Serotonin 2C receptor in a rat model of temporal lobe epilepsy: From brainstem expression to pharmacological blockade in relation to ventilatory function.

Because of its involvement in breathing control and neuronal excitability, dysregulation of the serotonin (5-HT) 2C receptor (5-HT2C) might play a key role in sudden unexpected death in epilepsy. Seizure-induced respiratory arrest is thus prevented by a 5-HT2B/C agonist in different seizure model. However, the specific contribution of 5-HT2C in chronic epilepsy-related respiratory dysfunction remains unknown. In a rat model of temporal lobe epilepsy (EPI rats), in which we previously reported interictal respiratory dysfunctions and a reduction of brainstem 5-HT tone, quantitative reverse transcriptase polymerase chain reaction showed overexpression of TPH2 (5-HT synthesis enzyme), SERT (5-HT reuptake transporter), and 5-HT2C transcript levels in the brainstem of EPI rats, and of RNA-specific adenosine deaminase (ADAR1, ADAR2) involved in the production of 5-HT2C isoforms. Interictal ventilation was assessed with whole-body plethysmography before and 2 h after administration of SB242084 (2 mg/kg), a specific antagonist of 5-HT2C. As expected, SB242084 administration induced a progressive decrease in ventilatory parameters and an alteration of breathing stability in both control and EPI rats. However, the size of the SB242084 effect was lower in EPI rats than in controls. Increased 5-HT2C gene expression in the brainstem of EPI rats could be part of a compensatory mechanism against epilepsy-related low 5-HT tone and expression of 5-HT2C isoforms for which 5-HT affinity might be lower.

Neonatal brain injury unravels transcriptional and signaling changes underlying the reactivation of cortical progenitors.

Germinal activity persists throughout life within the ventricular-subventricular zone (V-SVZ) of the postnatal forebrain due to the presence of neural stem cells (NSCs). Accumulating evidence points to a recruitment for these cells following early brain injuries and suggests their amenability to manipulations. We used chronic hypoxia as a rodent model of early brain injury to investigate the reactivation of cortical progenitors at postnatal times. Our results reveal an increased proliferation and production of glutamatergic progenitors within the dorsal V-SVZ. Fate mapping of V-SVZ NSCs demonstrates their contribution to de novo cortical neurogenesis. Transcriptional analysis of glutamatergic progenitors shows parallel changes in methyltransferase 14 (Mettl14) and Wnt/ß-catenin signaling. In agreement, manipulations through genetic and pharmacological activation of Mettl14 and the Wnt/ß-catenin pathway, respectively, induce neurogenesis and promote newly-formed cell maturation. Finally, labeling of young adult NSCs demonstrates that pharmacological NSC activation has no adverse effects on the reservoir of V-SVZ NSCs and on their germinal activity.

Phosphatidylserine enriched with polyunsaturated n-3 fatty acid supplementation for attention-deficit hyperactivity disorder in children and adolescents with epilepsy: A randomized placebo-controlled trial.

BACKGROUND: Attention-deficit hyperactivity disorder (ADHD) is a frequent comorbidity in children with epilepsy, which management mostly relies on the usual treatments of ADHD, especially methylphenidate. Supplementation with polyunsaturated n-3 Fatty Acid (PUFA) has been proposed as an alternative therapeutic approach in ADHD without epilepsy but has never been evaluated in epilepsy-associated ADHD. METHODS: A multicenter double blind randomized placebo-controlled trial evaluating supplementation with PUFA, in eicosapentaenoic- and docosahexaenoic-acid form, conjugated to a phospholipid vector (PS-Omega3) in children aged >6 and <16-years old, and suffering from any type of epilepsy and ADHD (inattentive or combined type) according to DSM-V. After a 4-week baseline period, patients were allocated (1:1) either to placebo group or to PS-Omega 3 group and entered a 12 week-double-blind treatment period which was followed by a 12 week-open-label treatment period. The primary outcome was the reduction of the ADHD-rating scale IV attention-deficit subscore after 12 weeks of treatment. RESULTS: The study was stopped early because of lack of eligible participants and the expected sample size was not reached. Seventy-four patients were randomized, 44 in PS-Omega3, and 30 in the placebo group. The reduction after 12 weeks of treatment in the inattention subscore of the ADHD-IV scale was -1.57 in the PS-Omega3 group, and -2.90 in the placebo group (p = 0.33, α = 5%). Results were similar after 24 weeks of treatment and for all other ADHD-related secondary outcomes, with no difference between placebo and PS-Omega3. CONCLUSION: Our study remaining underpowered, no formal conclusion about the effect of Ps-Omega3 could be drawn. However, our data strongly suggested that the PS-Omega 3 formulation used in the current study did not improve ADHD symptoms in children with epilepsy. PLAIN LANGUAGE SUMMARY: Supplementation with polyunsaturated n-3 Fatty Acid (PUFA) has been proposed in ADHD but has never been evaluated in patients with both epilepsy and ADHD. To address this issue, we conducted a multicenter double blind randomized placebo-controlled trial evaluating supplementation with PUFA in children with epilepsy and ADHD. The study was stopped early because of lack of eligible participants, hampering formal conclusion. However, the evolution of the ADHD symptoms at 12 and 24 weeks did not differ between placebo and PUFA supplementation, strongly suggesting that PUFA did not improve ADHD symptoms in children with epilepsy.

