Srinivasan Tupal

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.

Atoh1 governs the migration of postmitotic neurons that shape respiratory effectiveness at birth and chemoresponsiveness in adulthood.

Hindbrain neuronal networks serving respiratory, proprioceptive, and arousal functions share a developmental requirement for the bHLH transcription factor Atoh1. Loss of Atoh1 in mice results in respiratory failure and neonatal lethality; however, the neuronal identity and mechanism by which Atoh1-dependent cells sustain newborn breathing remains unknown. We uncovered that selective loss of Atoh1 from the postmitotic retrotrapezoid nucleus (RTN) neurons results in severely impaired inspiratory rhythm and pronounced neonatal death. Mice that escape neonatal death develop abnormal chemoresponsiveness as adults. Interestingly, the expression of Atoh1 in the RTN neurons is not required for their specification or maintenance, but is important for their proper localization and to establish essential connections with the preBötzinger Complex (preBötC). These results provide insights into the genetic regulation of neonatal breathing and shed light on the labile sites that might contribute to sudden death in newborn infants and altered chemoresponsiveness in adults.

Adenosine A1 Receptor Protects Against Cisplatin Ototoxicity by Suppressing the NOX3/STAT1 Inflammatory Pathway in the Cochlea

Cisplatin is a commonly used antineoplastic agent that produces ototoxicity that is mediated in part by increasing levels of reactive oxygen species (ROS) via the NOX3 NADPH oxidase pathway in the cochlea. Recent studies implicate ROS generation in mediating inflammatory and apoptotic processes and hearing loss by activating signal transducer and activator of transcription (STAT1). In this study, we show that the adenosine A1 receptor (A1AR) protects against cisplatin ototoxicity by suppressing an inflammatory response initiated by ROS generation via NOX3 NADPH oxidase, leading to inhibition of STAT1. Trans-tympanic administration of the A1AR agonist R-phenylisopropyladenosine (R-PIA) inhibited cisplatin-induced ototoxicity, as measured by auditory brainstem responses and scanning electron microscopy in male Wistar rats. This was associated with reduced NOX3 expression, STAT1 activation, tumor necrosis factor-α (TNF-α) levels, and apoptosis in the cochlea. In vitro studies in UB/OC-1 cells, an organ of Corti immortalized cell line, showed that R-PIA reduced cisplatin-induced phosphorylation of STAT1 Ser727 (but not Tyr701) and STAT1 luciferase activity by suppressing the ERK1/2, p38, and JNK mitogen-activated protein kinase (MAPK) pathways. R-PIA also decreased the expression of STAT1 target genes, such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced cisplatin-mediated apoptosis. These data suggest that the A1AR provides otoprotection by suppressing NOX3 and inflammation in the cochlea and could serve as an ideal target for otoprotective drug therapy. SIGNIFICANCE STATEMENT Cisplatin is a widely used chemotherapeutic agent for the treatment of solid tumors. Its use results in significant and permanent hearing loss, for which no US Food and Drug Administration-approved treatment is currently available. In this study, we targeted the cochlear adenosine A1 receptor (A1AR) by trans-tympanic injections of the agonist R-phenylisopropyladenosine (R-PIA) and showed that it reduced cisplatin-induced inflammation and apoptosis in the rat cochlea and preserved hearing. The mechanism of protection involves suppression of the NOX3 NADPH oxidase enzyme, a major target of cisplatin-induced reactive oxygen species (ROS) generation in the cochlea. ROS initiates an inflammatory and apoptotic cascade in the cochlea by activating STAT1 transcription factor, which is attenuated by R-PIA. Therefore, trans-tympanic delivery of A1AR agonists could effectively treat cisplatin ototoxicity.

