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J Appl Physiol 97: 2248-2257, 2004. First published June 25, 2004; doi:10.1152/japplphysiol.00297.2004
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Functional magnetic resonance imaging during hypotension in the developing animal

Luke A. Henderson,1,2 Paul M. Macey,1 Chris A. Richard,1 Matthew L. Runquist,3 and Ronald M. Harper1,4

1Department of Neurobiology and 4Brain Research Institute, University of California at Los Angeles, Los Angeles, California 90095-1763; 2Department of Anatomy and Histology, University of Sydney, Sydney, New South Wales 2006, Australia; and 3Department of Radiology, University of Arizona, Tucson, Arizona 85724

Submitted 18 March 2004 ; accepted in final form 20 June 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Hypotension in adult animals recruits brain sites extending from cerebellar cortex to the midbrain and forebrain, suggesting a range of motor and endocrine reactions to maintain perfusion. We hypothesized that comparable neural actions during development rely more extensively on localized medullary processes. We used functional MRI to assess neural responses during sodium nitroprusside challenges in 14 isoflurane-anesthetized kittens, aged 14–25 days, and seven adult cats. Baseline arterial pressure increased with age in kittens, and basal heart rates were higher. The magnitude of depressor responses increased with age, while baroreceptor reflex sensitivity initially increased over those of adults. In contrast to a decline in adult cats, functional MRI signal intensity increased significantly in dorsal and ventrolateral medullary regions and the midline raphe in the kittens during the hypotensive challenges. In addition, significant signal intensity differences emerged in cerebellar cortex and deep nuclei, dorsolateral pons, midbrain tectum, hippocampus, thalamus, and insular cortex. The altered neural responses in medullary baroreceptor reflex sites may have resulted from disinhibitory or facilitatory influences from cerebellar and more rostral structures as a result of inadequately developed myelination or other neural processes. A comparable immaturity of blood pressure control mechanisms in humans would have significant clinical implications.

cat; heart rate; respiration; blood pressure; baroreceptor reflex

HYPOTENSIVE CHALLENGES IN the adult cat elicit functional magnetic resonance imaging (MRI; fMRI) signal intensity declines in multiple cardiovascular-related sites in medullary, cerebellar, insular, and pontine regions, and signal intensity increases in amygdala and hypothalamic areas (23). Recruitment to blood pressure challenges of structures as diverse as the forebrain and cerebellum emphasizes the widespread compensatory neural actions necessary to maintain vital perfusion. Participation of these diverse areas in mediating appropriate responses relies on the integrity of afferent and efferent projections from primary medullary afferent and effector sites. This integrity is compromised in very young animals that have not yet completed myelination and other axonal growth processes, with considerable evidence that the processes for mediating blood pressure challenges are not complete at birth (26). Resting blood pressure levels are lower in the very young animal, and baroreceptor reflex sensitivity requires a period of time to stabilize (16, 26).

The involvement of extramedullary areas that participate in compensatory actions to hypotension is a significant issue in developing humans. Infants are frequently exposed to circumstances that result in profound losses of blood pressure, including exposure to toxins, infection, foreign fluid stimulation of airways, or visceral pain (5, 17, 25). The necessity for participation of extramedullary structures in mediating hypotensive responses is unclear, but presumably those structures exert protective or additional compensatory roles not adequately managed by medullary reflexes. At least some clinical conditions suggest that mechanisms underlying restoration of arterial pressure (AP) can be deficient in infants; some fatal scenarios of the sudden infant death syndrome (SIDS) are associated with a profound loss of AP that is not adequately restored (27). Late or inadequate development of neural processes mediating AP restoration might compromise recovery from hypotension.

Because projections from medullary cardiovascular sites to extramedullary structures require a period of time to develop, participation of forebrain, cerebellar, and other regions distant from primary afferent and effector nuclei may not approach adult levels for some time after birth. Even certain medullary cardiovascular sites involved in AP regulation, such as the ventral medullary surface (VMS), require 20–30 days to respond at adult levels to pressor challenges in the kitten (16), although it is unclear whether that pattern reflects maturation of the VMS, as opposed to processes that affect the VMS. We hypothesized that AP regulation by extramedullary sites has not developed to adult levels in the kitten, that responses to depressor challenges would be mediated principally by local medullary reflexes in these young animals, and that developmental neural patterns would be revealed by fMRI of responses to a hypotensive challenge.


