluorescein spread, which we used to indicate drug spread, was not visible in the brainstem, one because of a defective microdialysis probe, and one because of an absent pre-drug 5% CO2 response. Of the remaining 16 animals, 12 had probe placements in the medullary raphe´; nine were in the midline, and three were located in the parapyramidal region (Fig. 1). Eleven of these 12 animals were treated with 10 mM muscimol; the twelfth animal received only aCSF and was part of the aCSF control group. Eight of the remaining 11 animals had a decreased CO2 response with muscimol dialysis. Seven of the eight probe placements were located at the level of the facial nucleus and one was found at the level of the middle inferior olive. Of the three animals that did not have a decreased CO2 response during muscimol dialysis, one probe was located parapyramidally at the level of the facial nucleus, one in the midline at the level of the caudal superior olive, and the third was located midline and dorsal to the ventral surface at the level of the rostral inferior olive. Four of the 16 piglets included in data analysis had probe placements located outside of the raphe´ and were considered to be anatomical controls since muscimol was dialyzed outside of our region of interest; two probes were too rostral, one was too caudal, and one was within the rostrocaudal axis of the raphe´ but lateral to the midline and dorsal to the ventral surface. 3.2. Typical experiment Fig. 2 shows a representative plethysmograph pressure tracing from one piglet for a typical experiment. Breathing in room air and in 5% CO2 before muscimol dialysis is depicted in Fig. 2a. Absolute V˙ E measured in 5% CO2 was

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Fig. 1. Twelve microdialysis probe locations in the medullary raphe´. (A) Schematic series of cross-sections taken from a piglet brainstem from rostral (top) to caudal (bottom). Scale bar is 1 mm. Closed circles (m) indicate a decreased CO2 response during 10 mM muscimol dialysis. Open circles (k) indicate no change in the CO2 response during dialysis. One of the 12 animals was treated with aCSF only and did not receive muscimol (w) (SO, superior olive; VII, facial nucleus; IO, inferior olive). (B) Sagittal section of the piglet brainstem illustrating the corresponding location from which the cross sections are derived. The facial nucleus is colored gray.

significantly greater than absolute V˙ E measured in room air (paired t-test, P B/0.001). Increases in both VT (130% of control) and fR (172% of control) contributed to the increase in V˙ E (234% of control). Fig. 2b shows the CO2 response from the same animal during 10 mM muscimol microdialysis into the medullary raphe´. The increase in V˙ E from room air to 5% CO2 during muscimol dialysis was only 174% of control; VT was increased by 121% of control and fR was increased by 135% of control. This was indicative of a 29% decrease in the VT response to CO2, a 52% decrease in the fR response to CO2, and a 46% decrease in the V˙ E response to CO2 with muscimol treatment in this one representative animal.

3.3. Ventilatory data 3.3.1. Ventilatory response to 5% CO2 during wakefulness with 10 mM muscimol microdialysis The averaged group ventilatory data (n /11) are shown in Fig. 3. The pre-drug 5% CO2 challenge was done with aCSF (vehicle) dialyzing through the microdialysis probe. Absolute V˙ E measured in 5% CO2 was significantly greater than absolute V˙ E measured in room air (Fig. 3a) (paired t-test, P B/0.001). This increase was due to increases in both VT and fR (Fig. 3b). Absolute V˙ E measured in 5% CO2 during muscimol dialysis was also significantly greater than the absolute V˙ E measured in room air during muscimol dialysis

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Fig. 2. Respiratory pressure deflections (Resp) representative of breathing in room air and in 5% CO2 from one piglet in quiet wakefulness. Right: records obtained in 5% CO2. Left: records obtained in room air. (A) Control period with aCSF dialysis. (B) Plethysmograph tracings obtained during microdialysis of 10 mM muscimol into the medullary raphe´.

