The postnatal development of ultrasonic vocalization-associated breathing is altered in glycine transporter 2-deficient mice.

KEY POINTS: Newborn mice produce ultrasonic vocalization to communicate with their mother. The neuronal glycine transporter (GlyT2) is required for efficient loading of synaptic vesicles in glycinergic neurons. Mice lacking GlyT2 develop a phenotype that resembles human hyperekplexia and the mice die in the second postnatal week. In the present study, we show that GlyT2-knockout mice do not acquire adult ultrasonic vocalization-associated breathing patterns. Despite the strong impairment of glycinergic inhibition, they can produce sufficient expiratory airflow to produce ultrasonic vocalization. Because mouse ultrasonic vocalization is a valuable read-out in translational research, these data are highly relevant for a broad range of research fields. ABSTRACT: Mouse models are instrumental with respect to determining the genetic basis and neural foundations of breathing regulation. To test the hypothesis that glycinergic synaptic inhibition is required for normal breathing and proper post-inspiratory activity, we analysed breathing and ultrasonic vocalization (USV) patterns in neonatal mice lacking the neuronal glycine transporter (GlyT2). GlyT2-knockout (KO) mice have a profound reduction of glycinergic synaptic currents already at birth, develop a severe motor phenotype and survive only until the second postnatal week. At this stage, GlyT2-KO mice are smaller, have a reduced respiratory rate and still display a neonatal breathing pattern with active expiration for the production of USV. By contrast, wild-type mice acquire different USV-associated breathing patterns that depend on post-inspiratory control of air flow. Nonetheless, USVs per se remain largely indistinguishable between both genotypes. We conclude that GlyT2-KO mice, despite the strong impairment of glycinergic inhibition, can produce sufficient expiratory airflow to produce ultrasonic vocalization.

A neuronal role of the Alanine-Serine-Cysteine-1 transporter (SLC7A10, Asc-1) for glycine inhibitory transmission and respiratory pattern.

The Alanine-Serine-Cysteine-1 transporter (SLC7A10, Asc-1) has been shown to play a role in synaptic availability of glycine although the exact mechanism remains unclear. We used electrophysiological recordings and biochemical experiments to investigate the role of Asc-1 transporter in glycinergic transmission in the brainstem respiratory network. Using both the Asc-1 substrate and transportable inhibitor D-isoleucine (D-Ile), and the non-transportable Asc-1 blocker Lu AE00527 (Lu), we found that D-Ile reduces glycinergic transmission and increases glycine release via hetero-exchange, whereas Lu has no acute effect on glycinergic synaptic transmission. Furthermore, D-Ile increases the frequency and reduces amplitude of the phrenic nerve activity in the arterially-perfused working heart brainstem preparation. These results suggest a role of Asc-1 in modulating presynaptic glycine levels that can impact on the respiratory network.

Breathing disturbances in a model of Rett syndrome: A potential involvement of the glycine receptor α3 subunit?

The glycine receptor α3 subunit is known to be a target for cAMP/PKA-mediated phosphorylation and regulation. Mice that lack this subunit are apparently normal but the 5-HT1A-receptor mediated modulation of respiratory network activity is disturbed. Since the intracellular cAMP-concentration is reduced in mice that lack the transcriptional modulator methyl-CpG-binding protein 2 (MeCP2) gene, we aimed to test if the α3 subunit of the glycine receptor is involved in the development of the breathing phenotype of MeCP2-deficient mice (Mecp2-/y). Therefore, we generated a double knock-out mouse line that lacks both the Mecp2 gene as well as the gene (Glra3) for the α3 subunit of the ionotropic glycine receptor. As compared to WT and Glra3-/- mice, both Mecp2-/y mice and Mecp2-/y; Glra3-/- mice (DKO) showed a slower respiratory rate and a tendency towards higher numbers of apneas. Interestingly, the irregularity of the breathing was significantly reduced in DKO as compared to Mecp2-/y littermates. In the light of the unaltered survival of DKO mice, however, the contribution of the glycine receptor α3 subunit for development and progression of the breathing disturbances in the mouse model of Rett syndrome appears to be only of minor relevance.

GlyT2-Dependent Preservation of MECP2-Expression in Inhibitory Neurons Improves Early Respiratory Symptoms but Does Not Rescue Survival in a Mouse Model of Rett Syndrome.

