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J Appl Physiol 92: 331-342, 2002;
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Vol. 92, Issue 1, 331-342, January 2002

Neural responses to intravenous serotonin revealed by functional magnetic resonance imaging

Luke A. Henderson1,2, Pearl L. Yu1, Robert C. Frysinger1, Jean-Philippe Galons3, Richard Bandler2, and Ronald M. Harper1

1 Department of Neurobiology, University of California at Los Angeles, California 90095-1763; 2 Department of Anatomy and Histology and Pain Management and Research Center, Royal North Shore Hospital, University of Sydney, Sydney, New South Wales 2006, Australia; and 3 Department of Radiology, University of Arizona, Tucson, Arizona 85724


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the sequence of neural responses to the hypotension, bradycardia, and apnea evoked by intravenous administration of 5-hydroxytryptamine (serotonin). Functional magnetic resonance imaging signal changes were assessed in nine isoflurane-anesthetized cats during baseline and after a bolus intravenous low dose (10 µg/kg) or high dose (20-30 µg/kg) of 5-hydroxytryptamine. In all cats, high-dose challenges elicited rapid-onset, transient signal declines in the intermediate portion of the solitary tract nucleus, caudal midline and caudal and rostral ventrolateral medulla, and fastigial nucleus of the cerebellum. Slightly delayed phasic declines appeared in the dentate and interpositus nuclei and dorsolateral pons. Late-developing responses also emerged in the solitary tract nucleus, parapyramidal region, periaqueductal gray, spinal trigeminal nucleus, inferior olivary nucleus, cerebellar vermis, and fastigial nucleus. Amygdala and hypothalamic sites showed delayed and prolonged signal increases. Intravenous serotonin infusion recruits cerebellar, amygdala, and hypothalamic sites in addition to classic brain stem cardiopulmonary areas and exhibits site-specific temporal patterns.

myocardial ischemia; Bezold-Jarisch reflex; bradycardia; apnea; hypotension


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MECHANICAL OR CHEMICAL STIMULATION of cardiopulmonary afferents, such as the aortic plexus, myocardial receptors, or bronchopulmonary C fibers, elicits a profound hypotension, bradycardia, and apnea known as the Bezold-Jarisch depressor reflex. The reflex is of considerable clinical interest, because loss of perfusion from the accompanying hypotension may be life threatening and rapid intervention is often necessary for survival (9, 29). The integrity of the reflex may play a role in compensatory cardiovascular responses in myocardial ischemia or aortic stenosis (38, 42). Although the focus of attention for eliciting the reflex has been directed principally on stimulation of afferents associated with myocardial damage, other conditions, such as acute hypovolemia (19) or deep pain (24), may trigger similar physiological sequences.

Since the first description of the Bezold-Jarisch reflex by Von Bezold and Hirt in the late 19th century and Jarisch in the early 20th century (23), a range of substances have been identified that elicit this reflex, including plant alkaloids; nicotine; capsaicin; venoms from snakes, insects, and marine animals; histamine; and serotonin [5-hydroxytryptamine (5-HT)] (1). Studies employing electrophysiological recording, stimulation, and lesion techniques have begun to define the neural structures responsible for the reflex expression. Components of the circuitry responsible for the baroreceptor reflex appear to mediate hypotensive and bradycardic components of the Bezold-Jarisch reflex [e.g., nucleus of the solitary tract (NTS), caudal ventrolateral medulla (CVLM), and rostral ventrolateral medulla (RVLM)] (11, 48). However, certain medullary structures, such as the caudal midline medulla (CMM, including the nucleus raphe pallidus and obscurus), not normally associated with baroreflex control, appear to be recruited during stimulation by 5-HT (46). The anatomic organization of medullary and more rostral sites involved in airway reflexes has been outlined earlier (12). However, the sequence of activation of these structures responsible for expression of apnea during airway reflexes and the extent of contributions from these areas remain unknown. Although single-cell recording and stimulation and lesion studies can reveal specific functions of a localized brain region, determination of the relative onset of activity within activated neural sites during this challenge requires simultaneous assessment of widespread neural areas, a difficult task for microelectrode recording or stimulation/lesion studies.

