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The pre-Bötzinger complex (preBötC), a key primary generator of the inspiratory breathing rhythm, contains neurons that project directly to facial nucleus (7n) motoneurons to coordinate orofacial and nasofacial activity. To further understand the identity of 7n-projecting preBötC neurons, we used a combination of optogenetic viral transgenic approaches to demonstrate that selective photoinhibition of these neurons affects mystacial pad activity, with minimal effects on breathing. These effects are altered by the type of anesthetic employed and also between anesthetized and conscious states. The population of 7n-projecting preBötC neurons we transduced consisted of both excitatory and inhibitory neurons that also send collaterals to multiple brainstem nuclei involved with the regulation of autonomic activity. We show that modulation of subgroups of preBötC neurons, based on their axonal projections, is a useful strategy to improve our understanding of the mechanisms that coordinate and integrate breathing with different motor and physiological behaviors. This is of fundamental importance, given that abnormal respiratory modulation of autonomic activity and orofacial behaviors have been associated with the development and progression of diseases., Competing Interests: MM, AW, AC, JB, SC, SM, CM, RB, AA No competing interests declared, (© 2023, Melo et al.)

Spinal cord injury (SCI) leads not only to major impairments in sensorimotor control but also to dramatic dysregulation of autonomic functions including major cardiovascular disturbances. Consequently, individuals with SCI endure daily episodic hypo/hypertension and are at increased risk for cardiovascular disease. Several studies have suggested that an intrinsic spinal coupling mechanism between motor and sympathetic neuronal networks exist and that propriospinal cholinergic neurons may be responsible for a synchronized activation of both somatic and sympathetic outputs. We therefore investigated in the present study, the effect of cholinergic muscarinic agonists on cardiovascular parameters in freely moving adult rats after SCI. Female Sprague-Dawley rats were implanted with radiotelemetry sensors for long-term in vivo monitoring of blood pressure (BP). From BP signal, we calculated heart rate (HR) and respiratory frequency. We first characterized the physiological changes occurring after a SCI performed at the T3-T4 level in our experimental model system. We then investigated the effects on BP, HR and respiration, of the muscarinic agonist oxotremorine using one variant that crossed the blood brain barrier (Oxo-S) and one that does not (Oxo-M) in both Pre- and Post-SCI animals. After SCI, both HR and respiratory frequency increased. BP values exhibited an immediate profound drop before progressively increasing over the three-week post-lesion period but remained below control values. A spectral analysis of BP signal revealed the disappearance of the low frequency component of BP (0.3-0.6 Hz) referred to as Mayer waves after SCI. In Post-SCI animals, central effects mediated by Oxo-S led to an increase in HR and MAP, a slowdown in respiratory frequency and to an increased power in the 0.3-0.6 Hz frequency band. This study unravels some of the mechanisms by which muscarinic activation of spinal neurons could contribute to partial restoration of BP after SCI., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Rett syndrome (RTT) is a severe neurodevelopmental disorder that arise from de novo mutations in the X-linked gene MECP2 (methyl-CpG-binding protein 2). Circulating levels of the adipocyte hormone leptin are elevated in RTT patients and rodent models of the disease. Leptin targets a large number of brain structures and regulates a wide range of developmental and physiological functions which are altered in RTT. We hypothesized that elevated leptin levels might contribute to RTT pathogenesis. Accordingly, we show that pharmacological antagonism of leptin or genetic reduction of leptin production prevents the degradation of health status, weight loss and the progression of breathing and locomotor deficits. At the neuronal level, the anti-leptin strategies rescue the hippocampal excitatory/inhibitory imbalance and synaptic plasticity impairment. Targeting leptin might therefore represent a new approach for RTT treatment.