Are we there yet? A critical evaluation of sudden and unexpected death in epilepsy models.

Although animal models have helped to elaborate meaningful hypotheses about the pathophysiology of sudden and unexpected death in epilepsy (SUDEP), specific prevention strategies are still lacking, potentially reflecting the limitations of these models and the intrinsic difficulties of investigating SUDEP. The interpretation of preclinical data and their translation to diagnostic and therapeutic developments in patients thus require a high level of confidence in their relevance to model the human situation. Preclinical models of SUDEP are heterogeneous and include rodent and nonrodent species. A critical aspect is whether the animals have isolated seizures exclusively induced by a specific trigger, such as models where seizures are elicited by electrical stimulation, pharmacological intervention, or DBA mouse strains, or whether they suffer from epilepsy with spontaneous seizures, with or without spontaneous SUDEP, either of nongenetic epilepsy etiology or from genetically based developmental and epileptic encephalopathies. All these models have advantages and potential disadvantages, but it is important to be aware of these limitations to interpret data appropriately in a translational perspective. The majority of models with spontaneous seizures are of a genetic basis, whereas SUDEP cases with a genetic basis represent only a small proportion of the total number. In almost all models, cardiorespiratory arrest occurs during the course of the seizure, contrary to that in patients observed at the time of death, potentially raising the issue of whether we are studying models of SUDEP or models of periseizure death. However, some of these limitations are impossible to avoid and can in part be dependent on specific features of SUDEP, which may be difficult to model. Several preclinical tools are available to address certain gaps in SUDEP pathophysiology, which can be used to further validate current preclinical models.

CB2 receptor in the CNS: From immune and neuronal modulation to behavior.

Despite low levels of cannabinoid receptor type 2 (CB2R) expression in the central nervous system in human and rodents, a growing body of evidence shows CB2R involvement in many processes at the behavioral level, through both immune and neuronal modulations. Recent in vitro and in vivo evidence have highlighted the complex role of CB2R under physiological and inflammatory conditions. Under neuroinflammatory states, its activation seems to protect the brain and its functions, making it a promising target in a wide range of neurological disorders. Here, we provide a complete and updated overview of CB2R function in the central nervous system of rodents, spanning from modulation of immune function in microglia but also in other cell types, to behavior and neuronal activity, in both physiological and neuroinflammatory contexts.

Update on the controversial identity of cells expressing cnr2 gene in the nervous system.

The function of cannabinoid receptor type 2 (CB2R), mainly expressed by leukocytes, has long been limited to its peripheral immunomodulatory role. However, the use of CB2R-specific ligands and the availability of CB2R-Knock Out mice revealed that it could play a functional role in the CNS not only under physiological but also under pathological conditions. A direct effect on the nervous system emerged when CB2R mRNA was detected in neural tissues. However, accurate mapping of CB2R protein expression in the nervous system is still lacking, partly because of the lack of specificity of antibodies available. This review examines the regions and cells of the nervous system where CB2R protein is most likely present by cross-referencing mRNA and protein data published to date. Of the many antibodies developed to target CB2R, only a few have partially passed specificity tests and detected CB2R in the CNS. Efforts must be continued to support the development of more specific and better validated antibodies in each of the species in which CB2R protein is sought or needs to be quantified.