Functional magnetic resonance imaging (fMRI) procedures provide a means to evaluate activity changes in widespread brain sites without use of systemic contrast agents, ionizing radiation, implanted electrodes, or other brain intervention. The origin and sequence of neural activity responses to challenges can be assessed rapidly over the entire brain, assisting determination of functional organization of responses to physiological challenges. We used the blood oxygen level-dependent (BOLD) technique, which is based on the principle that activated brain areas undergo increased local blood flow and volume, becoming relatively more oxygenated than surrounding areas. Because oxygenated blood exhibits different paramagnetic properties, activated regions show small magnetic signal changes (33). Unlike functional neuroanatomy procedures, such as c-Fos or 2-deoxyglucose mapping techniques, fMRI procedures allow repeatable assessments with minimal injury to the subject.

We used BOLD imaging techniques in adult cats to visualize brain regions that respond to activation of the cardiopulmonary afferents by intravenous administration of 5-HT. Intravenous 5-HT administration has been used to stimulate cardiopulmonary afferents while avoiding concerns of agent transmission through the blood-brain barrier (13, 44). Stimulation effects from intravenous 5-HT administration are very short lasting (50), thus minimizing accumulation of effects from multiple trials. The primary objectives were to 1) determine the neural substrates involved in mediating arterial pressure, heart rate, and respiratory responses to stimulation of cardiopulmonary afferents and 2) evaluate the temporal sequence of neural activation in different areas after the afferent stimulation.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Under isoflurane anesthesia, nine adult cats (3 male, 6 female; 2-5.9 kg) were intubated, and the left or right carotid artery and jugular vein were cannulated for the measurement of arterial pressure and administration of pharmacological agents. The tip of the jugular cannula was inserted to the right atrium. Silver wire was placed subcutaneously in the left and right thoracic walls for recording of electrocardiographic (ECG) activity. Magnetic resonance imaging of the brain requires head immobility and a means to provide consistent interanimal head positioning within the scanner to ensure comparable neural views across different subjects with reference to a standard stereotactic atlas. Under isoflurane anesthesia, cats were placed in a Kopf stereotactic device, the dorsal surface of the skull was exposed via a midline incision, and a headpiece was attached to the skull by nylon screws and by dental cement in an opened frontal sinus. The headpiece consisted of a Plexiglas tube that later slipped over a mating rod in a larger carrier to support the animal. This carrier fit comfortably into a 60-mm-diameter Doty head coil (Doty Scientific, Columbia, SC) in the scanner (17).

Echo-planar fMRI procedures are especially sensitive to magnetic field inhomogeneities. Such inhomogeneities are created by the bony tentorium between the cerebellum and cerebrum, cerebrospinal-brain tissue interfaces, air-filled sinuses, and ferrous particles produced by the drill bits during surgery. This sensitivity can lead to excessive signal drop-out or to very large (>15%) signal changes, which were excluded from analysis in this series of studies. To reduce magnetic field inhomogeneities from ferrous particles, the frontal sinuses were well flushed with saline, and, later in the series, a diamond-tipped drill bit replaced the steel bit. A small bag of deuterium oxide (heavy water, 2H2O) was placed immediately dorsal to the cerebellum on the back of the neck to reduce magnetic field inhomogeneities created by skull structures near the caudal brain. These two procedures substantially reduced artifacts.

The ECG leads were led to a high common mode rejection and low-noise operational amplifier inside the scanner area and were coupled by fiber-optic cables to a receiver located outside of the magnetic resonance imaging room (34). In the absence of a magnetic resonance-compatible pneumotachometer, an indication of respiratory activity was provided by assessing thoracic wall breathing movements using a small, sealed, air-filled bag attached to nondistensible plastic tubing, placed between the thoracic wall of the cat and the wall of the carrier. The plastic tubing was then led external to the scanner to a pressure transducer (PX138 series, Omega Engineering, Stamford, CT). Low-compliance arterial cannulas were led outside the scanner shielding for connection to an arterial pressure monitor. Body temperature was assessed by using an optically coupled rectal probe and was maintained between 37.0 and 38.0°C by using a magnetic resonance imaging-compatible heating blanket (Gaymar, Orchard Park, NY). Animals were maintained on 1% isoflurane during the entire procedure. A magnetic resonance imaging-compatible pulse oximeter was used to record O2 saturation. All physiological signals were recorded digitally by using the DaqEz analog-to-digital system (Quatech). The externalization of physiological signal transduction with fluid- or air-filled transfer media or optic coupling minimized noise introduced into the fMRI images and, conversely, reduced noise introduced into the physiological signals produced by the changing scanner magnetic fields.

