Computer-aided neurophysiology and imaging with open-source PhysImage.

Improved integration between imaging and electrophysiological data has become increasingly critical for rapid interpretation and intervention as approaches have advanced in recent years. Here, we present PhysImage, a fork of the popular public-domain ImageJ that provides a platform for working with these disparate sources of data, and we illustrate its utility using in vitro preparations from murine embryonic and neonatal tissue. PhysImage expands ImageJ's core features beyond an imaging program by facilitating integration, analyses, and display of 2D waveform data, among other new features. Together, with the Micro-Manager plugin for image acquisition, PhysImage substantially improves on closed-source or blended approaches to analyses and interpretation, and it furthermore aids post hoc automated analysis of physiological data when needed as we demonstrate here. Developing a high-throughput approach to neurophysiological analyses has been a major challenge for neurophysiology as a whole despite data analytics methods advancing rapidly in other areas of neuroscience, biology, and especially genomics. NEW & NOTEWORTHY High-throughput analyses of both concurrent electrophysiological and imaging recordings has been a major challenge in neurophysiology. We submit an open-source solution that may be able to alleviate, or at least reduce, many of these concerns by providing an institutionally proven mechanism (i.e., ImageJ) with the added benefits of open-source Python scripting of PhysImage data that eases the workmanship of 2D trace data, which includes electrophysiological data. Together, with the ability to autogenerate prototypical figures shows this technology is a noteworthy advance.

Robust network oscillations during mammalian respiratory rhythm generation driven by synaptic dynamics.

How might synaptic dynamics generate synchronous oscillations in neuronal networks? We address this question in the preBötzinger complex (preBötC), a brainstem neural network that paces robust, yet labile, inspiration in mammals. The preBötC is composed of a few hundred neurons that alternate bursting activity with silent periods, but the mechanism underlying this vital rhythm remains elusive. Using a computational approach to model a randomly connected neuronal network that relies on short-term synaptic facilitation (SF) and depression (SD), we show that synaptic fluctuations can initiate population activities through recurrent excitation. We also show that a two-step SD process allows activity in the network to synchronize (bursts) and generate a population refractory period (silence). The model was validated against an array of experimental conditions, which recapitulate several processes the preBötC may experience. Consistent with the modeling assumptions, we reveal, by electrophysiological recordings, that SF/SD can occur at preBötC synapses on timescales that influence rhythmic population activity. We conclude that nondeterministic neuronal spiking and dynamic synaptic strengths in a randomly connected network are sufficient to give rise to regular respiratory-like rhythmic network activity and lability, which may play an important role in generating the rhythm for breathing and other coordinated motor activities in mammals.

Functional Interactions between Mammalian Respiratory Rhythmogenic and Premotor Circuitry.

UNLABELLED: Breathing in mammals depends on rhythms that originate from the preBötzinger complex (preBötC) of the ventral medulla and a network of brainstem and spinal premotor neurons. The rhythm-generating core of the preBötC, as well as some premotor circuits, consist of interneurons derived from Dbx1-expressing precursors (Dbx1 neurons), but the structure and function of these networks remain incompletely understood. We previously developed a cell-specific detection and laser ablation system to interrogate respiratory network structure and function in a slice model of breathing that retains the preBötC, the respiratory-related hypoglossal (XII) motor nucleus and XII premotor circuits. In spontaneously rhythmic slices, cumulative ablation of Dbx1 preBötC neurons decreased XII motor output by ∼50% after ∼15 cell deletions, and then decelerated and terminated rhythmic function altogether as the tally increased to ∼85 neurons. In contrast, cumulatively deleting Dbx1 XII premotor neurons decreased motor output monotonically but did not affect frequency nor stop XII output regardless of the ablation tally. Here, we couple an existing preBötC model with a premotor population in several topological configurations to investigate which one may replicate the laser ablation experiments best. If the XII premotor population is a "small-world" network (rich in local connections with sparse long-range connections among constituent premotor neurons) and connected with the preBötC such that the total number of incoming synapses remains fixed, then the in silico system successfully replicates the in vitro laser ablation experiments. This study proposes a feasible configuration for circuits consisting of Dbx1-derived interneurons that generate inspiratory rhythm and motor pattern. SIGNIFICANCE STATEMENT: To produce a breathing-related motor pattern, a brainstem core oscillator circuit projects to a population of premotor interneurons, but the assemblage of this network remains incompletely understood. Here we applied network modeling and numerical simulation to discover respiratory circuit configurations that successfully replicate photonic cell ablation experiments targeting either the core oscillator or premotor network, respectively. If premotor neurons are interconnected in a so-called "small-world" network with a fixed number of incoming synapses balanced between premotor and rhythmogenic neurons, then our simulations match their experimental benchmarks. These results provide a framework of experimentally testable predictions regarding the rudimentary structure and function of respiratory rhythm- and pattern-generating circuits in the brainstem of mammals.

