Sheree M. Johnson

Respiratory rhythm generation in neonatal and adult mammals: the hybrid pacemaker–network model

We review a new unified model of respiratory rhythm generation - the hybrid pacemaker-network model. This model represents a comprehensive synthesis of cellular and network mechanisms that can theoretically account for rhythm generation in different functional states, from the most reduced states in the neonatal nervous system in vitro to the intact adult system in vivo. The model incorporates a critical neuronal kernel consisting of a network of excitatory neurons with state-dependent, oscillatory bursting or pacemaker properties. This kernel, located in the pre-Bötzinger complex of the ventrolateral medulla, provides a rudimentary pacemaker network mechanism for generating an inspiratory rhythm, revealed predominately in functionally reduced states in vitro. In vivo the kernel is embedded in a larger network that interacts with the kernel via inhibitory synaptic connections that provide the dynamic control required for the evolution of the complete pattern of inspiratory and expiratory network activity. The resulting hybrid of cellular pacemaker and network properties functionally endows the system with multiple mechanisms of rhythm generation. New biophysically realistic mathematical models of the hybrid pacemaker-network have been developed that illustrate these concepts and provide a computational framework for investigating interactions of cellular and network processes that must be analyzed to understand rhythm generation.

GFP-expressing locus ceruleus neurons from Prp57 transgenic mice exhibit CO2/H+ responses in primary cell culture

The locus ceruleus (LC) contains neurons that increase their firing rate (FR) in vitro when exposed to elevated CO(2)/H(+) and have been proposed to influence the respiratory network to make compensatory adjustments in ventilation. Prp57 transgenic mice express green fluorescent protein (GFP) in the LC and were used to isolate, culture, and target LC neurons for electrophysiological recording. We hypothesized that GFP-LC neurons would exhibit CO(2)/H(+) chemosensitivity under primary culture conditions, evidenced as a change in FR. This is the first study to quantify CO(2)/H(+) responses in LC neuron FR in cell culture. Neurons were continuously bathed with solutions containing antagonists of glutamate and GABA receptors, and the acid-base status was changed from control (5% CO(2); pH approximately 7.4) to hypercapnic acidosis (9% CO(2); pH approximately 7.2) and hypocapnic alkalosis (3% CO(2); pH approximately 7.6). FR was quantified during perforated patch current clamp recordings. Approximately 86% of GFP-LC neurons were stimulated, and approximately 14% were insensitive to changes in CO(2)/H(+). The magnitude of the response of these neurons depended on the baseline FR, ranging from 155.9 +/- 6% when FR started at 2.95 +/- 0.49 Hz to 381 +/- 55.6% when FR started at 1.32 +/- 0.31 Hz. These results demonstrate that cultured LC neurons from Prp57 transgenic mice retain functional sensing molecules necessary for CO(2)/H(+) responses. Prp57 transgenic mice will serve as a valuable model to delineate mechanisms involved in CO(2)/H(+) responsiveness in catecholaminergic neurons.

Effect of hyperoxic exposure during early development on neurotrophin expression in the carotid body and nucleus tractus solitarii

Synaptic activity can modify expression of neurotrophins, which influence the development of neuronal circuits. In the newborn rat, early hyperoxia silences the synaptic activity and input from the carotid body, impairing the development and function of chemoreceptors. The purpose of this study was to determine whether early hyperoxic exposure, sufficient to induce hypoplasia of the carotid body and decrease the number of chemoafferents, would also modify neurotrophin expression within the nucleus tractus solitarii (nTS). Rat pups were exposed to hyperoxia (fraction of inspired oxygen 0.60) or normoxia until 7 or 14 days of postnatal development (PND). In the carotid body, hyperoxia decreased brain-derived neurotrophic factor (BDNF) protein expression by 93% (P = 0.04) after a 7-day exposure, followed by a decrease in retrogradely labeled chemoafferents by 55% (P = 0.004) within the petrosal ganglion at 14 days. Return to normoxia for 1 wk after a 14-day hyperoxic exposure did not reverse this effect. In the nTS, hyperoxia for 7 days: 1) decreased BDNF gene expression by 67% and protein expression by 18%; 2) attenuated upregulation of BDNF mRNA levels in response to acute hypoxia; and 3) upregulated p75 neurotrophic receptor, truncated tropomyosin kinase B (inactive receptor), and cleaved caspase-3. These effects were not observed in the locus coeruleus (LC). Hyperoxia for 14 days also decreased tyrosine hydroxylase levels by 18% (P = 0.04) in nTS but not in the LC. In conclusion, hyperoxic exposure during early PND reduces neurotrophin levels in the carotid body and the nTS and shifts the balance of neurotrophic support from prosurvival to proapoptotic in the nTS, the primary brain stem site for central integration of sensory and autonomic inputs.

