Why considering sexual differences is necessary when studying encephalopathy of prematurity through rodent models.

Preterm birth is a high-risk factor for the development of gray and white matter abnormalities, referred to as "encephalopathy of prematurity," that may lead to life-long motor, cognitive, and behavioral impairments. The prevalence and clinical outcomes of encephalopathy of prematurity differ between sexes, and elucidating the underlying biological basis has become a high-priority challenge. Human studies are often limited to assessment of brain region volumes by MRI, which does not provide much information about the underlying mechanisms of lesions related to very preterm birth. However, models using KO mice or pharmacological manipulations in rodents allow relevant observations to help clarify the mechanisms of injury sustaining sex-differential vulnerability. This review focuses on data obtained from mice aged P1-P5 or rats aged P3 when submitted to cerebral damage such as hypoxia-ischemia, as their brain lesions share similarities with lesion patterns occurring in very preterm human brain, before 32 gestational weeks. We first report data on the mechanisms underlying the development of sexual brain dimorphism in rodent, focusing on the hippocampus. In the second part, we describe sex specificities of rodent models of encephalopathy of prematurity (RMEP), focusing on mechanisms underlying differences in hippocampal vulnerability. Finally, we discuss the relevance of these RMEP. Together, this review highlights the need to systematically search for potential effects of sex when studying the mechanisms underlying deficits in RMEP in order to design effective sex-specific medical interventions in human preterms.

Time- and sex-dependent efficacy of magnesium sulfate to prevent behavioral impairments and cerebral damage in a mouse model of cerebral palsy.

Cerebral lesions acquired in the perinatal period can induce cerebral palsy (CP), a multifactorial pathology leading to lifelong motor and cognitive deficits. Several risk factors, including perinatal hypoxia-ischemia (HI), can contribute to the emergence of CP in preterm infants. Currently, there is no international consensus on treatment strategies to reduce the risk of developing CP. A meta-analysis showed that magnesium sulfate (MgSO4) administration to mothers at risk of preterm delivery reduces the risk of developing CP (Crowther et al., 2017). However, only a few studies have investigated the long-term effects of MgSO4 and it is not known whether sex would influence MgSO4 efficacy. In addition, the search for potential deleterious effects is essential to enable broad use of MgSO4 in maternity wards. We used a mouse model of perinatal HI to study MgSO4 effects until adolescence, focusing on cognitive and motor functions, and on some apoptosis and inflammation markers. Perinatal HI at postnatal day 5 (P(5)) induced (1) sensorimotor deficits in pups; (2) increase in caspase-3 activity 24 h after injury; (3) production of proinflammatory cytokines from 6 h to 5 days after injury; (4) behavioral and histological alterations in adolescent mice with considerable interindividual variability. MgSO4 prevented sensorimotor alterations in pups, with the same efficacy in males and females. MgSO4 displayed anti-apoptotic and anti-inflammatory effects without deleterious side effects. Perinatal HI led to motor coordination impairments in female adolescent mice and cognitive deficits in both sexes. MgSO4 tended to prevent these motor and cognitive deficits only in females, while it prevented global brain tissue damage in both sexes. Moreover, interindividual and intersexual differences appeared regarding the lesion size and neuroprotection by MgSO4 in a region-specific manner. These differences, the partial prevention of disorders, as well as the mismatch between histological and behavioral observations mimic clinical observations. This underlines that this perinatal HI model is suitable to further analyze the mechanisms of sex-dependent perinatal lesion susceptibility and MgSO4 efficacy.

Magnesium Sulfate Prevents Neurochemical and Long-Term Behavioral Consequences of Neonatal Excitotoxic Lesions: Comparison Between Male and Female Mice.