The Echo-planar technique (8) was used to visualize rapid brain signal changes. A set of anatomic images was acquired by a 4.7-T Bruker scanner using a transverse relaxation time rapid acquisition with relaxation enhancement (T2 RARE) protocol, followed by a time series of 45 gradient-echo image sets, composed of 19 coronal sections, acquired during each 5-HT or saline challenge (repetition time = 1 s, 8 interleaved scans per image set, echo time = 25 ms, flip angle = 90°, field of view = 8 cm, thickness = 2 mm, no interslice gap). Voxel sizes were 0.625 × 0.625 × 2 mm (the latter, slice thickness).

The 45 image sets were acquired over an uninterrupted 6-min period. The first 3 min consisted of baseline image acquisition, after which a challenge of high-dose (20 or 30 µg/kg) or low-dose (10 µg/kg) 5-HT was delivered intravenously as a single bolus; the challenge was followed by an additional 3 min of image collection. All doses were dissolved in 0.5 ml of saline and were followed immediately by a 5-ml saline flush (5.5 ml total). An equivalent bolus volume of normal saline (5.5 ml) was administered over an equivalent time period as the 5-HT bolus (4 s), as a control dose. At the end of the studies, the animals were euthanized with an overdose of pentobarbital sodium. All procedures were approved by the University of Arizona Institutional Review Board.

The arterial pressure, ECG, and thoracic wall movement traces were digitized at 1 kHz. Inspiratory and expiratory onset and offset times and amplitude of thoracic wall movements were determined by a peak-searching algorithm, and the extent of thoracic wall movements and respiratory rates were determined from those calculations. Mean values for 100-ms successive epochs of arterial pressure, heart rate, respiratory rate, and thoracic wall movement extent were calculated over the entire baseline and challenge periods and are averaged over animals.

The first image set (8 s) was excluded from analysis because the technical characteristics of the scan procedure required a settling time for adequate signal assessment, leaving 44 image sets for analysis, 22 during baseline and 22 during challenge. The MedX image analysis package (Sensor Systems, Sterling, VA) was used to evaluate functional image signal changes during each challenge, relative to the corresponding baseline period. Animation of successive image sets was used to provide an initial visual assessment of movement during the scanning period; movement was also tracked by plotting values for the center of intensity of one slice per volume over time to assess whether significant motion emerged. No detectable motion artifact, defined as sudden changes twice the baseline noise level, was apparent, largely because the animals were anesthetized and because the head restraint minimized movement (17); therefore, no motion correction was performed. All image sets were ratio intensity normalized so that mean intensity and standard deviation values were equalized. Gaussian smoothing was performed with X = 1.3, Y = 1.3, Z = 1 mm. Significant changes in voxel intensity were determined by using a t-test. Nonsignificant voxels were removed from the volume, leaving only those voxels reaching a probability of P < 0.005. The percent changes in signal intensity (threshold values: ±0.5 to ±15% change from baseline) of these significant voxels were then displayed onto a set of T2 RARE anatomic images.

Regions of interest were selected, the boundaries of which were determined by anatomic criteria. In each animal, signal intensities of all voxels within a region of interest were averaged for each time point, and these signal intensities were then averaged across all animals. The mean signal intensity values (±SE) at each time point were expressed as a percent change from baseline. Statistical comparisons with baseline were calculated for each time point using repeated-measures ANOVA (P < 0.005).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Physiology

Intravenous saline. The mean resting arterial pressure, heart rate, and respiratory rate values before saline administration were 109 ± 9 mmHg, 147 ± 10 beats/min, and 23 ± 5 breaths/min, respectively. For all cats, intravenous saline resulted in a small perturbation in arterial pressure and heart rate but not in respiratory rate (Fig. 1C).


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  		Fig. 1.   Traces of arterial pressure, heart rate (in beats/min), respiratory rate (in breaths/min), and extent of thoracic wall movement during baseline and after bolus intravenous delivery of 20-30 µg/kg 5-hydroxytryptamine (5-HT) (A), 10 µg/kg 5-HT (B), and saline (C). Vertical dashed lines indicate start of challenge. Values are means ± SE.

Intravenous 5-HT.  ARTERIAL PRESSURE. In seven of eight animals, high-dose (n = 11) and low-dose (n = 7) 5-HT evoked a triphasic arterial pressure response (Fig. 1, A and B) (1 of the 9 animals was excluded because of scanner-induced artifactual noise). Within 5 s of high-dose 5-HT administration onset, arterial pressure fell 18 ± 5 mmHg (baseline = 120 ± 6 mmHg); the fall was transient (~10 s to maximum decrease) and returned to 3 ± 3 mmHg below baseline. A prolonged hypotension followed, reaching 25 ± 5 mmHg below baseline and returning to baseline ~500 s after challenge onset. The remaining animal showed a similar initial decrease but no late hypotension. Patterns after low-dose administration were similar but with reduced magnitude (Fig. 1B).

