Brainstem Respiratory Oscillators Develop Independently of Neuronal Migration Defects in the Wnt/PCP Mouse Mutant looptail

The proper development and maturation of neuronal circuits require precise migration of component neurons from their birthplace (germinal zone) to their final positions. Little is known about the effects of aberrant neuronal position on the functioning of organized neuronal groups, especially in mammals. Here, we investigated the formation and properties of brainstem respiratory neurons in looptail (Lp) mutant mice in which facial motor neurons closely apposed to some respiratory neurons fail to migrate due to loss of function of the Wnt/Planar Cell Polarity (PCP) protein Vangl2. Using calcium imaging and immunostaining on embryonic hindbrain preparations, we found that respiratory neurons constituting the embryonic parafacial oscillator (e-pF) settled at the ventral surface of the medulla in Vangl2Lp/+ and Vangl2Lp/Lp embryos despite the failure of tangential migration of its normally adjacent facial motor nucleus. Anatomically, the e-pF neurons were displaced medially in Lp/+ embryos and rostro-medially Lp/Lp embryos. Pharmacological treatments showed that the e-pF oscillator exhibited characteristic network properties in both Lp/+ and Lp/Lp embryos. Furthermore, using hindbrain slices, we found that the other respiratory oscillator, the preBötzinger complex, was also anatomically and functionally established in Lp mutants. Importantly, the displaced e-pF oscillator established functional connections with the preBötC oscillator in Lp/+ mutants. Our data highlight the robustness of the developmental processes that assemble the neuronal networks mediating an essential physiological function

Transcriptional landscape of bone marrow-derived very small embryonic-like stem cells during hypoxia

<p>Abstract</p> <p>Background</p> <p>Hypoxia is a ubiquitous feature of many lung diseases and elicits cell-specific responses. While the effects of hypoxia on stem cells have been examined under <it>in vitro </it>conditions, the consequences of <it>in vivo </it>oxygen deprivation have not been studied.</p> <p>Methods</p> <p>We investigated the effects of <it>in vivo </it>hypoxia on a recently characterized population of pluripotent stem cells known as very small embryonic-like stem cells (VSELs) by whole-genome expression profiling and measuring peripheral blood stem cell chemokine levels.</p> <p>Results</p> <p>We found that exposure to hypoxia in mice mobilized VSELs from the bone marrow to peripheral blood, and induced a distinct genome-wide transcriptional signature. Applying a computationally-intensive methodology, we identified a hypoxia-induced gene interaction network that was functionally enriched in a diverse array of programs including organ-specific development, stress response, and wound repair. Topographic analysis of the network highlighted a number of densely connected hubs that may represent key controllers of stem cell response during hypoxia and, therefore, serve as putative targets for altering the pathophysiologic consequences of hypoxic burden.</p> <p>Conclusions</p> <p>A brief exposure to hypoxia recruits pluripotent stem cells to the peripheral circulation and actives diverse transcriptional programs that are orchestrated by a selective number of key genes.</p

Genetic inactivation of the vesicular glutamate transporter 2 (VGLUT2) in the mouse: What have we learnt about functional glutamatergic neurotransmission?

During the past decade, three proteins that possess the capability of packaging glutamate into presynaptic vesicles have been identified and characterized. These three vesicular glutamate transporters, VGLUT1–3, are encoded by solute carrier genes Slc17a6–8. VGLUT1 (Slc17a7) and VGLUT2 (Slc17a6) are expressed in glutamatergic neurons, while VGLUT3 (Slc17a8) is expressed in neurons classically defined by their use of another transmitter, such as acetylcholine and serotonin. As glutamate is both a ubiquitous amino acid and the most abundant neurotransmitter in the adult central nervous system, the discovery of the VGLUTs made it possible for the first time to identify and specifically target glutamatergic neurons. By molecular cloning techniques, different VGLUT isoforms have been genetically targeted in mice, creating models with alterations in their glutamatergic signalling. Glutamate signalling is essential for life, and its excitatory function is involved in almost every neuronal circuit. The importance of glutamatergic signalling was very obvious when studying full knockout models of both VGLUT1 and VGLUT2, none of which were compatible with normal life. While VGLUT1 full knockout mice die after weaning, VGLUT2 full knockout mice die immediately after birth. Many neurological diseases have been associated with altered glutamatergic signalling in different brain regions, which is why conditional knockout mice with abolished VGLUT-mediated signalling only in specific circuits may prove helpful in understanding molecular mechanisms behind such pathologies. We review the recent studies in which mouse genetics have been used to characterize the functional role of VGLUT2 in the central nervous system

