Organization of the core respiratory network: Insights from optogenetic and modeling studies.

The circuit organization within the mammalian brainstem respiratory network, specifically within and between the pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes, and the roles of these circuits in respiratory pattern generation are continuously debated. We address these issues with a combination of optogenetic experiments and modeling studies. We used transgenic mice expressing channelrhodopsin-2 under the VGAT-promoter to investigate perturbations of respiratory circuit activity by site-specific photostimulation of inhibitory neurons within the pre-BötC or BötC. The stimulation effects were dependent on the intensity and phase of the photostimulation. Specifically: (1) Low intensity (≤ 1.0 mW) pulses delivered to the pre-BötC during inspiration did not terminate activity, whereas stronger stimulations (≥ 2.0 mW) terminated inspiration. (2) When the pre-BötC stimulation ended in or was applied during expiration, rebound activation of inspiration occurred after a fixed latency. (3) Relatively weak sustained stimulation (20 Hz, 0.5-2.0 mW) of pre-BötC inhibitory neurons increased respiratory frequency, while a further increase of stimulus intensity (> 3.0 mW) reduced frequency and finally (≥ 5.0 mW) terminated respiratory oscillations. (4) Single pulses (0.2-5.0 s) applied to the BötC inhibited rhythmic activity for the duration of the stimulation. (5) Sustained stimulation (20 Hz, 0.5-3.0 mW) of the BötC reduced respiratory frequency and finally led to apnea. We have revised our computational model of pre-BötC and BötC microcircuits by incorporating an additional population of post-inspiratory inhibitory neurons in the pre-BötC that interacts with other neurons in the network. This model was able to reproduce the above experimental findings as well as previously published results of optogenetic activation of pre-BötC or BötC neurons obtained by other laboratories. The proposed organization of pre-BötC and BötC circuits leads to testable predictions about their specific roles in respiratory pattern generation and provides important insights into key circuit interactions operating within brainstem respiratory networks.

Structural-functional properties of identified excitatory and inhibitory interneurons within pre-Botzinger complex respiratory microcircuits.

We comparatively analyzed cellular and circuit properties of identified rhythmic excitatory and inhibitory interneurons within respiratory microcircuits of the neonatal rodent pre-Bötzinger complex (pre-BötC), the structure generating inspiratory rhythm in the brainstem. We combined high-resolution structural-functional imaging, molecular assays for neurotransmitter phenotype identification in conjunction with electrophysiological property phenotyping, and morphological reconstruction of interneurons in neonatal rat and mouse slices in vitro. This approach revealed previously undifferentiated structural-functional features that distinguish excitatory and inhibitory interneuronal populations. We identified distinct subpopulations of pre-BötC glutamatergic, glycinergic, GABAergic, and glycine-GABA coexpressing interneurons. Most commissural pre-BötC inspiratory interneurons were glutamatergic, with a substantial subset exhibiting intrinsic oscillatory bursting properties. Commissural excitatory interneurons projected with nearly planar trajectories to the contralateral pre-BötC, many also with axon collaterals to areas containing inspiratory hypoglossal (XII) premotoneurons and motoneurons. Inhibitory neurons as characterized in the present study did not exhibit intrinsic oscillatory bursting properties, but were electrophysiologically distinguished by more pronounced spike frequency adaptation properties. Axons of many inhibitory neurons projected ipsilaterally also to regions containing inspiratory XII premotoneurons and motoneurons, whereas a minority of inhibitory neurons had commissural axonal projections. Dendrites of both excitatory and inhibitory interneurons were arborized asymmetrically, primarily in the coronal plane. The dendritic fields of inhibitory neurons were more spatially compact than those of excitatory interneurons. Our results are consistent with the concepts of a compartmental circuit organization, a bilaterally coupled excitatory rhythmogenic kernel, and a role of pre-BötC inhibitory neurons in shaping inspiratory pattern as well as coordinating inspiratory and expiratory activity.

Yushaniadezhui (Poaceae, Bambusoideae), a new bamboo species from Yunnan, China.

