GABAergic neurons in the medullary raphé possess network independent chemosensitivity in situ

The identity and location of central pH/CO2 sensitive chemoreceptors are not fully understood. Serotonin (5-HT) and γ-aminobutyric acid (GABA) synthesizing neurons in the medullary raphé have demonstrated intrinsic chemosensitivity in vitro. This evidence forms the basis for our "push-pull" model of raphé contributions to chemosensitivity. We have previously shown that CO2-stimulated 5-HT neurons occur in the medullary raphé in situ. Here, we test the hypothesis that the medullary raphé contains GABA synthesizing CO2-inhibited neurons that retain their chemosensitivity after pharmacological blockade of major fast synaptic inputs. To assess chemosensitivity, we record extracellular single neuron discharge during normocapnic and hypercapnic conditions within the medullary raphé of the unanesthetized juvenile rat in situ perfused decerebrate brainstem preparation. Network dependence of chemosensitivity is assessed by application of antagonists for AMPA, NMDA, glycine, and GABAa receptors that disrupt fast-synaptic network properties. Juxtacellular labeling and immunohistochemistry establish neurotransmitter phenotypes of recorded neurons. Results support independence of CO2-inhibited GABA neuron chemosensitivity from fast synaptic inputs.Supported by NIH 54NS041069-06A1

The Rostral Medulla of Bullfrog Tadpoles Contains Critical Lung Rhythmogenic and Chemosensitive Regions Across Metamorphosis

The development of amphibian breathing provides insight into vertebrate respiratory control mechanisms. Neural oscillators in the rostral and caudal medulla drive ventilation in amphibians, and previous reports describe ventilatory oscillators and CO2 sensitive regions arise during different stages of amphibian metamorphosis. However, inconsistent findings have been enigmatic, and make comparisons to potential mammalian counterparts challenging. In the current study we assessed amphibian central CO2responsiveness and respiratory rhythm generation during two different developmental stages. Whole-nerve recordings of respiratory burst activity in cranial and spinal nerves were made from intact or transected brainstems isolated from tadpoles during early or late stages of metamorphosis. Brainstems were transected at the level of the trigeminal nerve, removing rostral structures including the nucleus isthmi, midbrain, and locus coeruleus, or transected at the level of the glossopharyngeal nerve, removing the putative buccal oscillator and caudal medulla. Removal of caudal structures stimulated the frequency of lung ventilatory bursts and revealed a hypercapnic response in normally unresponsive preparations derived from early stage tadpoles. In preparations derived from late stage tadpoles, removal of rostral or caudal structures reduced lung burst frequency, while CO2 responsiveness was retained. Our results illustrate that structures within the rostral medulla are capable of sensing CO2 throughout metamorphic development. Similarly, the region controlling lung ventilation appears to be contained in the rostral medulla throughout metamorphosis. This work offers insight into the consistency of rhythmic respiratory and chemosensitive capacities during metamorphosis

Sex-specific vagal and spinal modulation of swallow and its coordination with breathing.

Swallow-breathing coordination is influenced by changes in lung volume, which is modulated by feedback from both vagal and spinal sensory afferents. The purpose of this study was to manipulate feedback from these afferents, with and without a simultaneous mechanical challenge (chest compression), in order to assess the influence of each sensory pathway on swallow in rats. We hypothesized that manipulation of afferent feedback would shift the occurrence of swallow toward the inspiratory phase of breathing. Afferent feedback was perturbed by lidocaine nebulization, extra-thoracic vagotomy, or lidocaine administration to the pleural space in sodium pentobarbital anesthetized rats (N = 43). These different afferent perturbations were performed both in control conditions (no chest compression), and with chest compression. Manipulating pulmonary stretch receptor-mediated volume feedback in male animals decreased swallow occurrence. Female rats appear to rely more on spinal afferent feedback, as swallow occurrence shifted to late expiration with chest compression and vagotomy or lidocaine injections. Results suggest that sex-specific mechanisms modulate swallow-breathing coordination, and that vagal feedback is inhibitory to swallow-related muscles, while spinal feedback from pleural afferents has excitatory effects. This study supports the theory that a balance of vagal and spinal afferent feedback is necessary to maintain an optimal swallow pattern and swallow-breathing coordination

Sex-specific vagal and spinal modulation of breathing with chest compression.