Magnesium sulfate (MgSO4) administration to mothers at risk of preterm delivery is proposed as a neuroprotective strategy against neurological alterations such as cerebral palsy in newborns. However, long-term beneficial or adverse effects of MgSO4 and sex-specific sensitivity remain to be investigated. We conducted behavioral and neurochemical studies of MgSO4 effects in males and females, from the perinatal period to adolescence in a mouse model of cerebral neonatal lesion. The lesion was produced in 5-day-old (P5) pups by ibotenate intracortical injection. MgSO4 (600 mg/kg, i.p.) prior to ibotenate prevented lesion-induced sensorimotor alterations in both sexes at P6 and P7. The lesion increased glutamate level at P10 in the prefrontal cortex, which was prevented by MgSO4 in males. In neonatally lesioned adolescent mice, males exhibited more sequelae than females in motor and cognitive functions. In the perirhinal cortex of adolescent mice, the neonatal lesion induced an increase in vesicular glutamate transporter 1 density in males only, which was negatively correlated with cognitive scores. Long-term sequelae were prevented by neonatal MgSO4 administration. MgSO4 never induced short- or long-term deleterious effect on its own. These results also strongly suggest that sex-specific neuroprotection should be foreseen in preterm infants.

Experimental and clinical evidence of differential effects of magnesium sulfate on neuroprotection and angiogenesis in the fetal brain.

Clinical studies showed beneficial effects of magnesium sulfate regarding the risk of cerebral palsy. However, regimen protocols fluctuate worldwide and risks of adverse effects impacting the vascular system have been reported for human neonates, keeping open the question of the optimal dosing. Using clinically relevant concentrations and doses of magnesium sulfate, experiments consisted of characterizing, respectively, ex vivo and in vivo, the effects of magnesium sulfate on the nervous and vascular systems of mouse neonates by targeting neuroprotection, angiogenesis, and hemodynamic factors and in measuring, in human fetuses, the impact of a 4-g neuroprotective loading dose of magnesium sulfate on brain hemodynamic parameters. Preclinical experiments using cultured cortical slices from mouse neonates showed that the lowest and highest tested concentrations of magnesium sulfate were equally potent to prevent excitotoxic-induced cell death, cell edema, cell burst, and intracellular calcium increase, whereas no side effects were found regarding apoptosis. In contrast, in vivo data revealed that magnesium sulfate exerted dose-dependent vascular effects on the fetal brain. In particular, it induced brain hypoperfusion, stabilization of Hif-1α, long-term upregulation of VEGF-R2 expression, impaired endothelial viability, and altered cortical angiogenesis. Clinically, in contrast to 6-g loading doses used in some protocols, a 4-g bolus of magnesium sulfate did not altered fetal brain hemodynamic parameters. In conclusion, these data provide the first mechanistic evidence of double-sword and dose-dependent actions of magnesium sulfate on nervous and vascular systems. They strongly support the clinical use of neuroprotection protocols validated for the lowest (4-g) loading dose of magnesium sulfate.

Age-dependent alterations of the NMDA receptor developmental profile and adult behavior in postnatally ketamine-treated mice.

Ketamine is a NMDA receptor (NMDAR) antagonist used in pediatric anesthesia. Given the role of glutamatergic signaling during brain maturation, we studied the effects of a single ketamine injection (40 mg/kg s.c) in mouse neonates depending on postnatal age at injection (P2, P5, or P10) on cortical NMDAR subunits expression and association with Membrane-Associated Guanylate Kinases PSD95 and SAP102. The effects of ketamine injection at P2, P5, or P10 on motor activity were compared in adulthood. Ketamine increased GluN2A and GluN2B mRNA levels in P2-treated mice without change in proteins, while it decreased GluN2B protein in P10-treated mice without change in mRNA. Ketamine reduced GluN2A mRNA and protein levels in P5-treated mice without change in GluN2B and GluN1. Ketamine affected the GluN2A/PSD95 association regardless of the age at injection, while GluN2B/PSD95 association was enhanced only in P5-treated mice. Microdissection of ketamine-treated mouse cortex showed a decrease in GluN2A mRNA level in superficial layers (I-IV) and an increase in all subunit expressions in deep layers (V-VI) in P5- and P10-treated mice, respectively. Our data suggest that ketamine impairs cortical NMDAR subunit developmental profile and delays the synaptic targeting of GluN2A-enriched NMDAR. Ketamine injection at P2 or P10 resulted in hyperlocomotion in adult male mice in an open field, without change in females. Voluntary running-wheel exercise showed age- and sex-dependent alterations of the mouse activity, especially during the dark phase. Overall, a single neonatal ketamine exposure led to short-term NMDAR cortical developmental profile impairments and long-term motor activity alterations persisting in adulthood.