Bicarbonate-Regulated Soluble Adenylyl Cyclase (sAC) mRNA Expression and Activity in Peripheral Chemoreceptors

UNLABELLED Peripheral arterial chemoreceptors in the carotid body (CB) are modulated by pH/CO(2). Soluble adenylyl cyclase (sAC) is directly stimulated by bicarbonate ions (HCO(3)). Because CO(2)/HCO(3) mediates depolarization in chemoreceptors, we hypothesized that sAC mRNA would be expressed in the CB, and its expression and function would be regulated by CO(2)/HCO(3).Sprague-Dawley rats at postnatal days 16-17 were used to compare sAC mRNA gene expression between CB and non-chemosensitive tissues: superior cervical (SCG), petrosal (PG) and nodose ganglia (NG) by quantitative real time-PCR. Rat sAC gene expression was standardized to the expression of GAPDH (housekeeping gene) and the data were analyzed with the Pfaffl method. Gene and protein expression, and sAC regulation in the testis was used as a positive control. To determine the regulation of sAC mRNA expression and activity, all tissues were exposed to increasing concentrations of bicarbonate (0, 24, 44 mM, titrated with CO(2) and maintained a constant pH of 7.40). RESULTS sAC mRNA expression was between 2-11% of CB expression in the SCG, PG and NG. Furthermore, only in the CB did HCO(3) upregulate sAC gene expression and increase cAMP levels. CONCLUSION sAC mRNA and protein expression is present in peripheral arterial chemoreceptors and non-chemoreceptors. In the CB, CO(2)/HCO(3) not only activated sAC but also regulated its expression, suggesting that sAC may be involved in the regulation of cAMP levels in response to hyper/hypocapnia.

Alpha-7 and alpha-4 nicotinic receptor subunit immunoreactivity in genioglossus muscle motoneurons

In the present study, immunohistochemistry combined with retrograde labeling techniques were used to determine if hypoglossal motoneurons (HMNs), retrogradely labeled after cholera toxin B subunit (CTB) injection to the genioglossus muscle in rats, show immunoreactivity for alpha-7 and alpha-4 subunits of nicotinic acetylcholine receptors (nAChRs). CTB-positive HMNs projecting to the genioglossus muscle were consistently labeled throughout the rostrocaudal extent of the hypoglossal nuclei with the greatest labeling at and caudal to area postrema. Alpha-7 subunit immunoreactivity was found in 39.44+/-5.10% of 870 CTB-labeled motoneurons and the alpha-4 subunit in 51.01+/-3.71% of 983 CTB-positive neurons. Rostrally, the number of genioglossal motoneurons demonstrating immunoreactivity for the alpha-7 subunit was 45.85+/-10.04% compared to 34.96+/-5.11% at and caudal to area postrema (P>0.1). The number of genioglossal motoneurons that showed immunoreactivity for the alpha-4 subunit was 55.03+/-4.83% at and caudal to area postrema compared to 42.98+/-3.90% in rostral areas (P=0.074). These results demonstrate that nAChR immunoreactivity is present in genioglossal motoneurons and suggest a role for alpha-7 and alpha-4 subunits containing nAChRs in the regulation of upper airway patency.

Hypercapnic and hypoxic responses require intact neural transmission from the pre-Bötzinger complex

The central respiratory network that includes the pre-Bötzinger complex (pre-BötC), a region believed to contain rhythmogenic neurons, is capable of responding to fluctuations in CO2 and pH. However, the role of inputs from this site in mediating ventilatory responses to hypercapnia and/or hypoxia in nonsedated animals is not well established. Therefore, in the present study we tested the hypothesis that altered transmission from the pre-BötC to its target sites would decrease chemosensory responsiveness to acute hypercapnia and modulate the ventilatory response to hypoxia. Colchicine was used to block axonal transport. At 48 h after bilateral microinjections of colchicine into the pre-BötC (100 microg/uL, 100 nL/site), but not saline, the baseline frequency of breathing decreased; however, rhythmicity was not altered. In addition, there was a significant fall in the ventilatory response to hypercapnia (5 and 12% CO2) and hypoxia (8% O2). These findings indicate that, inputs from pre-BötC neurons are of critical importance in providing the normal ventilatory response to both hypercapnia and hypoxia.