   METHODS
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 ABSTRACT
 METHODS
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Seven adult cats (six females; 2.5–3.5 kg) and 17 kittens aged 14–25 days (eight females; 228–367 g) were anesthetized with 5% isoflurane in 100% O2. Data from a subset of the challenges of the adult cat group have been previously reported (23). Following anesthetic induction, isoflurane levels were reduced to 2% for surgical procedures. A tracheotomy was performed, and a tracheal tube inserted. A cannula was placed into the right or left carotid or femoral artery and extended outside the MRI scanner room to a force transducer for AP measurement. The left or right jugular or femoral vein was cannulated for drug and fluid administration. Silver-silver chloride ECG electrodes were placed on either side of the thorax. ECG signals were amplified by using a MRI-compatible amplifier and passed through an optic fiber to a recording system outside the magnetic field (36). Thoracic wall movements were detected by a sealed air-filled bag connected by low-compliance tubing to a pressure transducer located external to the scanner shield; because no MRI-compatible pneumotachograph was available, this indirect measure of breathing was necessary. Each animal was anchored inside the magnetic resonance coil with a Plexiglas stabilization device (22). Body temperature was measured by using an optically coupled rectal probe and maintained between 37.0 and 38.0°C with a MRI-compatible heating blanket. At surgical completion, and before scanning, the isoflurane level was further reduced and maintained between 1.0 and 1.2%.

Depressor responses were elicited by intravenous injection of sodium nitroprusside (NTP; 1–5 µg/kg). Each NTP injection was delivered as a small bolus (adult cats, 0.5 ml; kittens, 0.1 ml) followed by a saline flush (adult cats, 1 ml; kittens, 0.25 ml), over ~8 s. In each animal, up to seven separate administrations of NTP were delivered. To determine baroreceptor reflex sensitivity in each animal, three successive cardiac R-wave-to-R-wave (RR) intervals and corresponding systolic AP values were measured and averaged, resulting in a single value for the three intervals. The calculation was repeated at time points before and during the hypotensive challenge, resulting in a total of seven RR and systolic AP values per animal. The RR intervals were then plotted against systolic AP, and a linear regression was used to measure the slope of this relationship, with the slope being defined as baroreceptor reflex sensitivity. The baroreceptor reflex sensitivity of each animal was plotted against age. The magnitudes of the hypotensive responses to 5 µg/kg NTP administration were also determined at each age.

A gradient echo echo-planar imaging (EPI) protocol using blood oxygen level-dependent (BOLD) contrast (13) was used to visualize signal changes in the brain. Each EPI series consisted of 60 brain volumes [repetition time = 4 s, matrix size = 128 x 128, phase encode steps/segment = 32, time to echo = 12 ms, flip angle = 90°, field of view = 7 x 7 cm (adults) and 6 x 6 cm (kittens), voxel sizes = 0.55 x 0.55 x 2.25 mm (adults) and 0.47 x 0.47 x 1.75 mm (kittens), no interslice gap]. During each EPI series, images were acquired continuously during a 60-s baseline and 180-s challenge period. A rest period of at least 13 min was allowed between each series to provide adequate recovery from the challenge. A set of high-resolution T2-weighted anatomical images was collected before EPI imaging. At completion of the studies, the animals were euthanized with an overdose of pentobarbital sodium (50 mg/kg). All procedures were approved by the University of Arizona Institutional Animal Care and Use Committee.

SPM2 (Statistical Parametric Mapping) (15) and custom software were used to analyze all image sets. Adjustments were made to SPM2 to account for sub-millimeter voxel dimensions. The head stabilization device and anesthesia effectively prevented movement; thus motion correction was unnecessary. Regions surrounding the brain were manually removed, and the image sets were corrected for differences in acquisition time across slices. The T2-weighted anatomical image set from each animal was then spatially normalized to that of one animal. The resulting normalized images were averaged and smoothed [isotropic Gaussian filter, full width at half-maximum (FWHM) = 1.5 mm], creating a T2-weighted anatomical template. For each animal, every EPI series was first spatially normalized to its own T2-weighted anatomical set and then normalized to the T2-weighted anatomical template by using the parameters from the anatomical normalization. All white matter was selected from the normalized T2-weighted anatomical images and removed from the EPI image sets. The resulting gray-matter-only image sets were then spatially smoothed (FWHM = 1.5 mm), intensity normalized, and temporally smoothed (FWHM = 12 s). The first five scans were excluded from analysis in each series to allow for intensity stabilization.