Respiratory dysfunction in two rodent models of chronic epilepsy and acute seizures and its link with the brainstem serotonin system.

Patients with drug-resistant epilepsy can experience respiratory alterations, notably during seizures. The mechanisms underlying long-term alterations in respiratory function remain unclear. As the brainstem 5-HT system is a prominent modulator of respiratory function, this study aimed at determining whether epilepsy is associated with alterations in both the respiratory function and brainstem serotonin (5-HT) system in rats. Epilepsy was triggered by pilocarpine-induced status epilepticus in rats. Our results showed that 30-50% of epileptic (EPI) rats exhibited a sharp decrease in oxygen consumption (SDOC), low metabolic rate of oxygen, and slow regular ventilation (EPI/SDOC + rats). These alterations were detected only in rats with chronic epilepsy, independent of behavioral seizures, were persistent over time, and not associated with death. In these rats, 5-HT fiber density in the nucleus tractus solitarius was lower than that in the control and EPI/SDOC- rats. Both EPI/SDOC + rats and DBA/2 mice that present with audiogenic-induced seizure followed by fatal respiratory arrest-a model of sudden and expected death in epilepsy-had increased transcript levels of tryptophan hydroxylase 2 and 5-HT presynaptic transporter. Thus, our data support that 5-HT alterations are associated with chronic and acute epilepsy-related respiratory dysfunction.

Relation between coffee consumption and risk of seizure-related respiratory dysfunction in patients with drug-resistant focal epilepsy.

OBJECTIVE: Caffeine is an antagonist of the adenosine pathway, which is involved in regulation of breathing. Extracellular concentrations of adenosine are increased in the immediate aftermath of a seizure. Seizure-related overstimulation of adenosine receptors might promote peri-ictal apnea. However, the relation between caffeine consumption and risk of seizure-related respiratory dysfunction in patients with drug-resistant focal epilepsy remains unknown. METHODS: We performed a cross-sectional analysis of data collected in patients included in the SAVE study in Lyon's epilepsy monitoring unit at the Adult Epilepsy Department of the Lyon University Hospital between February 2016 and October 2018. The video-electroencephalographic recordings of 156 patients with drug-resistant focal epilepsy included in the study were reviewed to identify those with ≥1 focal seizure (FS), valid pulse oximetry (SpO2 ) measurement, and information about usual coffee consumption. This latter was collected at inclusion using a standardized self-questionnaire and further classified into four groups: none, rare (≤3 cups/week), moderate (4 cups/week to 3 cups/day), and high (≥4 cups/day). Peri-ictal hypoxemia (PIH) was defined as SpO2 < 90% for at least 5 s occurring during the ictal period, the post-ictal period, or both. RESULTS: Ninety patients fulfilled inclusion criteria, and 323 seizures were analyzed. Both the level of usual coffee consumption (p = .033) and the level of antiepileptic drug withdrawal (p = .004) were independent risk factors for occurrence of PIH. In comparison with FS in patients with no coffee consumption, risk of PIH was four times lower in FS in patients with moderate consumption (odds ratio [OR] = .25, 95% confidence interval [CI] = .07-.91, p = .036) and six times lower in FS in patients with high coffee consumption (OR = .16, 95% CI = .04-.66, p = .011). However, when PIH occurred, its duration was longer in patients with moderate or high consumption than in those with no coffee consumption (p = .042). SIGNIFICANCE: Coffee consumption may be a protective factor for seizure-related respiratory dysfunction, with a dose-dependent effect.

Mitigation of Expression of Virulence Genes in Legionella pneumophila Internalized in the Free-Living Amoeba Willaertia magna C2c Maky.

Legionella pneumophila is a human pathogen responsible for a severe form of pneumonia named Legionnaire disease. Its natural habitat is aquatic environments, being in a free state or intracellular parasites of free-living amoebae, such as Acanthamoeba castellanii. This pathogen is able to replicate within some amoebae. Willaertia magna C2c Maky, a non-pathogenic amoeba, was previously demonstrated to resist to L. pneumophila and even to be able to eliminate the L. pneumophila strains Philadelphia, Lens, and Paris. Here, we studied the induction of seven virulence genes of three L. pneumophila strains (Paris, Philadelphia, and Lens) within W. magna C2c Maky in comparison within A. castellanii and with the gene expression level of L. pneumophila strains alone used as controls. We defined a gene expression-based virulence index to compare easily and without bias the transcript levels in different conditions and demonstrated that W. magna C2c Maky did not increase the virulence of L. pneumophila strains in contrast to A. castellanii. These results confirmed the non-permissiveness of W. magna C2c Maky toward L. pneumophila strains.