Glycinergic interneurons are functionally integrated into the inspiratory network of mouse medullary slices

Neuronal activity in the respiratory network is functionally dependent on inhibitory synaptic transmission. Using two-photon excitation microscopy, we analyzed the integration of glycinergic neurons in the isolated inspiratory pre-Bötzinger complex-driven network of the rhythmic slice preparation. Inspiratory (96%) and ‘tonic’ expiratory neurons (4%) were identified via an increase or decrease, respectively, of the cytosolic free calcium concentration during the inspiratory-related respiratory burst. Furthermore, in BAC-transgenic mice expressing EGFP under the control of the GlyT2-promoter, 50% of calcium-imaged inspiratory neurons were glycinergic. Inspiratory bursting of glycinergic neurons in the slice was confirmed by whole-cell recording. We also found glycinergic neurons that receive phasic inhibition from other glycinergic neurons. Our calcium imaging data show that glycinergic neurons comprise a large population of inspiratory neurons in the pre-Bötzinger complex-driven network of the rhythmic slice preparation

Synaptic and Intrinsic Activation of GABAergic Neurons in the Cardiorespiratory Brainstem Network

GABAergic pathways in the brainstem play an essential role in respiratory rhythmogenesis and interactions between the respiratory and cardiovascular neuronal control networks. However, little is known about the identity and function of these GABAergic inhibitory neurons and what determines their activity. In this study we have identified a population of GABAergic neurons in the ventrolateral medulla that receive increased excitatory post-synaptic potentials during inspiration, but also have spontaneous firing in the absence of synaptic input. Using transgenic mice that express GFP under the control of the Gad1 (GAD67) gene promoter, we determined that this population of GABAergic neurons is in close apposition to cardioinhibitory parasympathetic cardiac neurons in the nucleus ambiguus (NA). These neurons fire in synchronization with inspiratory activity. Although they receive excitatory glutamatergic synaptic inputs during inspiration, this excitatory neurotransmission was not altered by blocking nicotinic receptors, and many of these GABAergic neurons continue to fire after synaptic blockade. The spontaneous firing in these GABAergic neurons was not altered by the voltage-gated calcium channel blocker cadmium chloride that blocks both neurotransmission to these neurons and voltage-gated Ca2+ currents, but spontaneous firing was diminished by riluzole, demonstrating a role of persistent sodium channels in the spontaneous firing in these cardiorespiratory GABAergic neurons that possess a pacemaker phenotype. The spontaneously firing GABAergic neurons identified in this study that increase their activity during inspiration would support respiratory rhythm generation if they acted primarily to inhibit post-inspiratory neurons and thereby release inspiration neurons to increase their activity. This population of inspiratory-modulated GABAergic neurons could also play a role in inhibiting neurons that are most active during expiration and provide a framework for respiratory sinus arrhythmia as there is an increase in heart rate during inspiration that occurs via inhibition of premotor parasympathetic cardioinhibitory neurons in the NA during inspiration

A Sodium Leak Current Regulates Pacemaker Activity of Adult Central Pattern Generator Neurons in Lymnaea Stagnalis