Twenty-five years ago, a new physiological preparation called the working heart-brainstem preparation (WHBP) was introduced with the claim it would provide a new platform allowing studies not possible before in cardiovascular, neuroendocrine, autonomic and respiratory research. Herein, we review some of the progress made with the WHBP, some advantages and disadvantages along with potential future applications, and provide photographs and technical drawings of all the customised equipment used for the preparation. Using mice or rats, the WHBP is an in situ experimental model that is perfused via an extracorporeal circuit benefitting from unprecedented surgical access, mechanical stability of the brain for whole cell recording and an uncompromised use of pharmacological agents akin to in vitro approaches. The preparation has revealed novel mechanistic insights into, for example, the generation of distinct respiratory rhythms, the neurogenesis of sympathetic activity, coupling between respiration and the heart and circulation, hypothalamic and spinal control mechanisms, and peripheral and central chemoreceptor mechanisms. Insights have been gleaned into diseases such as hypertension, heart failure and sleep apnoea. Findings from the in situ preparation have been ratified in conscious in vivo animals and when tested have translated to humans. We conclude by discussing potential future applications of the WHBP including two-photon imaging of peripheral and central nervous systems and adoption of pharmacogenetic tools that will improve our understanding of physiological mechanisms and reveal novel mechanisms that may guide new treatment strategies for cardiorespiratory diseases., (© 2022 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Behavioral phenotyping devices have been successfully used to build ethograms, but many aspects of behavior remain out of reach of available phenotyping systems. We now report on a novel device, which consists in an open-field platform resting on highly sensitive piezoelectric (electromechanical) pressure-sensors, with which we could detect the slightest movements (up to individual heart beats during rest) from freely moving rats and mice. The combination with video recordings and signal analysis based on time-frequency decomposition, clustering, and machine learning algorithms provided non-invasive access to previously overlooked behavioral components. The detection of shaking/shivering provided an original readout of fear, distinct from but complementary to behavioral freezing. Analyzing the dynamics of momentum in locomotion and grooming allowed to identify the signature of gait and neurodevelopmental pathological phenotypes. We believe that this device represents a significant progress and offers new opportunities for the awaited advance of behavioral phenotyping., (© 2021. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused mainly by mutations in the MECP2 gene. Mouse models of RTT show reduced expression of the cation-chloride cotransporter KCC2 and altered chloride homeostasis at presymptomatic stages. However, whether these alterations persist to late symptomatic stages has not been studied. Here we assess KCC2 and NKCC1 expressions and chloride homeostasis in the hippocampus of early [postnatal (P) day 30-35] and late (P50-60) symptomatic male Mecp2-null (Mecp2 -/ y ) mice. We found (i) no difference in the relative amount, but an over-phosphorylation, of KCC2 and NKCC1 between wild-type (WT) and Mecp2 -/ y hippocampi and (ii) no difference in the inhibitory strength, nor reversal potential, of GABA A -receptor-mediated responses in Mecp2 -/ y CA3 pyramidal neurons compared to WT at any stages studied. Altogether, these data indicate the presence of a functional chloride extrusion mechanism in Mecp2 -/ y CA3 pyramidal neurons at symptomatic stages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Belaïdouni, Diabira, Zhang, Graziano, Bader, Montheil, Menuet, Wayman and Gaiarsa.)

Huntington's disease (HD) is a progressive, autosomal dominant neurodegenerative disorder affecting striatal neurons beginning in young adults with loss of muscle coordination and cognitive decline. Less appreciated is the fact that patients with HD also exhibit cardiac and respiratory dysfunction, including pulmonary insufficiency and cardiac arrhythmias. The underlying mechanism for these symptoms is poorly understood. In the present study we provide insight into the cause of cardiorespiratory dysfunction in HD and identify a potentially novel therapeutic target. We now show that intracellular calcium (Ca2+) leak via posttranslationally modified ryanodine receptor/intracellular calcium release (RyR) channels plays an important role in HD pathology. RyR channels were oxidized, PKA phosphorylated, and leaky in brain, heart, and diaphragm both in patients with HD and in a murine model of HD (Q175). HD mice (Q175) with endoplasmic reticulum Ca2+ leak exhibited cognitive dysfunction, decreased parasympathetic tone associated with cardiac arrhythmias, and reduced diaphragmatic contractile function resulting in impaired respiratory function. Defects in cognitive, motor, and respiratory functions were ameliorated by treatment with a novel Rycal small-molecule drug (S107) that fixes leaky RyR. Thus, leaky RyRs likely play a role in neuronal, cardiac, and diaphragmatic pathophysiology in HD, and RyRs are a potential novel therapeutic target.