Cumulative lesioning of respiratory interneurons disrupts and precludes motor rhythms in vitro.

How brain functions degenerate in the face of progressive cell loss is an important issue that pertains to neurodegenerative diseases and basic properties of neural networks. We developed an automated system that uses two-photon microscopy to detect rhythmic neurons from calcium activity, and then individually laser ablates the targets while monitoring network function in real time. We applied this system to the mammalian respiratory oscillator located in the pre-Bötzinger Complex (preBötC) of the ventral medulla, which spontaneously generates breathing-related motor activity in vitro. Here, we show that cumulatively deleting preBötC neurons progressively decreases respiratory frequency and the amplitude of motor output. On average, the deletion of 120 ± 45 neurons stopped spontaneous respiratory rhythm, and our data suggest ≈82% of the rhythm-generating neurons remain unlesioned. Cumulative ablations in other medullary respiratory regions did not affect frequency but diminished the amplitude of motor output to a lesser degree. These results suggest that the preBötC can sustain insults that destroy no more than ≈18% of its constituent interneurons, which may have implications for the onset of respiratory pathologies in disease states.

Clonal analysis by distinct viral vectors identifies bona fide neural stem cells in the adult zebrafish telencephalon and characterizes their division properties and fate.

Neurogenesis is widespread in the zebrafish adult brain through the maintenance of active germinal niches. To characterize which progenitor properties correlate with this extensive neurogenic potential, we set up a method that allows progenitor cell transduction and tracing in the adult zebrafish brain using GFP-encoding retro- and lentiviruses. The telencephalic germinal zone of the zebrafish comprises quiescent radial glial progenitors and actively dividing neuroblasts. Making use of the power of clonal viral vector-based analysis, we demonstrate that these progenitors follow different division modes and fates: neuroblasts primarily undergo a limited amplification phase followed by symmetric neurogenic divisions; by contrast, radial glia are capable at the single cell level of both self-renewing and generating different cell types, and hence exhibit bona fide neural stem cell (NSC) properties in vivo. We also show that radial glial cells predominantly undergo symmetric gliogenic divisions, which amplify this NSC pool and may account for its long-lasting maintenance. We further demonstrate that blocking Notch signaling results in a significant increase in proliferating cells and in the numbers of clones, but does not affect clone composition, demonstrating that Notch primarily controls proliferation rather than cell fate. Finally, through long-term tracing, we illustrate the functional integration of newborn neurons in forebrain adult circuitries. These results characterize fundamental aspects of adult progenitor cells and neurogenesis, and open the way to using virus-based technologies for stable genetic manipulations and clonal analyses in the zebrafish adult brain.

Evidence for peripheral and central actions of codeine to dysregulate swallowing in the anesthetized cat.

