The breath shape controls intonation of mouse vocalizations.

Intonation in speech is the control of vocal pitch to layer expressive meaning to communication, like increasing pitch to indicate a question. Also, stereotyped patterns of pitch are used to create distinct sounds with different denotations, like in tonal languages and, perhaps, the 10 sounds in the murine lexicon. A basic tone is created by exhalation through a constricted laryngeal voice box, and it is thought that more complex utterances are produced solely by dynamic changes in laryngeal tension. But perhaps, the shifting pitch also results from altering the swiftness of exhalation. Consistent with the latter model, we describe that intonation in most vocalization types follows deviations in exhalation that appear to be generated by the re-activation of the cardinal breathing muscle for inspiration. We also show that the brainstem vocalization central pattern generator, the iRO, can create this breath pattern. Consequently, ectopic activation of the iRO not only induces phonation, but also the pitch patterns that compose most of the vocalizations in the murine lexicon. These results reveal a novel brainstem mechanism for intonation.

The breath shape controls intonation of mouse vocalizations.

Intonation in speech is the control of vocal pitch to layer expressive meaning to communication, like increasing pitch to indicate a question. Also, stereotyped patterns of pitch are used to create distinct "words", like the ten sounds in the murine lexicon. A basic tone is created by exhalation through a constricted laryngeal voice box, and it is thought that more complex utterances are produced solely by dynamic changes in laryngeal tension. But perhaps, the shifting pitch also results from altering the power of exhalation. Consistent with the latter model, we describe that intonation in many adult murine vocalizations follows deviations in exhalation and that the brainstem vocalization central pattern generator, the iRO, can create this breath pattern. Consequently, ectopic activation of the iRO not only induces phonation, but also the pitch patterns that compose most of the vocalizations in the murine lexicon. These results reveal a novel brainstem mechanism for intonation.

Transformation of Our Understanding of Breathing Control by Molecular Tools.

The rhythmicity of breath is vital for normal physiology. Even so, breathing is enriched with multifunctionality. External signals constantly change breathing, stopping it when under water or deepening it during exertion. Internal cues utilize breath to express emotions such as sighs of frustration and yawns of boredom. Breathing harmonizes with other actions that use our mouth and throat, including speech, chewing, and swallowing. In addition, our perception of breathing intensity can dictate how we feel, such as during the slow breathing of calming meditation and anxiety-inducing hyperventilation. Heartbeat originates from a peripheral pacemaker in the heart, but the automation of breathing arises from neural clusters within the brainstem, enabling interaction with other brain areas and thus multifunctionality. Here, we document how the recent transformation of cellular and molecular tools has contributed to our appreciation of the diversity of neuronal types in the breathing control circuit and how they confer the multifunctionality of breathing.

A novel reticular node in the brainstem synchronizes neonatal mouse crying with breathing.

Human speech can be divided into short, rhythmically timed elements, similar to syllables within words. Even our cries and laughs, as well as the vocalizations of other species, are periodic. However, the cellular and molecular mechanisms underlying the tempo of mammalian vocalizations remain unknown. Furthermore, even the core cells that produce vocalizations remain ill-defined. Here, we describe rhythmically timed neonatal mouse vocalizations that occur within single breaths and identify a brainstem node that is necessary for and sufficient to structure these cries, which we name the intermediate reticular oscillator (iRO). We show that the iRO acts autonomously and sends direct inputs to key muscles and the respiratory rhythm generator in order to coordinate neonatal vocalizations with breathing, as well as paces and patterns these cries. These results reveal that a novel mammalian brainstem oscillator embedded within the conserved breathing circuitry plays a central role in the production of neonatal vocalizations.

ß-arrestin 2 germline knockout does not attenuate opioid respiratory depression.