(Fig. 3a) (P B/0.001). However, absolute V˙ E measured in 5% CO2 during muscimol dialysis was significantly decreased compared to the absolute V˙ E measured in 5% CO2 during aCSF (vehicle) dialysis (paired t-test, P /0.012). Absolute V˙ E in 5% CO2 was decreased by 17% (paired ttest, P /0.012) and the change in ventilation from room air to 5% CO2 during muscimol dialysis was 18% less than the change in ventilation from room air to 5% CO2 prior to drug treatment (paired ttest, P /0.036). Decreases in both absolute VT (7%, paired t-test, P /0.157) and absolute fR (11%, paired t-test, P /0.070) contributed to the decrease in absolute V˙ E in 5% CO2 during

muscimol dialysis, although these differences were not statistically significant. Room air breathing during aCSF (vehicle) dialysis was not different from room air breathing during muscimol dialysis (paired t-test, P /0.173). However, in 9 of the 11 animals, V˙ E did decrease by 16% in room air breathing during muscimol dialysis. This was due to decreases in both VT and fR. 3.3.2. Ventilatory response to 5% CO2 during NREM sleep with 10 mM muscimol microdialysis The ventilatory data that we have during NREM sleep are fragmentary because the piglets

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Fig. 3. Mean9/SEM absolute values for minute ventilation (/V˙ E ); tidal volume (VT), and respiratory frequency (fR) as a function of plethysmograph CO2; n/11. Ventilatory measurements were made during room air breathing and in 5% CO2 before (with aCSF microdialysis) (m) and during 10 mM muscimol dialysis (k) into the medullary raphe´. (A) 10 mM muscimol decreased significantly both absolute /V˙ E in 5% CO2 (P /0.012), as well as the ventilatory response to CO2 (P /0.036) in quiet wakefulness. * Statistical significance (P B/0.05). (B) Reductions in both VT and fR contributed to the decreased ventilatory response to CO2.

tended to arouse with the 5% CO2 stimulus, and also because muscimol itself disrupted sleep (see below). Thus, it was difficult to examine the CO2 response during sleep. Five of nine animals cycled through NREM sleep in both room air and in 5% CO2 during baseline measurements with aCSF

dialysis. Two of nine cycled through NREM sleep in room air and in 5% CO2 during 10 mM muscimol dialysis. Ventilation was compared in these animals. Pre-drug absolute V˙ E measured in room air was 658.39/30.7 ml/kg/min and increased to 1226.09/84.9 ml/kg/min in 5% CO2 (n/5).

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Absolute V˙ E measured in room air during muscimol dialysis was 527.49/5.5 ml/kg/min and increased to 1206.59/290.8 ml/kg/min in 5% CO2 (n/2). Analysis of these limited data shows that there was no effect of muscimol on the absolute V˙ E in 5% CO2 during NREM sleep compared to the pre-drug absolute V˙ E measured in 5% CO2 (unpaired t-test, P /0.930).

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3.3.3. Ventilation measured in the aCSF control group aCSF was continuously microdialyzed throughout the entire experimental protocol in five animals during which time piglets were subjected to two 5% CO2 challenges. The ventilatory data for these control experiments are shown in Fig. 4. V˙ E measured during the first 5% CO2 challenge (vehicle) was significantly increased by 233% of control, VT by 145% of control, and fR by 160% of control in 5% CO2. Absolute V˙ E measured during the second 5% CO2 challenge (aCSF) was significantly increased by 243% of control; VT increased by 150% and fR increased by 158%. The two 5% CO2 challenges were not significantly different from each other. Eupneic breathing was not altered in these animals (paired t-test, P /0.404). 3.3.4. Ventilation measured in the anatomical control group Neither room air breathing (paired t-test, P / 0.299) nor ventilation measured in 5% CO2 (paired t-test, P /0.835) were affected in the anatomical control group, in which muscimol was dialyzed outside of the medullary raphe´ (n /4). Baseline pre-drug V˙ E measured in room air during vehicle (aCSF) dialysis was 762.59/64.8 ml/kg/min and increased to 1699.19/224.1 ml/kg/min in 5% CO2. V˙ E measured in room air during muscimol dialysis was 653.399/56.7 ml/kg/min and increased to 1674.539/168.4 in 5% CO2. There was no significant change in VT in room air (paired t-test, P / 0.803) or in 5% CO2 (paired t-test, P /0.293) during drug treatment. Neither was there a significant difference in fR in room air (paired t-test, P /0.262) or in 5% CO2 (paired t-test, P /0.444). The pre-drug CO2 response was not significantly different from the CO2 response seen during 10 mM muscimol dialysis outside of the raphe´.