To ensure that the magnitude of the hypotensive response was similar across all ages, only image sets from NTP challenges in which the fall in AP was between 15 and 35% were used, resulting in available data from 7 adult cats and 14 kittens. Multiple trials in each animal were averaged so that only one image set and one mean physiology trace from each animal were used for statistical analysis. Two statistical procedures were used: cluster and volumes-of-interest (VOI) analysis. Cluster analysis consisted of a voxel-by-voxel procedure to search for regions in which signal intensity changes covaried with the changes in AP following NTP administration. Significant differences between adult cats and kittens were determined (random effects; P < 0.05), and the resulting statistical maps were overlaid onto the T2-weighted anatomical template image set. For selected clusters, the average (±SE) signal intensity at each time point was extracted from the processed images and plotted over time by group. VOI analysis used anatomically defined regions to explore changes in signal intensity, irrespective of the pattern of change. These VOI were selected from the EPI image sets by using anatomical landmarks on an animal-by-animal basis. The VOI included regions in the medulla [nucleus of the solitary tract (NTS), caudal ventrolateral medulla (CVLM), rostral ventrolateral medulla (RVLM)], cerebellum (vermis, fastigial/interpositus, and dentate nuclei), diencephalon (amygdala, dorsal and ventral hypothalamus), and cerebral cortex (left and right insula). The average voxel intensity across the VOI was calculated for each volume (every 4 s), resulting in a time trend for each animal. Significant differences between baseline and challenge periods and between adults and kittens during the challenge were determined by using repeated-measures ANOVA (P < 0.05).


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
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Physiology.   Typical patterns of response in AP, heart rate (HR), and breathing rate (BR; assessed as thoracic wall movement) to a 5 µg/kg NTP administration in a 23-day-old kitten are shown in Fig. 1A. Baseline AP increased with age from 50 mmHg at 15 days to 75 mmHg by 25 days; adult cats showed APs of 100 mmHg (Fig. 1B). In the kitten group, baseline HR was higher (~235 beats/min) than in adults (~200 beats/min) (Fig. 1C). The magnitude of AP change to a 5 µg/kg NTP dose also increased with age (Fig. 1D). Baroreceptor reflex sensitivity was more variable in the kitten group and tended to increase from day 14 to day 25 (Fig. 1E); adult levels were substantially lower and more consistent.



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  		Fig. 1. A: typical response of arterial pressure (AP), heart rate (HR), relative thoracic wall movement, and breathing rate (BR) to a 5 µg/kg sodium nitroprusside (NTP) administration (onset at vertical dashed line, time 0). B: mean baseline AP with age. C: mean baseline HR with age. bpm, Beats/min. D: mean percent change in AP (relative to 60-s baseline) for a 5 µg/kg NTP dose with age. E: baroreceptor reflex sensitivity with age.

 
For the trials used in the fMRI analysis (14 kittens and 7 adults, selected on the basis of having a similar mean AP percent change), mean AP, HR, and BR responses are shown in Fig. 2. The change in peak HR emerged earlier and to a greater extent in kittens over adult cats. BR changes over the first 2 min of the response were similar.



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  		Fig. 2. Mean (±SE) percent change ({Delta}) from baseline AP (A), HR (B), and BR (C) in adults and kittens chosen for functional MRI (fMRI) analysis. NTP administration is indicated by the vertical dashed lines.

 
Cluster and VOI differences.   Brain regions in which signal intensity changes significantly differed in kittens from adult cats are shown in Fig. 3. The changes in signal intensity for six of these regions are shown in Fig. 4. The principal response differences emerged in the cerebellar cortex and deep nuclei, ventral and midline medulla, including the medullary raphe nuclei, dorsolateral pons, midbrain tectum, hippocampus, thalamus, right insula, and hypothalamus. VOI analysis revealed additional response differences in the NTS, RVLM, CVLM, deep cerebellar nuclei, and left insula (Fig. 5).



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  		Fig. 3. Regions of significant (P < 0.05, random effects model) fMRI signal intensity differences between kittens and adult cats; the signal patterns in these clusters were correlated to the mean AP decline. Signal intensity changes are pseudocolored, according to their significance level (t value), and overlaid on sequential coronal-plane anatomical brain sections. Stereotaxic levels (in mm) relative to the interaural line (47) are indicated on the top left of each row of images.

