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Am J Physiol Regul Integr Comp Physiol 294: R884-R894, 2008. First published December 19, 2007; doi:10.1152/ajpregu.00655.2007
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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Activation of 5-HT1A receptors in medullary raphé disrupts sleep and decreases shivering during cooling in the conscious piglet

J. W. Brown,1 E. A. Sirlin,1 A. M. Benoit,1 J. M. Hoffman,1 and R. A. Darnall1,2

Departments of 1Physiology and 2Pediatrics, Dartmouth Medical School, Lebanon, New Hampshire

Submitted 10 September 2007 ; accepted in final form 13 December 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of 5-HT1A receptors in the medullary raphé decreases sympathetically mediated brown adipose tissue (BAT) thermogenesis and peripheral vasoconstriction when previously activated with leptin, LPS, prostaglandins, or cooling. It is not known whether shivering is also modulated by medullary raphé 5-HT1A receptors. We previously showed in conscious piglets that activation of 5-HT1A receptors with (±)-8-hydroxy-2-(dipropylamino)-tetralin (8-OH-DPAT) in the paragigantocellularis lateralis (PGCL), a medullary region lateral to the raphé that contains substantial numbers of 5-HT neurons, eliminates rapid eye movement (REM) sleep and decreases shivering in a cold environment, but does not attenuate peripheral vasoconstriction. Hoffman JM, Brown JW, Sirlin EA, Benoit AM, Gill WH, Harris MB, Darnall RA. Am J Physiol Regul Integr Comp Physiol 293: R518–R527, 2007. We hypothesized that, during cooling, activation of 5-HT1A receptors in the medullary raphé would also eliminate REM sleep and, in contrast to activation of 5-HT1A receptors in the PGCL, would attenuate both shivering and peripheral vasoconstriction. In a continuously cool environment, dialysis of 8-OH-DPAT into the medullary raphé resulted in alternating brief periods of non-REM sleep and wakefulness and eliminated REM sleep, as observed when 8-OH-DPAT is dialyzed into the PGCL. Moreover, both shivering and peripheral vasoconstriction were significantly attenuated after 8-OH-DPAT dialysis into the medullary raphé. The effects of 8-OH-DPAT were prevented after dialysis of the selective 5-HT1A receptor antagonist WAY-100635. We conclude that, during cooling, exogenous activation of 5-HT1A receptors in the medullary raphé decreases both shivering and peripheral vasoconstriction. Our data are consistent with the hypothesis that neurons expressing 5-HT1A receptors in the medullary raphé facilitate spinal motor circuits involved in shivering, as well as sympathetic stimulation of other thermoregulatory effector mechanisms.

thermoregulation; serotonin; brain stem; raphé; the sudden infant death syndrome

MANY NEURONS IN THE MEDULLARY raphé, including serotonergic (5-HT) neurons, project to the intermediolateral cell column (IML) of the spinal cord and modulate sympathetic outflow to thermoregulatory effector mechanisms, including brown adipose tissue (BAT) thermogenesis, vasoconstriction of thermoregulatory cutaneous vascular beds, and heart rate (HR) (14, 53, 58, 86, 89). Studies in anesthetized and conscious animals have shown that activation of 5-HT1A receptors in the medullary raphé with the selective agonist, (±)-8-hydroxy-2-(dipropylamino)-tetralin (8-OH-DPAT), attenuates sympathetic outflow to BAT and peripheral vessels when previously elevated by LPS, leptin, or cooling (11, 52, 59, 63, 64).

There is mounting evidence that medullary raphé 5-HT neurons modulate sympathetically mediated thermoregulatory mechanisms; however, little is known about the role of medullary raphé 5-HT neurons in shivering thermogenesis. Shivering is an involuntary tremor that is caused by an oscillatory instability involving fusimotor innervation of skeletal muscle (77–79). Together with BAT thermogenesis, shivering is an important mechanism for thermoregulatory heat production. Importantly, in the human infant, shivering becomes the predominant form of thermoregulatory heat production beyond the newborn period. Moreover, piglets do not have BAT and use shivering as the primary mechanism for thermoregulatory heat production and thus are excellent models in which to study shivering. In recent experiments in conscious piglets, we showed that activation of 5-HT1A receptors in the juxtafacial paragigantocellularis lateralis (PGCL), a region lateral to the raphé within the rostrocaudal dimension of the facial nucleus containing substantial numbers of 5-HT neurons (17, 61), significantly reduced shivering during cooling, but had little or no effect on peripheral vasoconstriction (34). A role for medullary raphé neurons in modulating shivering is also suggested by recent evidence that the activity of fusimotor fibers innervating skeletal muscle during skin cooling can be attenuated by the microinjection of glycine into the medullary raphé (87). In another report, microinjection of 8-OH-DPAT or lidocaine into the raphé magnus attenuated shivering activity during cooling in conscious rats (7).

Sleep is a period of relative vulnerability, when many homeostatic control systems function at lower levels compared with wakefulness (WAKE). Our laboratory showed previously that dialysis of 8-OH-DPAT into the PGCL, both at thermoneutrality and during continuous cooling, results in fragmented sleep with alternating brief periods of non-rapid eye movement (NREM) sleep and WAKE, and an almost complete elimination of rapid eye movement (REM) sleep (17, 34). These effects were eliminated after blocking the 5-HT1A receptor with the selective 5-HT1A antagonist N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-2-pyridinylcyclohexane-carboxamide (WAY-100635) and attenuated after destroying PGCL 5-HT neurons with 5,7-dihydroxytryptamine, indicating that they were largely the result of activating 5-HT1A autoreceptors located on 5-HT neurons (17). In other experiments in conscious piglets focused on the cardiorespiratory function of the medullary raphé, microdialysis of 8-OH-DPAT produced an increase in WAKE and decreased sleep (50). These data suggest that neurons in the medulla expressing 5-HT1A receptors, including 5-HT neurons, play a role in sleep modulation, especially for REM.