Fig. 4. Mean9/S.E.M. absolute values for minute ventilation (/V˙ E ); tidal volume (VT), and respiratory frequency (fR) as a function of plethysmograph CO2 measured during quiet wakefulness in the aCSF control experiments; n/5. aCSF was continuously microdialyzed throughout the experimental protocol, during which time piglets were subjected to two CO2 challenges, the first denoted as vehicle (m) and the second as aCSF (k). No significant differences in the CO2 responses were detected in these control experiments.

3.4. /V˙ O2 ; MAP, HR and Tb measured during quiet wakefulness Table 1 shows metabolic rate (/V˙ O2 ); blood pressure (MAP), heart rate (HR) and body temperature (Tb) data measured during quiet wakefulness before and during 10 mM muscimol dialysis into the medullary raphe´ (n/9). Musci-

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Table 1 V˙ O2 ; MAP, HR and Tb measured in room air during quiet wakefulness

/

V˙ O2/ MAP HR Tb

/

10 mM muscimol group (n/9)

aCSF controls (n/4)

Anatomical controls (n/3)

Baseline

Test period

Baseline

Test period

Baseline

Test period

22.99/1.8 739/2 1999/13 37.89/0.4

24.19/1.6 749/2 1999/15 37.99/0.4

21.99/3.1 789/5 1909/10 38.49/0.2

24.49/2.3 789/6 2169/19 38.59/0.3

23.59/1.4 779/3 2329/34 38.29/1.4

25.79/3.1 759/4 2319/27 38.19/1.3

Values are means 9/ SEM. Metabolic rate (/V˙ O2 ); ml O2/kg/min; blood pressure (MAP), mm Hg; heart rate (HR), beats/min; body temperature (Tb), 8C. These parameters were not significantly affected by: 1) 10 mM muscimol microdialysis into the medullary raphe´ (10 mM muscimol group), 2) 10 mM muscimol dialysis outside the raphe´ (anatomical control group), or 3) aCSF microdialysis into the raphe´ (aCSF control group).

mol did not have a significant affect on any of these parameters. Data from 2 of the 11 animals were not included in the metabolic rate average because an O2 consumption measurement was not obtained in these animals. Two animals were excluded from the MAP average due to technical problems. The data from these two animals were also omitted from the HR average. Two animals were excluded from the Tb average because the temperature signal was out of range during the experiment due to problems with the thermister connection. In these two instances, rectal temperature was measured both before and at the end of the experiment and these readings were used so that ventilation could still be calculated in these animals. ˙ O ; MAP, HR and Tb (all measured in room /V 2 air during quiet wakefulness) were unaltered in the aCSF control animals (n/4) (Table 1). Likewise, neither V˙ O2 ; MAP, HR nor Tb were statistically different before or during muscimol dialysis in the anatomical control group (n /3) (Table 1). 3.5. Criteria for sleep analysis Wakefulness, NREM sleep, and REM sleep were identified as previously described (Darnall et al., 2001). Both active and quiet wakefulness were included together in the sleep analysis. In the present study, only those piglets who cycled through NREM sleep at least once during both of the pre-drug, room air baseline measurements (e.g. the initial baseline prior to the pre-drug CO2 challenge (baseline I) and the baseline subsequent

to the CO2 challenge but prior to muscimol dialysis (baseline II)), were included in sleep analysis. This criterion was established in order to show that an arousal stimulus, such as CO2, does not prevent a piglet from cycling through sleep once the stimulus has ended. Two of the 11 piglets treated with muscimol did not meet this criterion, and were excluded from sleep analysis. Data from the remaining nine animals, including data from animals that did not cycle through sleep at a particular condition, were included in group averages. 3.6. Sleep cycling 3.6.1. Sleep cycling prior to muscimol dialysis During the first pre-drug, room air measurement (baseline I), all animals (9 of 9) cycled through sleep (wakefulness 0/NREM sleep 0/ REM sleep 0/wakefulness). The average percentage of time spent in each state is shown in Fig. 5. There was a significant increase in wakefulness at the pre-drug 5% CO2 challenge (one way repeated measures ANOVA, Student
/Newman
/Keuls method, all pairwise multiple comparison, P B/ 0.05). During the pre-drug 5% CO2 challenge, five of nine animals cycled through NREM sleep and two of nine animals cycled through REM sleep. There were significant decreases in NREM sleep (one way repeated measures ANOVA, Tukey test, all pairwise multiple comparison, P B/0.05) and REM sleep (one way repeated measures ANOVA, Student
/Newman
/Keuls method, all pairwise multiple comparison, P B/0.05) at the