A new bamboo species, Yushaniadezhui, from Kunming, Yunnan, China is described and illustrated in this paper. The new species used to be misidentified as Y.polytricha. Based on careful comparison of morphological features and molecular phylogeny evidence, we confirmed its identity as a new member of the genus Yushania. Yushaniadezhui resembles Y.maculata, Y.polytricha and Y.weixiensis in several aspects, such as culm height and branch complement structure. However, the glabrous culm leaf sheaths and internodes, the absence of auricles and oral setae on most foliage leaves, except the one-year-old foliage leaves, the pubescence on the adaxial surface of the one-year-old foliage leaves and its limestone habitat preference can readily distinguish this new species from its related taxa. Moreover, we emphasise that individuals from various populations and molecular markers with different inheritance patterns for phylogeny reconstruction should be included in new species discovery, especially in plant groups with complex evolutionary histories.

Morphological deficits of glial cells in a transgenic mouse model for developmental stuttering.

Vocal production involves intricate neural coordination across various brain regions. Stuttering, a common speech disorder, has genetic underpinnings, including mutations in lysosomal-targeting pathway genes. Using a Gnptab-mutant mouse model linked to stuttering, we examined neuron and glial cell morphology in vocal production circuits. Our findings revealed altered astrocyte and microglia processes in these circuits in Gnptab-mutant mice, while control regions remained unaffected. Our results shed light on the potential role of glial cells in stuttering pathophysiology and highlight their relevance in modulating vocal production behaviors.

Inference technique for the synaptic conductances in rhythmically active networks and application to respiratory central pattern generation circuits.

Unraveling synaptic interactions between excitatory and inhibitory interneurons within rhythmic neural circuits, such as central pattern generation (CPG) circuits for rhythmic motor behaviors, is critical for deciphering circuit interactions and functional architecture, which is a major problem for understanding how neural circuits operate. Here we present a general method for extracting and separating patterns of inhibitory and excitatory synaptic conductances at high temporal resolution from single neuronal intracellular recordings in rhythmically active networks. These post-synaptic conductances reflect the combined synaptic inputs from the key interacting neuronal populations and can reveal the functional connectome of the active circuits. To illustrate the applicability of our analytic technique, we employ our method to infer the synaptic conductance profiles in identified rhythmically active interneurons within key microcircuits of the mammalian (mature rat) brainstem respiratory CPG and provide a perspective on how our approach can resolve the functional interactions and circuit organization of these interneuron populations. We demonstrate the versatility of our approach, which can be applied to any other rhythmic circuits where conditions allow for neuronal intracellular recordings.

The complete plastid genome of the endangered shrub Brassaiopsis angustifolia (Araliaceae): Comparative genetic and phylogenetic analysis.

Brassaiopsis angustifolia K.M. Feng belongs to the family Araliaceae, and is an endangered shrub species in southwest China. Despite the importance of this species, the plastid genome has not been sequenced and analyzed. In this study, the complete plastid genome of B. angustifolia was sequenced, analyzed, and compared to the eight species in the Araliaceae family. Our study reveals that the complete plastid genome of B. angustifolia is 156,534 bp long, with an overall GC content of 37.9%. The chloroplast genome (cp) encodes 133 genes, including 88 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. All protein-coding genes consisted of 21,582 codons. Among the nine species of Araliaceae, simple sequence repeats (SSRs) and five large repeat sequences were identified with total numbers ranging from 37 to 46 and 66 to 78, respectively. Five highly divergent regions were successfully identified that could be used as potential genetic markers of Brassaiopsis and Asian Palmate group. Phylogenetic analysis of 47 plastomes, representing 19 genera of Araliaceae and two related families, was performed to reconstruct highly supported relationships for the Araliaceae, which highlight four well-supported clades of the Hydrocotyle group, Greater Raukaua group, Aralia-Panax group, and Asian Palmate group. The genus Brassaiopsis can be divided into four groups using internal transcribed spacer (ITS) data. The results indicate that plastome and ITS data can contribute to investigations of the taxonomy, and phylogeny of B. angustifolia. This study provides a theoretical basis for species identification and future biological research on resources of the genus Brassaiopsis.

TASK channels contribute to the K+-dominated leak current regulating respiratory rhythm generation in vitro.