The resting membrane potential of the pacemaker neurons is one of the essential mechanisms underlying rhythm generation. In this study, we described the biophysical properties of an uncharacterized channel (U-type channel) and investigated the role of the channel in the rhythmic activity of a respiratory pacemaker neuron and the respiratory behaviour in adult freshwater snail Lymnaea stagnalis. Our results show that the channel conducts an inward leak current carried by Na+ (ILeak-Na). The ILeak-Na contributed to the resting membrane potential and was required for maintaining rhythmic action potential bursting activity of the identified pacemaker RPeD1 neurons. Partial knockdown of the U-type channel suppressed the aerial respiratory behaviour of the adult snail in vivo. These findings identified the Na+ leak conductance via the U-type channel, likely a NALCN-like channel, as one of the fundamental mechanisms regulating rhythm activity of pacemaker neurons and respiratory behaviour in adult animals

Silicon central pattern generators for cardiac diseases

Belgium Herbarium image of Meise Botanic Garden

Silicon central pattern generators for cardiac diseases

Cardiac rhythm management devices provide therapies for both arrhythmias and resynchronisation but not heart failure, which affects millions of patients worldwide. This paper reviews recent advances in biophysics and mathematical engineering that provide a novel technological platform for addressing heart disease and enabling beat-to-beat adaptation of cardiac pacing in response to physiological feedback. The technology consists of silicon hardware central pattern generators (hCPGs) that may be trained to emulate accurately the dynamical response of biological central pattern generators (bCPGs). We discuss the limitations of present CPGs and appraise the advantages of analog over digital circuits for application in bioelectronic medicine. To test the system, we have focused on the cardio-respiratory oscillators in the medulla oblongata that modulate heart rate in phase with respiration to induce respiratory sinus arrhythmia (RSA). We describe here a novel, scalable hCPG comprising physiologically realistic (Hodgkin–Huxley type) neurones and synapses. Our hCPG comprises two neurones that antagonise each other to provide rhythmic motor drive to the vagus nerve to slow the heart. We show how recent advances in modelling allow the motor output to adapt to physiological feedback such as respiration. In rats, we report on the restoration of RSA using an hCPG that receives diaphragmatic electromyography input and use it to stimulate the vagus nerve at specific time points of the respiratory cycle to slow the heart rate. We have validated the adaptation of stimulation to alterations in respiratory rate. We demonstrate that the hCPG is tuneable in terms of the depth and timing of the RSA relative to respiratory phase. These pioneering studies will now permit an analysis of the physiological role of RSA as well as its any potential therapeutic use in cardiac disease

Developmental basis of the rostro-caudal organization of the brainstem respiratory rhythm generator

The Hox genetic network plays a key role in the anteroposterior patterning of the rhombencephalon at pre- and early-segmental stages of development of the neural tube. In the mouse, it controls development of the entire brainstem respiratory neuronal network, including the pons, the parafacial respiratory group (pFRG) and the pre-Bötzinger complex (preBötC). Inactivation of Krox20/Egr2 eliminates the pFRG activity, thereby causing life-threatening neonatal apnoeas alternating with respiration at low frequency. Another respiratory abnormality, the complete absence of breathing, is induced when neuronal synchronization fails to develop in the preBötC. The present paper summarizes data on a third type of respiratory deficits induced by altering Hox function at pontine levels. Inactivation of Hoxa2, the most rostrally expressed Hox gene in the hindbrain, disturbs embryonic development of the pons and alters neonatal inspiratory shaping without affecting respiratory frequency and apnoeas. The same result is obtained by the Phox2a+/− mutation modifying the number of petrosal chemoafferent neurons, by eliminating acetylcholinesterase and by altering Hox-dependent development of the pons with retinoic acid administration at embryonic day 7.5. In addition, embryos treated with retinoic acid provide a mouse model for hyperpnoeic episodic breathing, widely reported in pre-term neonates, young girls with Rett's syndrome, patients with Joubert syndrome and adults with Cheyne–Stokes respiration. We conclude that specific respiratory deficits in vivo are assignable to anteroposterior segments of the brainstem, suggesting that the adult respiratory neuronal network is functionally organized according to the rhombomeric, Hox-dependent segmentation of the brainstem in embryos