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Fig. 5. Mean9/S.E.M. values for the percentage of time spent awake (black bars), in non rapid eye movement (NREM) sleep (white bars), and in rapid eye movement (REM) sleep (gray bars) before (with aCSF dialysis) and during 10 mM muscimol microdialysis into the medullary raphe´ (n/9). Duration of each experimental condition: baseline I, 249/3 min; pre-drug 5% CO2 challenge, 129/2 min; baseline II, 259/3 min; 10 mM muscimol dialyzed during room air breathing, 119/1 min; 10 mM muscimol dialyzed during the 5% CO2 challenge, 119/1 min. Note increased wakefulness and decreased NREM sleep in room air during 10 mM muscimol dialysis. * Significant increase in wakefulness compared to pre-drug baseline I (one way repeated measures ANOVA, Student
/Newman
/Keuls method, all pairwise multiple comparison, P B/0.05). $ Significant decrease in NREM sleep compared to pre-drug baseline II (one way repeated measures ANOVA, Tukey test, all pairwise multiple comparison, P B/0.05). § Significant decrease in REM sleep compared to pre-drug baseline I (one way repeated measures ANOVA, Student
/Newman
/Keuls method, all pairwise multiple comparison, P B/ 0.05).

pre-drug 5% CO2 challenge. Sleep cycling returned during the second, pre-drug, room air baseline (baseline II). The percentage of wakefulness at this condition was not different from the percentage of wakefulness measured in baseline I (repeated measures ANOVA, Student
/Newman
/Keuls method, all pairwise multiple comparison, P / 0.05). Nor were the percentages of NREM sleep (one-way repeated measures ANOVA, Tukey test, all pairwise multiple comparison, P /0.05) or REM sleep (one-way repeated measures ANOVA, Student
/Newman
/Keuls method, all pairwise multiple, P /0.05) significantly different at baseline II. A restoration in sleep cycling during this second baseline confirms that an arousal stimulus, such as CO2, does not prevent the piglets from cycling through sleep after the stimulus has ended.

3.6.2. Sleep cycling during muscimol dialysis */ effects on wakefulness A result of muscimol dialysis was to significantly increase the percentage of time spent awake. The percentage of wakefulness measured in room air breathing during muscimol dialysis was significantly increased compared to the percentage of wakefulness seen in either baseline I or baseline II (one-way repeated measures ANOVA, Student
/ Newman
/Keuls method, all pairwise multiple comparison, P B/0.05). Wakefulness was also significantly increased during the 5% CO2 challenge with muscimol dialysis (one-way repeated measures ANOVA, Student
/Newman
/Keuls method, all pairwise multiple comparison, P B/0.05). There was not a significant interaction between muscimol and CO2 (two way repeated measures ANOVA, P /0.716).

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3.6.3. Sleep cycling during muscimol dialysis */ effects on NREM and REM sleep All animals cycled through NREM sleep in both room air baselines (baselines I and II). Eight of nine continued to cycle through NREM sleep in room air during muscimol dialysis. At this condition there was a significant decrease in percentage of NREM sleep compared to the percentage of NREM sleep seen in baseline II (one-way repeated measures ANOVA, Tukey test, all pairwise multiple comparison, P B/0.05), but not in comparison to baseline I (one way repeated measures ANOVA, Tukey test, all pairwise multiple comparison, P /0.05). Only three of nine animals cycled through NREM sleep at 5% CO2 during muscimol dialysis. The percentage of NREM sleep at this condition was not significantly different from the percentage of NREM sleep measured at the predrug CO2 challenge (one-way repeated measures ANOVA, Tukey test, all pairwise multiple comparison, P /0.05). All animals cycled through REM sleep in both room air baselines (baselines I and II). Five of nine animals continued to cycle through REM sleep in room air during muscimol dialysis. There was not a significant difference between the percentage of REM sleep measured at this condition compared to the percentage of REM sleep seen in either baseline I or baseline II with aCSF dialysis (one way repeated measures ANOVA, Student
/Newman
/Keuls method, all pairwise multiple comparison, P /0.05). Only one animal cycled through REM sleep at 5% CO2 challenge during muscimol dialysis. The percentage of REM sleep at this condition was not significantly different from the percentage of REM sleep measured at the pre-drug CO2 challenge (one-way repeated measures ANOVA, Student
/Newman
/Keuls method, all pairwise multiple comparison, P /0.05). 3.6.4. Sleep cycling in the aCSF control group Sleep cycling was analyzed in three of the five aCSF control animals that cycled through sleep in both pre-drug baselines I and II (Table 2). Each treatment condition lasted for approximately the same duration as was reported for the muscimol data. The aCSF test period did not significantly disrupt the percentage of time spent awake (one