Chemogenetics enables manipulation of neuronal activity in experimental animals. While providing information about the transduced neuron expressing a ligand-activated molecule, chemogenetics does not provide understanding about the antecedent circuit that drives that neuron's activity. For current approaches, this is not feasible, because the activating molecules are not genetically encoded. The insect allatostatin/allatostatin receptor system, a highly specific, powerful inhibitory chemogenetic approach, has this advantage, because the ligand, being a peptide, is genetically encoded. We developed viral vector-based systems to express biologically active allatostatin in neurons in vivo and allatostatin receptors in subpopulations of postsynaptic neurons. We demonstrate that activity-dependent release of allatostatin induces inhibition of allatostatin receptor-expressing neurons. We validate the approach in the vagal viscerosensory system where inhibitory, rather than the usual excitatory, viscerosensory input leads to sustained decreases in baroreceptor reflex sensitivity and bodyweight., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Heart rate and blood pressure oscillate in phase with respiratory activity. A component of these oscillations is generated centrally, with respiratory neurons entraining the activity of pre-sympathetic and parasympathetic cardiovascular neurons. Using a combination of optogenetic inhibition and excitation in vivo and in situ in rats, as well as neuronal tracing, we demonstrate that preBötzinger Complex (preBötC) neurons, which form the kernel for inspiratory rhythm generation, directly modulate cardiovascular activity. Specifically, inhibitory preBötC neurons modulate cardiac parasympathetic neuron activity whilst excitatory preBötC neurons modulate sympathetic vasomotor neuron activity, generating heart rate and blood pressure oscillations in phase with respiration. Our data reveal yet more functions entrained to the activity of the preBötC, with a role in generating cardiorespiratory oscillations. The findings have implications for cardiovascular pathologies, such as hypertension and heart failure, where respiratory entrainment of heart rate is diminished and respiratory entrainment of blood pressure exaggerated., Competing Interests: CM, AC, JB, MM, SL, JK, NK, SM, SM, AA No competing interests declared, (© 2020, Menuet et al.)

Respiratory modulation of sympathetic nerve activity (respSNA) was studied in a hypertensive rodent model of chronic kidney disease (CKD) using Lewis Polycystic Kidney (LPK) rats and Lewis controls. In adult animals under in vivo anaesthetised conditions (n = 8-10/strain), respiratory modulation of splanchnic and renal nerve activity was compared under control conditions, and during peripheral (hypoxia), and central, chemoreceptor (hypercapnia) challenge. RespSNA was increased in the LPK vs. Lewis (area under curve (AUC) splanchnic and renal: 8.7 ± 1.1 vs. 3.5 ± 0.5 and 10.6 ± 1.1 vs. 7.1 ± 0.2 μV.s, respectively, P < 0.05). Hypoxia and hypercapnia increased respSNA in both strains but the magnitude of the response was greater in LPK, particularly in response to hypoxia. In juvenile animals studied using a working heart brainstem preparation (n = 7-10/strain), increased respSNA was evident in the LPK (thoracic SNA, AUC: 0.86 ± 0.1 vs. 0.42 ± 0.1 μV.s, P < 0.05), and activation of peripheral chemoreceptors (NaCN) again drove a larger increase in respSNA in the LPK with no difference in the response to hypercapnia. Amplified respSNA occurs in CKD and may contribute to the development of hypertension., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Prader-Willi syndrome (PWS) is a genetic neurodevelopmental disorder that presents with hypotonia and respiratory distress in neonates. The Necdin -deficient mouse is the only model that reproduces the respiratory phenotype of PWS (central apnea and blunted response to respiratory challenges). Here, we report that Necdin deletion disturbs the migration of serotonin (5-HT) neuronal precursors, leading to altered global serotonergic neuroarchitecture and increased spontaneous firing of 5-HT neurons. We show an increased expression and activity of 5-HT Transporter (SERT/Slc6a4) in 5-HT neurons leading to an increase of 5-HT uptake. In Necdin -KO pups, the genetic deletion of Slc6a4 or treatment with Fluoxetine, a 5-HT reuptake inhibitor, restored normal breathing. Unexpectedly, Fluoxetine administration was associated with respiratory side effects in wild-type animals. Overall, our results demonstrate that an increase of SERT activity is sufficient to cause the apneas in Necdin- KO pups, and that fluoxetine may offer therapeutic benefits to PWS patients with respiratory complications.