HEART RATE . In all animals, high-dose 5-HT administration resulted in a biphasic change in heart rate. Within 5 s of the high-dose challenge, heart rate began to decline, reaching a nadir of 118 ± 5 beats/min (baseline = 144 ± 6 beats/min). This transient bradycardia was followed by a prolonged tachycardia, reaching a maximal increase of 29 ± 6 beats/min above baseline. Heart rate then returned to baseline within 300 s of 5-HT delivery. Low-dose heart rate patterns were identical but were reduced in amplitude.

RESPIRATION . Breathing was markedly affected by 5-HT in a dose-dependent fashion. Within 5 s of high-dose administration, an apnea ensued, with cessation of breathing efforts for 30 ± 7 s (range 8-100 s). After the apnea, respiratory rate returned to baseline (27 ± 2 breaths/min) within 2 s and then increased to a level 37 ± 5 breaths/min above baseline for ~300 s. The magnitude of thoracic wall movements also returned to baseline, after an increase (34 ± 11% above baseline) for ~150 s. Low-dose administration resulted in a reduced apnea duration 12 ± 3 s (range 5-30 s) and a reduced compensatory later tachypnea (22 ± 6% above baseline) for ~150 s.

Distribution of fMRI Signal Changes

Intravenous saline. Intravenous administration of saline resulted in no significant change in signal intensity in any area of the brain (Fig. 2).


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  		Fig. 2.   Signal-intensity changes in regions of the medulla (A), cerebellum (B), pons and midbrain (C), and hypothalamus and amygdala (D) after an equivalent volume of intravenous saline. Vertical dashed lines indicate start of challenge. Values are means ± SE.

Intravenous 5-HT. In contrast to intravenous saline, 5-HT evoked significant changes in signal intensity in a number of brain structures. These changes were not confined to medullary regions classically associated with mediation of changes in arterial pressure, heart rate, and breathing, but they also included rostral brain structures and sites within the cerebellum. The signal changes, in most cases, were dose dependent.

Three general trends were observed to high-dose 5-HT challenge. 1) Medullary and some cerebellar sites showed a rapid-onset transient decline in signal intensity, followed by a fast recovery and sustained signal increase; in some cases, activity remained below baseline. 2) In the dentate and interpositus cerebellar nuclei and dorsolateral pons, the initial transient change was delayed. 3) In rostral brain sites, as well as the inferior olive, parapyramidal region, and spinal trigeminal nucleus, activity showed a delayed onset, sustained increase.

EARLY-ONSET TRANSIENT DECLINE. The changes in signal intensity are shown as pseudocolored overlays on anatomic views as well as mean ± SE values across baseline and challenge for different areas in Figs. 3-5. In medullary sites of the intermediate NTS (NTS region at the level of and rostral to the obex), RVLM, CVLM, and CMM (Fig. 3), an initial decline in signal intensity occurred (intermediate NTS maximum fall = 2.3 ± 1%; RVLM maximum fall = 0.8 ± 0.8%); the decline emerged slightly later (first 16 s) within the CVLM (maximum fall = 1.7 ± 0.9%). A large transient decrease in signal intensity also appeared within the CMM, falling 2.2 ± 1% within the first 8 s and returning to baseline at 24 s. This transient decline was the only response observed in the CMM; no significant late change emerged. The fastigial nucleus also showed an immediate phasic decline (maximum decrease = 1.4 ± 0.4%); however, this decline was more prolonged than other medullary sites.


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  		Fig. 3.   Signal-intensity changes in the medulla after administration of high-dose (20 or 30 µg/kg) or low-dose (10 µg/kg) intravenous 5-HT, relative to the initial 3-min baseline period in all cats. A: caudal ventrolateral medulla. B: rostral ventrolateral medulla. C: nucleus of the solitary tract, caudal. D: nucleus of the solitary tract, intermediate. E: caudal midline medulla. F: spinal trigeminal nucleus. Signal changes were combined from the left and right sides of the outlined areas (white outlines) and at the 1 or 2 stereotactic levels (e.g., P13, P11) indicated on the brain outline in E. The P level refers to distance in mm posterior to the interaural line (41). Significant signal changes, calculated from 2 or 3 scans in the region of the arrows, are encoded by extent of change (percent change from baseline), pseudocolored according to the color scale (-15 to -0.5% and 0.5 to 15%), and superimposed on anatomic images. Vertical dashed lines indicate start of challenge. *Significance difference compared with baseline, P < 0.005.