way repeated measures ANOVA, Tukey test, all pairwise multiple comparison, P /0.05), the percentage of time spent in NREM sleep (one-way repeated measures ANOVA, Tukey test, all pairwise multiple comparison, P /0.05) or the percentage of time spent in REM sleep (one-way repeated measures ANOVA, P /0.094). Although the aCSF control animals spent more time awake during both of the pre-drug, room air baseline measurements than did the muscimol group, the data show that the aCSF test period did not further increase wakefulness as was seen during muscimol treatment. 3.6.5. Sleep cycling in the anatomical control group Of the four anatomical controls, three animals cycled through NREM sleep in both baseline I and baseline II, and these data were analyzed for percentage of time spent in each sleep state (Table 2). 10 mM Muscimol dialysis outside of the medullary raphe´ did not significantly disrupt the percentage of time spent awake (one-way repeated measures ANOVA, P /0.174), the percentage of time spent in NREM sleep (one-way repeated measures ANOVA, P /0.164) or the percentage of time spent in REM sleep (one-way repeated measures ANOVA, P /0.350).

4. Discussion 4.1. Main findings Focal and reversible inhibition of the medullary raphe´ with 10 mM muscimol significantly reduced both absolute V˙ E in 5% CO2 by 17% and the change in V˙ E from room air to 5% CO2 by 18%. Additionally, muscimol disrupted sleep cycling, as seen by an increase in the percentage of time spent awake and a decrease in the percentage of time spent in NREM sleep. Our interpretation, at present, is that these are two independent effects. 4.2. Neuroanatomy 4.2.1. Probe location Most of our probe placements were located in the raphe´ at the level of the facial nucleus.

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Table 2 Sleep cycling in the control experiments Sleep state

Pre-drug Room air */baseline I (%)

aCSF controls Awake 579/12 NREM 349/6 REM 99/6

aCSF dialysate 5% CO2 (%)

Room air */baseline II (%)

Room air (%)

5% CO2 (%)

909/6 49/4 69/6

459/9 399/4 169/5

479/14 269/12 279/5

659/10 129/7 239/15

10 mM muscimol Anatomical controls Awake 389/20 NREM 529/15 REM 109/5

779/13 199/13 49/4

179/4 719/5 129/2

449/18 549/17 29/2

649/26 289/20 89/5

Values are means9/S.E.M.; n/3 in the aCSF control group; n/3 in the anatomical control group. Data are expressed as the percentage of time spent awake, in non rapid eye movement (NREM) sleep, and in rapid eye movement (REM) sleep at each experimental condition. The top panel shows data from the aCSF control group in which aCSF was continuously microdialyzed into the medullary raphe´. The bottom panel shows data from the anatomical control group in which 10 mM muscimol was dialyzed outside of the raphe´. Muscimol did not significantly affect sleep cycling in the control experiments.