Lung volume is modulated by sensory afferent feedback via vagal and spinal pathways. The purpose of this study was to systematically alter afferent feedback with and without a mechanical challenge (chest compression). We hypothesized that manipulation of afferent feedback by nebulization of lidocaine, extra-thoracic vagotomy, or lidocaine administration to the pleural space would produce differential effects on the motor pattern of breathing during chest compression in sodium pentobarbital anesthetized rats (N = 43). Our results suggest that: 1) pulmonary stretch receptors are not the sole contributor to breathing feedback in adult male and female rats; 2) of our manipulations, chest compression had the largest effect on early expiratory diaphragm activity ("yield"); 3) reduction of spinally-mediated afferent feedback modulates breathing patterns most likely via inhibition; and 4) breathing parameters demonstrate large sex differences. Compared to males, female animals had lower respiratory rates (RR), which were further depressed by vagotomy, while chest compression increased RR in males, and decreased yield in females without changing RR. Collectively, our results suggest that balance between tonic vagal inhibition and spinal afferent feedback maintains breathing characteristics, and that it is important to specifically evaluate sex differences when studying control of breathing

Functional Connectivity in Raphé-Pontomedullary Circuits Supports Active Suppression of Breathing During Hypocapnic Apnea

Hyperventilation is a common feature of disordered breathing. Apnea ensues if CO2 drive is sufficiently reduced. We tested the hypothesis that medullary raphé, ventral respiratory column (VRC), and pontine neurons have functional connectivity and persistent or evoked activities appropriate for roles in the suppression of drive and rhythm during hyperventilation and apnea. Phrenic nerve activity, arterial blood pressure, end-tidal CO2, and other parameters were monitored in 10 decerebrate, vagotomized, neuromuscularly-blocked, and artificially ventilated cats. Multielectrode arrays recorded spiking activity of 649 neurons. Loss and return of rhythmic activity during passive hyperventilation to apnea were identified with the S-transform. Diverse fluctuating activity patterns were recorded in the raphé-pontomedullary respiratory network during the transition to hypocapnic apnea. The firing rates of 160 neurons increased during apnea; the rates of 241 others decreased or stopped. VRC inspiratory neurons were usually the last to cease firing or lose rhythmic activity during the transition to apnea. Mayer wave-related oscillations (0.04–0.1 Hz) in firing rate were also disrupted during apnea. Four-hundred neurons (62%) were elements of pairs with at least one hyperventilation-responsive neuron and a correlational signature of interaction identified by cross-correlation or gravitational clustering. Our results support a model with distinct groups of chemoresponsive raphé neurons contributing to hypocapnic apnea through parallel processes that incorporate disfacilitation and active inhibition of inspiratory motor drive by expiratory neurons. During apnea, carotid chemoreceptors can evoke rhythm reemergence and an inspiratory shift in the balance of reciprocal inhibition via suppression of ongoing tonic expiratory neuron activity. breathing is a remarkably robust behavior that is activated at birth and continues until death, yet the brain stem neural network controlling it is even more remarkable in its malleability. For example, talking, swallowing, and coughing are motor acts that alter the breathing pattern and, rather than simply inhibiting breathing, the neural substrate for these motor acts transiently appropriates and reconfigures the respiratory pattern generator (Bolser et al. 2011, 2013; Shannon et al. 2004). However, what about conditions when breathing stops? Hyperventilation is a component of dangerous underwater breath-holding behaviors (Boyd et al. 2015; Craig 1961) and a common feature of disordered breathing (for discussion, see Abdala et al. 2014; Dempsey 2005; Laffey and Kavanagh 2002). If the drive from CO2 is sufficiently reduced, hypocapnic apnea, a transient cessation of breathing, ensues with its attendant and potentially adverse consequences (Bitter et al. 2011; Harper et al. 2013; Javaheri and Dempsey 