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  		Fig. 4.   Signal-intensity changes at 2 dose levels of 5-HT, combined with signal changes overlaid on comparable anatomic images in cerebellum [fastigial (A), interpositus and dentate nuclei (B), and cerebellar vermis (C)], and cerebellum-related regions [inferior olivary nucleus (D) and dorsolateral pons (E)], and the parapyramidal region (F). Stereotactic designations and pseudocolor calculations are as for Fig. 3. Vertical dashed lines indicate start of challenge. *Significance difference compared with baseline, P < 0.005.



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  		Fig. 5.   Functional magnetic resonance imaging signal changes at 2 dose levels of 5-HT, combined with signal changes overlaid on comparable anatomic images in midbrain periaqueductal gray (A), hypothalamus (C-E), amygdala (B), and the internal capsule (F; white matter control area). Values are means ± SE. A and P levels refer to distance in mm anterior and posterior to the interaural line, respectively (41). Pseudocolor scale is as for Fig. 3. Vertical dashed lines indicate start of challenge. *Significance difference compared with baseline, P < 0.005.

After the initial phasic decline, and concomitant with the end of the apnea and the onset of the prolonged hypotensive phase (24 s after 5-HT), signal intensity within the intermediate NTS and RVLM rapidly increased above baseline (intermediate NTS maximum increase = 2.8 ± 0.9%; RVLM maximum increase = 2.5 ± 0.9%). This increase in signal intensity within the RVLM was delayed with low doses. After the initial decrease, signal intensity within the CVLM and fastigial nucleus also returned to baseline levels; however, unlike the NTS or RVLM, signal intensity then decreased toward the end of the scanning period.

DELAYED-ONSET DECLINE. In two areas (Fig. 4), the dentate and interpositus nuclei and dorsolateral pons (including the Kolliker-Fuse nucleus), onset of the initial decline was delayed, reaching a nadir by 32-40 s (cerebellar nuclei maximum decline = 1.6 ± 0.8%; dorsolateral pons maximum decline = 3.3 ± 1.4%). This decrease was maximal at approximately the time at which respiration returned to prestimulus levels (40 s after 5-HT).

LATE-ONSET SUSTAINED RESPONSE. In contrast to the initial decline of several brain stem sites, more rostral brain areas of the amygdala, regions within the hypothalamus and the midbrain periaqueductal gray matter (PAG), showed signal intensity elevations to 5-HT challenge (Fig. 5). Although there was a short-lived initial rise in the amygdala and PAG (~8 s after 5-HT), the major response was a slowly rising and prolonged increase in signal intensity (hypothalamus maximum increase = 2.9 ± 0.4%, amygdala maximum increase = 1.5 ± 0.4%, PAG maximum increase = 1.4 ± 0.2%). Within the parapyramidal region, signal intensity increased within 16 s of 5-HT administration to 2.6 ± 2% above baseline. The only other sites to show such a late sustained increase were the caudal NTS (NTS region caudal to the obex), spinal trigeminal nucleus and the inferior olive (caudal NTS maximum increase = 2.8 ± 0.9%; spinal trigeminal nucleus maximum increase = 1.8 ± 0.4; inferior olive maximum increase = 2.4 ± 1.5%). The cerebellar vermis exhibited a late fall in signal intensity (maximum decrease = 2.6 ± 0.7%); however, unlike the fastigial and deep cerebellar nuclei, the vermis did not show an early transient response.

A summary of the three general responses is shown in Fig. 6. The time sequence of traces emphasizes the correlation of the early- and mid-onset transient responses with the response to cardiopulmonary stimulation. In addition, a correlation appears between the late-onset sustained responses with the prolonged hypotensive phase. No significant changes were found in the internal capsule, a control site (Fig. 5).