The etiology of hypertension, the world's biggest killer, remains poorly understood, with treatments targeting the established symptom, not the cause. The development of hypertension involves increased sympathetic nerve activity that, in experimental hypertension, may be driven by excessive respiratory modulation. Using selective viral and cell lesion techniques, we identify adrenergic C1 neurons in the medulla oblongata as critical for respiratory-sympathetic entrainment and the development of experimental hypertension. We also show that a cohort of young, normotensive humans, selected for an exaggerated blood pressure response to exercise and thus increased hypertension risk, has enhanced respiratory-related blood pressure fluctuations. These studies pinpoint a specific neuronal target for ameliorating excessive sympathetic activity during the developmental phase of hypertension and identify a group of pre-hypertensive subjects that would benefit from targeting these cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Spinally projecting neurons in the rostral ventrolateral medulla (RVLM) play a critical role in the generation of vasomotor sympathetic tone and are thought to receive convergent input from neurons at every level of the neuraxis; the factors that determine their ongoing activity remain unresolved. In this study we use a genetically restricted viral tracing strategy to definitively map their spatially diffuse connectome. We infected bulbospinal RVLM neurons with a recombinant rabies variant that drives reporter expression in monosynaptically connected input neurons and mapped their distribution using an MRI-based volumetric atlas and a novel image alignment and visualization tool that efficiently translates the positions of neurons captured in conventional photomicrographs to Cartesian coordinates. We identified prominent inputs from well-established neurohumoral and viscero-sympathetic sensory actuators, medullary autonomic and respiratory subnuclei, and supramedullary autonomic nuclei. The majority of inputs lay within the brainstem (88-94%), and included putative respiratory neurons in the pre-Bötzinger Complex and post-inspiratory complex that are therefore likely to underlie respiratory-sympathetic coupling. We also discovered a substantial and previously unrecognized input from the region immediately ventral to nucleus prepositus hypoglossi. In contrast, RVLM sympathetic premotor neurons were only sparsely innervated by suprapontine structures including the paraventricular nucleus, lateral hypothalamus, periaqueductal gray, and superior colliculus, and we found almost no evidence of direct inputs from the cortex or amygdala. Our approach can be used to quantify, standardize and share complete neuroanatomical datasets, and therefore provides researchers with a platform for presentation, analysis and independent reanalysis of connectomic data.

Changes in arterial Po2, Pco2, and pH are the strongest stimuli sensed by peripheral and central chemoreceptors to adjust ventilation to the metabolic demand. Erythropoietin (Epo), the main regulator of red blood cell production, increases the hypoxic ventilatory response, an effect attributed to the presence of Epo receptors in both carotid bodies and key brainstem structures involved in integration of peripheral inputs and control of breathing. However, it is not known whether Epo also has an effect on the hypercapnic chemoreflex. In a first attempt to answer this question, we tested the hypothesis that Epo alters the ventilatory response to increased CO2 levels. Basal ventilation and hypercapnic ventilatory response (HCVR) were recorded from control mice and from two transgenic mouse lines constitutively expressing high levels of human Epo in brain only (Tg21) or in brain and plasma (Tg6), the latter leading to polycythemia. To tease apart the potential effects of polycythemia and levels of plasma Epo in the HCVR, control animals were injected with an Epo analog (Aranesp), and Tg6 mice were treated with the hemolytic agent phenylhydrazine after splenectomy. Ventilatory parameters measured by plethysmography in conscious mice were consistent with data from electrophysiological recordings in anesthetized animals and revealed a blunted HCVR in Tg6 mice. Polycythemia alone and increased levels of plasma Epo blunt the HCVR. In addition, Tg21 mice with an augmented level of cerebral Epo also had a decreased HCVR. We discuss the potential implications of these findings in several physiopathological conditions., (Copyright © 2016 the American Physiological Society.)