Interestingly, both nonshivering and shivering thermogenesis and peripheral vasoconstriction are greatly attenuated, or even eliminated, during REM in most animal species (26, 67), a time when 5-HT neurons, including those located in the caudal raphé, are at their lowest level of activity (32, 35). These observations support the hypothesis of a state-related modulatory role for medullary 5-HT neurons in controlling shivering and thermoregulatory sympathetic activity. Thus both the decrease in 5-HT neuronal activity associated with REM and exogenous activation of 5-HT1A receptors are associated with an attenuation of thermoregulatory effector mechanisms.

Understanding the role of medullary 5-HT neurons in sleep and thermoregulation may help us determine mechanisms involved in sudden infant death syndrome (SIDS), where abnormalities in thermoregulation and arousal have been implicated (21, 22, 38, 39, 57, 83, 91). Almost 75% of SIDS infants studied have decreased binding to 5-HT1A receptors and increased numbers of 5-HT neurons in all of the medullary serotonergic nuclei, including the medullary raphé and PGCL (41–43, 65, 69), groups of neurons homologous to those that have been found to be important in thermoregulation and sleep in animals (12, 17, 53). We hypothesize that dysfunction in these regions may increase the risk for SIDS by altering sleep architecture and protective reflexes to stressors encountered during sleep, including thermal challenges.

In this study, we tested the hypothesis that, during continuous cooling, shivering, a major thermoregulatory effector mechanism, as well as sympathetically mediated peripheral vasoconstriction, is modulated by the activation of 5-HT1A receptors in the medullary raphé. A major goal was to determine whether activation of 5-HT1A receptors in the medullary raphé of conscious piglets during continuous cooling would attenuate both shivering and peripheral vasoconstriction. In addition, we wanted to determine whether activation of 5-HT1A receptors would eliminate REM, as our laboratory has observed during 8-OH-DPAT dialysis in the PGCL (17).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experiments were performed on 11 piglets, 5–13 days old, of either sex, weighing 1.9–3.6 kg at the time of study. All surgery and experimental protocols were approved by the Institutional Animal Care and Use Committee of Dartmouth College. The piglets were housed with the sow and siblings in a farrowing crate located in the Dartmouth College Animal Resource Center and were maintained at a constant ambient temperature of 21°C and 12:12 h light-dark cycles. Piglets were brought to the laboratory 1 or more days before surgery to accustom them to the experimental environment.

Surgical instrumentation. Our laboratory's surgical procedures have been described in detail previously (15–17, 34). Briefly, under sterile conditions and using isofluorane anesthesia, a dual-lumen catheter was placed into the femoral artery and advanced into the abdominal aorta, and a telemetric thermistor was placed into the peritoneal cavity. In each animal, two microdialysis guide tubes, 1 mm apart in the rostrocaudal dimension, were stereotaxically placed with their tips in the midline, near the ventral surface, within the rostrocaudal dimension of the facial nuclei. The guide tubes were angled to avoid the midline venous sinus. Electroencephalogram (EEG) electrodes were screwed into the left frontal and right occipital regions of the skull and referenced to a right parietal electrode. Electrooculogram (EOG) electrodes were sewn into the musculature lateral to each eye, and bipolar electromyogram (EMG) electrodes were sewn into the neck muscles on the right side, near the midline. All wires were tunneled and connected to two plastic pedestals that were then cemented to the skull, along with the microdialysis guide tubes. The femoral catheter was tunneled through the skin and exited the subscapular region on the back. After surgery, the animals were provided with analgesia and antibiotics, allowed to recover, and returned to the sow and siblings in the animal care facility.

Measurements. The animals were first studied 24–48 h after surgery. The piglet was suspended in a sling inside an 85-liter double-walled barometric plethysmograph modified to allow continuous gas flow (5, 19, 66). Air flowing through the plethysmograph was heated (~38°C at the heater/humidifier) and fully humidified. HR and mean arterial pressure were calculated from continuous measurements of arterial pressure (World Precision Instruments, Sarasota, FL). Respiratory measurements were derived from plethysmograph pressure fluctuations (Validyne, Northridge, CA). EEG, EOG, and EMG signals were amplified and band-pass filtered (0.1–300 Hz for EEG and EOG and 10–300 Hz for EMG) (Grass Technologies, West Warwick, RI). The percent carbon dioxide (CO2) in the outlet air of the plethysmograph (Capstar-100; CWI, Ardmore, PA) was continuously measured to monitor inspired CO2, and flow rates were adjusted to maintain outflow CO2 concentration <0.5%. Inlet and outlet oxygen concentration and flow rates were also continuously measured (AEI Technologies, Naperville, IL; Teledyne-Hastings, Hampton, VA). Plethysmograph air, piglet right ear skin (as a surrogate for ear surface blood flow) (Tear), and core temperature were continuously measured (YSI, Yellow Springs, OH, and DSI, St. Paul, MN). All electronic signals were digitized at 1,000 Hz and recorded using a computerized data-acquisition system (PowerLab; ADInstruments, Sydney, Australia). Throughout the experiment, piglet behavior was also video recorded and digitized for later sleep scoring. Shivering was assessed by measuring the percent time shivering, the number of shivering bouts/minute, and changes in integrated neck EMG (nEMG) activity, as we have previously described (34).

Protocol. Studies were performed on a total of 11 animals, 1–10 days after surgery. Nine experiments in nine different animals were performed to determine the effects of 8-OH-DPAT on sleep, shivering, and peripheral vasoconstriction. Five additional experiments were done in which artificial cerebrospinal fluid (aCSF) replaced 8-OH-DPAT in the dialysate and served as time controls. Three of these experiments were done in the same animals used for the 8-OH-DPAT experiments, but on different days. To confirm that the effects of 8-OH-DPAT were due to activation of 5-HT1A receptors (25), three experiments were done in which the dialysis of WAY-100635, a selective 5-HT1A receptor antagonist, immediately preceded the dialysis of DPAT. Two of these experiments were done in animals that were used for 8-OH-DPAT experiments, and one was done in an aCSF control animal, all on separate days. All animals were studied in a continuously cool environment in which the wall temperature was adjusted to ~5°C below the lower critical threshold of the thermoneutral zone, as determined previously (13), and maintained for the duration of the experiment.