Transient hypoxemia induced by cortical electrical stimulation: A mapping study in 75 patients.

OBJECTIVE: To identify which cortical regions are associated with direct electrical stimulation (DES)-induced alteration of breathing significant enough to impair pulse oximetry (SpO2). METHODS: Evolution of SpO2 after 1,352 DES was analyzed in 75 patients with refractory focal epilepsy who underwent stereo-EEG recordings. For each DES, we assessed the change in SpO2 from 30 seconds prior to DES onset to 120 seconds following the end of the DES. The primary outcome was occurrence of stimulation-induced transient hypoxemia as defined by decrease of SpO2 ≥5% within 60 seconds after stimulation onset as compared to pre-DES SpO2 or SpO2 nadir <90% during at least 5 seconds. Localization of the stimulated contacts was defined according to MarsAtlas brain parcellation and Freesurfer segmentation. RESULTS: A stimulation-induced transient hypoxemia was observed after 16 DES (1.2%) in 10 patients (13%), including 6 in whom SpO2 nadir was <90%. Among these 16 DES, 7 (44%) were localized within the perisylvian cortex. After correction for individual effects and the varying number of DES contributed by each person, significant decrease of SpO2 was significantly associated with the localization of DES (p = 0.019). CONCLUSION: Though rare, a significant decrease of SpO2 could be elicited by cortical direct electrical stimulation outside the temporo-limbic structures, most commonly after stimulation of the perisylvian cortex.

Short erythropoietin-derived peptide enhances memory, improves long-term potentiation, and counteracts amyloid beta-induced pathology.

Neurodegenerative disorders such as Alzheimer's disease (AD) are characterized by the irreversible neuronal loss and memory impairment, and current treatments are merely symptomatic. Erythropoietin (EPO) has been shown to possess neurotrophic, neuroprotective, anti-inflammatory, and memory-enhancing effects, which could be therapeutically beneficial in the different aspects of AD. However, the hematopoietic effect of EPO has hampered its potential as a neuroprotective and procognitive agent. In this study, we characterized a novel small peptide, NL100, derived from a conserved C-helix region of EPO. NL100 was shown to bind to the EPO receptor, induce neuritogenesis, and protect hippocampal neurons from oxidative- and Aß25-35-induced neurodegeneration in vitro. Importantly, long-term NL100 treatment did not induce hematopoiesis, overcoming this challenge associated with EPO. Memory-enhancing effects were demonstrated after NL100 treatment in social recognition test for short-term memory, in both healthy rats and rats challenged centrally with Aß25-35 peptide, and in the Morris water maze test for spatial memory. Moreover, NL100 was shown to reverse Aß25-35-induced hippocampal degeneration and gliosis as well as pilocarpine-induced suppression of long-term potentiation in rats. In conclusion, NL100 is a novel EPO-derived nonhematopoietic peptide with neuroprotective and memory-enhancing effects and could therefore be a potential candidate for the development of new treatments for neurodegenerative disorders and dementia.

Hypoxemia following generalized convulsive seizures: Risk factors and effect of oxygen therapy.

OBJECTIVE: To analyze the factors that determine the occurrence or severity of postictal hypoxemia in the immediate aftermath of a generalized convulsive seizure (GCS). METHODS: We reviewed the video-EEG recordings of 1,006 patients with drug-resistant focal epilepsy included in the REPO2MSE study to identify those with ≥1 GCS and pulse oximetry (SpO2) measurement. Factors determining recovery of SpO2 ≥ 90% were investigated using Cox proportional hazards models. Association between SpO2 nadir and person- or seizure-specific variables was analyzed after correction for individual effects and the varying number of seizures. RESULTS: A total of 107 GCS in 73 patients were analyzed. A transient hypoxemia was observed in 92 GCS (86%). Rate of GCS with SpO2 <70% dropped from 40% to 21% when oxygen was administered early (p = 0.046). Early recovery of SpO2 ≥90% was associated with early administration of oxygen (p = 0.004), absence of postictal generalized EEG suppression (PGES) (p = 0.014), and extratemporal lobe epilepsy (p = 0.001). Lack of early administration of O2 (p = 0.003), occurrence of PGES (p = 0.018), and occurrence of ictal hypoxemia during the focal phase (p = 0.022) were associated with lower SpO2 nadir. CONCLUSION: Postictal hypoxemia was observed in the immediate aftermath of nearly all GCS but administration of oxygen had a strong preventive effect. Severity of postictal hypoxemia was greater in temporal lobe epilepsy and when hypoxemia was already observed before the onset of secondary GCS.