Mutations in methyl-CpG-binding protein 2 (MECP2) gene have been shown to manifest in a neurodevelopmental disorder that is called Rett syndrome. A typical problem that occurs during development is a disturbance of breathing. To address the role of inhibitory neurons, we generated a mouse line that restores MECP2 in inhibitory neurons in the brainstem by crossbreeding a mouse line that expresses the Cre-recombinase (Cre) in inhibitory neurons under the control of the glycine transporter 2 (GlyT2, slc6a5) promotor (GlyT2-Cre) with a mouse line that has a floxed-stop mutation of the Mecp2 gene (Mecp2 (stop/y)). Unrestrained whole-body-plethysmography at postnatal day P60 revealed a low respiratory rate and prolonged respiratory pauses in Mecp2 (stop/y) mice. In contrast, GlyT2-Cre positive Mecp2 (stop/y) mice (Cre(+) ; Mecp2 (stop/y)) showed greatly improved respiration and were indistinguishable from wild type littermates. These data support the concept that alterations in inhibitory neurons are important for the development of the respiratory phenotype in Rett syndrome.

microRNA targeting of the P2X7 purinoceptor opposes a contralateral epileptogenic focus in the hippocampus.

The ATP-gated ionotropic P2X7 receptor (P2X7R) modulates glial activation, cytokine production and neurotransmitter release following brain injury. Levels of the P2X7R are increased in experimental and human epilepsy but the mechanisms controlling P2X7R expression remain poorly understood. Here we investigated P2X7R responses after focal-onset status epilepticus in mice, comparing changes in the damaged, ipsilateral hippocampus to the spared, contralateral hippocampus. P2X7R-gated inward currents were suppressed in the contralateral hippocampus and P2rx7 mRNA was selectively uploaded into the RNA-induced silencing complex (RISC), suggesting microRNA targeting. Analysis of RISC-loaded microRNAs using a high-throughput platform, as well as functional assays, suggested the P2X7R is a target of microRNA-22. Inhibition of microRNA-22 increased P2X7R expression and cytokine levels in the contralateral hippocampus after status epilepticus and resulted in more frequent spontaneous seizures in mice. The major pro-inflammatory and hyperexcitability effects of microRNA-22 silencing were prevented in P2rx7(-/-) mice or by treatment with a specific P2X7R antagonist. Finally, in vivo injection of microRNA-22 mimics transiently suppressed spontaneous seizures in mice. The present study supports a role for post-transcriptional regulation of the P2X7R and suggests therapeutic targeting of microRNA-22 may prevent inflammation and development of a secondary epileptogenic focus in the brain.

P2X7 receptor inhibition interrupts the progression of seizures in immature rats and reduces hippocampal damage.

AIMS: Early-life seizures, particularly when prolonged, may be harmful to the brain. Current pharmacotherapy is often ineffective; therefore, novel neuro- and/or glio-transmitter systems should be explored for targeting. The P2X7 receptor is a cation-permeable channel with trophic and excitability effects on neurons and glia which is activated by high amounts of ATP that may be released in the setting of injury after severe seizures. Here, we tested the effects of A-438079, a potent and selective P2X7 receptor antagonist in a lesional model of early-life status epilepticus. METHODS: Seizures were induced by intra-amygdala kainic acid in 10-day-old rat pups. Electrographic seizure severity, changes to P2X7 receptor expression, inflammatory responses and histological effects were evaluated. RESULTS: Seizures induced by intra-amygdala kainic acid increased levels of P2X7 receptor protein and interleukin-1ß and caused significant cell death within the ipsilateral hippocampus. A-438079 rapidly reached the brain following systemic injection in P10 rats. Intraperitoneal injection of A-438079 (5 and 15 mg/kg) 60 min after triggering seizures reduced seizure severity and neuronal death within the hippocampus. A-438079 had superior neuroprotective effects compared with an equally seizure-suppressive dose of phenobarbital (25 mg/kg). CONCLUSIONS: These results suggest P2X7 receptor antagonists may be suitable as frontline or adjunctive treatments of pediatric status epilepticus or other early-life seizures, particularly when associated with brain damage.

Increased neocortical expression of the P2X7 receptor after status epilepticus and anticonvulsant effect of P2X7 receptor antagonist A-438079.

