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.

The role of PHOX2B-derived astrocytes in chemosensory control of breathing and sleep homeostasis.

KEY POINTS: The embryonic PHOX2B-progenitor domain generates neuronal and glial cells which together are involved in chemosensory control of breathing and sleep homeostasis. Ablating PHOX2B-derived astrocytes significantly contributes to secondary hypoxic respiratory depression as well as abnormalities in sleep homeostasis. PHOX2B-derived astrocyte ablation results in axonal pathologies in the retrotrapezoid nucleus. ABSTRACT: We identify in mice a population of ∼800 retrotrapezoid nucleus (RTN) astrocytes derived from PHOX2B-positive, OLIG3-negative progenitor cells, that interact with PHOX2B-expressing RTN chemosensory neurons. PHOX2B-derived astrocyte ablation during early life results in adult-onset O2 chemoreflex deficiency. These animals also display changes in sleep homeostasis, including fragmented sleep and disturbances in delta power after sleep deprivation, all without observable changes in anxiety or social behaviours. Ultrastructural evaluation of the RTN demonstrates that PHOX2B-derived astrocyte ablation results in features characteristic of degenerative neuro-axonal dystrophy, including abnormally dilated axon terminals and increased amounts of synapses containing autophagic vacuoles/phagosomes. We conclude that PHOX2B-derived astrocytes are necessary for maintaining a functional O2 chemosensory reflex in the adult, modulate sleep homeostasis, and are key regulators of synaptic integrity in the RTN region, which is necessary for the chemosensory control of breathing. These data also highlight how defects in embryonic development may manifest as neurodegenerative pathology in an adult.

Fenfluramine, a serotonin-releasing drug, prevents seizure-induced respiratory arrest and is anticonvulsant in the DBA/1 mouse model of SUDEP.

OBJECTIVE: Prevention of sudden unexpected death in epilepsy (SUDEP) is a critical goal for epilepsy therapy. The DBA/1 mouse model of SUDEP exhibits an elevated susceptibility to seizure-induced death in response to electroconvulsive shock, hyperthermia, convulsant drug, and acoustic stimulation. The serotonin hypothesis of SUDEP is based on findings that treatments which modify serotonergic function significantly alter susceptibility to seizure-induced sudden death in several epilepsy models, including DBA/1 mice. Serotonergic abnormalities have also recently been observed in human SUDEP. Fenfluramine is a drug that enhances serotonin release in the brain. Recent studies have found that the addition of fenfluramine improved seizure control in patients with Dravet syndrome, which has a high incidence of SUDEP. Therefore, we investigated the effects of fenfluramine on seizures and seizure-induced respiratory arrest (S-IRA) in DBA/1 mice. METHODS: The dose and time course of the effects of fenfluramine (i.p.) on audiogenic seizures (Sz) induced by an electric bell in DBA/1 mice were determined. Videos of Sz-induced behaviors were recorded for analysis. Statistical significance (P < 0.05) was evaluated using the chi-square test. RESULTS: Sixteen hours after administration of 15 mg/kg of fenfluramine, a high incidence of selective block of S-IRA susceptibility (P < 0.001) occurred in DBA/1 mice without blocking any convulsive behavior. Thirty minutes after 20-40 mg/kg of fenfluramine, significant reductions of seizure incidence and severity, as well as S-IRA susceptibility occurred, which were long-lasting (≥48 hours). The median effective dose (ED50 ) of fenfluramine for significantly reducing Sz at 30 minutes was 21 mg/kg. SIGNIFICANCE: This study presents the first evidence for the effectiveness of fenfluramine in reducing seizure incidence, severity, and S-IRA susceptibility in a mammalian SUDEP model. The ability of fenfluramine to block S-IRA selectively suggests the potential usefulness of fenfluramine in prophylaxis of SUDEP. These results further confirm and extend the serotonin hypothesis of SUDEP.

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 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 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 byR-PIA. Therefore, trans-tympanic delivery of A1AR agonists could effectively treat cisplatin ototoxicity.

