Impaired hypercarbic and hypoxic responses from developmental loss of cerebellar Purkinje neurons: implications for sudden infant death syndrome.

Impaired responsivity to hypercapnia or hypoxia is commonly considered a mechanism of failure in sudden infant death syndrome (SIDS). The search for deficient brain structures mediating flawed chemosensitivity typically focuses on medullary regions; however, a network that includes Purkinje cells of the cerebellar cortex and its associated cerebellar nuclei also helps mediate responses to carbon dioxide (CO2) and oxygen (O2) challenges and assists integration of cardiovascular and respiratory interactions. Although cerebellar nuclei contributions to chemoreceptor challenges in adult models are well described, Purkinje cell roles in developing models are unclear. We used a model of developmental cerebellar Purkinje cell loss to determine if such loss influenced compensatory ventilatory responses to hypercapnic and hypoxic challenges. Twenty-four Lurcher mutant mice and wild-type controls were sequentially exposed to 2% increases in CO2 (0-8%) or 2% reductions in O2 (21-13%) over 4 min, with return to room air (21% O2/79% N2/0% CO2) between each exposure. Whole body plethysmography was used to continuously monitor tidal volume (TV) and breath frequency (f). Increased f to hypercapnia was significantly lower in mutants, slower to initiate, and markedly lower in compensatory periods, except for very high (8%) CO2 levels. The magnitude of TV changes to increasing CO2 appeared smaller in mutants but only approached significance. Smaller but significant differences emerged in response to hypoxia, with mutants showing smaller TV when initially exposed to reduced O2 and lower f following exposure to 17% O2. Since cerebellar neuropathology appears in SIDS victims, developmental cerebellar neuropathology may contribute to SIDS vulnerability.

Food-intake dysregulation in type 2 diabetic Goto-Kakizaki rats: hypothesized role of dysfunctional brainstem thyrotropin-releasing hormone and impaired vagal output.

Thyrotropin-releasing hormone (TRH), a neuropeptide contained in neural terminals innervating brainstem vagal motor neurons, enhances vagal outflow to modify multisystemic visceral functions and food intake. Type 2 diabetes (T2D) and obesity are accompanied by impaired vagal functioning. We examined the possibility that impaired brainstem TRH action may contribute to the vagal dysregulation of food intake in Goto-Kakizaki (GK) rats, a T2D model with hyperglycemia and impaired central vagal activation by TRH. Food intake induced by intracisternal injection of TRH analog was reduced significantly by 50% in GK rats, compared to Wistar rats. Similarly, natural food intake in the dark phase or food intake after an overnight fast was reduced by 56-81% in GK rats. Fasting (48h) and refeeding (2h)-associated changes in serum ghrelin, insulin, peptide YY, pancreatic polypeptide and leptin, and the concomitant changes in orexigenic or anorexigenic peptide expression in the brainstem and hypothalamus, all apparent in Wistar rats, were absent or markedly reduced in GK rats, with hormone release stimulated by vagal activation, such as ghrelin and pancreatic polypeptide, decreased substantially. Fasting-induced Fos expression accompanying endogenous brainstem TRH action decreased by 66% and 91%, respectively, in the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMV) in GK rats, compared to Wistar rats. Refeeding abolished fasting-induced Fos-expression in the NTS, while that in the DMV remained in Wistar but not GK rats. These findings indicate that dysfunctional brainstem TRH-elicited vagal impairment contributes to the disturbed food intake in T2D GK rats, and may provide a pathophysiological mechanism which prevents further weight gain in T2D and obesity.

Selectively diminished corpus callosum fibers in congenital central hypoventilation syndrome.