Leak channels regulate neuronal activity and excitability. Determining which leak channels exist in neurons and how they control electrophysiological behavior is fundamental. Here we investigated TASK channels, members of the two-pore domain K(+) channel family, as a component of the K(+)-dominated leak conductance that controls and modulates rhythm generation at cellular and network levels in the mammalian pre-Bötzinger complex (pre-BötC), an excitatory network of neurons in the medulla critically involved in respiratory rhythmogenesis. By voltage-clamp analyses of pre-BötC neuronal current-voltage (I-V) relations in neonatal rat medullary slices in vitro, we demonstrated that pre-BötC inspiratory neurons have a weakly outward-rectifying total leak conductance with reversal potential that was depolarized by approximately 4 mV from the K(+) equilibrium potential, indicating that background K(+) channels are dominant contributors to leak. This K(+) channel component had I-V relations described by constant field theory, and the conductance was reduced by acid and was augmented by the volatile anesthetic halothane, which are all hallmarks of TASK. We established by single-cell RT-PCR that pre-BötC inspiratory neurons express TASK-1 and in some cases also TASK-3 mRNA. Furthermore, acid depolarized and augmented bursting frequency of pre-BötC inspiratory neurons with intrinsic bursting properties. Microinfusion of acidified solutions into the rhythmically active pre-BötC network increased network bursting frequency, halothane decreased bursting frequency, and acid reversed the depressant effects of halothane, consistent with modulation of network activity by TASK channels. We conclude that TASK-like channels play a major functional role in chemosensory modulation of respiratory rhythm generation in the pre-Bötzinger complex in vitro.

Characterization of the complete chloroplast genome of medical plant Curculigo orchioides Gaertn. (Amaryllidaceae).

Curculigo orchioides Gaertn. distributed in subtropical regions of Asia including southern China and India. The plant is used as a traditional medicine in China for the treatment of menorrhagia, osteoporosis, and other gynecological problems. The complete chloroplast genome was reported in this study using the Illumina NovaSeq platform. The whole genome of this species was 157,472 bp in length, with a total GC content of 37.44%. The large single copy (LSC) was 86,507 bp, the small single copy (SSC) was 16,867 bp, and both of the two inverted repeats (IRs) were 27,049 bp, respectively. A total of 132 unique genes were identified, among which are 86 protein-coding genes, 38 tRNA genes and 8 rRNA genes. The phylogenetic analysis revealed that C. orchioides was highly clustered with C. capitulata. Our study will provide useful fundamental data for further phylogenetic and evolutionary studies of C. orchioides.

The complete chloroplast genome sequence of Ficus altissima (Moraceae).

Ficus altissima plays an important role on biodiversity in tropical forests. In this study, the complete chloroplast genome sequence and the genome features of F. altissima were analyzed using the Illumina NovaSeq platform. The whole chloroplast genome sequence of F. altissima is 160,251 including a large single-copy region (LSC, 88,468 bp), a small single-copy region (SSC, 20,009 bp), and a pair of repeat regions (IRs, 25,887 bp, each). Further gene annotation revealed the chloroplast genome contains 124 genes, including 79 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. A total of 82 simple sequence repeats (SSRs) were identified in the chloroplast genome. Phylogenetic development was analyzed based on F. altissima with other species of Moraceae. This information will be useful for study on the evolution and genetic diversity of F. altissima in the future.

Raphé neurons stimulate respiratory circuit activity by multiple mechanisms via endogenously released serotonin and substance P.