Alcohol withdrawal in epileptic rats - Effects on postictal depression, respiration, and death.

Patients with epilepsy are at risk of sudden unexpected death in epilepsy (SUDEP). The most common series of events in witnessed cases of SUDEP is a generalized convulsive seizure followed by terminal apnea. Risk factors for SUDEP include prolonged postictal depression (PID), as well as alcohol abuse. The present study examined these issues in a genetic epilepsy model that exhibits generalized convulsive audiogenic seizures (AGSz) but rarely exhibits seizure-induced death, the genetically epilepsy-prone rats (GEPR-9s). We evaluated the effect of ethanol withdrawal (ETX) in GEPR-9s on respiration patterns, duration of PID, and the incidence of seizure-induced death. Audiogenic seizures were induced in GEPR-9s and in normal Sprague-Dawley rats, which were subjected to a 4-day binge ethanol protocol, 18-24h after the last ethanol dose. Following the tonic seizures, all GEPR-9s exhibited PID, characterized by loss of the righting reflex and respiratory distress (RD), which were absent during ETX seizures in the normal rats. During ETX, GEPR-9s exhibited significant increases in the duration of PID and RD, compared with vehicle-treated GEPR-9s. A significant increase in the incidence of death following seizure in GEPR-9s subjected to ETX was observed, compared with that in vehicle-treated GEPR-9s and normal rats subjected to ETX. Death in GEPR-9s was preceded by prolonged seizures because, in part, of the emergence of post-tonic generalized clonus. These results indicate that ETX induced significant increases in the duration of PID and RD, which contributed to the greater incidence of mortality in GEPR-9s compared with that in vehicle-treated GEPR-9s and normal rats. These experiments observed an elevated risk of sudden death associated with alcohol withdrawal in a genetic epilepsy model that had previously been identified as a risk factor in human SUDEP.

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 (

Novel mutations in ATP1A3 associated with catastrophic early life epilepsy, episodic prolonged apnea, and postnatal microcephaly.

OBJECTIVE: Mutations of ATP1A3 have been associated with rapid onset dystonia-parkinsonism and more recently with alternating hemiplegia of childhood. Here we report one child with catastrophic early life epilepsy and shortened survival, and another with epilepsy, episodic prolonged apnea, postnatal microcephaly, and severe developmental disability. Novel heterozygous mutations (p.Gly358Val and p.Ile363Asn) were identified in ATP1A3 in these children. METHODS: Subjects underwent next-generation sequencing under a research protocol. Clinical data were collected retrospectively. The biochemical effects of the mutations on ATP1A3 protein function were investigated. Postmortem neuropathologic specimens from control and affected subjects were studied. RESULTS: The mutations localized to the P domain of the Na,K-ATPase α3 protein, and resulted in significant reduction of Na,K-ATPase activity in vitro. We demonstrate in both control human brain tissue and that from the subject with the p.Gly358Val mutation that ATP1A3 immunofluorescence is prominently associated with interneurons in the cortex, which may provide some insight into the pathogenesis of the disease. SIGNIFICANCE: The findings indicate these mutations cause severe phenotypes of ATP1A3-related disorder spectrum that include catastrophic early life epilepsy, episodic apnea, and postnatal microcephaly.

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;VGlut2(F/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.

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.

Prevention of seizure-induced sudden death in a chronic SUDEP model by semichronic administration of a selective serotonin reuptake inhibitor.

DBA/1 mice are a chronically susceptible model of sudden unexpected death in epilepsy (SUDEP) that exhibit chronic audiogenic generalized convulsive seizures (GCSs), leading to death from respiratory arrest (RA) if not resuscitated. Serotonin (5-HT) normally enhances respiration in response to elevated CO(2) levels, which occur during GCSs in humans. Selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, increase 5-HT availability. We examined whether fluoxetine can block GCS-induced sudden death in DBA/1 mice. Fluoxetine (15-70 mg/kg ip) was administered acutely with seizure induction at 30minutes or semichronically in five daily doses (20mg/kg/day) with induction after 5 days. Acute fluoxetine (45 or 70 mg/kg) significantly reduced the incidence of RA without blocking seizure susceptibility. Semichronic fluoxetine did not block seizures, but significantly reduced seizure-induced RA, which is consistent with effects of SSRIs on respiration in patients with epilepsy [Bateman LM, Li DS,LiN TC, Seyal M. Epilepsia 2010;51:2211-4]. These findings suggest that treatment with SSRIs should be evaluated for reducing the incidence of SUDEP in patients.