Age-dependent neonatal intracerebral hemorrhage in plasminogen activator inhibitor 1 knockout mice.

Intracerebral-intraventricular hemorrhages (ICH/IVH) in very preterm neonates are responsible for high mortality and subsequent disabilities. In humans, tissue plasminogen activator (t-PA) initiates fibrinolysis and activates endoluminal-endothelial receptors; dysfunction of the t-PA inhibitor (PAI-1) results in recurrent hemorrhages. We used PAI-1 knockout (PAI-1) mice to examine the role of t-PA in age-dependent intracranial hemorrhages as a possible model of preterm ICH/IVH. Intracortical injection of 2 µL of phosphate-buffered saline produced a small traumatic injury and a high rate of hemorrhage in PAI-1 pups at postnatal day 3 (P3) or P5, whereas it had no effect in wild-type neonates. This resulted in white matter and cortical lesions, ventricle enlargement, hyperlocomotion, and altered cortical levels of serotonin and dopamine in the adult PAI mice. N-methyl-D-aspartate receptor blockers, plasmin- and matrix metalloproteinases inhibitors reduced hemorrhage and tissue lesions. In contrast to P3 to P5, no significant hemorrhages were induced in P10 PAI-1 pups and there were no behavioral or neurochemical alterations in adulthood. These data suggest that microvascular immaturity up to P5 in mice is a determinant factor required for t-PA-dependent vascular rupture. Neonatal PAI-1 mice could be a useful ICH/IVH model for studying the ontogenic window of vascular immaturity and vascular protection against later neurodisabilities.

The efficiency of glutamate uptake differs between neonatal and adult cortical microvascular endothelial cells.

Glutamate transporters (excitatory amino-acid transporters (EAATs)) are essential for brain homeostasis. While previous studies indicate that the vascular endothelium contributes to glutamate efflux in the adult brain, little information is available regarding glutamate uptake in the immature brain. The present study shows a differential expression pattern of EAATs between cortical microvessels in adults and newborns. In addition, adult cortical endothelial cells take up glutamate more efficiently than neonatal cells. Our findings indicate age-specific changes in extracellular glutamate regulation by brain endothelial cells, suggesting differences in the efficiency of glutamate efflux during an excitotoxic process that, in turn, may contribute to age-specific brain vulnerability.

Acute and subchronic treatments with selective serotonin reuptake inhibitors increase Nociceptin/Orphanin FQ (NOP) receptor density in the rat dorsal raphe nucleus; interactions between nociceptin/NOP system and serotonin.

Nociceptin/Orphanin FQ is the endogenous ligand of NOP receptor, formerly referred to as the Opioid Receptor-Like 1 receptor. We have previously shown that NOP receptors were located on serotonergic neurons in the rat dorsal raphe nucleus, suggesting possible direct interactions between nociceptin and serotonin in this region, which is a target for antidepressant action. In the present study, we investigated further the link between Selective Serotonin Reuptake Inhibitor (SSRI) antidepressant treatments and the nociceptin/NOP receptor system. Intraperitoneal administration of the SSRI citalopram induced an increase in NOP-receptor density, measured by autoradiographic [(3)H] nociceptin binding, in the rat dorsal raphe nucleus, from the first to the 21st day of treatment. This effect was also observed with other SSRIs (sertraline, fluoxetine), but not with two tricyclic antidepressants (imipramine, clomipramine) and was abolished by pre-treatment with para-chlorophenylalanine, an inhibitor of serotonin synthesis. Using microdialysis experiments, we demonstrated that NOP-receptor activation by infusion of nociceptin 10(-6) M or 10(-5) M increased the level of extracellular serotonin in the dorsal raphe nucleus. This effect was abolished by co-infusion of the NOP-receptor antagonist UFP 101. These results confirm the existence of reciprocal interactions between serotonin and nociceptin/NOP transmissions in the dorsal raphe nucleus.

Serotonin1B heteroreceptor activation induces an antidepressant-like effect in mice with an alteration of the serotonergic system.