 


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  		Fig. 4. Selected time trends for clusters shown in Fig. 3, including the ventral medulla/medullary raphe, dorsolateral pons, cerebellar cortex, insula, posterior thalamus, and lateral hypothalamus. Onset of the NTP injection is indicated by the vertical dashed line. Coronal and sagittal views are shown above the traces. White arrows indicate cluster from which the time trend was calculated.

 


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  		Fig. 5. Time trends (means ± SE) of selected volumes of interest (VOI) in response to the NTP challenge. VOI are outlined in white on anatomical images, and stereotaxic levels (in mm) relative to the interaural line (47) are indicated above each image. Onset of challenge is at vertical dashed line. Time points of significant difference between baseline and challenge periods are indicated by the gray lines above the x-axis. *Time points of significant difference between kittens and adults during the challenge periods, P < 0.05.

 
Both the dorsal and ventral medulla showed substantial differences in responses in kittens from those of adults. Signal intensity in kittens increased in both areas, unlike the declines found in adults, findings confirmed in the VOI analysis of the NTS and both the CVLM and RVLM (Fig. 5). Moreover, the fastigial nucleus showed significantly greater declines in activity in kittens compared with adults. Signal intensity in the cerebellar vermis decreased in both adults and kittens, and no change in signal was found in either group in the dentate nuclei. In the adults, no change in signal was found in the dorsolateral pons, but a substantial transient decline occurred in kittens. In the posterior thalamus, a transient increase occurred at 50 s in the kittens, unlike a late sustained rise in the adults (Fig. 4). The kittens also showed an increased response in the insula (Figs. 4 and 5), with a decline in adults (Fig. 4). Both adults and kittens showed similar increases in signal intensity in the amygdala and ventral hypothalamus (Fig. 5).

A remarkable difference emerged in the hippocampus, with virtually the entire extent of the structure showing a significant response difference between adult cats and kittens (Fig. 3); the adult cats increased signal, and almost no response occurred in the kittens (Fig. 5).

Laterality of response differences.   Although signal intensity changes within the brain stem were for the most part bilateral (Fig. 3), in more rostral structures, lateralization of responses appeared in regions such as the thalamus, hypothalamus, insula, and hippocampus (Figs. 3 and 5).


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
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GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
These studies show that, in the presence of lower baseline AP, the baroreceptor reflex response to hypotension is often enhanced in the developing kitten over the adult cat and that structures that normally mediate depressor responses in adults demonstrate activity patterns suggestive of underdeveloped modulation of baroreceptor reflex control. Responses of medullary structures were inverse to those of adults, and multiple supramedullary patterns were altered in the kitten; the group differences were often unilateral.

Baroreceptor reflex sensitivity.   Resting AP increased with age, reaching near-adult cat levels by the 25th day of life, a pattern previously demonstrated in other species (24, 26). In contrast, sensitivity of the baroreceptor reflex initially rose in the kittens but was consistently lower in adult cats. Considerable interspecies variability exists in the development of the baroreceptor reflex, with both increased (10, 18) and decreased (35) sensitivity with age. Volatile anesthetics significantly alter adult baroreceptor reflex sensitivity (4, 45); however, data on the developing feline baroreceptor reflex sensitivity under comparable conditions are lacking. Baroreceptor reflex sensitivity changes reported here may be influenced by use of isoflurane anesthesia. Furthermore, the mode of baroreceptor activation may also exert additional effects (46).

Medulla.   The decrease in baroreceptor afferent activity that accompanies lowering of AP diminishes tonic synaptic input onto NTS neurons, and, in turn, neurons in the CVLM and the tonically active pressor region of the RVLM. The AP decline causes an associated increase in HR and sympathetic drive, resulting in a return of AP toward baseline. Because BOLD signal changes closely relate to synaptic activity rather than neuronal firing (29), the diminished synaptic input should result in a decrease in BOLD signal intensity in these three medullary regions, which was the case in the adult cats. These BOLD signal changes in the adult cat are supported by c-Fos data and are discussed in detail in a previous study (23). Baroreceptor reflex sensitivity was often greater in the kittens, and, therefore, signal intensity in the NTS (the site of primary baroreceptor afferent termination) would be expected to decline to the same or greater extent than in the adult. However, NTP administration in the kittens elicited a signal intensity increase in the NTS, CVLM, and RVLM. It is unlikely that the signal intensity differences between kittens and adults resulted from dissimilar baroreceptor afferent firing rates, as such an interpretation would imply that an increase in NTS signal intensity would result from an increase in baroreceptor firing rate during AP lowering. Instead, the results suggest that these medullary structures are modulated differently by "higher" structures in kittens.

