Leanne C. McKay

Sleep-disordered breathing after targeted ablation of preBötzinger complex neurons

Ablation of preBötzinger complex (preBötC) neurons, critical for respiratory rhythm generation, resulted in a progressive, increasingly severe disruption of respiratory pattern, initially during sleep and then also during wakefulness in adult rats. Sleep-disordered breathing is highly prevalent in elderly humans and in some patients with neurodegenerative disease. We propose that sleep-disordered breathing results from loss of preBötC neurons and could underlie death during sleep in these populations.

Morphological and functional reversal of phenotypes in a mouse model of Rett syndrome

Rett syndrome is a neurological disorder caused by mutation of the X-linked MECP2 gene. Mice lacking functional Mecp2 display a spectrum of Rett syndrome-like signs, including disturbances in motor function and abnormal patterns of breathing, accompanied by structural defects in central motor areas and the brainstem. Although routinely classified as a neurodevelopmental disorder, many aspects of the mouse phenotype can be effectively reversed by activation of a quiescent Mecp2 gene in adults. This suggests that absence of Mecp2 during brain development does not irreversibly compromise brain function. It is conceivable, however, that deep-seated neurological defects persist in mice rescued by late activation of Mecp2. To test this possibility, we have quantitatively analysed structural and functional plasticity of the rescued adult male mouse brain. Activation of Mecp2 in ∼70% of neurons reversed many morphological defects in the motor cortex, including neuronal size and dendritic complexity. Restoration of Mecp2 expression was also accompanied by a significant improvement in respiratory and sensory-motor functions, including breathing pattern, grip strength, balance beam and rotarod performance. Our findings sustain the view that MeCP2 does not play a pivotal role in brain development, but may instead be required to maintain full neurological function once development is complete.

A bilateral cortico-bulbar network associated with breath holding in humans, determined by functional magnetic resonance imaging

Few tasks are simpler to perform than a breath hold; however, the neural basis underlying this voluntary inhibitory behaviour, which must suppress spontaneous respiratory motor output, is unknown. Here, using blood oxygen level-dependent functional magnetic resonance imaging (BOLD fMRI), we investigated the neural network responsible for volitional breath holding in 8 healthy humans. BOLD images of the whole brain (156 brain volumes, voxel resolution 3x3x3 mm) were acquired every 5.2 s. All breath holds were performed for 15 s at resting expiratory lung volume when respiratory musculature was presumed to be relaxed, which ensured that the protocol highlighted the inhibitory components underlying the breath hold. An experimental paradigm was designed to dissociate the time course of the whole-brain BOLD signal from the time course of the local, neural-related BOLD signal associated with the inhibitory task. We identified a bilateral network of cortical and subcortical structures including the insula, basal ganglia, frontal cortex, parietal cortex and thalamus, which are in common with response inhibition tasks, and in addition, activity within the pons. From these results we speculate that the pons has a role in integrating information from supra-brainstem structures, and in turn it exerts an inhibitory effect on medullary respiratory neurones to inhibit breathing during breath holding.

Episodic hypoxia evokes long‐term facilitation of genioglossus muscle activity in neonatal rats

The aim of this study was to determine if episodic hypoxia evokes persistent increases of genioglossus muscle (GG) activity, termed long‐term facilitation (LTF), in neonatal rats in vivo. Experiments were performed on anaesthetized, spontaneously breathing, intubated neonatal rats (postnatal days (P) 3–7), divided into three groups. The first group (n= 8) was subjected to three 5‐min periods of hypoxia (5% O2–95% N2) alternating with 5 min periods of room air. The second group (n= 8) was exposed to 15 min of continuous hypoxia. The third (n= 4) group was not exposed to hypoxia and served as a control. GG EMG activity and airflow were recorded before, during and for 60 min after episodic and continuous hypoxic exposure. During hypoxia, GG EMG burst amplitude and tidal volume (VT) significantly increased compared to baseline levels (episodic protocol: mean ±s.e.m; 324 ± 59% of control and 0.13 ± 0.007 versus 0.09 ± 0.005 ml, respectively; continuous protocol: 259 ± 30% of control and 0.16 ± 0.005 versus 0.09 ± 0.007 ml, respectively; P < 0.05). After the episodic protocol, GG EMG burst amplitude transiently returned to baseline; over the next 60 min, burst amplitude progressively increased to levels significantly greater than baseline (238 ± 40% at 60 min; P < 0.05), without any significant increase in VT and respiratory frequency (P> 0.05). After the continuous protocol, there was no lasting increase in GG EMG burst amplitude. We conclude that LTF of upper airway muscles is an adaptive respiratory behaviour present from birth.