Systemic administration of opioids has been associated with aspiration and swallow dysfunction in humans. We speculated that systemic administration of codeine would induce dysfunctional swallowing and that this effect would have a peripheral component. Experiments were conducted in spontaneously breathing, anesthetized cats. The animals were tracheotomized and electromyogram (EMG) electrodes were placed in upper airway and chest wall respiratory muscles for recording swallow related motor activity. The animals were allocated into three groups: vagal intact (VI), cervical vagotomy (CVx), and supra-nodose ganglion vagotomy (SNGx). A dose response to intravenous codeine was performed in each animal. Swallowing was elicited by injection of 3 mL of water into the oropharynx. The number of swallows after vehicle was significantly higher in the VI group than in SNGx. Codeine had no significant effect on the number of swallows induced by water in any of the groups. However, the magnitudes of water swallow-related EMGs of the thyropharyngeus muscle were significantly increased in the VI and CVx groups by 2-4 fold in a dose-related manner. In the CVx group, the geniohyoid muscle EMG during water swallows was significantly increased. There was a significant dose-related increase in spontaneous swallowing in each group from codeine. The spontaneous swallow number at the 10 mg/kg dose of codeine was significantly larger in the CVx group than that in the SNGx group. During water-evoked swallows, intravenous codeine increased upper airway motor drive in a dose-related manner, consistent with dysregulation. The data support the existence of both central and peripheral actions of codeine on spontaneous swallowing. At the highest dose of codeine, the reduced spontaneous swallow number in the SNGx group relative to CVx is consistent with a peripheral excitatory action of codeine either on pharyngeal/laryngeal receptors or in the nodose ganglion itself. The higher number of swallows in the CVx group than the VI group supports disinhibition of this behavior by elimination of inhibitory vagal sensory afferents.

Automated cell-specific laser detection and ablation of neural circuits in neonatal brain tissue.

A key feature of neurodegenerative disease is the pathological loss of neurons that participate in generating behaviour. To investigate network properties of neural circuits and provide a complementary tool to study neurodegeneration in vitro or in situ, we developed an automated cell-specific laser detection and ablation system. The instrument consists of a two-photon and visible-wavelength confocal imaging setup, controlled by executive software, that identifies neurons in preparations based on genetically encoded fluorescent proteins or Ca(2+) imaging, and then sequentially ablates cell targets while monitoring network function concurrently. Pathological changes in network function can be directly attributed to ablated cells, which are logged in real time. Here, we investigated brainstem respiratory circuits to demonstrate single-cell precision in ablation during physiological network activity, but the technique could be applied to interrogate network properties in neural systems that retain network functionality in reduced preparations in vitro or in situ.

Dendritic calcium activity precedes inspiratory bursts in preBotzinger complex neurons.

Medullary interneurons of the preBötzinger complex assemble excitatory networks that produce inspiratory-related neural rhythms, but the importance of somatodendritic conductances in rhythm generation is still incompletely understood. Synaptic input may cause Ca(2+) accumulation postsynaptically to evoke a Ca(2+)-activated inward current that contributes to inspiratory burst generation. We measured Ca(2+) transients by two-photon imaging dendrites while recording neuronal somata electrophysiologically. Dendritic Ca(2+) accumulation frequently precedes inspiratory bursts, particularly at recording sites 50-300 µm distal from the soma. Preinspiratory Ca(2+) transients occur in hotspots, not ubiquitously, in dendrites. Ca(2+) activity propagates orthodromically toward the soma (and antidromically to more distal regions of the dendrite) at rapid rates (300-700 µm/s). These high propagation rates suggest that dendritic Ca(2+) activates an inward current to electrotonically depolarize the soma, rather than propagate as a regenerative Ca(2+) wave. These data provide new evidence that respiratory rhythmogenesis may depend on dendritic burst-generating conductances activated in the context of network activity.

Calcium-activated nonspecific cation current and synaptic depression promote network-dependent burst oscillations.