Signal intensity response differences between kittens and adult cats within the ventrolateral medullary regions were reminiscent of age-dependent optical activity patterns on the VMS elicited by AP elevation in pentobarbital-anesthetized animals. Activity in the VMS increased during phenylephrine administration in kittens <25 days old but decreased in developing animals >30 days old, a pattern that persists in the adult cat (16, 39). Because, in this study, baroreceptor reflex sensitivity was often enhanced in kittens, the medullary circuitry responsible for mediating this reflex appears to be intact. Thus either facilitatory or disinhibitory influences on the medullary reflex arc appear to be operating differently in the kittens compared with adult cats, and the patterns found in cerebellar and more rostral sites may provide insights into those processes.

Cluster analysis also revealed significant signal intensity differences in the region of the caudal raphe nuclei, with the kitten pattern showing an early and large signal increase but an opposite response in adults. The cluster included both the midline raphe and an immediately adjacent area; thus precise differentiation of raphe areas, which contain multiple neuron groups, is difficult. Neurons in the raphe pallidus project directly to the intermediolateral column of the spinal cord (2) and also to the tonically active sympathoexcitatory region of the RVLM (49). Although medullary raphe inhibition does not alter the maximum HR increase evoked by NTP administration in the urethane-chloralose anesthetized rat, medullary raphe disinhibition results in sympathoexcitation (6). The adult response found here, with a raphe signal intensity decline to hypotension, may reflect disinhibition and would be consistent with a sympathoexcitatory role. The kittens also increased HR in response to lowered blood pressure, although the magnitude of the HR change per unit change in AP was more varied compared with that in the adults. Despite a seemingly successful baroreceptor reflex expression, the kitten raphe response, like those of sites in the baroreflex arc (NTS, CVLM, RVLM), was opposite to that of adults. Some processes, functioning in an inverse manner to those of the adult, are apparently able to accomplish the cardiac rate changes and sympathetic outflow modification in kittens and a successful resolution of the AP disturbance.

Activation of a discrete site within the adult caudal raphe of the rat also evokes profound declines in respiratory frequency and often apnea (50). Because respiratory influences exert significant effects on blood pressure (21, 33), the late decline in respiratory rate may emerge from the sustained signal increase in the kitten caudal raphe. We could not assess tidal volume in these subjects, making complete assessment of breathing contributions to the blood pressure response not possible. It is the case that enhanced volume, rather than rate, may have contributed to the blood pressure response, and raphe structures may participate in that maintenance.

Cerebellum.   Of the nonmedullary regions showing significant response differences, the cerebellar structures are of particular interest. Electrical and chemical stimulation of the fastigial nucleus alters AP and HR (8, 51), whereas bilateral fastigial nucleus lesions significantly reduce the reflexive increases in HR during NTP administration, i.e., reduce baroreceptor reflex sensitivity (7) and lead to an inability to compensate for marked hypotension resulting from blood loss (30). Because fastigial depressor responses are abolished by RVLM lesions, but the fastigial nucleus does not receive afferents from or project directly to the RVLM, its influence on the RVLM must be via a multisynaptic pathway (8). The fastigial nucleus projects directly to the NTS, and lesions of this latter region augment the AP and HR changes evoked by fastigial nucleus stimulation (12). This direct route to the medullary cardiovascular reflex pathway may provide a means for fastigial nucleus-mediated alterations in baroreceptor reflex sensitivity. However, the fastigial nucleus also projects to multiple rostral sites, including the dorsolateral pons, anterior and posterior thalamus, and insula. The fastigial nucleus is normally under inhibitory control by the cerebellar cortex. Underdevelopment of afferent fibers to the cerebellar cortex or fastigial nucleus, or of efferent fibers from the fastigial nucleus, would modify influences on sites that could modulate basal AP levels or transient responses to AP challenges.