Unilateral Ablation of Pre-Bötzinger Complex Disrupts Breathing during Sleep but Not Wakefulness

RATIONALE In adult rats, bilateral ablation of pre-Bötzinger complex (preBötC) neurokinin 1-expressing (NK1R) neurons leads to a progressive and irreversible disruption in breathing pattern, initially during sleep, eventually resulting in an ataxic breathing pattern during wakefulness. OBJECTIVES Here we determine whether ablation of fewer preBötC NK1R neurons leads to a persistent pattern of disordered breathing during sleep but not during wakefulness. METHODS Adult male Sprague-Dawley rats (n = 12) were instrumented to record diaphragmatic, abdominal, and neck EMG, and EEG. Fourteen days later, a second surgery was performed to stereotaxically microinject into the preBötC on one side the toxin saporin conjugated to substance P (SP-SAP), which selectively ablates NK1R neurons. MEASUREMENTS AND MAIN RESULTS Postinjection, rats were monitored within a plethysmograph until they were killed (Days 21-51). At Days 6-9 post-unilateral SP-SAP injection, respiratory pattern during sleep, particularly REM sleep, became increasingly disordered, characterized by an increase in frequency of central sleep apnea and hypopneas (36.8 +/- 7.4 episodes/h of REM vs. 6 +/- 2.0 episodes/h in preinjection controls; P < 0.05), whereas breathing during resting wakefulness remained stable. Unlike bilateral SP-SAP-injected rats, an ataxic breathing pattern did not develop during wakefulness. Rats that were monitored up to 51 days post-SP-SAP injection continued to have sleep-disordered breathing; breathing during wakefulness remained relatively stable. Histologic analysis of the ventrolateral medulla confirmed that NK1R neurons within the preBötC on the injected but not on the contralateral side of the medulla were ablated. CONCLUSIONS Gradual loss of preBötC NK1R neurons may be an underlying factor of sleep-disordered breathing, in particular of central sleep apnea.

Sub-cortical and brainstem sites associated with chemo-stimulated increases in ventilation in humans

We investigated the neural basis for spontaneous chemo-stimulated increases in ventilation in awake, healthy humans. Blood oxygen level dependent (BOLD) functional MRI was performed in nine healthy subjects using T2 weighted echo planar imaging. Brain volumes (52 transverse slices, cortex to high spinal cord) were acquired every 3.9 s. The 30 min paradigm consisted of six, 5-min cycles, each cycle comprising 45 s of hypoxic-isocapnia, 45 s of isooxic-hypercapnia and 45 s of hypoxic-hypercapnia, with 55 s of non-stimulatory hyperoxic-isocapnia (control) separating each stimulus period. Ventilation was significantly (p<0.001) increased during hypoxic-isocapnia, isooxic-hypercapnia and hypoxic-hypercapnia (17.0, 13.8, 24.9 L/min respectively) vs. control (8.4 L/min) and was associated with significant (p<0.05, corrected for multiple comparisons) signal increases within a bilateral network that included the basal ganglia, thalamus, red nucleus, cerebellum, parietal cortex, cingulate and superior mid pons. The neuroanatomical structures identified provide evidence for the spontaneous control of breathing to be mediated by higher brain centres, as well as respiratory nuclei in the brainstem.

Regional Cerebrovascular Responses to Hypercapnia and Hypoxia

A limited number of studies using differing imaging approaches suggest that there are regional variation in the cerebrovascular response to hypercapnia and hypoxia. However there are limitations to these studies. In particular, it is not clear if existing studies of hypoxia have fully accounted for the confounding effects of the changes in arterial PCO2 on cerebral perfusion that, if uncontrolled, will accompany the hypoxic stimulus. We determined quantitative maps of grey matter cerebral blood flow using a multi-slice pulsed arterial spin labelling MRI method at 3 T at rest, during conditions of isocapnic euoxia, hypercapnia, and mild isocapnic hypoxia. From these data, we determined grey matter cerebrovascular reactivity maps which show the spatial distribution of the responses to these interventions. Whilst, overall, cerebral perfusion increased with hypercapnia and hypoxia, hypoxia cerebrovascular reactivity maps showed very high variation both within and between individuals: most grey matter regions exhibiting a positive cerebrovascular reactivity, but some exhibiting a negative reactivity. The physiological explanation for this variation remains unclear and it is not known if these local differences will vary with state or with regional brain activity. The potential interaction between hypoxic or hypercapnic cerebrovascular changes and neurally related changes in brain perfusion is of particular interest for functional imaging studies of brain activation in which arterial blood gases are altered. We have determined the interaction between global hypoxia and hypercapnia-induced blood oxygen level-dependent (BOLD) MRI signal and local neurally related BOLD signal. Although statistically significant interactions were present, physiologically the effects were weak and, in practice, they did not change the statistical outcome related to the analysis of the neurally related signals. These data suggest that such respiratory-related confounds can be successfully accounted for in functional imaging studies.