Opioids are perhaps the most effective analgesics in medicine. However, between 1999 and 2018, over 400,000 people in the United States died from opioid overdose. Excessive opioids make breathing lethally slow and shallow, a side-effect called opioid-induced respiratory depression. This doubled-edged sword has sparked the desire to develop novel therapeutics that provide opioid-like analgesia without depressing breathing. One such approach has been the design of so-called 'biased agonists' that signal through some, but not all pathways downstream of the µ-opioid receptor (MOR), the target of morphine and other opioid analgesics. This rationale stems from a study suggesting that MOR-induced ß-arrestin 2 dependent signaling is responsible for opioid respiratory depression, whereas adenylyl cyclase inhibition produces analgesia. To verify this important result that motivated the 'biased agonist' approach, we re-examined breathing in ß-arrestin 2-deficient mice and instead find no connection between ß-arrestin 2 and opioid respiratory depression. This result suggests that any attenuated effect of 'biased agonists' on breathing is through an as-yet defined mechanism.

Opioid drugs are commonly prescribed due to their powerful painkilling properties. However, when misused, these compounds can cause breathing to become dangerously slow and shallow: between 1999 and 2018, over 400,000 people died from opioid drug overdoses in the United States alone. Exactly how the drugs affect breathing remains unclear. What is known is that opioids work by binding to specific receptors at the surface of cells, an event which has a ripple effect on many biochemical pathways. Amongst these, research published in 2005 identified the ß-arrestin 2 pathway as being responsible for altering breathing. This spurred efforts to find opioid-like drugs that would not interfere with the pathway, retaining their ability relieve pain but without affecting breathing. However, new evidence is now shedding doubt on the conclusions of this study. In response, Bachmutsky, Wei et al. attempted to replicate the original 2005 findings. Mice with carefully controlled genetic background were used, in which the genes for the ß-arrestin 2 pathway were either present or absent. Both groups of animals had similar breathing patterns under normal conditions and after receiving an opioid drug. The results suggest ß-arrestin 2 is not involved in opioid-induced breathing suppression. These findings demonstrate that research to develop opioid-like drugs that do not affect the ß-arrestin 2 pathway are based on a false premise. Precisely targeting a drug's molecular mechanisms to avoid suppressing breathing may still be a valid approach, but more research is needed to identify the right pathways.

Opioids depress breathing through two small brainstem sites.

The rates of opioid overdose in the United States quadrupled between 1999 and 2017, reaching a staggering 130 deaths per day. This health epidemic demands innovative solutions that require uncovering the key brain areas and cell types mediating the cause of overdose- opioid-induced respiratory depression. Here, we identify two primary changes to murine breathing after administering opioids. These changes implicate the brainstem's breathing circuitry which we confirm by locally eliminating the µ-Opioid receptor. We find the critical brain site is the preBötzinger Complex, where the breathing rhythm originates, and use genetic tools to reveal that just 70-140 neurons in this region are responsible for its sensitivity to opioids. Future characterization of these neurons may lead to novel therapies that prevent respiratory depression while sparing analgesia.

Opioids such as morphine or fentanyl are powerful substances used to relieve pain in medical settings. However, taken in too high a dose they can depress breathing ­ in other words, they can lead to slow, shallow breaths that cannot sustain life. In the United States, where the misuse of these drugs has been soaring in the past decades, about 130 people die each day from opioid overdose. Pinpointing the exact brain areas and neurons that opioids act on to depress breathing could help to create safer painkillers that do not have this deadly effect. While previous studies have proposed several brain regions that could be involved, they have not been able to confirm these results, or determine which area plays the biggest role. Opioids influence the brain of animals (including humans) by attaching to proteins known as opioid receptors that are present at the surface of neurons. Here, Bachmutsky et al. genetically engineered mice that lack these receptors in specific brain regions that control breathing. The animals were then exposed to opioids, and their breathing was closely monitored. The experiments showed that two small brain areas were responsible for breathing becoming depressed under the influence of opioids. The region with the most critical impact also happens to be where the breathing rhythms originate. There, a small group of 50 to 140 neurons were used by opioids to depress breathing. Crucially, these cells were not necessary for the drugs' ability to relieve pain. Overall, the work by Bachmutsky et al. highlights a group of neurons whose role in creating breathing rhythms deserves further attention. It also opens the possibility that targeting these neurons would help to create safer painkillers.

Neuronal heterogeneity and stereotyped connectivity in the auditory afferent system.