A standardized randomized 6-month aerobic exercise-training down-regulated pro-inflammatory genes, but up-regulated anti-inflammatory, neuron survival and axon growth-related genes.

There is considerable support for the view that aerobic exercise may confer cognitive benefits to mild cognitively impaired elderly persons. However, the biological mechanisms mediating these effects are not entirely clear. As a preliminary step towards informing this gap in knowledge, we enrolled older adults confirmed to have mild cognitive impairment (MCI) in a 6-month exercise program. Male and female subjects were randomized into a 6-month program of either aerobic or stretch (control) exercise. Data collected from the first 10 completers, aerobic exercise (n=5) or stretch (control) exercise (n=5), were used to determine intervention-induced changes in the global gene expression profiles of the aerobic and stretch groups. Using microarray, we identified genes with altered expression (relative to baseline values) in response to the 6-month exercise intervention. Genes whose expression were altered by at least two-fold, and met the p-value cutoff of 0.01 were inputted into the Ingenuity Pathway Knowledge Base Library to generate gene-interaction networks. After a 6-month aerobic exercise-training, genes promoting inflammation became down-regulated, whereas genes having anti-inflammatory properties and those modulating immune function or promoting neuron survival and axon growth, became up-regulated (all fold change≥±2.0, p<0.01). These changes were not observed in the stretch group. Importantly, the differences in the expression profiles correlated with significant improvement in maximal oxygen uptake (VO2max) in the aerobic program as opposed to the stretch group. We conclude that three distinct cellular pathways may collectively influence the training effects of aerobic exercise in MCI subjects. We plan to confirm these effects using rt-PCR and correlate such changes with the cognitive phenotype.

Dynamics of excitatory networks of bursting pacemaking neurons: Modeling and experimental studies of the respiratory central pattern generator

Abstract We have explored the dynamics of a computational model of an excitatory network of bursting pacemaker neurons with heterogeneous properties. The network generates synchronous bursts of activity, and the frequency of both single cells and the synaptically coupled pacemaker cell population may be controlled by varying the degree of depolarizing input (DI). The dynamic range of DI where stable bursting occurs is significantly larger for the coupled population than that of individual cells, suggesting a functional role of cellular heterogeneity in making biological rhythms more robust. Experimental evidence is presented from the pacemaker-network generating the respiratory rhythm in the mammalian brainstem.

Exaggerated vasopressor response to exercise and cerebral blood flow velocity.

We studied 10 young adults, normotensive at rest, comprising a control group (n = 5) with normal blood pressure responsiveness to exercise and an experimental group exhibiting greater percentage of body fat and body mass index (BMI) than the controls, with exaggerated blood pressure (vasopressor) responsiveness to exercise (EEBPR) (n = 5). Lower absolute and varying oxygen consumption/body weight normalized units of middle cerebral arterial blood flow velocity (MCAV) were found during exercise in the experimental group (P < .01). These findings support the hypothesis that the combination of EEBPR and high BMI is associated with low MCAV that may put such individuals at risk for cerebral hypoperfusion and cognitive deficits.

The Role of Hypoxia-Inducible Factor 1 in Mild Cognitive Impairment

Neuroinflammation and reactive oxygen species are thought to mediate the pathogenesis of Alzheimer’s disease (AD), suggesting that mild cognitive impairment (MCI), a prodromal stage of AD, may be driven by similar insults. Several studies document that hypoxia-inducible factor 1 (HIF-1) is neuroprotective in the setting of neuronal insults, since this transcription factor drives the expression of critical genes that diminish neuronal cell death. HIF-1 facilitates glycolysis and glucose metabolism, thus helping to generate reductive equivalents of NADH/NADPH that counter oxidative stress. HIF-1 also improves cerebral blood flow which opposes the toxicity of hypoxia. Increased HIF-1 activity and/or expression of HIF-1 target genes, such as those involved in glycolysis or vascular flow, may be an early adaptation to the oxidative stressors that characterize MCI pathology. The molecular events that constitute this early adaptation are likely neuroprotective, and might mitigate cognitive decline or the onset of full-blown AD. On the other hand, prolonged or overwhelming stressors can convert HIF-1 into an activator of cell death through agents such as Bnip3, an event that is more likely to occur in late MCI or advanced Alzheimer’s dementia.