Julian F. R. Paton

A working heart-brainstem preparation of the mouse.

An intra-arterially perfused working heart-brainstem preparation (WH-BP) was developed to allow studies into functionally identified cardiovascular and respiratory neurones in an in vitro milieu. This report provides that first description of this preparation. Evidence is presented indicating that the WH-BP: (1) spontaneously generates eupneic-like phrenic nerve activity, indicative of adequate oxygenation of the brainstem; (2) preserves the integrity of central coupling between the central respiratory rhythm generator and cardiac vagal motor neurones and, (3), allow an intracellular analysis of medullary cardio-respiratory neurones. The WH-BP may provide an advantaged environment for analysis of both synaptic and cellular mechanisms within the medulla that regulate cardio-respiratory activity.

Primer on the Autonomic Nervous System

Publisher Summary This chapter focuses on the central neural interconnectivity between the brainstem respiratory pattern generator and neural networks governing sympathetic and parasympathetic activity. The respiratory-related alterations in venous return/cardiac output described above also contribute to the respiratory modulation of arterial pressure. However, the fluctuations persist in the working heart brainstem preparation indicating again there is an important central neural component to the coupling. Loss of vagal tone in cardiovascular diseases can be clearly demonstrated by the diminished change in heart rate on administration a vagolytic drug like atropine and also by the loss of respiratory sinus arrhythmia (RSA). The burst of cardiac vagal activity seems to originate centrally at the level of the preganglionic neurons in the nucleus ambiguus that are inhibited during inspiration but excited during postinspiration. There are some key studies that need to be performed. Given its protective role, the site(s) and mechanisms of blockade of cardiac vagal transmission must be identified in cardiovascular disease to allow novel, targeted therapy to be devised.

Structural and functional architecture of respiratory networks in the mammalian brainstem

Neural circuits controlling breathing in mammals are organized within serially arrayed and functionally interacting brainstem compartments extending from the pons to the lower medulla. The core circuit components that constitute the neural machinery for generating respiratory rhythm and shaping inspiratory and expiratory motor patterns are distributed among three adjacent structural compartments in the ventrolateral medulla: the Bötzinger complex (BötC), pre-Bötzinger complex (pre-BötC) and rostral ventral respiratory group (rVRG). The respiratory rhythm and inspiratory–expiratory patterns emerge from dynamic interactions between: (i) excitatory neuron populations in the pre-BötC and rVRG active during inspiration that form inspiratory motor output; (ii) inhibitory neuron populations in the pre-BötC that provide inspiratory inhibition within the network; and (iii) inhibitory populations in the BötC active during expiration that generate expiratory inhibition. Network interactions within these compartments along with intrinsic rhythmogenic properties of pre-BötC neurons form a hierarchy of multiple oscillatory mechanisms. The functional expression of these mechanisms is controlled by multiple drives from more rostral brainstem components, including the retrotrapezoid nucleus and pons, which regulate the dynamic behaviour of the core circuitry. The emerging view is that the brainstem respiratory network has rhythmogenic capabilities at multiple hierarchical levels, which allows flexible, state-dependent expression of different rhythmogenic mechanisms under different physiological and metabolic conditions and enables a wide repertoire of respiratory behaviours.

Increased sympathetic outflow in juvenile rats submitted to chronic intermittent hypoxia correlates with enhanced expiratory activity

Chronic intermittent hypoxia (CIH) in rats produces changes in the central regulation of cardiovascular and respiratory systems by unknown mechanisms. We hypothesized that CIH (6% O2 for 40 s, every 9 min, 8 h day−1) for 10 days alters the central respiratory modulation of sympathetic activity. After CIH, awake rats (n= 14) exhibited higher levels of mean arterial pressure than controls (101 ± 3 versus 89 ± 3 mmHg, n= 15, P < 0.01). Recordings of phrenic, thoracic sympathetic, cervical vagus and abdominal nerves were performed in the in situ working heart–brainstem preparations of control and CIH juvenile rats. The data obtained in CIH rats revealed that: (i) abdominal (Abd) nerves exhibited an additional burst discharge in late expiration; (ii) thoracic sympathetic nerve activity (tSNA) was greater during late expiration than in controls (52 ± 5 versus 40 ± 3%; n= 11, P < 0.05; values expressed according to the maximal activity observed during inspiration and the noise level recorded at the end of each experiment), which was not dependent on peripheral chemoreceptors; (iii) the additional late expiratory activity in the Abd nerve correlated with the increased tSNA; (iv) the enhanced late expiratory activity in the Abd nerve unique to CIH rats was accompanied by reduced post‐inspiratory activity in cervical vagus nerve compared to controls. The data indicate that CIH rats present an altered pattern of central sympathetic–respiratory coupling, with increased tSNA that correlates with enhanced late expiratory discharge in the Abd nerve. Thus, CIH alters the coupling between the central respiratory generator and sympathetic networks that may contribute to the induced hypertension in this experimental model.