OBJECTIVE: We sought to demonstrate whether the specific activation of serotonin1B (5-HT1B) heteroreceptors by systemic or local administration of the selective 5-HT1B receptor agonist anpirtoline could mediate antidepressant-like effects in mice. METHODS: We confirmed the selectivity of action of anpirtoline in the forced swim test (FST) in 5-HT1B knockout mice. We then evaluated the behavioural effects of anpirtoline on 5-HT-lesioned (5,7-dihydroxytryptamine creatinine [5,7-DHT]) and 5-HT-depleted (p-CPA) mice. We estimated the depletion level and selectivity of action of 5,7-DHT and p-CPA by measuring the neurotransmitter levels and [3H]-citalopram binding. We investigated the antidepressant-like effect of anpirtoline when locally perfused in an area of the brain where the response is mainly attributable to presynaptic (cortex and hippocampus) or postsynaptic receptors (substantia nigra and caudate putamen). Furthermore, we evaluated the effect of the 5-HT1B receptor antagonist GR127935 on the activity of various antidepressants in the FST. RESULTS: Anpirtoline was devoid of effects in 5-HT1B receptor knockout mice. It induced a greater effect in p-CPA and 5,7-DHT pretreated mice compared with control subjects, suggesting that the antidepressant-like activity of anpirtoline mainly depends on 5-HT1B heteroreceptor stimulation (autoreceptors being destroyed by 5,7-DHT). This observation was confirmed by the results showing the antidepressant-like effect of anpirtoline when locally perfused in areas of the brain that contain postsynaptic receptors. The blockade of 5-HT1B receptors antagonizes the effect of selective serotonin reuptake inhibitors (SSRIs). CONCLUSION: Our results demonstrate that the antidepressant-like effect of SSRIs in the FST requires the activation of 5-HT1B heteroreceptors.

Brain-derived neurotrophic factor-deficient mice exhibit a hippocampal hyperserotonergic phenotype.

Growing evidence supports the involvement of brain-derived neurotrophic factor (BDNF) in mood disorders and the mechanism of action of antidepressant drugs. However, the relationship between BDNF and serotonergic signalling is poorly understood. Heterozygous mutants BDNF +/- mice were utilized to investigate the influence of BDNF on the serotonin (5-HT) system and the activity of the serotonin transporter (SERT) in the hippocampus. The zero net flux method of quantitative microdialysis revealed that BDNF +/- heterozygous mice have increased basal extracellular 5-HT levels in the hippocampus and decreased 5-HT reuptake capacity. In keeping with these results, the selective serotonin reuptake inhibitor paroxetine failed to increase hippocampal extracellular 5-HT levels in BDNF +/- mice while it produced robust effects in wild-type littermates. Using in-vitro autoradiography and synaptosome techniques, we investigated the causes of attenuated 5-HT reuptake in BDNF +/- mice. A significant decrease in [3H]citalopram-binding-site density in the CA3 subregion of the ventral hippocampus and a significant reduction in [3H]5-HT uptake in hippocampal synaptosomes, revealed mainly a decrease in SERT function. However, 5-HT1A autoreceptors were not desensitized in BDNF +/- mice. These results provide evidence that constitutive reductions in BDNF modulate SERT function reuptake in the hippocampus.

Consequences of changes in BDNF levels on serotonin neurotransmission, 5-HT transporter expression and function: studies in adult mice hippocampus.

In vivo intracerebral microdialysis is an important neurochemical technique that has been applied extensively in genetic and pharmacological studies aimed at investigating the relationship between neurotransmitters. Among the main interests of microdialysis application is the infusion of drugs through the microdialysis probe (reverse dialysis) in awake, freely moving animals. As an example of the relevance of intracerebral microdialysis, this review will focus on our recent neurochemical results showing the impact of Brain-Derived Neurotrophic Factor (BDNF) on serotonergic neurotransmission in basal and stimulated conditions. Indeed, although the elevation of 5-HT outflow induced by chronic administration of selective serotonin reuptake inhibitors (SSRIs) causes an increase in BDNF protein levels and expression (mRNA) in the hippocampus of rodents, the reciprocal interaction has not been demonstrated yet. Thus, the neurochemical sight of this question will be addressed here by examining the consequences of either a constitutive decrease or increase in brain BDNF protein levels on hippocampal extracellular levels of 5-HT in conscious mice.

Coupling of ORL1 (NOP) receptor to G proteins is decreased in the nucleus accumbens of anxious relative to non-anxious mice.