Congenital central hypoventilation syndrome (CCHS), a condition associated with mutations in the PHOX2B gene, is characterized by loss of breathing drive during sleep, insensitivity to CO2 and O2, and multiple somatomotor, autonomic, neuropsychological, and ophthalmologic deficits, including impaired intrinsic and extrinsic eye muscle control. Brain structural studies show injury in peri-callosal regions and the corpus callosum (CC), which has the potential to affect functions disturbed in the syndrome; however, the extent of CC injury in CCHS is unclear. Diffusion tensor imaging (DTI)-based fiber tractography procedures display fiber directional information and allow quantification of fiber integrity. We performed DTI in 13 CCHS children (age, 18.2±4.7 years; eight male) and 31 control (17.4±4.9 years; 18 male) subjects using a 3.0-Tesla magnetic resonance imaging scanner; CC fibers were assessed globally and regionally with tractography procedures, and fiber counts and densities compared between groups using analysis-of-covariance (covariates; age and sex). Global CC evaluation showed reduced fiber counts and densities in CCHS over control subjects (CCHS vs. controls; fiber-counts, 4490±854 vs. 5232±777, P<0.001; fiber-density, 10.0±1.5 vs. 10.8±0.9 fibers/mm2, P<0.020), and regional examination revealed that these changes are localized to callosal axons projecting to prefrontal (217±47 vs. 248±32, P<0.005), premotor (201±51 vs. 241±47, P<0.012), parietal (179±64 vs. 238±54, P<0.002), and occipital regions (363±46 vs. 431±82, P<0.004). Corpus callosum fibers in CCHS are compromised in motor, cognitive, speech, and ophthalmologic regulatory areas. The mechanisms of fiber injury are unclear, but may result from hypoxia or perfusion deficits accompanying the syndrome, or from consequences of PHOX2B action.

Central autonomic regulation in congenital central hypoventilation syndrome.

Congenital central hypoventilation syndrome (CCHS) patients show significant autonomic dysfunction in addition to the well-described loss of breathing drive during sleep. Some characteristics, for example, syncope, may stem from delayed sympathetic outflow to the vasculature; other symptoms, including profuse sweating, may derive from overall enhanced sympathetic output. The dysregulation suggests significant alterations to autonomic regulatory brain areas. Murine models of the genetic mutations present in the human CCHS condition indicate brainstem autonomic nuclei are targeted; however, the broad range of symptoms suggests more widespread alterations. We used functional magnetic resonance imaging (fMRI) to assess neural response patterns to the Valsalva maneuver, an autonomic challenge eliciting a sequence of sympathetic and parasympathetic actions, in nine CCHS and 25 control subjects. CCHS patients showed diminished and time-lagged heart rate responses to the Valsalva maneuver, and muted fMRI signal responses across multiple brain areas. During the positive pressure phase of the Valsalva maneuver, CCHS responses were muted, but were less so in recovery phases. In rostral structures, including the amygdala and hippocampus, the normal declining patterns were replaced by increasing trends or more modest declines. Earlier onset responses appeared in the hypothalamus, midbrain, raphé pallidus, and left rostral ventrolateral medulla. Phase-lagged responses appeared in cerebellar pyramis and anterior cingulate cortex. The time-distorted and muted central responses to autonomic challenges likely underlie the exaggerated sympathetic action and autonomic dyscontrol in CCHS, impairing cerebral autoregulation, possibly exacerbating neural injury, and enhancing the potential for cardiac arrhythmia.

Reduced caudate nuclei volumes in patients with congenital central hypoventilation syndrome.