Necdin shapes serotonergic development and SERT activity modulating breathing in a mouse model for Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a genetic neurodevelopmental disorder that presents with hypotonia and respiratory distress in neonates. The Necdin-deficient mouse is the only model that reproduces the respiratory phenotype of PWS (central apnea and blunted response to respiratory challenges). Here, we report that Necdin deletion disturbs the migration of serotonin (5-HT) neuronal precursors, leading to altered global serotonergic neuroarchitecture and increased spontaneous firing of 5-HT neurons. We show an increased expression and activity of 5-HT Transporter (SERT/Slc6a4) in 5-HT neurons leading to an increase of 5-HT uptake. In Necdin-KO pups, the genetic deletion of Slc6a4 or treatment with Fluoxetine, a 5-HT reuptake inhibitor, restored normal breathing. Unexpectedly, Fluoxetine administration was associated with respiratory side effects in wild-type animals. Overall, our results demonstrate that an increase of SERT activity is sufficient to cause the apneas in Necdin-KO pups, and that fluoxetine may offer therapeutic benefits to PWS patients with respiratory complications.

Altered hypermetabolic response to cortical spreading depolarizations after traumatic brain injury in rats.

Spreading depolarizations are waves of near-complete breakdown of neuronal transmembrane ion gradients, free energy starving, and mass depolarization. Spreading depolarizations in electrically inactive tissue are associated with poor outcome in patients with traumatic brain injury. Here, we studied changes in regional cerebral blood flow and brain oxygen (PbtO2), glucose ([Glc]b), and lactate ([Lac]b) concentrations in rats, using minimally invasive real-time sensors. Rats underwent either spreading depolarizations chemically triggered by KCl in naïve cortex in absence of traumatic brain injury or spontaneous spreading depolarizations in the traumatic penumbra after traumatic brain injury, or a cluster of spreading depolarizations triggered chemically by KCl in a remote window from which spreading depolarizations invaded penumbral tissue. Spreading depolarizations in noninjured cortex induced a hypermetabolic response characterized by a decline in [Glc]b and monophasic increases in regional cerebral blood flow, PbtO2, and [Lac]b, indicating transient hyperglycolysis. Following traumatic brain injury, spontaneous spreading depolarizations occurred, causing further decline in [Glc]b and reducing the increase in regional cerebral blood flow and biphasic responses of PbtO2 and [Lac]b, followed by prolonged decline. Recovery of PbtO2 and [Lac]b was significantly delayed in traumatized animals. Prespreading depolarization [Glc]b levels determined the metabolic response to clusters. The results suggest a compromised hypermetabolic response to spreading depolarizations and slower return to physiological conditions following traumatic brain injury-induced spreading depolarizations.

Cognitive Deficits and Inflammatory Response Resulting from Mild-to-Moderate Traumatic Brain Injury in Rats Are Exacerbated by Repeated Pre-Exposure to an Innate Stress Stimulus.

Traumatic brain injury (TBI) is common in both military and civilian populations, and often results in neurobehavioral sequelae that impair quality of life in both patients and their families. Although individuals who are chronically exposed to stress are more likely to experience TBI, it is still unknown whether pre-injury stress influences the outcome after TBI. The present study tested whether behavioral and cognitive long-term outcome after TBI in rats is affected by prior exposure to an innate stress stimulus. Young adult male Sprague-Dawley rats were exposed to the predator odor 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) or to water (WAT); exposure was repeated eight times at irregular intervals over a 2-week period. Rats were subsequently subjected to either mild-to-moderate bilateral brain injury (lateral fluid percussion [LFP]) or sham surgery (Sham). Four experimental groups were studied: Sham-WAT, Sham-TMT, LFP-WAT and LFP-TMT. Compared with Sham-WAT rats, LFP-WAT rats exhibited transient locomotor hyperactivity without signs of anxiety, minor spatial learning acquisition and hippocampal long-term potentiation deficits, and lower baseline activity of the hypothalamic-pituitary-adrenal axis with slightly stronger reactivity to restraint stress. Exposure to TMT had only negligible effects on Sham rats, whereas it exacerbated all deficits in LFP rats except for locomotor hyperactivity. Early brain inflammatory response (8 h post-trauma) was aggravated in rats pre-exposed to TMT, suggesting that increased brain inflammation may sustain functional deficits in these rats. Hence, these data suggest that pre-exposure to stressful conditions can aggravate long-term deficits induced by TBI, leading to severe stress response deficits, possibly due to dysregulated inflammatory response.