Serotonin 5-HT4 receptors play a critical role in the action of fenfluramine to block seizure-induced sudden death in a mouse model of SUDEP.

RATIONALE: Our previous study showed that the recently approved anticonvulsant drug, fenfluramine, which enhances the release of serotonin (5-hydroxytryptamine, 5-HT) in the brain, prevents seizure-induced respiratory arrest (S-IRA) in the DBA/1 mouse model of sudden unexpected death in epilepsy (SUDEP). The present study examined the role of 5-HT receptor subtypes in mediating the effect of this agent by combined administration of fenfluramine with selective 5-HT receptor antagonists prior to seizure in DBA/1 mice. METHODS: Fenfluramine (15 mg/kg, i.p.) was administered to primed DBA/1 mice, and audiogenic seizure (Sz) was induced 16 h later. Thirty min prior to Sz induction a selective antagonist acting on 5-HT1A, 5-HT2, 5-HT3 5-HT4, 5-HT5A, 5-HT6 or 5-HT7 receptors at a sub-toxic dose was administered, and changes in seizure-induced behaviors were evaluated. Follow-up studies examined the effect of administration of a 5-HT4 receptor agonist, BIMU 8, as well as the effect of co-administration of ineffective doses of fenfluramine and BIMU-8 on Sz behaviors. RESULTS: The 5-HT4 antagonist (GR125487) was the only 5-HT receptor antagonist that was able to reverse the action of fenfluramine to block Sz and S-IRA. Treatment with the 5-HT4 receptor agonist (BIMU-8), or co-administration of ineffective doses of BIMU-8 and fenfluramine significantly reduced the incidence of S-IRA and tonic Sz in DBA/1 mice. The antagonists for 5-HT3, 5-HT5A 5-HT6, and 5-HT7 receptors did not significantly affect the action of fenfluramine. However, the 5-HT1A and the 5-HT2 antagonists enhanced the anticonvulsant effects of fenfluramine. CONCLUSIONS: These findings suggest that the action of fenfluramine to prevent seizure-induced sudden death in DBA/1 mice is mediated primarily by activation of 5-HT4 receptors. These studies are the first to indicate the therapeutic potential of 5-HT4 receptor agonists either alone or in combination with fenfluramine for preventing SUDEP. Enhancement of the anticonvulsant effect of fenfluramine by 5-HT1A and 5-HT2 antagonists may involve presynaptic actions of these antagonists. Thus, the Sz and S-IRA blocking actions of fenfluramine involve complex interactions with several 5-HT receptor subtypes. These data also provide further support for the serotonin hypothesis of SUDEP.

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

PURPOSE: Kindling of audiogenic seizure (AGS) involves >or=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. METHODS: The effects of a single focal bilateral microinjection into LA of an AC activator, MPB forskolin {7-Deacetyl-7-[O-(N-methylpiperazino)-gamma-butyryl]-forskolin dihydrochloride} (25-100 pmol/side), on seizure behavior in GEPR-9s were evaluated. RESULTS: One-time bilateral microinjection of MPB forskolin in GEPR-9s precipitously induced an AGS kindling-like effect, which involved significant increases in seizure duration and long-lasting susceptibility to AGS that culminates in PTC. This effect occurred at 24 h after MPB forskolin microinjection and lasted >or=5 weeks. The effect was seen when AGS was initiated at 1 and 12 h after microinjection, but not if AGS was induced only at 24 h, indicating the importance of the temporal proximity of AGS induction to the MPB forskolin microinjection. DISCUSSION: These findings indicate that one-time activation of AC within the NMDA receptor-mediated molecular cascade results in precipitous induction of AGS kindling. These data further suggest that AC activation in the LA may be an important epileptogenic mechanism that subserves the long-lasting persistence of AGS kindling.