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  		Fig. 6.   Traces of arterial pressure, heart rate (in beats/min), and respiratory rate (in breaths/min) after high-dose 5-HT administration, together with signal values from each sampled area over the baseline and course of this challenge. Values are means ± SE. Signal changes are grouped by timing of initial changes to the challenges: early, mid, or late onset. Early-onset declines developed primarily from medullary sites, as well as the cerebellar fastigial nucleus. Sites slightly delayed in responses include the parapyramidal region, the interpositus and dentate cerebellar nuclei, and the dorsolateral pons. Late-onset response increases emerged principally from rostral brain stem and forebrain sites but also from inferior olive, spinal trigeminal nucleus, and caudal nucleus of the solitary tract. Note the late-onset decreased response from the cerebellar vermis. Vertical dashed lines indicate start of challenge, termination of apnea, and return of respiratory rate to baseline.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of cardiopulmonary fibers by intravenous 5-HT elicited discrete signal changes over multiple brain regions. In addition to medullary regions previously implicated by c-Fos or electrophysiological procedures in expression of responses to cardiopulmonary stimulation, the sites included neural areas in the cerebellum and regions rostral to the medulla; the latter sites have been implicated in anatomic studies of airway reflex and blood pressure control (12, 43). Whereas activation in medullary areas followed a sequence of an initial decline within the first 8 s and a later rise, rostral areas showed no such phasic initial decline but only a late-emerging increase; these increases were long lasting (Fig. 6). The temporal patterning of responses in different sites suggests specific roles for separate sites mediating the response. The marked apnea, hypotension, and bradycardia associated with 5-HT administration recruited an integrated pattern of neural responses in multiple brain areas, some of which are not traditionally associated with cardiovascular and respiratory control.

Physiology

As reported by others, intravenous 5-HT administration evoked a short-latency hypotension and apnea, which, as Meller et al. (30) have shown, is elicited from activation of 5-HT3 receptors located on cardiopulmonary vagal afferents. In addition, 5-HT administration evoked a late-onset prolonged hypotension. This later hypotension has been presumed to result from activation of 5-HT7 receptors, evoking a peripherally mediated vasodilation and a reflexive, baroreceptor-mediated increase in heart rate (39, 49). Thus 5-HT administration evoked two distinct hypotensive reflexes: an early-onset transient hypotension, bradycardia, and apnea and a late-developing, presumably peripherally mediated hypotension and baroreflex-mediated tachycardia.

Medulla

Consistent with previous c-Fos (11), electrophysiology (46), and lesion (26) evidence, 5-HT infusion evoked signal-intensity changes in several medullary regions. Signal intensity immediately decreased within the intermediate NTS and CVLM. The cardiopulmonary afferent activity ascends through the vagus to the NTS (22, 31, 45) from which CVLM neurons receive input (47). It is of interest that the initial signal change is a decline in both the NTS and CVLM; the decreases may reflect a combination of activation by cardiopulmonary afferent stimulation and deactivation by the baroreceptor reflex as arterial pressure falls. Although both the caudal and intermediate NTS increased signal intensity during the late hypotensive phase, only the intermediate NTS displayed an early fall. This may reflect the disparate anatomic projections of vagal afferents (12, 40) and vagal C fibers, particularly the specificity of termination of afferents within NTS regions at the level of, and slightly rostral to, the obex (25).

The direction of signal change in the CVLM and the initial decrease within the RVLM support a role originally hypothesized from optical imaging data obtained from the ventral medullary surface (VMS) of cats and goats. Optical studies reveal that stimulation of chemoreceptors by hypercapnia, simulated hypoxia with intravenous cyanide, or arterial pressure elevation elicits a decline in activity over regions of the VMS, with the extent of decline dependent on state (28, 36, 37). Transection of the carotid sinus nerve eliminates the signal decline to cyanide administration, suggesting that the VMS receives inhibitory or dysfacilitatory influences from carotid chemoreceptors. Stimulation of C-fiber afferents by 5-HT also elicits a transient decline in activity in multiple medullary areas bordering the VMS, as well as the CMM, but not (at least at high doses) in the NTS.

In contrast to the CVLM, CMM, and NTS, signal intensity in the parapyramidal region increased significantly, but it did so later in the cardiopulmonary phase. Retrograde tracing (both viral transneuronal and nonviral) indicates that the parapyramidal region contains parasympathetic neurons innervating both the phrenic motor nucleus and the peripheral airways (12, 16). Furthermore, the region responds to respiratory stimuli such as hypercapnia, hypoxia, and anoxia (4, 6). The slightly delayed-onset and maintained nature of the increase in parapyramidal signal intensity suggest a role in both the cessation of the apnea and compensatory increase in respiratory drive during the late-onset baroreflex phase. The region of the VMS, immediately lateral to the parapyramidal region, increases activity, as evaluated by optical procedures, to hypotension elicited by sodium nitroprusside administration or hypovolemia (14, 36).

