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 preBotzinger 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 in embryonic mice. 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

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

AbstractCisplatin-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 cisplatin's 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.</jats:p

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 A(1) receptor (A(1)AR) 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 A(1)AR 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 Ser(727) (but not Tyr(701)) 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 A(1)AR 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 A(1) receptor (A(1)AR) 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 A(1)AR 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.02265.00

Developmental Origin of PreBotzinger 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

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

Molecular Substrates And Neuronal Network Subserving Audiogenic Kindling In Genetically Epilepsy-Prone Rats

Genetically epilepsy-prone rats of the severe seizure strain (GEPR-9s) are a model of generalized tonic-clonic seizures. GEPR-9s exhibit inherited susceptibility to audiogenic seizures (AGS) in response to intense acoustic stimulation. Several lines of evidence indicate that the neuronal network of AGS is primarily localized in the brainstem in GEPR-9s. The audiogenic seizure kindling (AGS kindling) process involves periodically repeated induction of AGS. In GEPR-9s induction of 14 or more AGS over 1-2 week period leads to a gradual increase in seizure duration, cortical epileptiform EEG, and the emergence of post-tonic clonus (PTC), which are persistent features of subsequent AGS. AGS kindling results in the expansion of the requisite brainstem seizure network to recruit forebrain areas, particularly the amygdala, into the expanded network. The auditory pathway projects acoustic information from the primary auditory pathway to the lateral nucleus of amygdala (LA). The LA is important in mediating the changes in seizure behavior in AGS kindled GEPR-9s, which involves NMDA receptor activation. The persistence of AGS kindling appears to be dependent on the molecular mechanisms initiated by NMDA receptor activation in LA, which may involve adenylyl cyclase (AC). Therefore, I studied the ability of AC activation in LA to mimic AGS kindling in GEPR-9s. We performed a one-time focal bilateral microinjection of an AC activator, MPB forskolin {7-Deacetyl-7-[O-(N-methylpiperazino)-γ-butyryl] - forskolin dihydrochloride} (25-100 pmol/side) into the LA of non-kindled GEPR-9s. The one-time microinjection of MPB forskolin in GEPR-9s, across all the doses at 24 hr, precipitously induced an AGS kindling-like effect, which involved significant increases in seizure duration and long-lasting susceptibility to AGS that culminated in PTC. To further examine the role of AC in the emergence of PTC we performed bilateral focal microinjections of an AC inhibitor, SQ 22,356 (0.25-0.5 nmol/side), in AGS kindled GEPR-9s. Microinjection of SQ 22,536 significantly reduced PTC in AGS kindled GEPR-9s without blocking AGS. These data suggest that AC activation in the LA is a critical step in the NMDA receptor activation-dependent molecular cascade, mediating the appearance and persistence of AGS that culminate in PTC, which is induced by AGS kindling. The LA projects to the major amygdala output nucleus, the central nucleus of amygdala (CeA), which in turn has major projections to the periaqueductal gray (PAG). Previous studies have demonstrated the existence of projections from the CeA to the ventrolateral nucleus of periaqueductal gray (PAG). In GEPR-9s, the PAG, is known to play a critical role in the propagation of AGS. Therefore, extracellular single unit action potentials of PAG neurons were studied in awake behaving GEPR-9s in response to electrical stimulation in the CeA, acoustic stimulation, and during AGS. In response to electrical stimulation in the CeA a significantly greater increase in neuronal firing in PAG was seen in AGS kindled GEPR-9s as compared to non-kindled GEPR-9s. PAG neurons in AGS kindled GEPR-9s showed a precipitous increase in responses to CeA stimulation. The PAG neurons also displayed significantly increased responsiveness to acoustic stimulation as a result of AGS kindling in GEPR-9s. These PAG neurons displayed tonic pattern of activity during wild running and tonic hind-limb extension phases of AGS in both non-kindled and AGS kindled GEPR-9s. The tonic firing pattern in PAG transformed into a burst firing pattern coinciding temporally with the PTC behavior in kindled GEPR-9s that is absent in non-kindled GEPR-9s. The neuronal firing pattern in PAG is similar to that previously observed in the LA. Burst patterns of neuronal activity are known to be associated with increased synaptic efficiency and are indicative of neuroplasticity associated with AGS kindling. Gabapentin is an anticonvulsant drug that is also effective in treating non-epileptic conditions such as anxiety disorders, ethanol withdrawal and neuropathic pain. The pathway connecting CeA to PAG has been implicated in the networks subserving these conditions. Gabapentin reversed the increase in single unit firing seen in PAG of AGS kindled GEPR-9s in response to electrical stimulation in CeA at a dose (50 mg/kg) that significantly reduced the incidence of PTC during AGS. Therefore, the effectiveness of gabapentin in the treatment of the above-mentioned CNS conditions may also be due, in part, to its effect on the pathway from CeA to PAG. Focal bilateral microinjection of NMDA (2.5 nmol/side) into the PAG of non-kindled GEPR-9s did not induce susceptibility to AGS, culminating in PTC. Since previous studies found that microinjection of NMDA into the LA initiated transient susceptibility to PTC during AGS in non-kindled GEPR-9s, the present data suggest that, unlike the LA, neuroplasticity in the PAG is not likely to mediate AGS kindling in GEPR-9s. Taken together, the results of the present studies suggest that activation of AC within the molecular cascade initiated by NMDA receptor-activation in the LA may be an important epileptogenic mechanism that subserves AGS kindling in GEPR-9s. The PAG, which undergoes changes associated with AGS kindling, may serve as the brainstem site for re-entry of epileptic activity from the amygdala in kindled GEPR-9s. However, the amygdala may be more greatly involved than the PAG in the neuroplastic mechanisms subserving AGS kindling