Approximately 1.5 h before each experiment was started, the plethysmograph was sealed to allow the temperature and humidity to stabilize. After stabilization was complete, calibration was performed using sequential triplicate injections of 1, 2, 3, and 5 ml of air. The piglet was then placed in the plethysmograph and connected to the monitoring equipment. The microdialysis probes were inserted, and dialysis was started with aCSF [containing the following (in mM): 152.2 Na, 3.0 K, 131.1 Cl, and 1.5 Ca, adjusted to a pH of 7.4] at a flow rate of 8.5 µl/min. Recordings were begun after temperature, humidity, and outlet carbon dioxide and oxygen concentrations had reached stable values (~1 h).

Following a 60- to 90-min baseline period with aCSF dialysis, the dialysate was switched to 30 mM 8-OH-DPAT for 30 min and then switched back to aCSF for an additional 60–90 min. In the time control experiments, the identical protocol was used, except that aCSF was substituted for 8-OH-DPAT in the dialysate. In the WAY experiments, 8-OH-DPAT was dialyzed immediately after a 30-min period of 30 mM WAY-100635 dialysis.

Drugs. Relative high concentrations of 8-OH-DPAT were used in the dialysate in the present study compared with those used in dialysis experiments in the dorsal raphé (71). They are otherwise consistent with those used in our laboratory's previous experiments (17, 34, 50) and those done by other investigators in the caudal brain stem (7). Higher concentrations in the dialysate may be necessary for several reasons. First, the estimated tissue concentration near the dialysis probe is approximately 1/10th of the dialysate concentration (18) and decreases as the drug diffuses away from the probe. Second, compared with dorsal raphé 5-HT neurons, caudal medullary 5-HT neurons have faster firing rates (32), and their firing rates decrease less in response to serotonin and 5-HT receptor agonists (33, 90), perhaps suggesting a lower density of, or less dependency on, 5-HT1A autoreceptors. There is no information about the responsiveness of postsynaptic 5-HT1A receptors in the medullary raphé.

Data reduction and calculations. Data reduction and analysis, including sleep scoring, was done using custom programs written in MATLAB (MathWorks, Natick, MA) that have been described in detail previously (17). Briefly, sleep-state scoring was accomplished using a wavelet-based analysis that derived frequency information from the EEG. Periods of NREM sleep, REM sleep, and WAKE were identified using the combination of EEG, EOG, and nuchal EMG data. State identification was confirmed by observing the piglet's behavior using synchronized video recordings.

Body (Tbody), Tear, and plethysmograph temperature data were resampled at 1 Hz. Since shivering was suspended during REM, and shivering is difficult to separate from other body movements during WAKE, it was quantified only during NREM sleep by determining, for each NREM bout, the percentage of time shivering, the number of shivering bursts per minute, and the mean integrated nEMG activity. This was accomplished by first confirming the presence of shivering during a given bout of NREM sleep in the video recording and then determining the percentage of time spent in shivering activity for that epoch by manually measuring the duration of each rhythmic shivering burst (bout) of nEMG activity. To determine changes in nEMG activity, the raw signal was resampled at 100 Hz, rectified, and integrated over 5-s bins and averaged for each NREM period. The number of shivering bouts and the sum of their durations were used to calculate the number of shivering bouts per minute and percent time spent in shivering, respectively.

For cardiorespiratory variables, the original digitized data were resampled at rates appropriate for each variable. For respiratory calculations, the maxima and minima of breath-related pressure fluctuations were determined using an automated peak detector followed by manual correction, if necessary. Tidal volume (VT) was calculated from the pressure fluctuations using methods used previously (5). Minute ventilation was calculated as the product of VT and instantaneous respiratory rate, calculated from the interbreath interval. The peak of each blood pressure pulse was determined similarly and was used to calculate beat-to-beat HR. Mean arterial pressure was calculated from the arterial pressure waveform. Estimates of oxygen consumption (VO2) were calculated from continuously measured plethysmograph inlet and outlet oxygen concentrations and plethysmograph flow rate.

For each variable, artifact-free segments of the recording were averaged and archived as to the time before or after 8-OH-DPAT dialysis and state (NREM, REM, or WAKE). To determine the effects of 8-OH-DPAT dialysis, data were averaged in 20- or 30-min bins before and 15-min bins after the onset of 8-OH-DPAT dialysis. An identical process was used when aCSF was substituted for 8-OH-DPAT and when 8-OH-DPAT dialysis was preceded by dialysis of WAY-100635.

Analysis and statistics. Nine animals of either sex were studied to determine whether activating medullary raphé 5-HT1A receptors with 8-OH-DPAT would alter sleep, decrease shivering, and decrease peripheral vasoconstriction induced by a cold environment. Five animals received aCSF dialysis during the entire period and served as controls. Comparisons were made during NREM sleep with ANOVA for repeated measures, or single-value T-tests with appropriate adjustments for multiple comparisons. Values are expressed as means ± SE, and the criterion for statistical significance was set at P < 0.05.

Neuroanatomy. At the conclusion of experiments, microinjections of 20–50 µl of 1% potassium permanganate were made through a broken microdialysis probe to mark the location of the tip of each microdialysis probe (85). Each piglet was killed with a lethal intra-arterial injection of pentobarbital sodium followed by an intracardiac injection of 5–10 ml of saturated potassium chloride. The brain stem was removed and frozen in cryoembedding medium (Tissue-Tek OCT; Sakura Finetek, Torrance, CA). Brain stems were cryosectioned (50 µm) at ~18°C, and sections were slide mounted, fixed for 10 min in 37% phosphate-buffered formalin (pH 7.4), and stained with cresyl violet. We referenced the location of each probe with respect to three relevant internal medullary structures: the midline, the ventral surface, and the caudal pole of the facial nucleus (15). Probe tips were considered to be in the appropriate position if they were within the rostral-caudal dimensions of the facial nucleus and within 1 mm of the midline. The locations of the dialysis probe tips for experimental and control animals, as determined by permanganate, are shown in Fig. 1.