The change in signal intensity in the CMM was particularly remarkable, because of its immediate onset, profound extent of fall, and transient nature, with the signal returning to baseline within 24 s. Unlike other regions, such as NTS, RVLM, cerebellum, amygdala, or dorsal lateral pontine sites, only the initial phasic decline was apparent. The duration of signal-intensity decrease in CMM was concurrent with the initial physiological responses, but the rapid return to baseline did not parallel the long-lasting hypotension. The transient nature of the response suggests a signaling function, rather than a role in long-term maintenance of arterial pressure or respiration. The primary role of the CMM in regulation of breathing or arterial pressure control remains unclear. Although chemical activation of the CMM results in hypotension and bradycardia (15, 18), electrolytic lesions and chemical inactivation of this region have no effect on resting arterial pressure or heart rate or on the arterial pressure or heart rate responses to activation of either cardiopulmonary afferents or baroreceptor reflexes (19, 26). However, Vayssettes-Courchay and colleagues (46) reported that single neurons within the CMM increase or decrease firing rate after activation of cardiopulmonary afferents, suggesting that the structure plays a role in the apneic, rather than vasodepressor, components of the reflex. Chemical activation of the CMM alters respiratory patterning and enhances phrenic nerve discharge (3, 15), and, most strikingly, ibotenic acid lesions of the CMM results in respiratory failure (7), whereas comparable lesions in well-described respiratory regions (NTS, area postrema, lateral and ventrolateral medulla, including the nucleus ambiguus) fail to elicit such a breathing cessation. The signal-intensity decrease in CMM may indicate an activity decline that plays a role in the profound apnea after 5-HT infusion.

Cerebellum and Related Structures

The signal changes found in the inferior olive (a principal afferent source to cerebellar Purkinje cells), the fastigial, dentate, and interpositius cerebellar nuclei, and the vermis reinforce the significant role that cerebellar-related structures play in mediating breathing and arterial pressure challenges, at least for those of an extreme nature (27, 51). Signal changes in the inferior olive, and also the fastigial, dentate, and interpositus cerebellar nuclei reached their maximal values at approximately the termination of the initial arterial pressure fall and apnea from 5-HT stimulation (Fig. 6), suggesting participation with correction of the apnea (51) and/or the hypotension and bradycardia (27). Together with the inferior olive and fastigial nucleus, the vermis showed late-developing signal changes, possibly representing a role for error correcting the long-lasting hypotension accompanying the challenge (27). Cardiovascular and respiratory expression of fastigial action could be mediated through the dorsolateral pons, which receives significant projections from the fastigial nucleus (43). Dorsal lateral pontine areas showed both a delayed transient response and a later response. The delayed response to the initial 5-HT component may reflect action by this phase-switching site (5) to restore inspiration, whereas the late component may be acting on the late-phase hypotension.

Rostral Sites

In contrast to the initial signal declines in the brain stem, rostral brain areas showed only increased signal intensity after intravenous 5-HT. Apart from an initial, transient signal increase in the PAG and amygdala, the rise in signal intensity emerged late in the response for all rostral areas. This later activation likely represents action to restore breathing after the apnea from the 5-HT administration and, perhaps via the baroreceptor reflex, to increase heart rate.

Studies using c-Fos procedures indicate that neurons in the ventrolateral column of the PAG are selectively activated by intravenous 5-HT (24). Furthermore, stimulation of the lateral and ventrolateral regions of the PAG has been found to modify baroreflex sensitivity in anesthetized rats (21, 32). Thus the late sustained increase in PAG activity reported here may represent actions to alter baroreflex sensitivity and, at the same time, trigger late-phase cardiac responses.

Participation of both amygdala and hypothalamic sites as components of a neural network controlling the airway has been demonstrated anatomically (12). Hypothalamic sites all increased signal intensity to the challenge, and these responses were late and sustained. Expression of c-Fos within the dorsal hypothalamus (including paraventricular nucleus) and ventral hypothalamus (including the arcuate nucleus) increases after baroreceptor unloading (35). Furthermore, during baroreceptor unloading, c-Fos is expressed in norepinephrine-containing CVLM neurons that project directly to the paraventricular nucleus and are critical in mediating the vasopressin release during baroreceptor unloading (10). The late-onset increase in dorsal hypothalamic signal intensity reported here may reflect increased neural activity, possibly resulting in increased vasopressin release.