Autoradiography using [3H]-8-OH-DPAT, a 5HT1A receptor agonist, shows that there is a dense cluster of serotonergic autoreceptors in the midline raphe´ at this level of the newborn piglet brainstem (Kinney et al., Unpublished data). Interestingly, seven of eight animals that had probes located in this region showed a decreased ventilatory response to CO2 during muscimol dialysis (Fig. 1). Three additional probes were located outside this region, but were still included in data analysis. This is because immunohistochemistry for tryptophan hydroxylase (TPOH; the rate-limiting enzyme in serotonin biosynthesis) revealed TPOH-positive neurons distributed throughout the rostrocaudal axis of the medullary raphe´ in the newborn piglet (Messier et al., Unpublished data). Therefore, because these three probes were located in the medullary raphe´, as by our definition of its rostrocaudal extent, they were included in our study. 4.2.2. Region of tissue damage The area of tissue damage, which we consider to be an indicator of the size of the lesion produced by the probe, was measured in the 11 animals treated with 10 mM muscimol. This was done by examining the darkened area around the lesion site that was visible with cresyl violet staining. The

average area of tissue damage for each animal, measured on the sections that contained the middle-most fluorescein spread, was 0.007 cm2. In four of the five animals that were treated with aCSF one day prior to muscimol dialysis, the average area of tissue damage was 0.010 cm2. Even though the size of the lesion was greater in the aCSF control group, there was no significant affect on ventilation in this group, suggesting that the probe damage itself did not influence the experimental results. We believe that the tissue damage resulted from guidetube implantation, as well as from multiple insertions of the microdialysis probe for drug administration. 4.2.3. Volume of drug spread Fluorescein (10 mM in the dialysate) was used to estimate the spread of 10 mM muscimol from the microdialysis probe tip. A limitation of this dye is that it does not have an affinity to a specific receptor, so the fluorescein diffusion may be an overestimate of muscimol diffusion. This limitation has been discussed previously (Curran et al., 2001; Darnall et al., 2001). In the present study, the average cross sectional area of fluorescein was 0.017 cm2 and the average fluorescein length (rostrocaudal spread) was 4.7 mm. Dialysis of 10 mM fluorescein for approximately 20 min at a

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pump rate of either 8.5 ml/min (n /7) or 4.5 ml/min (n/4) produced a volume of spread of 5.8 ml. This is similar to measurements made by Curran et al. (2000), who estimated a volume of spread of 6.3 ml when 10 mM fluorescein was dialyzed for 10 min at a flow rate of 8.5 ml/min. 4.3. Medullary raphe´ involvement in the CO2 response The results of the present study show that inhibition of the medullary raphe´ with 10 mM muscimol significantly reduces the awake CO2 response by 17%. Dreshaj et al. (1998) examined the CO2 response in decorticate or anesthetized 14
/20-day-old piglets after reversible blockade of the raphe´ with either 2% lidocaine, a sodium channel blocker, or chemical lesioning of the raphe´ with ibotenic acid, an excitatory amino acid neurotoxin (Dreshaj et al., 1998). In their experiment, they made 3 to 5, 300nl microinjections 1 mm apart from the pontomedullary junction to the rostral root of the hypoglossal nerve and examined the effects of phrenic and hypoglossal nerve activities to progressive hyperoxic hypercapnia. Their lidocaine data showed a 47% decrease in the phrenic nerve response and a 71% decrease in the hypoglossal nerve response to 7.5% CO2 (n /10). Similarly, at 7.5% CO2, phrenic nerve activity was decreased by 69% and hypoglossal nerve activity decreased by 86% after ibotenic acid lesioning (n/12). A major difference between the Dreshaj et al. (1998) study and ours is that they microinjected at multiple sites along the raphe´, while we microdialyzed at one focal location. With the microdialysis technique there is a substantial drop in the dialysate concentration as the drug diffuses across the membrane tip into the central nervous system (CNS), resulting in an approximate 10
/100-fold reduction in the drug concentration (de Lange et al., 1995). Thus, although we dialyzed with 10 mM muscimol, the actual concentration in the brain tissue was likely to be 1 mM or less. There are a number of reasons why we may not have seen effects as large as those observed in the Dreshaj et al. (1998) experiment. First, we are studying unanesthetized piglets, as opposed to