Pons.   A region of the pons encompassing the parabrachial nucleus (PBN) also exhibited a different response pattern in kittens compared with adults. The PBN receives direct input from both the NTS and the fastigial nucleus (11, 34) and projects to other regions that also exhibited significant response differences: the insula cortex and lateral hypothalamus (44). Although the PBN appears to play no role in setting resting AP and HR levels, lesions enhance baroreceptor reflex sensitivity (43). In addition, PBN electrical stimulation prolongs inhibition of NTS neurons previously activated by carotid sinus stimulation (14). Thus NTS signal intensity may represent a combination of at least two competing inputs: decreased baroreceptor primary afferent input and increased PBN input. The PBN and NTS signal intensity differences in kittens may result from an underdeveloped PBN or PBN response, and thus, during NTP administration, reduced PBN input to the NTS may result in an undampened baroreceptor reflex and increased baroreceptor reflex sensitivity.

Rostral brain regions.   Signal intensity increased in the insular cortex of kittens and declined in adult cats. The insular cortex is an important site for autonomic control, evoking AP and HR changes on activation and containing baroreceptor responsive neurons (52, 53). Insular cortex functions are lateralized, with left side lesions increasing and right side lesions having little effect on baroreceptor reflex sensitivity (55). The structure projects directly to baroreceptor afferent recipient regions of the NTS (41) and possesses reciprocal connections with other regions that also exhibit signal intensity differences: the PBN and lateral hypothalamus (44). In particular, the posterior insula receives input from the ventral posterior thalamus (31), a region with similar signal intensity responses as the insula (increase in kittens, decrease in adult cats) and, like the insula, containing baroreceptive neurons (54). Furthermore, microinjection of cobalt chloride (a synaptic blocking agent) into the ventral posterior thalamus blocks activity in baroreceptor-activated neurons of the posterior insula (54), a finding that suggests that, despite more direct pathways from the NTS to the insula, the thalamus is critical for relaying baroreceptor-related input to the insula. Thus, like the PBN, the adult insular cortex may act via the thalamus to dampen reflexive changes in HR and sympathetic drive evoked by baroreceptor stimulation. Immaturity of these pathways may fail to dampen the baroreceptor reflex, resulting in increases in baroreceptor reflex sensitivity and the signal intensity differences in these regions.

The lateral hypothalamic area (LHA) directly projects to the RVLM, and LHA activation alters the discharge rates of RVLM barosensitive neurons (1, 3) and produces AP changes (1, 9). In addition, LHA lesions significantly depress the responsiveness of vasopressin-secreting neurons in the supraoptic region during baroreceptor reflex activation (32). Any age-related pattern difference in LHA action is of interest to pathologies such as SIDS, because of the recently demonstrated role of vasopressin in restoring AP in instances of shock (48).

The hippocampal response was remarkable in the extent of structure involved and the nature of the response difference between the adult and kitten. Electrical stimulation of the hippocampus elevated AP, and the structure showed marked activity changes to respiratory manipulations associated with AP changes (38). Strong correlations exist between hippocampal and ventrolateral medulla neural activity and the electrocardiogram (37). The hippocampus sends afferents directly to barorecipient regions of the NTS and also receives direct afferent inputs from the cerebellar deep nuclei (42). Despite the important role played by the hippocampus in AP regulation, the connections to the structure appear to be unformed in the kitten, suggesting that alternative mechanisms are employed to cope with AP changes in the young animal. These mechanisms may include the renin-angiotension system, vasopressin release, or processes like somatomotor activation or respiratory adjustments.

A schematic representation of selected neural sites involved in the expression or modulation of the baroreceptor reflex is shown in Fig. 6 with hypothesized interactions. As described earlier, regions such as the fastigial nucleus, dorsolateral pons, and insula cortex have ongoing activity that modulates the baroreceptor reflex arc within the medulla. Immaturity of these sites, or of regions that modulate activity within these sites (e.g., cerebellar cortex tonic inhibition of the fastigial nucleus), may underlie the increased variability in baroreceptor reflex sensitivity found in the kittens.