DBA/1 mice exhibit chronic susceptibility to audiogenic seizures followed by sudden death associated with respiratory arrest.

One proposed cause of sudden unexpected death in epilepsy (SUDEP) in patients is generalized convulsive seizures with respiratory malfunction. We evaluated DBA/1 mice as a chronic SUDEP model. In DBA/1 mice, audiogenic seizures induced by acoustic stimulation resulted in generalized convulsive seizures followed by respiratory arrest from postnatal day (PND) 21 to 100. The incidence of respiratory arrest susceptibility increased, reaching approximately 90-100% by three to seven daily seizures when testing began on PND 21-30. Respiratory arrest was reversible with resuscitation in approximately 98% of mice, which allows repeated seizure testing. Electrocardiographic activity in DBA/1 mice was detectable for approximately 4-6 minutes after respiratory arrest, indicating that death is likely due to respiratory cessation, as cardiac changes occur later. These findings suggest that DBA/1 mice are a useful chronic SUDEP model. These mice die suddenly from respiratory arrest after generalized convulsive seizures until reaching PND >or=100, allowing testing of chronic preventive treatments for SUDEP.

Deficient post-ictal cardiorespiratory compensatory mechanisms mediated by the periaqueductal gray may lead to death in a mouse model of SUDEP.

Post-ictal cardiorespiratory failure is implicated as a major cause of sudden unexpected death in epilepsy (SUDEP) in patients. The DBA/1 mouse model of SUDEP is abnormally susceptible to fatal seizure-induced cardiorespiratory failure (S-CRF) induced by convulsant drug, hyperthermia, electroshock, and acoustic stimulation. Clinical and pre-clinical studies have implicated periaqueductal gray (PAG) abnormalities in SUDEP. Recent functional neuroimaging studies observed that S-CRF resulted in selective changes in PAG neuronal activity in DBA/1 mice. The PAG plays a critical compensatory role for respiratory distress caused by numerous physiological challenges in non-epileptic individuals. These observations suggest that abnormalities in PAG-mediated cardiorespiratory modulation may contribute to S-CRF in DBA/1 mice. To evaluate this, electrical stimulation (20 Hz, 20-100 µA, 10 s) was presented in the PAG of anesthetized DBA/1 and C57BL/6 (non-epileptic) control mice, and post-stimulus changes in respiration [inter-breath interval (IBI)] and heart rate variability (HRV) were examined. The post-stimulus period was considered analogous to the post-ictal period when S-CRF occurred in previous DBA/1 mouse studies. PAG stimulation caused significant intensity-related decreases in IBI in both mouse strains. However, this effect was significantly reduced in DBA/1 vis-a-vis C57BL/6 mice. These changes began immediately following cessation of stimulation and remained significant for 10 s. This time period is critical for initiating resuscitation to successfully prevent seizure-induced death in previous DBA/1 mouse experiments. Significant post-stimulus increases in HRV were also seen at ≥60 µA in the PAG in C57BL/6 mice, which were absent in DBA/1 mice. These data along with previous neuroimaging findings suggest that compensatory cardiorespiratory modulation mediated by PAG is deficient, which may be important to the susceptibility of DBA/1 mice to S-CRF. These observations suggest that correcting this deficit pharmacologically or by electrical stimulation may help to prevent S-CRF. These findings further support the potential importance of PAG abnormalities to human SUDEP.

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 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.

Early investigational drugs for hearing loss.