anesthetized or decorticate animals. Second, we are only affecting a region of the raphe´ in the vicinity of the microdialysis probe tip. Thus, it may be possible that a substantial portion of the raphe´ must be inhibited before there is a large decrease in the CO2 response in the conscious animal. Third, in our experiments we gave CO2 systemically, and thus stimulated all chemosensitive brainstem regions. Thus, although we inhibited raphe´ chemosensitive neurons, other chemosensitive sites were still responsive to the CO2 challenge. Therefore, it is not surprising that we did not see a large depression in the CO2 response when we inhibited only one of many brainstem regions involved in central chemoreception. Fourth, only 8 of the 11 animals showed a decreased response to CO2 with muscimol dialysis, yet we included data from all 11 animals in our analysis since the 11 probe placements were verified anatomically to be within the raphe´. Analyzing data from only the eight animals that showed a decreased CO2 response during muscimol dialysis revealed a 21% reduction in absolute V˙ E and a 32% reduction in the change in V˙ E from room air to 5% CO2. 4.4. Medullary raphe´ chemoreception across the sleep/wake cycle Muscimol did not significantly affect the NREM CO2 response in the few animals from which we were able to obtain NREM sleep data. In support of this, Veasey et al. (1995) showed in freely moving cats that the discharge rates of four of six serotonergic raphe´ neurons, studied during both quiet wake and NREM sleep, were significantly increased in response to hypercapnia (8
/ 10% CO2) during wakefulness only. In contrast, Nattie and Li (2001) focally acidified the medullary raphe´ of the unanesthetized rat by microdialysis of acidic aCSF and saw an increase in respiratory frequency during NREM sleep only. Age, species differences, and differences in experimental design could explain the apparently inconsistent results. Data from our study suggest that the medullary raphe´ functions as a central chemoreceptor during wakefulness in the newborn piglet, however, our sleep data are fragmentary

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and only come from two animals. Additional studies are needed to test this hypothesis. 4.5. Blood pressure and heart rate Muscimol (10 mM) dialysis into the medullary raphe´ did not alter blood pressure or heart rate in room air (Table 1) or in 5% CO2 (data not shown). Dreshaj et al. (1998) did not see changes in blood pressure or heart rate with lidocaine, but they did observe an initial depressor response with ibotenic acid microinjections into the medullary raphe´ of decorticate or anesthetized piglets. In a separate study, electrolytic lesioning of the medullary raphe´ in anesthetized cats did not affect blood pressure or heart rate (McCall and Harris, 1987). Our data obtained from unanesthetized piglets agree with these data.

211

extended period of time in room air and in 5% CO2 is needed to support our finding. Inhibition of medullary raphe´ neurons containing the GABAA receptor was involved in a disruption of sleep cycling. The medullary raphe´ contains both GABAergic and serotonergic neurons that express the GABAA receptor (Gao et al., 1993). Furthermore, serotonergic medullary raphe´ neurons colocalize with a variety of peptides (substance P, thyrotropin releasing hormone (TRH), galanin, enkephalin) or neurotransmitters (GABA, glutamate) (see Ho¨kfelt et al., 2000). Due to the complexity of raphe´ organization, we do not know the exact phenotype of the raphe´ neurons affected in our experiments. 4.7. Anatomical and physiological relationship between the medullary raphe´ and the rostral ventral medulla (RVM)

4.6. Muscimol disruption of sleep cycling Sleep cycling in newborn piglets, suspended from a sling inside a plethysmograph, has been previously characterized (Darnall et al., 2001). Sleep was measured in room air before, during, and after 10
/40 mM muscimol microdialysis into the rostral ventral medulla (RVM). Sleep cycling, measured over a 40
/50-min control period with aCSF dialyzing through the microdialysis probe, took an average of 19 min to complete, as measured from the start of one REM episode to the start of the next REM event. Muscimol disrupted sleep cycling, producing a significant increase in the percentage of time spent in wakefulness and significant decreases in the percentages of time spent in NREM and REM sleep (Darnall et al., 2001). In our case, the control period over which we measured sleep cycling in our experiments was shorter, approximately 20 min, and our sleep cycle length was shorter, approximately 12 min. It is important to note that our experiments were not designed to study sleep cycling. However, we also unexpectedly noted a muscimol-induced disruption in sleep cycling, which was characterized by an increase in the average percentage of time spent awake and a decrease in the average percentage of time spent in NREM sleep. A more complete sleep analysis looking at sleep over an