Central pattern generators (CPGs) produce neural-motor rhythms that often depend on specialized cellular or synaptic properties such as pacemaker neurons or alternating phases of synaptic inhibition. Motivated by experimental evidence suggesting that activity in the mammalian respiratory CPG, the preBötzinger complex, does not require either of these components, we present and analyze a mathematical model demonstrating an unconventional mechanism of rhythm generation in which glutamatergic synapses and the short-term depression of excitatory transmission play key rhythmogenic roles. Recurrent synaptic excitation triggers postsynaptic Ca(2+)-activated nonspecific cation current (I(CAN)) to initiate a network-wide burst. Robust depolarization due to I(CAN) also causes voltage-dependent spike inactivation, which diminishes recurrent excitation and thus attenuates postsynaptic Ca(2+) accumulation. Consequently, activity-dependent outward currents-produced by Na/K ATPase pumps or other ionic mechanisms-can terminate the burst and cause a transient quiescent state in the network. The recovery of sporadic spiking activity rekindles excitatory interactions and initiates a new cycle. Because synaptic inputs gate postsynaptic burst-generating conductances, this rhythm-generating mechanism represents a new paradigm that can be dubbed a 'group pacemaker' in which the basic rhythmogenic unit encompasses a fully interdependent ensemble of synaptic and intrinsic components. This conceptual framework should be considered as an alternative to traditional models when analyzing CPGs for which mechanistic details have not yet been elucidated.

Developmental origin of preBötzinger complex respiratory neurons.

A subset of preBötzinger Complex (preBötC) neurokinin 1 receptor (NK1R) and somatostatin peptide (SST)-expressing neurons are necessary for breathing in adult rats, in vivo. Their developmental origins and relationship to other preBötC glutamatergic neurons are unknown. Here we show, in mice, that the "core" of preBötC SST(+)/NK1R(+)/SST 2a receptor(+) (SST2aR) neurons, are derived from Dbx1-expressing progenitors. We also show that Dbx1-derived neurons heterogeneously coexpress NK1R and SST2aR within and beyond the borders of preBötC. More striking, we find that nearly all non-catecholaminergic glutamatergic neurons of the ventrolateral medulla (VLM) are also Dbx1 derived. PreBötC SST(+) neurons are born between E9.5 and E11.5 in the same proportion as non-SST-expressing neurons. Additionally, preBötC Dbx1 neurons are respiratory modulated and show an early inspiratory phase of firing in rhythmically active slice preparations. Loss of Dbx1 eliminates all glutamatergic neurons from the respiratory VLM including preBötC NK1R(+)/SST(+) neurons. Dbx1 mutant mice do not express any spontaneous respiratory behaviors in vivo. Moreover, they do not generate rhythmic inspiratory activity in isolated en bloc preparations even after acidic or serotonergic stimulation. These data indicate that preBötC core neurons represent a subset of a larger, more heterogeneous population of VLM Dbx1-derived neurons. These data indicate that Dbx1-derived neurons are essential for the expression and, we hypothesize, are responsible for the generation of respiratory behavior both in vitro and in vivo.

Asymmetric control of inspiratory and expiratory phases by excitability in the respiratory network of neonatal mice in vitro.

Rhythmic motor behaviours consist of alternating movements, e.g. swing-stance in stepping, jaw opening and closing during chewing, and inspiration-expiration in breathing, which must be labile in frequency, and in some cases, in the duration of individual phases, to adjust to physiological demands. These movements are the expression of underlying neural circuits whose organization governs the properties of the motor behaviour. To determine if the ability to operate over a broad range of frequencies in respiration is expressed in the rhythm generator, we isolated the kernel of essential respiratory circuits using rhythmically active in vitro slices from neonatal mice. We show respiratory motor output in these slices at very low frequencies (0.008 Hz), well below the typical frequency in vitro (approximately 0.2 Hz) and in most intact normothermic mammals. Across this broad range of frequencies, inspiratory motor output bursts remained remarkably constant in pattern, i.e. duration, peak amplitude and area. The change in frequency was instead attributable to increased interburst interval, and was largely unaffected by removal of fast inhibitory transmission. Modulation of the frequency was primarily achieved by manipulating extracellular potassium, which significantly affects neuronal excitability. When excitability was lowered to slow down, or in some cases stop, spontaneous rhythm, brief stimulation of the respiratory network with a glutamatergic agonist could evoke (rhythmic) motor output. In slices with slow (<0.02 Hz) spontaneous rhythms, evoked motor output could follow a spontaneous burst at short (60 s. We observed during inspiration a large magnitude (approximately 0.6 nA) outward current generated by Na(+)/K(+) ATPase that deactivated in 25-100 ms and thus could contribute to burst termination and the latency of evoked bursts but is unlikely to control the interburst interval. We propose that the respiratory network functions over a broad range of frequencies by engaging distinct mechanisms from those controlling inspiratory duration and pattern that specifically govern the interburst interval.