Brainstem serotonin (5-HT) neurons modulate activity of many neural circuits in the mammalian brain, but in many cases endogenous mechanisms have not been resolved. Here, we analyzed actions of raphé 5-HT neurons on respiratory network activity including at the level of the pre-Bötzinger complex (pre-BötC) in neonatal rat medullary slices in vitro, and in the more intact nervous system of juvenile rats in arterially perfused brainstem-spinal cord preparations in situ. At basal levels of activity, excitation of the respiratory network via simultaneous release of 5-HT and substance P (SP), acting at 5-HT(2A/2C), 5-HT(4), and/or neurokinin-1 receptors, was required to maintain inspiratory motor output in both the neonatal and juvenile systems. The midline raphé obscurus contained spontaneously active 5-HT neurons, some of which projected to the pre-BötC and hypoglossal motoneurons, colocalized 5-HT and SP, and received reciprocal excitatory connections from the pre-BötC. Experimentally augmenting raphé obscurus activity increased motor output by simultaneously exciting pre-BötC and motor neurons. Biophysical analyses in vitro demonstrated that 5-HT and SP modulated background cation conductances in pre-BötC and motor neurons, including a nonselective cation leak current that contributed to the resting potential, which explains the neuronal depolarization that augmented motor output. Furthermore, we found that 5-HT, but not SP, can transform the electrophysiological phenotype of some pre-BötC neurons to intrinsic bursters, providing 5-HT with an additional role in promoting rhythm generation. We conclude that raphé 5-HT neurons excite key circuit components required for generation of respiratory motor output.

The complete chloroplast genome of Dendrocalamus hamiltonii (Poaceae, Bambuseae).

Dendrocalamus hamiltonii is one of the best bamboo species with bamboo shoots, and has higher economic value. The chloroplast genome is a circular molecule of 139404 bp in length, consisting of a 82938 bp large single copy region (LSC), a 12876 bp small single copy region (SSC), and a pair of inverted repeats region (IRa and IRb: 21795 bp each). The GC content of chloroplast genome is 38.9%. The cp genome contains a total of 133 genes, including 86 protein-coding genes, 8 rRNA genes, and 39 tRNA genes. Moreover, phylogenomic analysis showed that D. hamiltonii and D. brandisii clustered together in one branch.

Morphometric analysis of astrocytes in brainstem respiratory regions.

Astrocytes, the most abundant and structurally complex glial cells of the central nervous system, are proposed to play an important role in modulating the activities of neuronal networks, including respiratory rhythm-generating circuits of the preBötzinger complex (preBötC) located in the ventrolateral medulla of the brainstem. However, structural properties of astrocytes residing within different brainstem regions are unknown. In this study astrocytes in the preBötC, an intermediate reticular formation (IRF) region with respiratory-related function, and a region of the nucleus tractus solitarius (NTS) in adult rats were reconstructed and their morphological features were compared. Detailed morphological analysis revealed that preBötC astrocytes are structurally more complex than those residing within the functionally distinct neighboring IRF region, or the NTS, located at the dorsal aspect of the medulla oblongata. Structural analyses of the brainstem microvasculature indicated no significant regional differences in vascular properties. We hypothesize that high morphological complexity of preBötC astrocytes reflects their functional role in providing structural/metabolic support and modulation of the key neuronal circuits essential for breathing, as well as constraints imposed by arrangements of associated neurons and/or other local structural features of the brainstem parenchyma.

Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits.

Transient receptor potential channel, TRPM4, the putative molecular substrate for Ca2+-activated nonselective cation current (ICAN), is hypothesized to generate bursting activity of pre-Bötzinger complex (pre-BötC) inspiratory neurons and critically contribute to respiratory rhythmogenesis. Another TRP channel, TRPC3, which mediates Na+/Ca2+ fluxes, may be involved in regulating Ca2+-related signaling, including affecting TRPM4/ICAN in respiratory pre-BötC neurons. However, TRPM4 and TRPC3 expression in pre-BötC inspiratory neurons and functional roles of these channels remain to be determined. By single-cell multiplex RT-PCR, we show mRNA expression for these channels in pre-BötC inspiratory neurons in rhythmically active medullary in vitro slices from neonatal rats and mice. Functional contributions were analyzed with pharmacological inhibitors of TRPM4 or TRPC3 in vitro as well as in mature rodent arterially perfused in situ brainstem-spinal cord preparations. Perturbations of respiratory circuit activity were also compared with those by a blocker of ICAN. Pharmacologically attenuating endogenous activation of TRPM4, TRPC3, or ICANin vitro similarly reduced the amplitude of inspiratory motoneuronal activity without significant perturbations of inspiratory frequency or variability of the rhythm. Amplitude perturbations were correlated with reduced inspiratory glutamatergic pre-BötC neuronal activity, monitored by multicellular dynamic calcium imaging in vitro. In more intact circuits in situ, the reduction of pre-BötC and motoneuronal inspiratory activity amplitude was accompanied by reduced post-inspiratory motoneuronal activity, without disruption of rhythm generation. We conclude that endogenously activated TRPM4, which likely mediates ICAN, and TRPC3 channels in pre-BötC inspiratory neurons play fundamental roles in respiratory pattern formation but are not critically involved in respiratory rhythm generation.