WNT4 mediates the autocrine effects of growth hormone in mammary carcinoma cells.

The expression of Wingless and Int-related protein (Wnt) ligands is aberrantly high in human breast cancer. We report here that WNT4 is significantly upregulated at the mRNA and protein level in mammary carcinoma cells expressing autocrine human growth hormone (hGH). Depletion of WNT4 using small interfering (si) RNA markedly decreased the rate of human breast cancer cell proliferation induced by autocrine hGH. Forced expression of WNT4 in the nonmalignant human mammary epithelial cell line MCF-12A stimulated cell proliferation in low and normal serum conditions, enhanced cell survival and promoted anchorage-independent growth and colony formation in soft agar. The effects of sustained production of WNT4 were concomitant with upregulation of proliferative markers (c-Myc, Cyclin D1), the survival marker BCL-XL, the putative WNT4 receptor FZD6 and activation of ERK1 and STAT3. Forced expression of WNT4 resulted in phenotypic conversion of MCF-12A cells, such that they exhibited the molecular and morphological characteristics of mesenchymal cells with increased cell motility. WNT4 production resulted in increased mesenchymal and cytoskeletal remodeling markers, promoted actin cytoskeleton reorganization and led to dissolution of cell-cell contacts. In xenograft studies, tumors with autocrine hGH expressed higher levels of WNT4 and FZD6 when compared with control tumors. In addition, Oncomine data indicated that WNT4 expression is increased in neoplastic compared with normal human breast tissue. Accordingly, immunohistochemical detection of WNT4 in human breast cancer biopsies revealed higher expression in tumor tissue vs normal breast epithelium. WNT4 is thus an autocrine hGH-regulated gene involved in the growth and development of the tumorigenic phenotype.

Locus Coeruleus Dysfunction in Transgenic Rats with Low Brain Angiotensinogen.

AIMS: Transgenic TGR(ASrAOGEN)680 (TGR) rats with specific downregulation of glial angiotensinogen (AOGEN) synthesis develop cardiovascular deficits, anxiety, altered response to stress, and depression. Here, we evaluated whether these deficits are associated with alteration of the integrity of the noradrenergic system originating from locus coeruleus (LC) neurons. METHODS: Adult TGR rats were compared to control Sprague Dawley rats in terms of the following: tissue levels of transcripts encoding noradrenergic markers, tissue tyrosine hydroxylase (TH) protein level, in vivo TH activity, density of TH-containing fibers, behavioral response to novelty, locomotor activity, and polysomnography. RESULTS: TH expression was increased in the LC of TGR rats compared to controls. In LC terminal fields, there was an increase in density of TH-containing fibers in TGR rats that was associated with an elevation of in vivo TH activity. TGR rats also displayed locomotor hyperactivity in response to novelty. Moreover, polysomnographic studies indicated that daily paradoxical sleep duration was increased in TGR rats and that the paradoxical sleep rebound triggered by total sleep deprivation was blunted in these rats. CONCLUSIONS: Altogether, these results suggest that disruption of astroglial AOGEN synthesis leads to cardiovascular, cognitive, behavioral, and sleep disorders that might be partly due to LC dysfunction.

Neuronal loss as evidenced by automated quantification of neuronal density following moderate and severe traumatic brain injury in rats.