TRPV1: A Common Denominator Mediating Antinociceptive and Antiemetic Effects of Cannabinoids

The most common medicinal claims for cannabis are relief from chronic pain, stimulation of appetite, and as an antiemetic. However, the mechanisms by which cannabis reduces pain and prevents nausea and vomiting are not fully understood. Among more than 450 constituents in cannabis, the most abundant cannabinoids are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Cannabinoids either directly or indirectly modulate ion channel function. Transient receptor potential vanilloid 1 (TRPV1) is an ion channel responsible for mediating several modalities of pain, and it is expressed in both the peripheral and the central pain pathways. Activation of TRPV1 in sensory neurons mediates nociception in the ascending pain pathway, while activation of TRPV1 in the central descending pain pathway, which involves the rostral ventral medulla (RVM) and the periaqueductal gray (PAG), mediates antinociception. TRPV1 channels are thought to be implicated in neuropathic/spontaneous pain perception in the setting of impaired descending antinociceptive control. Activation of TRPV1 also can cause the release of calcitonin gene-related peptide (CGRP) and other neuropeptides/neurotransmitters from the peripheral and central nerve terminals, including the vagal nerve terminal innervating the gut that forms central synapses at the nucleus tractus solitarius (NTS). One of the adverse effects of chronic cannabis use is the paradoxical cannabis-induced hyperemesis syndrome (HES), which is becoming more common, perhaps due to the wider availability of cannabis-containing products and the chronic use of products containing higher levels of cannabinoids. Although, the mechanism of HES is unknown, the effective treatment options include hot-water hydrotherapy and the topical application of capsaicin, both activate TRPV1 channels and may involve the vagal-NTS and area postrema (AP) nausea and vomiting pathway. In this review, we will delineate the activation of TRPV1 by cannabinoids and their role in the antinociceptive/nociceptive and antiemetic/emetic effects involving the peripheral, spinal, and supraspinal structures

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.11 page(s

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 Dbx1 derived neurons heterogeneously co-express 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 preBötC core neurons represent a subset of a larger, more heterogeneous population of VLM Dbx1 derived neurons. These data indicate 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