4-Aminopyridine-sensitive outward currents in preBötzinger complex neurons influence respiratory rhythm generation in neonatal mice.

We measured a low-threshold, inactivating K+ current, i.e. A-current (I(A)), in respiratory neurons of the preBötzinger complex (preBötC) in rhythmically active slice preparations from neonatal C57BL/6 mice. The majority of inspiratory neurons (21/34 = 61.8%), but not expiratory neurons (1/8 = 12.5%), expressed I(A). In whole-cell and somatic outside-out patches I(A) activated at -60 mV (half-activation voltage measured -16.3 mV) and only fully inactivated above -40 mV (half-inactivation voltage measured -85.6 mV), indicating that I(A) can influence membrane trajectory at baseline voltages during respiratory rhythm generation in vitro. 4-Aminopyridine (4-AP, 2 mm) attenuated I(A) in both whole-cell and somatic outside-out patches. In the context of rhythmic network activity, 4-AP caused irregular respiratory-related motor output on XII nerves and disrupted rhythmogenesis as detected with whole-cell and field recordings in the preBötC. Whole-cell current-clamp recordings showed that 4-AP changed the envelope of depolarization underlying inspiratory bursts (i.e. inspiratory drive potentials) from an incrementing pattern to a decrementing pattern during rhythm generation and abolished current pulse-induced delayed excitation. These data suggest that I(A) opposes excitatory synaptic depolarizations at baseline voltages of approximately -60 mV and influences the inspiratory burst pattern. We propose that I(A) promotes orderly recruitment of constituent rhythmogenic neurons by minimizing the activity of these neurons until they receive massive coincident synaptic input, which reduces the periodic fluctuations of inspiratory activity.

What role do pacemakers play in the generation of respiratory rhythm?

The pacemaker hypothesis that specialized neurons with conditional oscillatory- bursting properties are obligatory for respiratory rhythm generation in vitro has gained widespread acceptance, despite lack of direct proof. Here we critique the pacemaker hypothesis and provide an alternative explanation for rhythmogenesis based on emergent network properties. Pacemaker neurons in the preBötC depend on either persistent Na+ current I(NaP) or Ca(2+)-activated nonspecific cationic current (I(CAN)). Activity in slice preparations and synaptically- isolated pacemaker neurons undergo similar frequency modulation by perturbations including hypoxia and changes in external K+. These data have been used to argue that pacemaker cells must be rhythmogenic, but may simply reflect the action of these perturbations on intrinsic membrane properties throughout the preBötC and does not constitute proof that pacemakers necessarily drive the rhythm with synaptic coupling in place. Likewise, bath-applied drugs, such as riluzole (RIL) and flufenamic acid (FFA), attenuate I(NaP) and I(CAN), respectively, throughout the slice. Thus, when these drugs stop the rhythm, a widespread depression of excitability is likely the underlying cause, not selective blockade of bursting-pacemaker activity. We propose that rhythmogenesis is an emergent network property, wherein recurrent synaptic excitation initiates a positive feedback cycle among interneurons and that intrinsic currents like I(CAN) and I(NaP) promote inspiratory burst generation by augmenting synaptic excitation in the context of network activity. In this group-pacemaker framework, individual pacemaker neurons can be embedded but play the same role as every other network constituent.

Transcriptome of neonatal preBötzinger complex neurones in Dbx1 reporter mice.