Atoh1-dependent rhombic lip neurons are required for temporal delay between independent respiratory oscillators in embryonic mice

All motor behaviors require precise temporal coordination of different muscle groups. Breathing, for example, involves the sequential activation of numerous muscles hypothesized to be driven by a primary respiratory oscillator, the preBötzinger Complex, and at least one other as-yet unidentified rhythmogenic population. We tested the roles of Atoh1-, Phox2b-, and Dbx1-derived neurons (three groups that have known roles in respiration) in the generation and coordination of respiratory output. We found that Dbx1-derived neurons are necessary for all respiratory behaviors, whereas independent but coupled respiratory rhythms persist from at least three different motor pools after eliminating or silencing Phox2b- or Atoh1-expressing hindbrain neurons. Without Atoh1 neurons, however, the motor pools become temporally disorganized and coupling between independent respiratory oscillators decreases. We propose Atoh1 neurons tune the sequential activation of independent oscillators essential for the fine control of different muscles during breathing. DOI: http://dx.doi.org/10.7554/eLife.02265.001

Dysregulation of locus coeruleus development in congenital central hypoventilation syndrome

Human congenital central hypoventilation syndrome (CCHS), resulting from mutations in transcription factor PHOX2B, manifests with impaired responses to hypoxemia and hypercapnia especially during sleep. To identify brainstem structures developmentally affected in CCHS, we analyzed two postmortem neonatal-lethal cases with confirmed polyalanine repeat expansion (PARM) or Non-PARM (PHOX2B∆8) mutation of PHOX2B. Both human cases showed neuronal losses within the locus coeruleus (LC), which is important for central noradrenergic signaling. Using a conditionally active transgenic mouse model of the PHOX2B∆8 mutation, we found that early embryonic expression (<E10.5) caused failure of LC neuronal specification and perinatal respiratory lethality. In contrast, later onset (E11.5) of PHOX2B∆8 expression was not deleterious to LC development and perinatal respiratory lethality was rescued, despite failure of chemosensor retrotrapezoid nucleus formation. Our findings indicate that early-onset mutant PHOX2B expression inhibits LC neuronal development in CCHS. They further suggest that such mutations result in dysregulation of central noradrenergic signaling, and therefore, potential for early pharmacologic intervention in humans with CCHS.

Precipitous induction of audiogenic kindling by activation of adenylyl cyclase in the amygdala

Purpose:  Kindling of audiogenic seizure (AGS) involves ≥14 AGS over 1–2 weeks in genetically epilepsy‐prone rats (GEPR‐9s) and induces gradual seizure duration increases, epileptiform electroencephalography (EEG), and emergence of post tonic clonus (PTC), which are long‐lasting. N‐methyl‐d‐aspartate (NMDA)–receptor activation in lateral amygdala (LA) is implicated in AGS kindling initiation. However, the persistence of AGS kindling appears to be dependent on molecular mechanisms initiated by NMDA‐receptor activation, which may involve adenylyl cyclase (AC). This study attempted to mimic AGS kindling persistently in nonkindled GEPR‐9s by one‐time activation of AC in LA.

Testing the role of preBötzinger Complex somatostatin neurons in respiratory and vocal behaviors

Identifying neurons essential for the generation of breathing and related behaviors such as vocalisation is an important question for human health. The targeted loss of preBötzinger Complex (preBötC) glutamatergic neurons, including those that express high levels of somatostatin protein (SST neurons), eliminates normal breathing in adult rats. Whether preBötC SST neurons represent a functionally specialised population is unknown. We tested the effects on respiratory and vocal behaviors of eliminating SST neuron glutamate release by Cre‐Lox‐mediated genetic ablation of the vesicular glutamate transporter 2 (VGlut2). We found the targeted loss of VGlut2 in SST neurons had no effect on viability in vivo, or on respiratory period or responses to neurokinin 1 or μ‐opioid receptor agonists in vitro. We then compared medullary SST peptide expression in mice with that of two species that share extreme respiratory environments but produce either high or low frequency vocalisations. In the Mexican free‐tailed bat, SST peptide‐expressing neurons extended beyond the preBötC to the caudal pole of the VII motor nucleus. In the naked mole‐rat, however, SST‐positive neurons were absent from the ventrolateral medulla. We then analysed isolation vocalisations from SST‐Cre;VGlut2F/F mice and found a significant prolongation of the pauses between syllables during vocalisation but no change in vocalisation number. These data suggest that glutamate release from preBötC SST neurons is not essential for breathing but play a species‐ and behavior‐dependent role in modulating respiratory networks. They further suggest that the neural network generating respiration is capable of extensive plasticity given sufficient time.