Figure 1
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  		Fig. 1. Dialysis probe tip locations for all of the animals in the study (n = 11). Left: probe tip locations are superimposed on a photograph of the ventral surface of a piglet brain stem. In the diagram of the medullary ventral surface, the black shaded area on the left and the grid on the right are approximations of location of the facial nucleus. The grid was used to normalize the locations for variations in age and size of the animals by plotting the locations as a fraction of the distance from the caudal to rostral border of the facial nucleus. The lines (A–E) represent the rostral-caudal levels of the cross-sectional areas shown on the right. Each adjacent pair of probe tip locations for animals in the (±)-8-hydroxy-2-(dipropylamino)-tetralin (8-OH-DPAT) group (n = 9) are illustrated by a solid circle and a triangle, representing the more rostrally and more caudally placed probe, respectively. The paired open circles and triangles are the locations for animals in the artificial cerebrospinal fluid (aCSF) group (n = 5). Note that three animals received 8-OH-DPAT and aCSF on different days. 7N, seventh nerve; VII, facial nucleus; IO, inferior olive; SO, superior olive.

 

   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Activation of raphé 5-HT1A receptors disrupts sleep. As our laboratory found previously after dialysis of 8-OH-DPAT into the PGCL (17, 34), dialysis of 8-OH-DPAT into the medullary raphé produced fragmented sleep with short bouts of NREM, alternating with periods of WAKE, and an almost complete elimination of REM sleep. An example of sleep before and after 8-OH-DPAT dialysis is shown in Fig. 2A. The mean sleep data derived from experiments in which either 8-OH-DPAT or aCSF was dialyzed into the medullary raphé is shown in Fig. 3. The average duration of NREM sleep bouts was decreased after 8-OH-DPAT dialysis (353 ± 68 vs. 122 ± 36 s, P = 0.007), but not after aCSF dialysis [419 ± 90 vs. 471 ± 49 s; nonsignificant (NS); group (aCSF or DPAT) x time, P = 0.006]. The number of NREM bouts per hour also increased significantly after 8-OH-DPAT dialysis (6.8 ± 0.7 vs. 15.0 ± 1.4, P = 0.001), but not after aCSF dialysis (5.0 ± 0.9 vs. 5.2 ± 1.9, NS; group x time, P = 0.004). The increase in bouts per hour and the decrease in bout duration resulted in no significant change in the percent time spent in NREM. In contrast, the percent time spent in REM was dramatically decreased (22.1 ± 2.1 vs. 1.3 ± 1.4%, P < 0.001) after 8-OH-DPAT dialysis, whereas there was no effect on the percent time in REM after aCSF dialysis (30.0 ± 2.8 vs. 22.8 ± 1.9%, NS; group x time, P = 0.003). The decrease in time spent in REM was almost entirely due to a decrease in the number of REM bouts per hour after 8-OH-DPAT dialysis (3.8 ± 0.6 vs. 0.2 ± 0.5, P < 0.001) compared with no decrease after aCSF dialysis (4.4 ± 0.8 vs. 4.3 ± 0.6, NS; group x time, P = 0.006). The duration of the few isolated periods of REM after 8-OH-DPAT dialysis observed in two animals was not different from those before 8-OH-DPAT dialysis. Both the number of WAKE bouts per hour and the percent time spent in WAKE were increased after 8-OH-DPAT dialysis (6.6 ± 0.7 vs. 14.7 ± 1.5, P = 0.002; and 24.9 ± 4.3 vs. 49.0 ± 4.3%, P < 0.001, respectively), but not after aCSF dialysis (5.1 ± 0.9 vs. 5.2 ± 2.0, NS, group x time, P = 0.007; and 15.7 ± 5.8 vs. 13.2 ± 5.8%, NS, group x time, P < 0.001, respectively).


Figure 2
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  		Fig. 2. A: sleep and shivering before and after 8-OH-DPAT (DPAT) dialysis into the medullary raphé. An example of EEG, EOG, and integrated neck EMG (nEMG) recordings before and after 8-OH-DPAT dialysis are given. There is regular cycling of sleep before 8-OH-DPAT dialysis, with easily identifiable periods of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep, accompanied by shivering during NREM. Note that shivering is suspended during REM. After 8-OH-DPAT dialysis, there is fragmented sleep that, on closer inspection, is mainly alternating periods of NREM and wakefulness (WAKE), with no identifiable periods of REM. B: dialysis of the selective 5-HT1A antagonist, WAY-100635, eliminates the sleep and shivering effects of subsequent 8-OH-DPAT dialysis. An example is given of a single experiment in which WAY-100635 was dialyzed immediately before starting 8-OH-DPAT dialysis. Note that sleep cycling and shivering are not different before and after 8-OH-DPAT dialysis.

 

Figure 3
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  		Fig. 3. The average duration, percent time, and bouts per hour of NREM, REM, and WAKE before and after either 8-OH-DPAT (n = 9) or aCSF (n = 5) dialysis into the medullary raphé. The solid and open bars indicate periods before and after 8-OH-DPAT dialysis, and the shaded and white hatched bars indicate periods before and after aCSF dialysis, respectively. *Significant change after 8-OH-DPAT dialysis. #Changes after 8-OH-DPAT dialysis were significantly different from those after aCSF dialysis (see text for values and P values).

 
During NREM sleep after 8-OH-DPAT dialysis, delta power (EEG power at 0.5–4 Hz) was significantly lower compared with baseline periods before 8-OH-DPAT dialysis, suggesting "lighter" sleep, as our laboratory has observed previously after 8-OH-DPAT dialysis into the PGCL (17). On average, during NREM, after 8-OH-DPAT dialysis, delta power was 57.4 ± 5.9% of NREM delta power before 8-OH-DPAT dialysis, whereas there was no change in delta power during NREM after aCSF dialysis. Figure 4 shows the significant changes in delta power during NREM sleep after 8-OH-DPAT dialysis (P < 0.001) compared with no changes after aCSF dialysis (group x time, P < 0.001). Although 8-OH-DPAT dialysis resulted in a significant decrease in NREM delta power, the level of delta power during REM sleep before 8-OH-DPAT dialysis was 39.6 ± 5.9% of delta power during NREM after 8-OH-DPAT dialysis (P < 0.05).


Figure 4
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  		Fig. 4. NREM delta power before and after 8-OH-DPAT dialysis into the medullary raphé. bullet, Data from experiments in which 8-OH-DPAT was dialyzed into the medullary raphé (n = 9). {circ}, Data from experiments in which aCSF was substituted for 8-OH-DPAT in the experimental protocol (n = 5). Values are means ± SE. "Group x time" is the interaction term in a repeated-measures ANOVA, where time is the within-group comparison, and group (DPAT or aCSF) is the grouping variable.