Respiratory rhythm generation during gasping depends on persistent sodium current

In severe hypoxia, homeostatic mechanisms maintain function of the brainstem respiratory network. We hypothesized that hypoxia involves a transition from neuronal mechanisms of normal breathing (eupnea) to a rudimentary pattern of inspiratory movements (gasping). We provide evidence for hypoxia-driven transformation within the central respiratory oscillator, in which gasping relies on persistent sodium current, whereas eupnea does not depend on this cellular mechanism.

Adenoviral vector demonstrates that angiotensin II‐induced depression of the cardiac baroreflex is mediated by endothelial nitric oxide synthase in the nucleus tractus solitarii of the rat

1 Angiotensin II (ANGII) acting on ANGII type 1 (AT1) receptors in the solitary tract nucleus (NTS) depresses the baroreflex. Since ANGII stimulates the release of nitric oxide (NO), we tested whether the ANGII‐mediated depression of the baroreflex in the NTS depended on NO release. 2 In a working heart‐brainstem preparation (WHBP) of rat NTS microinjection of either ANGII (500 fmol) or a NO donor (diethylamine nonoate, 500 pmol) both depressed baroreflex gain by ‐56 and ‐67 %, respectively (P < 0.01). In contrast, whilst ANGII potentiated the peripheral chemoreflex, the NO donor was without effect. 3 NTS microinjection of non‐selective NO synthase (NOS) inhibitors (l‐NAME; 50 pmol) or (l‐NMMA; 200 pmol) prevented the ANGII‐induced baroreflex attenuation (P > 0.1). In contrast, a neurone‐specific NOS inhibitor, TRIM (50 pmol), was without effect. 4 Using an adenoviral vector, a dominant negative mutant of endothelial NOS (TeNOS) was expressed bilaterally in the NTS. Expression of TeNOS affected neither baseline cardiovascular parameters nor baroreflex sensitivity. However, ANGII microinjected into the transfected region failed to affect the baroreflex. 5 Immunostaining revealed that eNOS‐positive neurones were more numerous than those labelled for AT1 receptors. Neurones double labelled for both AT1 receptors and eNOS comprised 23 ± 5.4 % of the eNOS‐positive cells and 57 ± 9.2 % of the AT1 receptor‐positive cells. Endothelial cells were also double labelled for eNOS and AT1 receptors. 6 We suggest that ANGII activates eNOS located in either neurones and/or endothelial cells to release NO, which acts selectively to depress the baroreflex.

Brainstem respiratory networks: building blocks and microcircuits

Breathing movements in mammals are driven by rhythmic neural activity generated within spatially and functionally organized brainstem neural circuits comprising the respiratory central pattern generator (CPG). This rhythmic activity provides homeostatic regulation of gases in blood and tissues and integrates breathing with other motor acts. We review new insights into the spatial-functional organization of key neural microcircuits of this CPG from recent multidisciplinary experimental and computational studies. The emerging view is that the microcircuit organization within the CPG allows the generation of multiple rhythmic breathing patterns and adaptive switching between them, depending on physiological or pathophysiological conditions. These insights open the possibility for site- and mechanism-specific interventions to treat various disorders of the neural control of breathing.