We studied the involvement of endogenous ORL1 (NOP) receptors in the anxiety state. In mice selected as "anxious" and "non-anxious", ORL1 (NOP) receptor has been analysed by means of two autoradiographic approaches: [3H]nociceptin binding and nociceptin-stimulated [35S]GTPgammaS binding. We show that differences in anxiety state are associated with differences in G protein coupling efficiency of ORL1 (NOP) receptor in the nucleus accumbens, without any change in the density of the receptors.

Comparisons between anxiety tests for selection of anxious and non anxious mice.

Male Swiss albinos mice were submitted to two behavioural tests intended to determine their anxiety level: the elevated plus-maze test as well as the black and white compartments test. In addition they were submitted to the hole-board test. It was observed: (i) that the correlation between scores in the two first tests was weak, suggesting that they explore different components of anxiety; (ii) that the score on the latter test was better correlated with the response in the elevated plus-maze test than in the black and white compartments test. From these data three groups of animals were constituted, considered, respectively, as anxious, non anxious and intermediates. It was observed that both horizontal and vertical locomotion in an unfamiliar environment differed between groups, with higher activity in non anxious than in anxious. In the hole-board test, only animals classified as anxious displayed an obvious response to the anxiolytic drug diazepam (0.5mg/kg). Finally in the forced-swimming test, the three groups demonstrated a similar immobility time, suggesting that the operated segregation was not depending on a helpless component. It is proposed that the selection of mice from a combination of either elevated plus-maze and black and white compartments tests or a combination of hole-board test and black and white compartments test, allows to distinguish high or low anxiety animals among a population of mice.

Opioid receptor-like 1 (NOP) receptors in the rat dorsal raphe nucleus: evidence for localization on serotoninergic neurons and functional adaptation after 5,7-dihydroxytryptamine lesion.

A high density of opioid receptor-like 1 (ORL1) receptor (also referred to as NOP receptor) is found in limbic areas and in regions containing monoamines, which are implicated in emotional activity and physiopathology of depression and anxiety. We aimed at defining precisely the localization of ORL1 receptors in dorsal raphe nucleus, by means of a lesion strategy and autoradiographic studies. In control rats, [3H]nociceptin and nociceptin-stimulated [35S]GTPgammaS bindings were found to be correlated in several brain regions. We performed in rats a selective destruction of serotoninergic neurons by surgical stereotaxic injection of 5,7-dihydroxytryptamine (5,7-DHT) in dorsal raphe nucleus. This led to a marked decrease in serotonin contents in striata and frontal cortices (about -60%) and in autoradiographic [3H]citalopram binding in posterior regions. In dorsal raphe nucleus, [3H]nociceptin binding was decreased to the same extent as [3H]citalopram binding, whereas it was unchanged in the other regions studied. Nevertheless, in the dorsal raphe, nociceptin-stimulated [35S]GTPgammaS binding was decreased to a lesser extent than [3H]nociceptin binding in 5,7-DHT-lesioned rats. The ratio between nociceptin-stimulated [35S]GTPgammaS binding and [3H]nociceptin binding was significantly increased in 5,7-DHT-lesioned rats compared with controls in this region. These data demonstrate 1) that ORL1 receptors are located on serotoninergic neurons in the dorsal raphe nucleus and 2) that, after a lesion, the functionality of remaining ORL1 receptors appears to be up-regulated, which could correspond to a compensatory mechanism.

Behavioral, neurochemical, and electrophysiological characterization of a genetic mouse model of depression.