 
Blocking medullary 5-HT1A receptors with WAY-100635, a selective 5-HT1A receptor antagonist, completely eliminated any effects of subsequent 8-OH-DPAT dialysis on sleep. Figure 2B shows an example of sleep before and after experiments where WAY was dialyzed immediately preceding DPAT. We observed in three experiments that sleep patterns after 8-OH-DPAT dialysis, if preceded by WAY-100635 dialysis, were indistinguishable from sleep patterns before 8-OH-DPAT dialysis. Moreover, we did not observe any changes in sleep during the 30 min of WAY-100635 dialysis before 8-OH-DPAT dialysis.

Activation of medullary raphé 5-HT1A receptors in a cold environment decreases shivering during NREM sleep. As we had observed previously (34) during baseline periods, shivering in a continuously cool environment was largely suspended during REM sleep, but was consistently observed during NREM and WAKE. Dialysis of 8-OH-DPAT into the medullary raphé was associated with a decrease in shivering during NREM. We were unable to analyze shivering during WAKE because of movement artifacts. An example of an experiment in a single piglet was shown in Fig. 2A. As illustrated, shivering is suspended both during REM sleep before 8-OH-DPAT dialysis and during NREM sleep after 8-OH-DPAT dialysis. Figure 5, A and B, compares the effects of 8-OH-DPAT dialysis and aCSF dialysis on the mean percent time spent shivering and integrated nEMG, respectively. During NREM, 8-OH-DPAT dialysis resulted in a significant decrease in the percent time spent shivering (P < 0.01), whereas shivering continued after aCSF dialysis (group x time, P < 0.02). The decrease in shivering after 8-OH-DPAT dialysis was also accompanied by a decrease in the mean integrated nEMG activity (P < 0.001), but not after aCSF dialysis (group x time, P < 0.001).


Figure 5
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  		Fig. 5. A: percentage of time spent shivering over time before and after dialysis of either 8-OH-DPAT or aCSF. Over the first hour after the onset of 8-OH-DPAT dialysis, there is a decrease in the percent time spent shivering, but not after dialysis of aCSF. "Group x time" is the interaction term of a repeated-measures ANOVA, where time is the within-subject comparison, and group (DPAT or aCSF) is the grouping variable. B: changes in integrated nEMG activity, expressed as percentage of control after medullary raphé dialysis of either 8-OH-DPAT or aCSF. After 8-OH-DPAT dialysis, nEMG decreases, but there is no change after aCSF dialysis. Values are means ± SE.

 
In addition to a decrease in shivering activity, there was a general decrease in muscle tone, as indicated by a decrease in the nonphasic nEMG (nEMG activity in between shivering bursts) during NREM after 8-OH-DPAT dialysis. An analysis of the integrated nonphasic nEMG activity before and after 8-OH-DPAT dialysis revealed that nonphasic nEMG activity after 8-OH-DPAT dialysis was 54.1 ± 11.6% of that before 8-OH-DPAT dialysis (P = 0.016). After aCSF dialysis, nonphasic nEMG activity remained at 94.9 ± 9.4% of baseline (NS) (changes after 8-OH-DPAT vs. changes after aCSF, P = 0.02).

Activation of medullary raphé 5-HT1A receptors in a cold environment decreases peripheral vasoconstriction, HR, and Tbody. During a continuously cold environment, 8-OH-DPAT dialysis into the medullary raphé consistently produced increases in Tear and decreases in HR and Tbody. Figure 6 shows the changes in integrated nEMG activity, Tbody, Tear, and HR in a single experiment. Note the typical decreases in Tbody, HR, and integrated nEMG during REM sleep (open triangles) before the onset of 8-OH-DPAT dialysis, as we have shown previously (34). During the 45 min after the onset of 8-OH-DPAT dialysis, there were small but significant decreases in Tbody, HR, and nEMG, and a progressive increase in Tear, suggesting decreases in heat production (decrease in shivering), heat conservation (peripheral vasoconstriction), and sympathetic outflow to the heart. The mean data for Tbody, Tear, and HR are shown in Fig. 7. After 8-OH-DPAT dialysis, there was a decrease in Tbody (P < 0.001), whereas there was no change after aCSF dialysis (group x time, P = 0.007). HR also decreased ~25 beats/min (P < 0.001). Although the HR means trended lower after aCSF dialysis, this change was not significant, and the decreases in HR after 8-OH-DPAT dialysis were significantly greater than any changes after aCSF dialysis (group x time, P = 0.019). During cooling and before 8-OH-DPAT dialysis, mean Tear was ~10°C less than Tbody and very close to environmental temperature (29.8 ± 1.8°C), suggesting significant peripheral vasoconstriction (74). In the absence of changes in environmental temperature or blood pressure over the relatively brief period of 8-OH-DPAT dialysis, increases in Tear provide a reasonable estimate for increases in ear surface blood flow. After 8-OH-DPAT dialysis, Tear increased by ~1°C (P < 0.01), whereas, after aCSF dialysis, there was no change (group x time, P = 0.018). Dialysis of WAY-100635 before 8-OH-DPAT dialysis eliminated any effects on shivering (see Fig. 2B), Tbody, HR, and Tear. Tbody, Tear, and HR before and after 8-OH-DPAT dialysis preceded by WAY dialysis were 40.3 ± 0.4 vs. 40.1 ± 0.5, 31.2 ± 2.0 vs. 31.1 ± 2.1, and 188.3 ± 22.9 vs. 190.3 ± 26.3, respectively (all P > 0.5, ANOVA for repeated measures).