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  		Fig. 6. A sagittal schematic of the brain stem and cerebellum showing selected neural sites involved in the expression and modulation of the baroreceptor reflex in the adult cat (A) and kitten (B). The sites labeled in black represent the fundamental baroreceptor reflex arc, which relays information from arterial baroreceptors to the intermediolateral cell column of the spinal cord. Neural sites (gray) and their relationships to the baroreceptor arc (solid gray lines) have previously been shown to modulate the medullary baroreceptor reflex arc in the adult. In the adult cat, an AP decline will result in decreased synaptic activity (fMRI signal intensity) in the nucleus tractus solitarius (NTS), caudal ventrolateral medulla (CVLM), and rostral ventrolateral medulla (RVLM), producing an increase in sympathetic outflow. Alternatively, in the kitten, immaturity of regions modulating the NTS, CVLM, or RVLM result in a net increase in synaptic activity in the baroreceptor reflex arc during AP declines. Despite these changes, a successful outcome is still achieved. The dashed gray lines indicate possible interactions that are "immature" in the kittens. dlPons, dorsolateral pons; +, tonic excitatory input; –, tonic inhibitory input.

 
Lateralized responses.   As in adult animals (23) and humans (19), neural responses to AP changes were typically lateralized in the developing kittens. The organization of neural regulation of AP with respect to left or right regions in the brain is apparently established very early in life. Therefore, the differences between kittens and adults with respect to baseline AP and responses to transient AP manipulation do not derive from a change from unilateral to bilateral organization.

Relationship to human conditions.   The enhanced baroreceptor reflex sensitivity in the developing kitten has importance for clinical human issues. Developing infants can experience life-threatening hypotensive scenarios induced by infectious processes or other insults and must overcome these hypotensive challenges for survival. The enhanced baroreceptor reflex sensitivity, which results in more marked HR responses to hypotension, may place an infant at special risk to challenges. The marked hypotension and bradycardia that accompany the fatal scenario in some cases of the SIDS (27) may be especially related to these findings of early baroreceptor reflex sensitivity.

In addition to enhanced baroreceptor reflex sensitivity, baseline AP was lower in young kittens and required a period of time to develop to adult levels. The influences that modify transient responses to AP alterations may be exerting control of baseline AP. These levels may be especially affected by insular processes, with the right insula modulating levels of sympathetic outflow, largely by inhibitory mechanisms.

Limitations.   The possibility exists that some of the AP characteristics and the neural responses during early development result from immature properties of peripheral receptors or afferent processes or from inadequate development of efferent components. That possibility appears to be unlikely, because baroreceptor reflex sensitivity was often higher in the very young kittens and declined with age.

The findings presented here should be considered in the context that the data were collected entirely under isoflurane anesthesia, an agent that exerts considerable influence on the vasculature. It is likely that both the neural and physiological responses may be significantly altered in the unanesthetized state. Examination of VMS responses to blood pressure manipulation in the anesthetized and unanesthetized goat shows substantial state differences (20), and responses to such challenges significantly differ between sleep stages in the cat VMS (39, 40). Although volatile anesthetics depress baroreceptor reflex sensitivity (4, 45), isoflurane was chosen because its effects on baroreceptor reflex sensitivity appear less dramatic than those of other volatile anesthetics. The neural pathways by which isoflurane alters baroreflex sensitivity are unknown, although sites above the midbrain do not appear to be involved in altering this sensitivity (28). The changes reported here may differ in the unanesthetized state; thus the findings must be considered within this context.

Conclusions.   Baroreceptor reflex sensitivity is enhanced, and AP levels are lower in kittens, and the depressor response to a fixed dose of NTP approaches adult levels by 25 days of age. The physiological characteristics of kittens are associated with medullary and cerebellar responses nearly opposite to those of adults and show altered supramedullary responses, with many differences lateralized. The altered neural responses may result from disinhibitory or facilitatory responses mediated by cerebellar influences on other neural sites, with the early cerebellar influences mediated by inadequately developed myelination or other neural processes. If comparable changes in AP control occur in human infants, these processes would have significant clinical implications.


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This research was supported by National Institutes of Health (NIH) Grants HL-22418 and HD-22506, the Flinn Foundation, Phoenix, AZ, and NIH/National Cancer Institute Grant CA-83148.


   ACKNOWLEDGMENTS
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We thank Dr. Rajesh Kumar for comments on the manuscript and Rebecca Harper and Khanh Nguyen for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. M. Harper, Dept. of Neurobiology, Univ. of California at Los Angeles, 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.


   REFERENCES
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 ACKNOWLEDGMENTS
 REFERENCES
 

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