INTRODUCTION: Sensorineural hearing loss (HL) is becoming a global phenomenon at an alarming rate. Nearly 600 million people have been estimated to have significant HL in at least one ear. There are several different causes of sensorineural HL included in this review of new investigational drugs for HL. They are noise-induced, drug-induced, sudden sensorineural HL, presbycusis and HL due to cytomegalovirus infections. AREAS COVERED: This review presents trends in research for new investigational drugs encompassing a variety of causes of HL. The studies presented here are the latest developments either in the research laboratories or in preclinical, Phase 0, Phase I or Phase II clinical trials for drugs targeting HL. EXPERT OPINION: While it is important that prophylactic measures are developed, it is extremely crucial that rescue strategies for unexpected or unavoidable cochlear insult be established. To achieve this goal for the development of drugs for HL, innovative strategies and extensive testing are required for progress from the bench to bedside. However, although a great deal of research needs to be done to achieve the ultimate goal of protecting the ear against acquired sensorineural HL, we are likely to see exciting breakthroughs in the near future.

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.

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.

Audiogenic kindling induces plastic changes in the neuronal firing patterns in periaqueductal gray.

The ventrolateral periaqueductal gray (vlPAG) is a critical structure in the requisite neuronal network for audiogenic seizures (AGS), which is confined to the brainstem, in genetically epilepsy-prone rats (GEPR-9s). Periodic repetition of AGS in GEPR-9s induces AGS kindling resulting in expansion of the seizure network to the forebrain, epileptiform electrocorticographic activity, and prolonged seizure duration due to emergence of a previously unexpressed seizure behavior-generalized clonus (post-tonic clonus, PTC). The pathway that mediates AGS kindling has been shown to involve the amygdala, which is known to have extensive projections to the periaqueductal gray. The present study investigated whether AGS kindling results in changes in vlPAG neuronal firing by chronically implanting microwire electrodes to record vlPAG extracellular action potentials in awake-behaving GEPR-9s. Acoustic responses and neuronal firing patterns during AGS were compared in non-kindled and AGS kindled GEPR-9s. AGS kindling induced significant increases in acoustic responsiveness of vlPAG neurons. During AGS in both AGS kindled and non-kindled GEPR-9s, vlPAG neurons displayed tonic firing during wild running and tonic extension behaviors. However, the tonic firing pattern changed to burst firing exclusively during PTC, and this vlPAG neuronal firing change was seen only in kindled GEPR-9s. Burst firing is a hallmark of neuroplastic changes known to mediate increased synaptic efficiency. These results suggest that the amygdala to vlPAG pathway may be a critical element of the expanded seizure network that contributes importantly to the emergence of PTC induced by AGS kindling in GEPR-9s, which is supported by recent preliminary studies of this pathway.

Inhibition of adenylyl cyclase in amygdala blocks the effect of audiogenic seizure kindling in genetically epilepsy-prone rats.

Genetically epilepsy-prone rats of the severe seizure strain (GEPR-9s) exhibit audiogenic seizures (AGS) beginning with wild running and ending with tonic hind limb extension (TE). AGS kindling in GEPR-9s involves periodic repetition of >/=14 seizures over 7-21 days and results in prolonged seizures and an additional phase of generalized post-tonic clonus (PTC) that follows TE. AGS kindling behavior changes are long-lasting and involve expansion of the requisite seizure neuronal network from the brainstem to include the amygdala, mediated by neuroplasticity in lateral amygdala. Recent evidence indicates that focal activation of adenylyl cyclase (AC) in lateral amygdala leads to precipitous acquisition of AGS-kindled seizure behaviors, suggesting that activation of AC activity is important in development and maintenance of AGS kindling. The present study further examined the role of AC in AGS-kindled seizures in GEPR-9s by focally inhibiting AC in the amygdala. Bilateral microinjection of an AC inhibitor, SQ22,536 (0.25 and 0.50 nmol/side), in AGS-kindled GEPR-9s selectively blocked PTC during AGS at 1 h after microinjection, but the pre-kindled AGS behaviors remained intact. The incidence of PTC during AGS returned to pre-drug levels 12 h after the lower dose of SQ22,536 (0.25 nmol/side). However, after the higher dose of SQ22,536 (0.5 nmol/side), complete return to AGS with PTC was seen in all GEPR-9s at 120 h. These results indicate that maintenance of AGS kindling-mediated PTC in GEPR-9s may involve activation of AC. These data provide further evidence for the involvement of AC in the epileptogenic mechanisms subserving AGS kindling.