The significant projections from the amygdala central nucleus to the NTS and parabrachial pons, as well as areas surrounding the nucleus ambiguus (20), provide considerable potential for this limbic structure to modulate concurrent responses in brain stem sites to 5-HT stimulation. The principal influences from the amygdala appear to arise later in the challenge (although a short-lasting initial activation was present). The late-developing response may influence compensatory responses for recovery from the late-developing hypotension.

Relationship to Anatomic Descriptions

The sites demonstrating signal changes to 5-HT administration closely overlap with neural regions identified by transneuronal viral and nonviral retrograde labeling as innervating airway structures and the phrenic motor nucleus (12, 16). Regions displaying early-onset (CMM, CVLM, VMS, intermediate NTS) or late-onset sustained (parapyramidal region, dorsolateral pons, amygdala, hypothalamus, PAG) changes in signal intensity contain neurons innervating both the phrenic motor nucleus and airway structures. Neurons supplying the peripheral airways are also identified within the lateral paragigantocellular nucleus, an area in which we also observed signal changes with cardiopulmonary activation and that aspect will be closely examined in future studies. Labeling within cerebellar sites was not examined in the anatomic studies.

Limitations

Although we used a relatively fast acquisition procedure, image collection was slower than signal changes observed on ventral medullary surface areas with optical procedures (14). However, the focus of this study was to survey brain areas involved in stimulation of cardiopulmonary afferents and to provide a summary view of the temporal patterning of those responses. More rapid image acquisition can be obtained with higher field scanners or by using the current instrumentation with fewer slices, i.e., by limiting scans to brain areas shown by this initial study to be recruited during the challenge.

Cerebral perfusion is autoregulated so that little change in flow occurs between arterial pressures ranging from 50 to 150 mmHg (2). After high-dose administration of 5-HT, arterial pressure fell from 120 to 95 mmHg, well within the autoregulatory range. Magnetic resonance studies directly addressing this concern show that, between 120 and 60 mmHg, there is no significant change in either blood volume or blood flow within the striatum, cortex, or the brain at the level of bregma (52). Furthermore, we found that no region displayed signal changes paralleling the time course of arterial pressure changes and that opposite changes in signal intensity emerged in regions immediately bordering one another, e.g., the caudal ventrolateral medulla and the inferior olivary nucleus, an unlikely event if signal changes resulted from overall perfusion alterations. The changes we report here most likely resulted from local blood flow changes in response to changes in neural activity and were not due to overall changes in cerebral perfusion following changes in arterial pressure.

The initial apnea induced by 5-HT could modify arterial PO2 and end-tidal PCO2, with both modifications having the potential to alter activity in medullary and cerebellar sites. Because onset of the neural signal changes was coincident with apnea onset, and changes in arterial PO2 and end-tidal PCO2 require a finite time to develop, it appears unlikely that the initial, transient changes in neural activity resulted from O2 and CO2 alterations. The late-developing characteristics, however, could well interact with blood-gas levels. Partitioning of those late changes from contributions by gas levels, blood pressure changes, and other sources will be a necessary effort for future evaluation after this initial overview description.

The mode of delivery of 5-HT, via an intravenous route, has the potential to stimulate sensory afferents other than C-fiber cardiopulmonary sensors located in the atrium or thorax. Actions of 5-HT on the airway, such as bronchoconstriction and decreased lung compliance, can affect sensory activity other than direct C-fiber stimulation. Some of the issues, particularly of timing and localization of stimulation, could have been more readily accommodated with administration of phenyldiguanide, a 5-HT3 agonist, rather than 5-HT, and direct administration of the agent onto primary receptors of interest, e.g., the right atrium.


    ACKNOWLEDGEMENTS

We thank Drs. A. Gmitro and J. Alger for assistance with MR scanning and Dr. P. Macey for assistance with analysis.


    FOOTNOTES

This research was supported by National Heart, Lung, and Blood Institute Grant HL-22418; the Flinn Foundation (Phoenix, AZ); and National Institutes of Health/National Cancer Institute Grant CA-83148. Pearl L. Yu is a National Institute of Child Health and Human Development Fellow of the Pediatric Scientist Development Program (Grant K12-HD-00850).

Address for reprint requests and other correspondence: R. M. Harper, Dept. of Neurobiology, Univ. of California, Los Angeles, CA 90095-1763 (E-mail: rharper{at}ucla.edu).

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.

Received 29 August 2001; accepted in final form 17 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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