Situated in close proximity to the raphe´ is the RVM, which is comprised of the retrotrapezoid nucleus (RTN), portions of the nucleus paragigantocellularis lateralis (PGCL), and the parapyramidal region of the medullary raphe´ (Curran et al., 2000, 2001; Darnall et al., 2001). By definition, the RVM extends from the rostral chemosensitive area to the intermediate area (Curran et al., 2000). The raphe´ and portions of the RVM are connected anatomically in animals (Zagon, 1993; Lindsey et al., 1994), as well as in human infants (Zec et al., 1997). Many similarities exist between the medullary raphe´ and the RVM. Both are involved in the modulation of homeostatic processes, including cardiorespiratory control (Bernard, 1998; Madden et al., 1999; Schreihofer et al., 2000; Curran et al., 2001). Both are chemosensitive (Coates et al., 1993; Nattie and Li, 1996; Li and Nattie, 1997; Li et al., 1999, Curran et al., 2000, 2001; Richerson et al., 2001), and both the raphe´ and the RVM have been shown to be abnormal in many SIDS victims (Kinney et al., 1995; Panigrahy et al., 1997, 2000). Muscimol inhibition of both chemosensitive regions does not affect eupneic breathing or resting levels of MAP, HR, Tb or V˙ O2 in the conscious newborn piglet (Curran et al., 2001; Darnall et al., 2001). Muscimol dialysis into both

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regions significantly reduces the CO2 response; absolute V˙ E in 5% CO2 is decreased by 17% in the raphe´, and is decreased by 20% in the RVM (Curran et al., 2001). Furthermore, muscimol dialysis in both regions disrupts sleep (Curran et al., 2001; Darnall et al., 2001). Differences exist between the raphe´ and the RVM. While muscimol inhibition of these regions reduced the ventilatory response to CO2, the effect was entirely due to a decrease in VT in the RVM (Curran et al., 2001), while both VT and fR were affected in the raphe´. A second major difference between the raphe´ and RVM is that the raphe´ appears to have chemoreceptor function during wakefulness, while the RVM is chemosensitive during both wakefulness and NREM sleep (Curran et al., 2001) in the newborn piglet. Differences in brainstem location, anatomical projections, stimulus intensity needed to activate each site, and arousal state-dependence may explain the observed differences (Nattie, 2000). Nonetheless, it is evident that abnormalities in the raphe´ and RVM may be detrimental if the system is stressed at a time in which these sites should normally be playing an active role in maintaining homeostasis. 4.8. Possible physiological functions of the medullary raphe´: implications for SIDS Through its extensive innervation of the brainstem and spinal cord, the medullary raphe´ and its serotonergic system are thought to modulate a wide range of physiological processes. In this respect, the raphe´ has been termed a ‘gain-setter,’ whose function is to integrate ongoing activity and generate an appropriate physiological response (see Lovick, 1997). In terms of respiration, the raphe´ is involved in central chemoreception (Coates et al., 1993, Bernard et al., 1996; Dreshaj et al., 1998; Wang et al., 1998; Nattie and Li, 2001; Richerson et al., 2001; Wang et al., 2001), motor facilitation of breathing (Jelev et al., 2001), plasticity and long term facilitation (Millhorn, 1986), maturation of the respiratory network (Burnet et al., 2001), and upper airway patency (Haxhiu et al., 1998). Additionally, the raphe´ functions to modulate thermoregulation (Morrison, 1999; Morrison et al., 1999), blood flow (Rathner and

McAllen, 1999; Blessing and Nalivaiko, 2000), nociception (Leung and Mason, 1999), and sensory and motor output (Veasey et al., 1995, Jacobs and Fornal, 1991, 1997). Thus, disturbances of medullary raphe´ function, such as reductions in serotonergic receptor binding (Panigrahy et al., 2000), may lead to a decrease in many of these reflexes, which could result in homeostatic instability and a failure to overcome a challenge to the system. Understanding the physiological importance that medullary raphe´ neurons play in the control of breathing in both sleep and wakefulness in the newborn piglet may provide insight into how abnormalities in this brainstem region could contribute to the pathogenesis of SIDS.

Acknowledgements This research was supported by NICHD 36379 and HL 07449. We thank Dr. James J. Filiano, Dr. Mary M. Niblock, and Hong Gao for providing the piglet brainstem figures. We recognize Dr. Karen Moodie for her help with surgical and postoperative care, Laurie Hildebrandt for her technical assistance, and Jennifer Cawthern for her assistance with data analysis. We also gratefully acknowledge the direction and support of Dr. Hannah C. Kinney, the program project director.

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