We sequenced the transcriptome of brainstem interneurons in the specialized respiratory rhythmogenic site dubbed preBötzinger Complex (preBötC) from newborn mice. To distinguish molecular characteristics of the core oscillator we compared preBötC neurons derived from Dbx1-expressing progenitors that are respiratory rhythmogenic to neighbouring non-Dbx1-derived neurons, which support other respiratory and non-respiratory functions. Results in three categories are particularly salient. First, Dbx1 preBötC neurons express κ-opioid receptors in addition to µ-opioid receptors that heretofore have been associated with opiate respiratory depression, which may have clinical applications. Second, Dbx1 preBötC neurons express the hypoxia-inducible transcription factor Hif1a at levels three-times higher than non-Dbx1 neurons, which links core rhythmogenic microcircuits to O2-related chemosensation for the first time. Third, we detected a suite of transcription factors including Hoxa4 whose expression pattern may define the rostral preBötC border, Pbx3 that may influence ipsilateral connectivity, and Pax8 that may pertain to a ventrally-derived subset of Dbx1 preBötC neurons. These data establish the transcriptomic signature of the core respiratory oscillator at a perinatal stage of development.

Sodium and calcium current-mediated pacemaker neurons and respiratory rhythm generation.

The breathing motor pattern in mammals originates in brainstem networks. Whether pacemaker neurons play an obligatory role remains a key unanswered question. We performed whole-cell recordings in the preBotzinger Complex in slice preparations from neonatal rodents and tested for pacemaker activity. We observed persistent Na+ current (I(NaP))-mediated bursting in approximately 5% of inspiratory neurons in postnatal day 0 (P0)-P5 and in P8-P10 slices. I(NaP)-mediated bursting was voltage dependent and blocked by 20 mum riluzole (RIL). We found Ca2+ current (I(Ca))-dependent bursting in 7.5% of inspiratory neurons in P8-P10 slices, but in P0-P5 slices these cells were exceedingly rare (0.6%). This bursting was voltage independent and blocked by 100 microm Cd2+ or flufenamic acid (FFA) (10-200 microm), which suggests that a Ca2+-activated inward cationic current (I(CAN)) underlies burst generation. These data substantiate our observation that P0-P5 slices exposed to RIL contain few (if any) pacemaker neurons, yet maintain respiratory rhythm. We also show that 20 nm TTX or coapplication of 20 microm RIL + FFA (100-200 microm) stops the respiratory rhythm, but that adding 2 mum substance P restarts it. We conclude that I(NaP) and I(CAN) enhance neuronal excitability and promote rhythmogenesis, even if their magnitude is insufficient to support bursting-pacemaker activity in individual neurons. When I(NaP) and I(CAN) are removed pharmacologically, the rhythm can be maintained by boosting neural excitability, which is inconsistent with a pacemaker-essential mechanism of respiratory rhythmogenesis by the preBotzinger complex.

Methyl tetra-O-acetyl-α-D-glucopyranuronate: crystal structure and influence on the crystallisation of the ß anomer.

Methyl tetra-O-acetyl-ß-D-glucopyranuronate (1) and methyl tetra-O-acetyl-α-D-glucopyranuronate (3) were isolated as crystalline solids and their crystal structures were obtained. That of the ß anomer (1) was the same as that reported by Root et al., while anomer (3) was found to crystallise in the orthorhombic space group P212121 with two independent molecules in the asymmetric unit. No other crystal forms were found for either compound upon recrystallisation from a range of solvents. The α anomer (3) was found to be an impurity in initially precipitated batches of ß-anomer (1) in quantities <3%; however, it was possible to remove the α impurity either by recrystallisation or by efficient washing, i.e. the α anomer is not incorporated inside the ß anomer crystals. The ß anomer (1) was found to grow as prisms or needles elongated in the a crystallographic direction in the absence of the α impurity, while the presence of the α anomer (3) enhanced this elongation.

Transient Suppression of Dbx1 PreBötzinger Interneurons Disrupts Breathing in Adult Mice.