Voltage-Dependent Rhythmogenic Property of Respiratory Pre-Bötzinger Complex Glutamatergic, Dbx1-Derived, and Somatostatin-Expressing Neuron Populations Revealed by Graded Optogenetic Inhibition.

The rhythm of breathing in mammals, originating within the brainstem pre-Bötzinger complex (pre-BötC), is presumed to be generated by glutamatergic neurons, but this has not been directly demonstrated. Additionally, developmental expression of the transcription factor Dbx1 or expression of the neuropeptide somatostatin (Sst), has been proposed as a marker for the rhythmogenic pre-BötC glutamatergic neurons, but it is unknown whether these other two phenotypically defined neuronal populations are functionally equivalent to glutamatergic neurons with regard to rhythm generation. To address these problems, we comparatively investigated, by optogenetic approaches, the roles of pre-BötC glutamatergic, Dbx1-derived, and Sst-expressing neurons in respiratory rhythm generation in neonatal transgenic mouse medullary slices in vitro and also more intact adult perfused brainstem-spinal cord preparations in situ. We established three different triple-transgenic mouse lines with Cre-driven Archaerhodopsin-3 (Arch) expression selectively in glutamatergic, Dbx1-derived, or Sst-expressing neurons for targeted photoinhibition. In each line, we identified subpopulations of rhythmically active, Arch-expressing pre-BötC inspiratory neurons by whole-cell recordings in medullary slice preparations in vitro, and established that Arch-mediated hyperpolarization of these inspiratory neurons was laser power dependent with equal efficacy. By site- and population-specific graded photoinhibition, we then demonstrated that inspiratory frequency was reduced by each population with the same neuronal voltage-dependent frequency control mechanism in each state of the respiratory network examined. We infer that enough of the rhythmogenic pre-BötC glutamatergic neurons also have the Dbx1 and Sst expression phenotypes, and thus all three phenotypes share the same voltage-dependent frequency control property.

Perturbations of Respiratory Rhythm and Pattern by Disrupting Synaptic Inhibition within Pre-Bötzinger and Bötzinger Complexes.

The pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes are the brainstem compartments containing interneurons considered to be critically involved in generating respiratory rhythm and motor pattern in mammals. Current models postulate that both generation of the rhythm and coordination of the inspiratory-expiratory pattern involve inhibitory synaptic interactions within and between these regions. Both regions contain glycinergic and GABAergic neurons, and rhythmically active neurons in these regions receive appropriately coordinated phasic inhibition necessary for generation of the normal three-phase respiratory pattern. However, recent experiments attempting to disrupt glycinergic and GABAergic postsynaptic inhibition in the pre-BötC and BötC in adult rats in vivo have questioned the critical role of synaptic inhibition in these regions, as well as the importance of the BötC, which contradicts previous physiological and pharmacological studies. To further evaluate the roles of synaptic inhibition and the BötC, we bilaterally microinjected the GABAA receptor antagonist gabazine and glycinergic receptor antagonist strychnine into the pre-BötC or BötC in anesthetized adult rats in vivo and in perfused in situ brainstem-spinal cord preparations from juvenile rats. Muscimol was microinjected to suppress neuronal activity in the pre-BötC or BötC. In both preparations, disrupting inhibition within pre-BötC or BötC caused major site-specific perturbations of the rhythm and disrupted the three-phase motor pattern, in some experiments terminating rhythmic motor output. Suppressing BötC activity also potently disturbed the rhythm and motor pattern. We conclude that inhibitory circuit interactions within and between the pre-BötC and BötC critically regulate rhythmogenesis and are required for normal respiratory motor pattern generation.