Epigallocatechin-3-gallate, a prototypic chemopreventative agent for protection against cisplatin-based ototoxicity

Cisplatin-induced ototoxicity is one of the major factors limiting cisplatin chemotherapy. Ototoxicity results from damage to outer hair cells (OHCs) and other regions of the cochlea. At the cellular level, cisplatin increases reactive oxygen species (ROS) leading to cochlear inflammation and apoptosis. Thus, ideal otoprotective drugs should target oxidative stress and inflammatory mechanisms without interfering with cisplatins chemotherapeutic efficacy. In this study, we show that epigallocatechin-3-gallate (EGCG) is a prototypic agent exhibiting these properties of an effect otoprotective agent. Rats administered oral EGCG demonstrate reduced cisplatin-induced hearing loss, reduced loss of OHCs in the basal region of the cochlea and reduced oxidative stress and apoptotic markers. EGCG also protected against the loss of ribbon synapses associated with inner hair cells and Na+/K+ ATPase α1 in the stria vascularis and spiral ligament. In vitro studies showed that EGCG reduced cisplatin-induced ROS generation and ERK1/2 and signal transducer and activator of transcription-1 (STAT1) activity, but preserved the activity of STAT3 and Bcl-xL. The increase in STAT3/STAT1 ratio appears critical for mediating its otoprotection. EGCG did not alter cisplatin-induced apoptosis of human-derived cancer cells or cisplatin antitumor efficacy in a xenograft tumor model in mice because of its inability to rescue the downregulation of STAT3 in these cells. These data suggest that EGCG is an ideal otoprotective agent for treating cisplatin-induced hearing loss without compromising its antitumor efficacy.

Neuronal Network Interactions in the Startle Reflex, Learning Mechanisms, and CNS Disorders, Including Sudden Unexpected Death in Epilepsy

Abstract As discussed in this book, there are many primary neuronal networks that mediate important physiological functions of the central nervous system (CNS), including vision, hearing, locomotion, and respiration. Each of these normal networks can interact with other primary networks to mediate important cross-network functions, and the interaction can be either additive or competitive. An example of an additive or positive form of network interaction is seen in sensorimotor integration, including the visual-motor interactions that mediate eye movements in response to visual stimuli. Primary networks can also interact with nonprimary conditional multireceptive networks, leading to the emergence of more complex phenomena. These phenomena include certain forms of learning as well as CNS disorders, including, for example, those that can lead to death in epilepsy. Neuronal networks can compete with each other to reduce the network function. This occurs, for example, when an acoustic stimulus can induce cessation of locomotion in a learning paradigm such as fear conditioning. A negative interaction is also seen in the prototypical competitive network interaction that forms the basis of the original and conceptually important network interaction hypothesis, the gate control theory of pain. This network competition mechanism is proposed to be a critical mechanism mediating the therapeutic stimulation paradigms currently used to treat many CNS disorders. Network interactions are mediated by (1) direct electrophysiological connections between network neurons that release endogenous neuroactive substances via synaptic transmission and (2) indirect interactions that release these substances via volume transmission. These network interactions can occur essentially instantaneously or sequentially over a finite time course. The interactions can involve two networks, as in the somatosensory-motor network interactions, which occur in the tactile startle response, and are also seen in the interaction of the pain network with the therapeutic network initiated by certain pain-relieving electrical stimulation therapies. The network interactions can be more complex, involving three or more networks, and examples of multinetwork interaction include fear-potentiated startle, seizure-induced analgesia, and audiogenic seizure kindling. An example of such a multinetwork interaction with fatal consequences is proposed to occur in the CNS disorder of epilepsy called sudden unexpected death in epilepsy (SUDEP). The DBA/1 and DBA/2 mouse models of SUDEP involve additive interactions between the auditory and locomotor networks and a subsequent negative interaction with the respiratory network, which results in respiratory arrest. These network interactions may be mediated, in part, by the release of endogenous neuroactive substances that inhibit (e.g. adenosine) or enhance (e.g. serotonin) the activity of the respiratory network via synaptic and/or volume transmission. Thus, investigations of network interactions in normal situations and in CNS disorders may provide the keys to understanding learning mechanisms and also for treating CNS disorders with stimulation and/or pharmacological paradigms.