2013; Leung et al. 2012; Sankri-Tarbichi et al. 2009; Sankri-Tarbichi 2012). During the transition from eupneic-like breathing to hyperventilatory apnea, phrenic motoneurons (Prabhakar et al. 1986) and phasic respiratory-modulated brain stem neurons either cease to discharge or assume a tonic pattern of activity (Bainton and Kirkwood 1979; Batsel 1967; Cohen 1968; Haber et al. 1957; Nesland and Plum 1965; Orem and Vidruk 1998; St. John 1998; Sun et al. 2001, 2005). With one exception (Cohen 1968), these studies recorded neurons in the ventral respiratory column (VRC; Smith et al. 2013), one at a time. This approach precludes assessment of local connectivity within the VRC and of distributed interactions with pontine and raphé neurons of the respiratory network (Nuding et al. 2009a; Segers et al. 2008). The circuit mechanisms for hypocapnic apnea remain poorly understood. Both reduced excitatory chemoreceptor drive and active inhibitory processes may contribute to the suspended state of the respiratory central pattern generator. Peripheral chemoreceptors of the carotid body monitor changes in arterial O2 and CO2-pH (Kumar and Prabhakar 2012), and central chemoreceptors, distributed among various brain stem sites, sense brain CO2-pH (Nattie and Li 2012). Mechanisms of their joint and separate influences on pattern-generating circuits are subjects of active research (Duffin and Mateika 2013a,b; Phillipson et al. 1981; Teppema and Smith 2013a,b; Wilson and Day 2013a,b). Central chemoreceptors and their follower neurons, collectively termed chemoresponsive, may be either functionally excited or inhibited by an increase in PaCO2 (e.g., Bochorishvili et al. 2012; Dean et al. 1989; Guyenet et al. 2010; Marina et al. 2010; Nuding et al. 2009b; Ott et al. 2011, 2012; Richerson et al. 2001). Medullary raphé neurons have diverse responses to hypercapnia and acidosis: firing rates of serotonergic neurons increase (Brust et al. 2014; Iceman et al. 2013; Severson et al. 2003; Veasey et al. 1995; Wang et al. 1998, 2001), whereas GABAergic raphé neurons are functionally inhibited (Iceman et al. 2014). These results are consistent with the hypothesis that distinct populations of chemoresponsive raphé neurons produce an additive “push-pull” enhancement of breathing via excitation and disinhibition, respectively (Richerson et al. 2001), a notion similar to that proposed for baroreceptor-evoked modulation of breathing via raphé-mediated excitation and disinhibition of ventral respiratory column expiratory neurons (Lindsey et al. 1998). The distinct chemoresponsive profiles of different raphé neuron populations led us to conjecture that cells effectively excited during hypercapnia would exhibit decreased firing rates during hypocapnia and vice versa for neurons inhibited during hypercapnia. This possibility and gaps in our knowledge of network interactions motivated us to test the hypothesis that medullary raphé, VRC, and pontine neurons have functional connectivity as well as persistent and evoked activities appropriate for roles in the suppression of respiratory drive and rhythm during hyperventilation and hypocapnic apnea. Sears et al. (1982) demonstrated reciprocal tonic activation of inspiratory and expiratory motor neurons during hypocapnic apnea. A PaCO2 drive below the apneic threshold may promote expiratory activity and functionally suppress inspiration. Hypoxia associated with apnea evokes increased peripheral chemoreceptor activity, enhances or elicits tonic inspiratory motor neuron activities, and can reestablish respiratory rhythmogenesis. Fluctuations in this peripheral chemoreceptor-mediated “inspiratory shift,” operating through unknown circuit mechanisms, may contribute to periodic breathing in heart failure and central sleep apnea (Lovering et al. 2012). Our multielectrode arrays allow concurrent single-unit recordings from multiple brain stem nuclei that generate and modulate breathing. This approach is well suited for testing our hypothesis and assessment of the activity patterns of many neurons under the same conditions. Thus we recorded changes in firing rates during different chemoreceptor-evoked perturbations of breathing and evaluated spike trains for correlation features indicative of functional connectivity. Preliminary accounts of this work have been presented (Lindsey et al. 2014; Nuding et al. 2005, 2013)