Congenital central hypoventilation syndrome (CCHS) children show cognitive and affective deficits, in addition to state-specific loss of respiratory drive. The caudate nuclei serve motor, cognitive, and affective roles, and show structural deficits in CCHS patients, based on gross voxel-based analytic procedures. However, the magnitude and regional sites of caudate injury in CCHS are unclear. We assessed global caudate nuclei volumes with manual volumetric procedures, and regional volume differences with three-dimensional surface morphometry in 14 CCHS (mean age+/-SD: 15.1+/-2.3 years; 8 male) and 31 control children (15.1+/-2.4 years; 17 male) using brain magnetic resonance imaging (MRI). Two high-resolution T1-weighted image series were collected using a 3.0 Tesla MRI scanner; images were averaged and reoriented (rigid-body transformation) to common space. Both left and right caudate nuclei were outlined in the reoriented images, and global volumes calculated; surface models were derived from manually-outlined caudate structures. Global caudate nuclei volume differences between groups were evaluated using a multivariate analysis of covariance (covariates: age, gender, and total intracranial volume). Both left and right caudate nuclei volumes were significantly reduced in CCHS over control subjects (left, 4293.45+/-549.05 vs. 4626.87+/-593.41 mm(3), P<0.006; right, 4376.29+/-565.42 vs. 4747.81+/-578.13 mm(3), P<0.004). Regional deficits in CCHS caudate volume appeared bilaterally, in the rostral head, ventrolateral mid, and caudal body. Damaged caudate nuclei may contribute to CCHS neuropsychological and motor deficits; hypoxic processes, or maldevelopment in the condition may underlie the injury.

Cerebellar fastigial nuclei activity during blood pressure challenges.

The cerebellar fastigial nuclei (FN) assist in regulating compensatory responses to large blood pressure changes and show structural injury and functional impairment to cardiovascular challenges in syndromes with sleep-disordered breathing. The patterned time course of FN responses to elevation or lowering of blood pressure and location of responsive regions within the nuclei are unclear. We evaluated FN neural activity in six anesthetized rats using optical imaging procedures during elevation and lowering of arterial pressure by phenylephrine and nitroprusside, respectively. Hypertension diminished optical correlates of FN neural activity, while measures of activity increased to hypotension, with peak neural responses occurring 5-10 s later than peak blood pressure changes. Blood pressure responses were followed by heart rate changes, and peak respiratory rates developed even later, in close temporal proximity to FN activity patterns. Although overall topographical response trends were similar, regional patterns of altered neural activity appeared to both hypertension and hypotension. The extent of neural change was greater during recovery from hypertension than for hypotension at high-dose levels. Blood pressure levels saturated with increasing phenylephrine doses, while FN activity continued to decline. No saturation appeared in heart or respiratory rate trends. The findings suggest that the FN compensate for large blood pressure changes by sympathoexcitatory and inhibitory processes, which accompany late-developing somatic or respiratory adjustments.

Hypercapnic exposure in congenital central hypoventilation syndrome reveals CNS respiratory control mechanisms.

Congenital central hypoventilation syndrome (CCHS) patients show impaired ventilatory responses and loss of breathlessness to hypercapnia, yet arouse from sleep to high CO2, suggesting intact chemoreceptor afferents. The syndrome provides a means to differentiate brain areas controlling aspects of breathing. We used functional magnetic resonance imaging to determine brain structures responding to inspired 5% CO2-95% O2 in 14 CCHS patients and 14 controls. Global signal changes induced by the challenge were removed on a voxel-by-voxel basis. A priori-defined volume-of-interest time trends (assessed with repeated measures ANOVA) and cluster analysis based on modeling each subject to a step function (individual model parameter estimates evaluated with t-test, corrected for multiple comparisons) revealed three large response clusters to hypercapnia distinguishing the two groups, extending from the 1) posterior thalamus through the medial midbrain to the dorsolateral pons, 2) right caudate nucleus, ventrolaterally through the putamen and ventral insula to the mid-hippocampus, and 3) deep cerebellar nuclei to the dorsolateral cerebellar cortex bilaterally. Smaller clusters and defined areas of group signal differences in the midline dorsal medulla, amygdala bilaterally, right dorsal-posterior temporal cortex, and left anterior insula also emerged. In most sites, early transient or sustained responses developed in controls, with little, or inverse change in CCHS subjects. Limbic and medullary structures regulating responses to hypercapnia differed from those previously shown to mediate loaded breathing ventilatory response processing. The findings show the significant roles of cerebellar and basal ganglia sites in responding to hypercapnia and the thalamic and midbrain participation in breathing control.