Figure 6
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  		Fig. 6. An example of a typical experiment showing the effects of dialyzing 8-OH-DPAT into the medullary raphé of a conscious piglet maintained in a continuously cool environment. Each point represents artifact-free periods during NREM sleep (bullet), REM sleep ({triangleup}), and WAKE ({square}). Cycling of sleep occurs during the baseline period, but becomes fragmented after 8-OH-DPAT dialysis (onset indicated by vertical line) with little or no REM sleep. During REM sleep before 8-OH-DPAT dialysis, there are decreases in mean heart rate (HR) and piglet core temperature (Tbody) and increases in ear skin temperature (Tear), consistent with a suspension of thermoregulatory effector mechanisms. After 8-OH-DPAT dialysis, there is a decrease in HR and Tbody and an increase in Tear, accompanied by a decrease in integrated (Int) nEMG activity during NREM sleep.

 

Figure 7
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  		Fig. 7. Changes in piglet Tbody, Tear, and HR over time after medullary raphé dialysis of either 8-OH-DPAT or aCSF. After 8-OH-DPAT dialysis, Tbody and HR decrease and Tear increases, whereas there are no significant changes after aCSF dialysis. "Group x time" is the interaction term of a repeated-measures ANOVA, where time is the within-subject factor, and group (DPAT or aCSF) is the grouping factor. Values are means ± SE.

 
8-OH-DPAT dialysis into the medullary raphé did not result in any significant changes in respiration, blood pressure, or VO2. VT, respiratory rate, and minute ventilation were 8.8 ± 1.5 ml/kg, 51.8 ± 6.4 breaths/min, and 347.0 ± 20.0 ml·kg–1·min–1 before 8-OH-DPAT dialysis, and 8.9 ± 1.1 ml/kg, 49.8 ± 5.0 breaths/min, and 355.5 ± 32.0 ml·kg–1·min–1 60 min after 8-OH-DPAT dialysis, respectively. Similarly, mean blood pressure was 85.0 ± 3.6 mmHg before and 85.7 ± 2.9 mmHg after 8-OH-DPAT dialysis. VO2 was 20.1 ± 1.8 before and 20.2 ± 1.9 ml·kg–1·min–1 after 8-OH-DPAT dialysis. The plethysmograph used in this study was relatively large (~85 liters), and the slow response time tended to average out any transient changes in VO2. Thus estimates of VO2 during the brief periods of NREM that occurred between periods of WAKE after 8-OH-DPAT would be overestimated. We would predict that measurements with a faster response time would have revealed a decrease in VO2 after 8-OH-DPAT dialysis because of the relative hypotonia and suspension of shivering.


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
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The major findings are that, during cooling in conscious piglets, activation of 5-HT1A receptors in the medullary raphé fragments sleep, almost completely eliminates REM, and attenuates the activity of two important thermoregulatory mechanisms: shivering and peripheral vasoconstriction. During cooling, the effects of local dialysis of 8-OH-DPAT on sleep and shivering were similar to those that we observed when 8-OH-DPAT was dialyzed into the more laterally located PGCL (34). In contrast, dialysis of 8-OH-DPAT into the medullary raphé significantly attenuated peripheral vasoconstriction, whereas this did not occur after 8-OH-DPAT dialysis into the PGCL.

The effects of exogenous activation of 5-HT1A receptors in the medullary raphé with 8-OH-DPAT are complex, because 5-HT1A receptors are located both on 5-HT neurons functioning as autoreceptors and on non-5-HT neurons, where they function as inhibitory heteroceptors involved in the release of other neurotransmitters (31). Thus activation of 5-HT1A receptors would inhibit 5-HT neurons and presumably any non-5-HT neurons expressing 5-HT1A receptors. The actions of an exogenous 5-HT1A agonist, such as 8-OH-DPAT, when locally applied to a given region, will, therefore, depend on the relative contribution of autoreceptor and heteroreceptor activation. In regions that do not contain 5-HT neurons, it is reasonable to postulate that any actions would be due to activation of inhibitory heteroreceptors located on non-5-HT neurons. Thus, in experiments in which 8-OH-DPAT was microinjected into the rostral ventrolateral medulla, a region containing few, if any, 5-HT neurons, the resulting decrease in blood pressure was most likely secondary to activating postsynaptic 5-HT1A receptors expressed by non-5-HT neurons (46, 62). In the rat, it appears that ~30–43% of neurons in the raphé pallidus and raphé obscurus that express 5-HT1A receptors are serotonergic, implying that, in these regions, more than one-half of the neurons expressing 5-HT1A receptors are nonserotonergic (31). However, the distribution of 5-HT1A receptors among 5-HT and non-5-HT neurons in the subgroup of neurons that projected to the IML was not reported. In the piglet, however, the distribution of 5-HT1A receptors among raphé 5-HT and non-5-HT neurons is unknown. Although a systematic quantitative analysis was not done, our laboratory previously estimated in the piglet PGCL that close to 90% of immuno-labeled 5-HT1A receptors colocalized with tryptophan hydroxylase immunoreactive neurons (17). During naturally occurring REM sleep, thermoregulatory mechanisms are suspended at the same time 5-HT neurons are at their lowest level of activity. Thus there is an association between a suspension of thermoregulatory mechanisms and low levels of 5-HT neuronal activity. Nevertheless, although there is abundant evidence that 8-OH-DPAT decreases the activity of medullary 5-HT neurons, whether given systemically or applied locally, the contribution of inhibition of non-5-HT neurons to our findings remains uncertain.

Effects of 8-OH-DPAT dialysis into the medullary raphé on sleep. Previous evidence from our laboratory examining cell groups in the PGCL with similar methodology showed that the effects of 8-OH-DPAT on sleep were largely abolished after destruction of 5-HT neurons (17), indicating that the effects on sleep were due to activation of 5-HT1A autoreceptors. In these studies, however, effects on HR and Tbody, although small, were not attenuated, suggesting that these effects may have been due to activation of postsynaptic 5-HT1A heteroreceptors. Although we did confirm that the effects of medullary raphé dialysis of 8-OH-DPAT on sleep were secondary to activation of 5-HT1A receptors, we have not evaluated the effects of 8-OH-DPAT after destruction of medullary raphé 5-HT neurons and, therefore, cannot be certain that the effects of 8-OH-DPAT on sleep were entirely due to a decrease in the activity of 5-HT neurons.