Depression is a multifactorial illness and genetic factors play a role in its etiology. The understanding of its physiopathology relies on the availability of experimental models potentially mimicking the disease. Here we describe a model built up by selective breeding of mice with strikingly different responses in the tail suspension test, a stress paradigm aimed at screening potential antidepressants. Indeed, "helpless" mice are essentially immobile in the tail suspension test, as well as the Porsolt forced-swim test, and they show reduced consumption of a palatable 2% sucrose solution. In addition, helpless mice exhibit sleep-wakefulness alterations resembling those classically observed in depressed patients, notably a lighter and more fragmented sleep, with an increased pressure of rapid eye movement sleep. Compared with "nonhelpless" mice, they display higher basal seric corticosterone levels and lower serotonin metabolism index in the hippocampus. Remarkably, serotonin(1A) autoreceptor stimulation induces larger hypothermia and inhibition of serotoninergic neuronal firing in the nucleus raphe dorsalis in helpless than in nonhelpless mice. Thus, helpless mice exhibit a decrease in serotoninergic tone, which evokes that associated with endogenous depression in humans. Finally, both the behavioral impairments and the serotoninergic dysfunction can be improved by chronic treatment with the antidepressant fluoxetine. The helpless line of mice may provide an opportunity to approach genes influencing susceptibility to depression and to investigate neurophysiological and neurochemical substrates underlying antidepressant effects.

Differential effects of chronic antidepressant treatments on micro- and delta-opioid receptors in rat brain.

We performed an autoradiographic study of [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin (DAMGO)-sensitive [(3)H]naloxone binding to micro-opioid receptors and of [(3)H][D-Pen(2),D-Pen(5)]enkephalin (DPDPE) binding to delta-opioid receptors in the rat brain after 4- or 21-day treatments with paroxetine, reboxetine and moclobemide to investigate the participation of these receptors in the adaptive mechanisms occurring during the delay of action of new generation antidepressants. Paroxetine increased micro-opioid receptor binding site density in cingulate and insular cortices, dorsal endopiriform nucleus (4 days) and olfactory tubercle (21 days) and decreased it in thalamus (21 days). Reboxetine increased it in amygdala (4 days), hippocampus and thalamus (21 days) and decreased it in dorsal raphe (4 days). Moclobemide increased it in hippocampus (4 days) and decreased it in anterior olfactory nucleus, frontal cortex, amygdala and hypothalamus (21 days). Moclobemide increased delta-opioid receptor binding site density in frontal cortex and amygdala (4 days) and decreased it in amygdala and colliculi (21 days). Opioid receptors displayed distinct patterns of adaptations in response to the three antidepressants studied.

A chronic treatment with fluoxetine decreases 5-HT(1A) receptors labeling in mice selected as a genetic model of helplessness.

Two lines of mice were bred for their opposite helpless behavior in the tail suspension test, i.e., helpless (HL) mice and non helpless (NHL) mice. The 5-HT(1A) receptor labeling was quantified by means of autoradiography with (3)H-8-OH-DPAT on brain sections from mice of these two lines. We observed a significantly higher level of (3)H-8-OH-DPAT binding sites density in HL mice comparatively to NHL mice, in the medial prefrontal, cingulate, motor and sensorial cortices, in several regions of the limbic system, such as CA3 field of hippocampus, dentate gyrus, medial and baso-medial amygdala, and in dorsal and median raphe nuclei. A chronic 21-day treatment with the antidepressant fluoxetine (10 mg/kg, i.p. daily) attenuated significantly the spontaneous helplessness in HL mice but did not alter the behavior of NHL mice. In the brain of HL mice chronically injected with fluoxetine, the elevated (3)H-8-OH-DPAT binding sites density was no longer observed after treatment in several regions, among which the raphe nuclei. Conversely, the antidepressant treatment did not modify the (3)H-8-OH-DPAT binding sites density in NHL mice. The variation of 5-HT(1A) receptors binding density in the HL mice in response to a chronic fluoxetine treatment parallels the attenuation of the spontaneous helplessness observed in the tail suspension test, and may underlie this behavior.

Chronic paroxetine increases [3H]nociceptin binding in rat dorsal raphe nucleus.

Opioid-receptor-like I (ORLI) receptors, ORLI mRNA and nociceptin are particularly abundant in the limbic system and in central monoaminergic areas, brain regions involved in mood regulation and response to antidepressants. To analyse whether ORLI receptors adaptations occur during the first 3 weeks of an antidepressant treatment, we administered paroxetine to rats (10 mg/kg, i.p., once a day) for 4, 7,14 or 21 days. A significant increase (22-50%) in [3H]nociceptin binding sites density appeared specifically in the dorsal raphe nucleus after 4, 7 or 21 days of treatment, whereas no change was observed at any time in any other brain regions studied. These data constitute the first evidence of a modulation of ORLI receptors by an antidepressant treatment.