Hypoxia reveals posterior thalamic, cerebellar, midbrain, and limbic deficits in congenital central hypoventilation syndrome.

Congenital central hypoventilation syndrome (CCHS) patients show deficient respiratory and cardiac responses to hypoxia and hypercapnia, despite apparently intact arousal responses to hypercapnia and adequate respiratory motor mechanisms, thus providing a model to evaluate functioning of particular brain mechanisms underlying breathing. We used functional magnetic resonance imaging to assess blood oxygen level-dependent signals, corrected for global signal changes, and evaluated them with cluster and volume-of-interest procedures, during a baseline and 2-min hypoxic (15% O(2), 85% N(2)) challenge in 14 CCHS and 14 age- and gender-matched control subjects. Hypoxia elicited significant (P < 0.05) differences in magnitude and timing of responses between groups in cerebellar cortex and deep nuclei, posterior thalamic structures, limbic areas (including the insula, amygdala, ventral anterior thalamus, and right hippocampus), dorsal and ventral midbrain, caudate, claustrum, and putamen. Deficient responses to hypoxia included no, or late, changes in CCHS patients with declining signals in control subjects, a falling signal in CCHS patients with no change in controls, or absent early transient responses in CCHS. Hypoxia resulted in signal declines but no group differences in hypothalamic and dorsal medullary areas, the latter being a target for PHOX2B, mutations of which occur in the syndrome. The findings extend previously identified posterior thalamic, midbrain, and cerebellar roles for normal mediation of hypoxia found in animal fetal and adult preparations and suggest significant participation of limbic structures in responding to hypoxic challenges, which likely include cardiovascular and air-hunger components. Failing structures in CCHS include areas additional to those associated with PHOX2B expression and chemoreceptor sites.

fMRI signal changes in response to forced expiratory loading in congenital central hypoventilation syndrome.

Congenital central hypoventilation syndrome (CCHS) patients show impaired ventilatory responses to CO2 and hypoxia and reduced drive to breathe during sleep but retain appropriate breathing patterns in response to volition or increased exercise. Breath-by-breath influences on heart rate are also deficient. Using functional magnetic resonance imaging techniques, we examined responses over the brain to voluntary forced expiratory loading, a task that CCHS patients can perform but that results in impaired rapid heart rate variation patterns normally associated with the loading challenge. Increased signals emerged in control (n = 14) over CCHS (n = 13; ventilator dependent during sleep but not waking) subjects in the cingulate and right parietal cortex, cerebellar cortex and fastigial nucleus, and basal ganglia, whereas anterior cerebellar cortical sites and deep nuclei, dorsal midbrain, and dorsal pons showed increased signals in the patient group. The dorsal and ventral medulla showed delayed responses in CCHS patients. Primary motor and sensory areas bordering the central sulcus showed comparable responses in both groups. The delayed responses in medullary sensory and output regions and the aberrant reactions in cerebellar and pontine sensorimotor coordination areas suggest that rapid cardiorespiratory integration deficits in CCHS may stem from defects in these sites. Additional autonomic and perceptual motor deficits may derive from cingulate and parietal cortex aberrations.

Functional magnetic resonance imaging responses to expiratory loading in obstructive sleep apnea.

Obstructive sleep apnea (OSA) is characterized by diminished upper airway muscle phasic and tonic activation during sleep, but enhanced activity during waking. We evaluated neural mechanisms underlying these patterns with functional magnetic resonance imaging procedures during baseline and expiratory loading conditions in nine medication-free OSA and 16 control subjects. Both groups developed similar expiratory loading pressures, but appropriate autonomic responses did not emerge in OSA cases. Reduced neural signals emerged in OSA cases within the frontal cortex, anterior cingulate, cerebellar dentate nucleus, dorsal pons, anterior insula and lentiform nuclei. Signal increases in OSA over control subjects developed in the dorsal midbrain, hippocampus, quadrangular cerebellar lobule, ventral midbrain and ventral pons. Fastigial nuclei and the amygdala showed substantially increased variability in OSA subjects. No group differences were found in the thalamus. OSA patients show aberrant responses in multiple brain areas and inappropriate cardiovascular responses to expiratory loading, perhaps as a consequence of previously-demonstrated limbic, cerebellar and motor area gray matter loss.