We postulate that the decrease in REM sleep after 8-OH-DPAT dialysis was secondary to the critical role played by medullary raphé neurons, including 5-HT neurons, in integrating multiple sensory inputs and modulating brain arousal and alerting systems. Neurons in both the medullary raphé and PGCL send efferent projections to the locus coeruleus, an important region involved in vigilance (3, 4, 80). The ventral oral pontine reticular nucleus, a region important for the generation and maintenance of REM sleep, also receives abundant serotonergic innervation that is not from the dorsal or median raphé (73). Serotonergic projections to the ventral oral pontine reticular nucleus may include those from the medullary raphé and PGCL. Although there has been considerable interest in the rostral groups of 5-HT neurons that play important roles in the regulation of sleep (51, 71, 76, 82, 84), less is known about the role of 5-HT neurons in the medullary raphé and PGCL in either regulating or modulating sleep. In this report, we present the novel finding that neurons expressing 5-HT1A receptors in the medullary raphé, like those in the PGCL (17), play a role in the modulation of sleep and, in particular, REM sleep.

Serotonergic neurons in the medullary raphé and adjacent reticular areas have major projections to the dorsal horn of the spinal cord (44, 81) and participate in the modulation of nociceptive (1, 6, 45) and nonnociceptive (27, 28) sensory inputs. Furthermore, 5,6-dihydroxytryptamine spinal cord lesions decrease REM in rats (9) and intrathecal administration of 8-OH-DPAT increases the amount of sleep and decreases the amount of WAKE (8, 10). These data support the hypothesis that 5-HT neurons in the raphé and the more lateral PGCL can modulate sleep state by damping sensory input at the spinal cord level. Thus inhibition of the activity of these neurons could result in an "undamping" of ascending sensory information, promoting more WAKE and the sleep fragmentation that we observed.

What we scored as short periods of NREM after 8-OH-DPAT dialysis might represent a dissociated state with REM-like hypotonia. However, there were no REMs or REM-like changes in respiration, blood pressure, or HR. In addition, although the levels of delta power, EEG amplitude, and nEMG activity were lower during NREM bouts after 8-OH-DPAT dialysis, they were significantly higher than during REM bouts before dialyzing DPAT. We, therefore, believe that the relative hypotonia during NREM after 8-OH-DPAT dialysis is more likely related to the role of 5-HT neurons and perhaps other neurons expressing 5-HT1A receptors in providing tonic state-related motor facilitation, important for the modulation of muscle tone and rhythmic motor activity (36).

Although environmental and Tbody changes can be associated with changes in sleep homeostasis, the changes in Tbody that we observed in our piglets after raphé dialysis of 8-OH-DPAT were small (~0.5°C). In our experience in piglets, sleep fragmentation and elimination of REM sleep have only been observed after 8-OH-DPAT dialysis. In multiple experiments, we have never observed these kinds of changes after cooling or heating, with Tbody ranging from 37 to 41°C.

8-OH-DPAT dialysis into the medullary raphé decreases peripheral vasoconstriction, HR, and Tbody during cooling. There are parallel lines of evidence that 5-HT neurons and neurons expressing the vesicular glutamate transporter 3 (VGLUT3) are among those that project to the IML and play a role in sympathetically related thermoregulatory and HR responses to cooling. Subsets of medullary 5-HT neurons and neurons expressing VGLUT3 are labeled by pseudorabies virus from BAT and tail injections (58, 89) and increase their activity in response to cooling administration (49, 58, 72), and, in the case of 5-HT neurons, the increase is positively correlated with BAT temperature (60). Both glutamate and 5-HT increase sympathetic outflow to BAT when injected into the IML (47, 58), and 5-HT enhances the excitatory effect of locally applied N-methyl-D-aspartate (47). It is not known whether raphé VGLUT3 expressing neurons also express 5-HT1A receptors. Finally, 8-OH-DPAT dialyzed into the medullary raphé produced a small but significant decrease in Tbody. With respect to peripheral vasoconstriction, our results in piglets are consistent with those of Ootsuka and Blessing in rabbits (63, 64) and further support a role for raphé neurons expressing 5-HT1A receptors in sympathetically mediated thermoregulatory mechanisms. A decrease in Tbody is a consistent finding when 8-OH-DAT is given either systemically or locally into the medullary raphé (75, 88). Based on our results and those of others, we speculate that this is secondary to an attenuation of both heat production (shivering) and heat conservation (peripheral vasoconstriction) necessary to maintain Tbody in a cold environment.

The dialysis of WAY-100635 eliminated the effects of 8-OH-DPAT on vasoconstriction, HR, and Tbody (and sleep), consistent with the findings of others (63, 64). Although our experiments were not designed to investigate the longer term effects of 5-HT1A receptor blockade, we did not appreciate any changes during the 30 min of WAY dialysis. This is not surprising since our animals were studied during continuous cooling where shivering, near maximum peripheral vasoconstriction, and elevations in HR were already present. Moreover, Ootsuka and Blessing (63) have reported in rabbits at room temperature that systemic administration of WAY-100635 into the raphé was not associated with any changes in ear pinna blood flow, suggesting that tonic activation of 5-HT1A receptors in the raphé does not contribute to tonic regulation of ear vasomotor tone. In addition, WAY-100635 has been described as a "silent" 5-HT1A antagonist, a term that has been used to distinguish true antagonists from partial agonists (23, 24). Although a few studies have shown effects of WAY-100635 alone on behavior (56) and 5-HT neuronal firing rate (29, 30) when microinjected into the dorsal raphé, the role of intrinsic 5-HT1A agonists in the tonic control of medullary raphé 5-HT neuronal firing rate remains unclear.

Dialysis of 8-OH-DPAT into the medullary raphé attenuates shivering and decreases muscle tone. The attenuation of shivering following activation of 5-HT1A receptors in the medullary raphé suggests that 5-HT neurons in this region have an excitatory effect on shivering thermogenesis, most likely at the level of the spinal cord, and play an important role in the thermoregulatory response to cold exposure. However, we cannot rule out the possibility that the effects of 8-OH-DPAT on shivering that we observed may have resulted from the activation of postsynaptic receptors on other spinally projecting neurons involved in shivering thermogenesis. Our data are consistent with recent studies showing that fusimotor activity is attenuated after "nonspecific" neuronal inhibition in the raphé with glycine (87). Our data further suggest that neurons expressing the 5-HT1A receptor in the raphé, as well as those in the more lateral PGCL, modulate cold shivering activity, but neurons in the raphé are more involved in sympathetically driven mechanisms than those in the PGCL.