Interneurons derived from Dbx1-expressing precursors located in the brainstem preBötzinger complex (preBötC) putatively form the core oscillator for inspiratory breathing movements. We tested this Dbx1 core hypothesis by expressing archaerhodopsin in Dbx1-derived interneurons and then transiently hyperpolarizing these neurons while measuring respiratory rhythm in vitro or breathing in vagus-intact adult mice. Transient illumination of the preBötC interrupted inspiratory rhythm in both slice preparations and sedated mice. In awake mice, light application reduced breathing frequency and prolonged the inspiratory duration. Support for the Dbx1 core hypothesis previously came from embryonic and perinatal mouse experiments, but these data suggest that Dbx1-derived preBötC interneurons are rhythmogenic in adult mice too. The neural origins of breathing behavior can be attributed to a localized and genetically well-defined interneuron population.

Mechanisms Leading to Rhythm Cessation in the Respiratory PreBötzinger Complex Due to Piecewise Cumulative Neuronal Deletions

The mammalian breathing rhythm putatively originates from Dbx1-derived interneurons in the preBötzinger complex (preBötC) of the ventral medulla. Cumulative deletion of ∼15% of Dbx1 preBötC neurons in an in vitro breathing model stops rhythmic bursts of respiratory-related motor output. Here we assemble in silico models of preBötC networks using random graphs for structure, and ordinary differential equations for dynamics, to examine the mechanisms responsible for the loss of spontaneous respiratory rhythm and motor output measured experimentally in vitro. Model networks subjected to cellular ablations similarly discontinue functionality. However, our analyses indicate that model preBötC networks remain topologically intact even after rhythm cessation, suggesting that dynamics coupled with structural properties of the underlying network are responsible for rhythm cessation. Simulations show that cumulative cellular ablations diminish the number of neurons that can be recruited to spike per unit time. When the recruitment rate drops below 1 neuron/ms the network stops spontaneous rhythmic activity. Neurons that play pre-eminent roles in rhythmogenesis include those that commence spiking during the quiescent phase between respiratory bursts and those with a high number of incoming synapses, which both play key roles in recruitment, i.e., recurrent excitation leading to network bursts. Selectively ablating neurons with many incoming synapses impairs recurrent excitation and stops spontaneous rhythmic activity and motor output with lower ablation tallies compared with random deletions. This study provides a theoretical framework for the operating mechanism of mammalian central pattern generator networks and their susceptibility to loss-of-function in the case of disease or neurodegeneration.

The retrotrapezoid nucleus neurons expressing Atoh1 and Phox2b are essential for the respiratory response to CO2.

Maintaining constant CO2 and H(+) concentrations in the arterial blood is critical for life. The principal mechanism through which this is achieved in mammals is the respiratory chemoreflex whose circuitry is still elusive. A candidate element of this circuitry is the retrotrapezoid nucleus (RTN), a collection of neurons at the ventral medullary surface that are activated by increased CO2 or low pH and project to the respiratory rhythm generator. Here, we use intersectional genetic strategies to lesion the RTN neurons defined by Atoh1 and Phox2b expression and to block or activate their synaptic output. Photostimulation of these neurons entrains the respiratory rhythm. Conversely, abrogating expression of Atoh1 or Phox2b or glutamatergic transmission in these cells curtails the phrenic nerve response to low pH in embryonic preparations and abolishes the respiratory chemoreflex in behaving animals. Thus, the RTN neurons expressing Atoh1 and Phox2b are a necessary component of the chemoreflex circuitry.

Supramolecular stacking motifs in the solid state of amide and triazole derivatives of cellobiose.

1-Acetamido-1-deoxy-(4-O-ß-d-glucopyranosyl-ß-d-glucopyranose) (5) and 1-deoxy-1-(4-phenyl-1,2,3-triazolyl)-(4-O-ß-d-glucopyranosyl-ß-d-glucopyranose) (7) were synthesised from 1-azido-1-deoxy-(4-O-ß-d-glucopyranosyl-ß-d-glucopyranose) (2) and crystallised as dihydrates. Crystal structural analysis of 5·2H2O displayed an acetamide C(4) chain and stacked cellobiose residues. The structure of 7·2H2O featured π-π stacking and stacking of the cellobiose residues.