Spiral ganglion (SG) neurons of the cochlea convey all auditory inputs to the brain, yet the cellular and molecular complexity necessary to decode the various acoustic features in the SG has remained unresolved. Using single-cell RNA sequencing, we identify four types of SG neurons, including three novel subclasses of type I neurons and the type II neurons, and provide a comprehensive genetic framework that define their potential synaptic communication patterns. The connectivity patterns of the three subclasses of type I neurons with inner hair cells and their electrophysiological profiles suggest that they represent the intensity-coding properties of auditory afferents. Moreover, neuron type specification is already established at birth, indicating a neuronal diversification process independent of neuronal activity. Thus, this work provides a transcriptional catalog of neuron types in the cochlea, which serves as a valuable resource for dissecting cell-type-specific functions of dedicated afferents in auditory perception and in hearing disorders.

Breathing control center neurons that promote arousal in mice.

Slow, controlled breathing has been used for centuries to promote mental calming, and it is used clinically to suppress excessive arousal such as panic attacks. However, the physiological and neural basis of the relationship between breathing and higher-order brain activity is unknown. We found a neuronal subpopulation in the mouse preBötzinger complex (preBötC), the primary breathing rhythm generator, which regulates the balance between calm and arousal behaviors. Conditional, bilateral genetic ablation of the ~175 Cdh9/Dbx1 double-positive preBötC neurons in adult mice left breathing intact but increased calm behaviors and decreased time in aroused states. These neurons project to, synapse on, and positively regulate noradrenergic neurons in the locus coeruleus, a brain center implicated in attention, arousal, and panic that projects throughout the brain.

Sighing.

A quick guide on sighing, a kind of breath with important physiological functions that is also used for emotional communication.

The peptidergic control circuit for sighing.

Sighs are long, deep breaths expressing sadness, relief or exhaustion. Sighs also occur spontaneously every few minutes to reinflate alveoli, and sighing increases under hypoxia, stress, and certain psychiatric conditions. Here we use molecular, genetic, and pharmacologic approaches to identify a peptidergic sigh control circuit in murine brain. Small neural subpopulations in a key breathing control centre, the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), express bombesin-like neuropeptide genes neuromedin B (Nmb) or gastrin-releasing peptide (Grp). These project to the preBötzinger Complex (preBötC), the respiratory rhythm generator, which expresses NMB and GRP receptors in overlapping subsets of ~200 neurons. Introducing either neuropeptide into preBötC or onto preBötC slices, induced sighing or in vitro sigh activity, whereas elimination or inhibition of either receptor reduced basal sighing, and inhibition of both abolished it. Ablating receptor-expressing neurons eliminated basal and hypoxia-induced sighing, but left breathing otherwise intact initially. We propose that these overlapping peptidergic pathways comprise the core of a sigh control circuit that integrates physiological and perhaps emotional input to transform normal breaths into sighs.

G-TRACE: rapid Gal4-based cell lineage analysis in Drosophila.

We combined Gal4-UAS and the FLP recombinase-FRT and fluorescent reporters to generate cell clones that provide spatial, temporal and genetic information about the origins of individual cells in Drosophila melanogaster. We named this combination the Gal4 technique for real-time and clonal expression (G-TRACE). The approach should allow for screening and the identification of real-time and lineage-traced expression patterns on a genomic scale.

Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes.

Using a large consortium of undergraduate students in an organized program at the University of California, Los Angeles (UCLA), we have undertaken a functional genomic screen in the Drosophila eye. In addition to the educational value of discovery-based learning, this article presents the first comprehensive genomewide analysis of essential genes involved in eye development. The data reveal the surprising result that the X chromosome has almost twice the frequency of essential genes involved in eye development as that found on the autosomes.

An efficient genetic screen in Drosophila to identify nuclear-encoded genes with mitochondrial function.

We conducted a screen for glossy-eye flies that fail to incorporate BrdU in the third larval instar eye disc but exhibit normal neuronal differentiation and isolated 23 complementation groups of mutants. These same phenotypes were previously seen in mutants for cytochrome c oxidase subunit Va. We have molecularly characterized six complementation groups and, surprisingly, each encodes a mitochondrial protein. Therefore, we believe our screen to be an efficient method for identifying genes with mitochondrial function.