The results of previous studies in our laboratory have shown decreases in skeletal muscle tone in NREM sleep after 8-OH-DPAT dialysis into the PGCL (17), even when the animals were maintained in a thermoneutral environment. As evidenced by significant decreases in nonphasic nEMG after 8-OH-DPAT dialysis, a general decrease in muscle tone was observed in the present study where 8-OH-DPAT was dialyzed into the medullary raphé. The mechanisms responsible may be similar to the muscle atonia commonly observed during REM sleep (37, 68). Magoun and Rhines (48) also observed an inhibitory effect of neurons in the caudal medulla on spinal motor activity. In newborn rats, both atonia-on and atonia-off neurons have been identified in both midline (raphé) and more lateral regions of the medulla, homologous to regions that we explored in the piglet, suggesting that neurons in this region modulate muscular tone (40). Moreover, there are major 5-HT projections from the medullary raphé to the ventral horn of the spinal cord (2), and excitatory 5-HT receptors in the cord appear to be important for the development of locomotion patterning (70). We hypothesize that the decrease in shivering and hypotonia after 8-OH-DPAT dialysis during cooling in the present study and the hypotonia we previously observed after 8-OH-DPAT dialysis into the PGCL during thermoneutrality are caused by a dysfacilitation of serotonergic excitatory modulation of muscle tone at the level of the spinal cord.

Other limitations of the study. Previous estimates of diffusion using agents such as fluorescein (17) suggest that 8-OH-DPAT dialyzed into the PGCL in our previous experiments did not diffuse into the raphé region. Our findings in previous studies that dialysis of 8-OH-DPAT into the medullary raphé, but not into the PGCL, attenuated peripheral vasoconstriction and HR support this contention. Therefore, we can say with some degree of confidence that neurons expressing 5-HT1A receptors in the PGCL modulate both sleep and shivering. Similarly, we can say with reasonable certainty that neurons expressing 5-HT1A receptors in the medullary raphé are more involved in sympathetically mediated thermoregulatory mechanisms than those in the PGCL. It is less certain that 8-OH-DPAT dialyzed into the raphé was confined to the raphé alone. It is possible that the effects on shivering and sleep we observed after raphé dialysis were due to diffusion of 8-OH-DPAT into more lateral PGCL regions. Alternatively, neurons expressing 5-HT1A receptors located both in the raphé and PGCL modulate sleep and shivering. Several lines of evidence suggest the latter: 1) microinjection of 8-OH-DPAT into the raphé magnus attenuates shivering in rats during cooling (7); 2) microinjection of glycine into the medullary raphé attenuates fusimotor fiber activity during cooling (87); and 3) the estimated lateral spread of 8-OH-DPAT as estimated by fluorescein dialysis is ~1.5 mm from the midline, with no overlap with the medial border of the PGCL (see Fig. 1 in Ref. 17).

Some of our dialysis probe tips were located rostrally in the medulla, near or in the raphé magnus. Although effects on shivering have been noted after microinjection of 8-OH-DPAT into this region, neurons involved in sympathetically mediated thermoregulatory mechanisms in the rodent, including BAT thermogenesis and peripheral vasoconstriction, are thought to be largely located in the raphé pallidus (52, 54, 55, 59). However, in the piglet, the raphé pallidus extends as a column near the ventral surface in the rostrocaudal dimension of the facial nucleus (see Fig. 1 in Ref. 17) (17, 61). It is, therefore, highly likely that 8-OH-DPAT diffused into the raphé pallidus, even from the most rostral locations.

Perspectives and significance. In summary, we have reported the important finding that neurons expressing 5-HT1A receptors located in the medullary raphé are involved in the modulation of both sleep and thermoregulatory effector mechanisms, including shivering and peripheral vasoconstriction. We postulate that the effects we observed were largely secondary to a decrease in 5-HT neuronal activity, but we cannot rule out a contribution from non-5-HT neurons expressing 5-HT1A receptors. We speculate that serotonergic neurons located in both the raphé and adjacent PGCL are important for sleep homeostasis and send excitatory projections to the spinal cord that modulate fusimotor activity essential for the generation of shivering tremor. In contrast, 5-HT neurons in the raphé have more influence on peripheral vasoconstriction than do 5-HT neurons located in the PGCL. These findings suggest a possible link between sleep and temperature homeostasis and the abnormalities in medullary serotonergic receptor binding and numbers found in the raphé and PGCL in substantial numbers of SIDS infants. Dysfunction in these regions may increase the risk for SIDS by altering sleep architecture and protective reflexes to stressors encountered during sleep, such as hypercapnia and thermal challenges (20).


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 ABSTRACT
 METHODS
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R. A. Darnall is supported by National Institute of Child Health and Human Development Grants PO1HD36379 and RO1HD045653 and a grant from the First Candle/SIDS Alliance.


   ACKNOWLEDGMENTS
 
The authors thank Drs. Eugene Nattie, Donald Bartlett, and Hannah Kinney for reviewing the manuscript and providing guidance; Laurie Hildebrandt for managing the laboratory; and Tracey Damon for identifying the location of our dialysis probe tips.

Present addresses: J. W. Brown, James Madison University, Department of Biology, BURR 306, MSC 7801, Harrisonburg, VA 22807 (e-mail: brown3jw@jmu.edu); E. A. Sirlin, Penn State University College of Medicine, 500 University Dr., Hershey, PA 17033 (e-mail: esirlin@hmc.psu.edu); A. M. Benoit, University of New England College of Osteopathic Medicine, 11 Hills Beach Rd., Biddiford, ME 04005 (e-mail: abenoit1@mail.une.edu); J. M. Hoffman, University of Vermont, Department of Neuroscience, 416 HSRF, 149 Beaumont Ave., Burlington, VT 05405 (e-mail: jill.hoffman@uvm.edu).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Darnall, Dept. of Physiology, Dartmouth Medical School, One Medical Center Dr., Borwell Bldg., Lebanon, NH 03756 (e-mail: robert.a.darnall{at}hitchcock.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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