Traumatic brain injury causes widespread neurological lesions that can be reproduced in animals with the lateral fluid percussion (LFP) model. The characterization of the pattern of neuronal death generated in this model remains unclear, involving both cortical and subcortical brain regions. Here, 7 days after moderate (3 atmospheres absolute [ATA]) or severe (3.8 ATA) LFP, we estimated neuronal loss by using immunohistochemistry together with a computer-assisted automated method for quantifying neuronal density in brain sections. Neuronal counts were performed ipsilateral to the impact, in the parietal cortex ventral to the site of percussion, in the temporal cortex, in the dorsal thalamus, and in the hippocampus. These results were compared with the counts observed at similar areas in sham animals. We found that neuronal density was severely decreased in the temporal cortex (-60%), in the dorsal thalamus (-63%), and in area CA3 of the hippocampus (-36%) of injured animals compared with controls but was not significantly modified in the cortices located immediately ventral to the impact. Total cellular density increased in brain structures displaying neuronal death, suggesting the presence of gliosis. The increase in the severity of LFP did not change the pattern of neuronal injury. This automated method simplified the study of neuronal loss following traumatic brain injury and allowed the identification of a pattern of neuronal loss that spreads from the dorsal thalamus to the temporal cortex, with the most severe lesions being in brain structures remote from the site of impact.

Automated immunohistochemical method to quantify neuronal density in brain sections: application to neuronal loss after status epilepticus.

BACKGROUND: To study neurotoxic processes, it is necessary to quantify the number of neurons in a given brain structure and estimate neuronal loss. Neuronal densities can be estimated by immunohistochemical quantitation of a neuronal marker such as the protein NeuN. However, NeuN expression may vary, depending on certain pathophysiological conditions and bias such quantifications. NEW METHOD: We have developed a simple automatic quantification of neuronal densities in brain sections stained with DAPI and antibody to NeuN. This method determines the number of DAPI-positive nuclei also positive for NeuN in at least two adjacent sections within a Z-stack of optical sections. RESULTS: We tested this method in animals with induced status epilepticus (SE) a state of intractable persistent seizure that produces extensive neuronal injury. We found that SE significantly reduced neuronal density in the piriform cortex, the amygdala, the dorsal thalamus, the CA3 area of the hippocampus, the dentate gyrus and the hilus, but not in the somatosensory cortex or the CA1 area. SE resulted in increases in the total density of cellular nuclei within these brain structures, suggesting gliosis. COMPARISON WITH EXISTING METHODS: This automated method was more accurate than simply estimating the overall NeuN fluorescence intensity in the brain section, and as accurate, but less time-consuming, than manual cell counts. CONCLUSION: This method simplifies and accelerates the unbiased quantification of neuronal density. It can be easily applied to other models of brain injury and neurodegeneration, or used to screen the efficacy of neuroprotective treatments.

Stochastic loss of silencing of the imprinted Ndn/NDN allele, in a mouse model and humans with prader-willi syndrome, has functional consequences.

Genomic imprinting is a process that causes genes to be expressed from one allele only according to parental origin, the other allele being silent. Diseases can arise when the normally active alleles are not expressed. In this context, low level of expression of the normally silent alleles has been considered as genetic noise although such expression has never been further studied. Prader-Willi Syndrome (PWS) is a neurodevelopmental disease involving imprinted genes, including NDN, which are only expressed from the paternally inherited allele, with the maternally inherited allele silent. We present the first in-depth study of the low expression of a normally silent imprinted allele, in pathological context. Using a variety of qualitative and quantitative approaches and comparing wild-type, heterozygous and homozygous mice deleted for Ndn, we show that, in absence of the paternal Ndn allele, the maternal Ndn allele is expressed at an extremely low level with a high degree of non-genetic heterogeneity. The level of this expression is sex-dependent and shows transgenerational epigenetic inheritance. In about 50% of mutant mice, this expression reduces birth lethality and severity of the breathing deficiency, correlated with a reduction in the loss of serotonergic neurons. In wild-type brains, the maternal Ndn allele is never expressed. However, using several mouse models, we reveal a competition between non-imprinted Ndn promoters which results in monoallelic (paternal or maternal) Ndn expression, suggesting that Ndn allelic exclusion occurs in the absence of imprinting regulation. Importantly, specific expression of the maternal NDN allele is also detected in post-mortem brain samples of PWS individuals. Our data reveal an unexpected epigenetic flexibility of PWS imprinted genes that could be exploited to reactivate the functional but dormant maternal alleles in PWS. Overall our results reveal high non-genetic heterogeneity between genetically identical individuals that might underlie the variability of the phenotype.