Scattered-light imaging in vivo tracks fast and slow processes of neurophysiological activation.

We imaged fast optical changes associated with evoked neural activation in the dorsal brainstem of anesthetized rats, using a novel imaging device. The imager consisted of a gradient-index (GRIN) lens, a microscope objective, and a miniature charged-coupled device (CCD) video camera. We placed the probe in contact with tissue above cardiorespiratory areas of the nucleus of the solitary tract and illuminated the tissue with 780-nm light through flexible fibers around the probe perimeter. The focus depth was adjusted by moving the camera and microscope objective relative to the fixed GRIN lens. Back-scattered light images were relayed through the GRIN lens to the CCD camera. Video frames were digitized at 100 frames per second, along with tracheal pressure, arterial blood pressure, and electrocardiogram signals recorded at 1 kHz per channel. A macroelectrode placed under the GRIN lens recorded field potentials from the imaged area. Aortic, vagal, and superior laryngeal nerves were dissected free of surrounding tissue within the neck. Separate shocks to each dissected nerve elicited evoked electrical responses and caused localized optical activity patterns. The optical response was modeled by four distinct temporal components corresponding to putative physical mechanisms underlying scattered light changes. Region-of-interest analysis revealed image areas which were dominated by one or more of the different time-course components, some of which were also optimally recorded at different tissue depths. Two slow optical components appear to correspond to hemodynamic responses to metabolic demand associated with activation. Two fast optical components paralleled electrical evoked responses.

A device for feline head positioning and stabilization during magnetic resonance imaging.

Minimization of head movement and reproduction of standard head positions are essential for reliable brain functional magnetic resonance imaging. Devices for stabilization and alignment of feline preparations are not available currently. We describe a system that involves minimal surgery, allows for both acute and chronic atraumatic positioning, and has the potential to be used for unanesthetized animals. The device uses non-metallic materials and stabilizes the head by means of an apparatus that fixes the head with nylon screws and dental cement in the frontal sinuses. Application of the head-stabilizing device decreases head movements by more than a factor of ten. Anatomical images show that this device provides 3 dimensional head placement at a precision comparable to that of a stereotactic frame, i.e. within 1 mm.

Autonomic control during sleep and risk for sudden death in infancy.

A variety of clinical pathology and experimental animal evidence suggests that cerebellar and vestibular structures mediate marked challenges to blood pressure and breathing, and are particularly involved in compensatory somatomotor and breathing efforts to overcome substantial losses in blood pressure. At least a subset of victims of the Sudden Infant Death Syndrome (SIDS), a sleep-related disorder, succumbs to a profound bradycardia and hypotension prior to respiratory cessation, suggesting a failure of autonomic control, or incompetent compensation of somatomotor and respiratory efforts, to overcome the cardiovascular collapse. The clinical and neurotransmitter evidence from SIDS victims implicates afferent and efferent components within vestibular/cerebellar blood pressure control systems in SIDS victims. Experimental evidence from animals suggests vestibular and cerebellar structures exercise critical roles in mediating autonomic responses to body position and extreme changes in blood pressure. The position-dependent risk for SIDS, together with the neuropathological evidence, suggests a significant role for vestibular/cerebellar structures in mediating the fatal outcome for the syndrome.

Visualization of sleep influences on cerebellar and brainstem cardiac and respiratory control mechanisms.