PURPOSE: ATP is an essential transmitter/cotransmitter in neuron function and pathophysiology and has recently emerged as a potential contributor to prolonged seizures (status epilepticus) through the activation of the purinergic ionotropic P2X7 receptor (P2X7R). Increased P2X7R expression has been reported in the hippocampus, and P2X7R antagonists reduced seizure-induced damage to this brain region. However, status epilepticus also produces damage to the neocortex. The present study was designed to characterize P2X7R in the neocortex and assess effects of P2X7R antagonists on cortical injury after status epilepticus. METHODS: Status epilepticus was induced in mice by intraamygdala microinjection of kainic acid. Specific P2X7R inhibitors were administered into the ventricle before seizure induction, and cortical electroencephalography and behavior was recorded to assess seizure severity. P2X7R expression was examined in neocortex up to 24 h after status epilepticus, in epileptic mice, and in resected neocortex from patients with pharmacoresistent temporal lobe epilepsy (TLE). In addition, the induction of P2X7R after status epilepticus was investigated using transgenic P2X7R reporter mice, which express enhanced green fluorescent protein under the control of the p2x7r promoter. KEY FINDINGS: Status epilepticus resulted in increased P2X7R protein levels in the neocortex of mice. Neocortical P2X7 receptor levels were also elevated in mice that developed epilepsy after status epilepticus and in resected neocortex from patients with pharmacoresistent TLE. Immunohistochemistry determined that neurons were the major cell population transcribing the P2X7R in the neocortex within the first 8 h after status epilepticus, whereas in epileptic mice, P2X7R up-regulation occurred in microglia as well as in neurons. Pretreatment of mice with the specific P2X7R inhibitor A-438079 reduced electrographic and clinical seizure severity during status epilepticus and reduced seizure-induced neuronal death in the neocortex. SIGNIFICANCE: Our findings identify neurons in the neocortex as an important site of P2X7R up-regulation after status epilepticus and in epilepsy, and provide support for the possible use of P2X7R antagonists for the treatment of status epilepticus and prevention of seizure-induced brain damage.

CHOP regulates the p53-MDM2 axis and is required for neuronal survival after seizures.

Hippocampal sclerosis is a frequent pathological finding in patients with temporal lobe epilepsy and can be caused by prolonged single or repeated brief seizures. Both DNA damage and endoplasmic reticulum stress have been implicated as underlying molecular mechanisms in seizure-induced brain injury. The CCAAT/enhancer-binding protein homologous protein (CHOP) is a transcriptional regulator induced downstream of DNA damage and endoplasmic reticulum stress, which can promote or inhibit apoptosis according to context. Recent work has proposed inhibition of CHOP as a suitable neuroprotective strategy. Here, we show that transcript and protein levels of CHOP increase in surviving subfields of the hippocampus after prolonged seizures (status epilepticus) in mouse models. CHOP was also elevated in the hippocampus from epileptic mice and patients with pharmacoresistant epilepsy. The hippocampus of CHOP-deficient mice was much more vulnerable to damage in mouse models of status epilepticus. Moreover, compared with wild-type animals, CHOP-deficient mice subject to status epilepticus developed more spontaneous seizures, displayed protracted hippocampal neurodegeneration and a deficit in a hippocampus-dependent object-place recognition task. The absence of CHOP was associated with a supra-maximal induction of p53 after status epilepticus, and inhibition of p53 abolished the cell death-promoting consequences of CHOP deficiency. The protective effect of CHOP could be partly explained by activating transcription of murine double minute 2 that targets p53 for degradation. These data demonstrate that CHOP is required for neuronal survival after seizures and caution against inhibition of CHOP as a neuroprotective strategy where excitotoxicity is an underlying pathomechanism.

Seizure suppression and neuroprotection by targeting the purinergic P2X7 receptor during status epilepticus in mice.

Prolonged seizures [status epilepticus (SE)] constitute a neurological emergency that can permanently damage the brain. SE results from a failure of the normal mechanisms to terminate seizures; in particular, γ-amino butyric acid-mediated inhibition, and benzodiazepine anticonvulsants are often incompletely effective. ATP acts as a fast neurotransmitter via ionotropic ligand-gated P2X receptors. Here we report that SE induced by intra-amygdala kainic acid in mice selectively increased hippocampal levels of P2X7 receptors relative to other P2X receptors. Using transgenic P2X7 reporter mice expressing enhanced green fluorescent protein, we identify dentate granule neurons as the major cell population transcribing the P2X7 receptor after SE. Pretreatment of mice with an intracerebroventricular microinjection of 1.75 nmol A438079, a P2X7 receptor antagonist, reduced seizure duration by 58% and reduced seizure-induced neuronal death by 61%. Injection of brilliant blue G (1 pmol), another selective antagonist, reduced seizure duration by 48% and was also neuroprotective. A438079 was seizure-suppressive when injected shortly after induction of SE, and coinjection of A438079 with lorazepam 60 min after triggering SE, when electrographic seizure-responsiveness to lorazepam had decreased, also terminated SE. Our results suggest that P2X7 receptor antagonists may be a promising class of drug for seizure abrogation and neuroprotection in SE.