Protein kinase C epsilon activation delays neuronal depolarization during cardiac arrest in the euthermic arctic ground squirrel

During the pre-hibernation season, arctic ground squirrels (AGS) can tolerate 8 minutes of asphyxial cardiac arrest (CA) without detectable brain pathology. Better understanding of the mechanisms regulating innate ischemia tolerance in AGS has the potential to facilitate the development of novel, prophylactic agents to induce ischemic tolerance in patients at risk of stroke or cardiac arrest. We hypothesized that neuroprotection in AGS involves robust maintenance of ion homeostasis similar to anoxia-tolerant turtles. Ion homeostasis was assessed by monitoring ischemic depolarization (ID) in cerebral cortex during CA in vivo and during oxygen glucose deprivation in vitro in acutely prepared hippocampal slices. In both models, the onset of ID was significantly delayed in AGS compared to rats. The epsilon protein kinase C (εPKC) is a key mediator of neuroprotection and inhibits both Na + /K + -ATPase and voltage-gated sodium channels, primary mediators of the collapse of ion homeostasis during ischemia. The selective peptide inhibitor of εPKC (εV1–2) shortened the time to ID in brain slices from AGS but not in rats despite evidence that εV1–2 decreased activation of εPKC in brain slices from both rats and AGS. These results support the hypothesis that εPKC activation delays the collapse of ion homeostasis during ischemia in AGS

Carotid Chemoreceptors Tune Breathing via Multipath Routing: Reticular Chain and Loop Operations Supported by Parallel Spike Train Correlations

We tested the hypothesis that carotid chemoreceptors tune breathing through parallel circuit paths that target distinct elements of an inspiratory neuron chain in the ventral respiratory column (VRC). Microelectrode arrays were used to monitor neuronal spike trains simultaneously in the VRC, peri-nucleus tractus solitarius (p-NTS)-medial medulla, the dorsal parafacial region of the lateral tegmental field (FTL-pF), and medullary raphe nuclei together with phrenic nerve activity during selective stimulation of carotid chemoreceptors or transient hypoxia in 19 decerebrate, neuromuscularly blocked, and artificially ventilated cats. Of 994 neurons tested, 56% had a significant change in firing rate. A total of 33,422 cell pairs were evaluated for signs of functional interaction; 63% of chemoresponsive neurons were elements of at least one pair with correlational signatures indicative of paucisynaptic relationships. We detected evidence for postinspiratory neuron inhibition of rostral VRC I-Driver (pre-Bötzinger) neurons, an interaction predicted to modulate breathing frequency, and for reciprocal excitation between chemoresponsive p-NTS neurons and more downstream VRC inspiratory neurons for control of breathing depth. Chemoresponsive pericolumnar tonic expiratory neurons, proposed to amplify inspiratory drive by disinhibition, were correlationally linked to afferent and efferent “chains” of chemoresponsive neurons extending to all monitored regions. The chains included coordinated clusters of chemoresponsive FTL-pF neurons with functional links to widespread medullary sites involved in the control of breathing. The results support long-standing concepts on brain stem network architecture and a circuit model for peripheral chemoreceptor modulation of breathing with multiple circuit loops and chains tuned by tegmental field neurons with quasi-periodic discharge patterns