Cerebellar and vestibular structures exert substantial influences on breathing and cardiovascular activity, particularly under conditions of extreme challenges. Influences from these structures, as well as from the ventral medullary surface, are greatly modified during sleep states. Vestibular lesions abolish the pronounced phasic autonomic variation found in the rapid eye movement sleep state, and spontaneous ventral medullary surface activity, as assessed by optical procedures, is greatly diminished in that state. Neural responses from the ventral medullary surface to hypotensive challenges are enhanced and appear "undampened" during the rapid eye movement sleep state. Functional magnetic resonance imaging reveals activation to blood pressure challenges in widespread brain areas of humans, and especially in cerebellar sites, such as the fastigial nucleus. A subset of victims of sudden infant death syndrome, a sleep-related disorder, appear to succumb from cardiovascular failure of a shock-like nature, and often show neurotransmitter receptor deficiencies in the ventral medullary surface, caudal midline raphe hypotensive regions, and the inferior olive, a major afferent relay to the cerebellum. Afferent and efferent vestibular/cerebellar structures, or sites within the cerebellum may mediate failure mechanisms in sudden infant death syndrome and a number of other sleep-disordered breathing and cardiovascular syndromes.

State influences on ventral medullary surface and physiological responses to sodium cyanide challenges.

Intravenous sodium cyanide (NaCN) administration lowers ventral medullary surface (VMS) activity in anesthetized cats. Sleep states modify spontaneous and blood pressure-evoked VMS activity and may alter VMS responses to chemoreceptor input. We studied VMS activation during peripheral chemoreceptor stimulation by intravenous NaCN using optical procedures in six cats instrumented for recording sleep physiology during sham saline and control site trials. Images of scattered 660-nm light were collected at 50 frames/s with an optical device after 80-100 microg total bolus intravenous NaCN delivery during waking and sleep states. Cyanide elicited an initial ventilatory decline, followed by large inspiratory efforts and an increase in respiratory rate, except in rapid eye movement sleep, in which an initial breathing increase occurred. NaCN evoked a pronounced decrease in VMS activity in all states; control sites and sham injections showed little effect. The activity decline was faster in rapid eye movement sleep, and the activity nadir occurred later in waking. Sleep states alter the time course but not the extent of decline in VMS activity.

Sudden infant death syndrome: a failure of compensatory cerebellar mechanisms?

The mechanisms underlying failure in sudden infant death syndrome may involve inadequate compensatory motor responses to a hypotensive challenge; the insult may result from a shock-like sequence, or from a ventilatory challenge that leads to a hypotensive event. Structures ordinarily not considered in mediating breathing or cardiovascular control, especially cerebellar-related structures, may play a critical role in compensatory responses, and underlie the position-dependent risk for SIDS. Dysfunction in affected brain areas appears to arise prenatally from a compromised fetal environment, with a nicotinic component contributing to the deficient mechanism. Physiologic characteristics of infants who later succumb to SIDS, and cardiovascular events associated with the fatal scenario suggest a failure of interaction between somatomotor and autonomic control mechanisms in infants at risk for the syndrome. A failure of compensatory motor actions to overcome a profound hypotension, perhaps mediated by cerebellar mechanisms that regulate blood pressure, may underlie-the fatal event.

Changes in ventral medullary light reflectance during hypercapnia in awake and sleeping cats.

Activity within rostral and intermediate ventral medullary surface areas, measured as 660 nm scattered light changes, was examined in six cats, (five experimental, one control site) following 5% CO(2) challenges during waking, quiet sleep, and rapid eye movement (REM) sleep states. Activity declined to hypercapnia in all states, with a smaller decline in quiet sleep compared to waking, and intermediate values in REM sleep. The decline occurred more rapidly, with a shorter latency, during wakefulness, but with a much slower return to baseline than during quiet sleep. During REM sleep, the latency to nadir and recovery were greater than in other states. Regional patterns of activation emerged which differed in extent of activation between states.