The Carotid Body as a Therapeutic Target for the Treatment of Sympathetically Mediated Diseases

Hypertension, heart failure (HF), type II diabetes mellitus, and chronic kidney disease represent significant and growing global health issues.1 The rates of control of blood pressure and the therapeutic efforts to prevent progression of HF, chronic kidney disease, diabetes mellitus, and their sequelae remain unsatisfactory.2–5 Although patient nonadherence and nonpersistence with medications participate in this failure, especially in asymptomatic disorders, the inherent complexity of drug titration, drug interactions, and both the real and perceived adverse events collectively contribute to the failure of lifelong polypharmacy. Furthermore, therapy targeting the potentially unique contribution of autonomic imbalance is limited by the poorly tolerated systemic adverse effects of adrenergic blocking agents. Recent introduction of medical procedures, such as renal denervation,6,7 and devices such as deep brain stimulation,8 baroreceptor stimulation,9 and direct vagus nerve stimulation10 begin to address these gaps in selective patients. The contribution of excessive sympathetic nerve activity to the development and progression of hypertension, insulin resistance, and HF has been demonstrated in both preclinical and human experiments. Preclinical experiments in models of these diseases have successfully used sympathetic or parasympathetic modifications to alter the time course of their progression.11,12 Reduction of blood pressure after dorsal rhizotomy in rats with renal hypertension and reduced total body noradrenaline and muscle sympathetic nerve activity in humans after renal denervation confirm that the afferent signals from the kidney underlie some of the excessive sympathetic drive seen in these states.13,14 However, additional afferent signals may arise from sites elsewhere in the body and in particular the carotid body (CB). We propose targeting the CB in patients with increased chemosensitivity to address the underlying autonomic imbalance seen in hypertension, HF, insulin resistance, and chronic kidney disorders. ### The CB: A Peripheral Chemosensor The CB (Figure 1), the dominant …

Amplified respiratory-sympathetic coupling in the spontaneously hypertensive rat: does it contribute to hypertension?

Sympathetic nerve activity (SNA) is elevated in established hypertension. We tested the hypothesis that SNA is elevated in neonate and juvenile spontaneously hypertensive (SH) rats prior to the development of hypertension, and that this may be due to augmented respiratory–sympathetic coupling. Using the working heart–brainstem preparation, perfusion pressure, phrenic nerve activity and thoracic (T8) SNA were recorded in male SH rats and normotensive Wistar–Kyoto (WKY) rats at three ages: neonates (postnatal day 9–16), 3 weeks old and 5 weeks old. Perfusion pressure was higher in SH rats at all ages reflecting higher vascular resistance. The amplitude of respiratory‐related bursts of SNA was greater in SH rats at all ages (P < 0.05). This was reflected in larger Traube–Hering pressure waves in SH rats (1.4 ± 0.8 versus 9.8 ± 1.5 mmHg WKY versus SH rat, 5 weeks old, n= 5 per group, P < 0.01). Recovery from hypocapnic‐induced apnoea and reinstatement of Traube–Hering waves produced a significantly greater increase in perfusion pressure in SH rats (P < 0.05). Differences in respiratory–sympathetic coupling in the SH rat were not secondary to changes in central or peripheral chemoreflex sensitivity, nor were they related to altered arterial baroreflex function. We have shown that increased SNA is already present in SH rats in early postnatal life as revealed by augmented respiratory modulation of SNA. This is reflected in an increased magnitude of Traube–Hering waves resulting in elevated perfusion pressure in the SH rat. We suggest that the amplified respiratory‐related bursts of SNA seen in the neonate and juvenile SH rat may be causal in the development of their hypertension.

Hypertension is critically dependent on the carotid body input in the spontaneously hypertensive rat

Peripheral chemoreflex sensitivity is enhanced in hypertension yet the role of these receptors in the development and maintenance of high blood pressure remains unknown. Carotid chemoreceptors were denervated in both young and adult spontaneously hypertensive rats (SHRs) by sectioning the carotid sinus nerves bilaterally while recording arterial blood pressure chronically using radio telemetry. Carotid sinus denervation (CSD) in the young animals prevented arterial pressure from reaching the hypertensive levels observed in sham‐operated animals whereas in adult SHRs arterial pressure fell by ∼20 mmHg. After CSD there was a decrease in sympathetic activity, measured indirectly using power spectral analysis and hexamethonium, and an improvement in baroreceptor reflex gain. Carotid bodies are active in the SHR and contribute to both the development and maintenance of hypertension; whether carotid body ablation is a useful anti‐hypertensive intervention in drug‐resistant hypertensive patients remains to be resolved.