Prenatal reduction of E14.5 embryonically fate-mapped pyramidal neurons in a mouse model of autism.

Although several observations suggest that the constitutive biological, genetic or physiological changes leading to autism spectrum disorders (ASD) start in utero, their early impact on the number and density of neurons in the brain remains unknown. Using genetic fate mapping associated with the immunollabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO) clearing method we identified and counted a selective population of neocortical and hippocampal pyramidal neurons in the in utero valproate (VPA) mouse model of autism. We report that 1 day before birth, the number of pyramidal neurons born at E14.5 in the neocortex and hippocampus of VPA mice is smaller than in age-matched controls. VPA also induced a reduction of the neocortical-but not hippocampal-volume 1 day before birth. Interestingly, VPA mice present an increase in both neocortical and hippocampal volumes 2 days after birth compared with controls. These results suggest that the VPA-exposed hippocampus and neocortex differ substantially from controls during the highly complex perinatal period, and specially 1 day before birth, reflecting the early pathogenesis of ASD.

Term or Preterm Cesarean Section Delivery Does Not Lead to Long-term Detrimental Consequences in Mice.

Epidemiological studies have provided contradictory data on the deleterious sequels of cesarean section (C-section) delivery and their links with developmental brain disorders such as Autism Spectrum Disorders. To gain better insight on these issues, we have now compared physiological, morphological, and behavioral parameters in vaginal, term, and preterm C-section delivered mice. We report that C-section delivery does not lead to long-term behavioral alterations though preterm C-section delivery modifies communicative behaviors in pups. Moreover, C-section delivery neither alters the gamma-aminobutyric acid (GABA) developmental excitatory to inhibitory shift nor the frequency or amplitude of glutamatergic and GABAergic postsynaptic currents in hippocampal pyramidal neurons. However, these neurons present an underdeveloped dendritic arbor at birth in pups born by C-section delivery, but this difference disappears 1 day later suggesting an accelerated growth after birth. Therefore, C-section delivery, with prematurity as an aggravating factor, induces transient developmental delays but neither impacts the GABA developmental sequence nor leads to long-term consequences in mice. The deleterious sequels of C-section delivery described in epidemiological studies might be due to a perinatal insult that could be aggravated by C-section delivery.

The GABA Developmental Shift Is Abolished by Maternal Immune Activation Already at Birth.

Epidemiological and experimental studies suggest that maternal immune activation (MIA) leads to developmental brain disorders, but whether the pathogenic mechanism impacts neurons already at birth is not known. We now report that MIA abolishes in mice the oxytocin-mediated delivery γ-aminobutyric acid (GABA) shift from depolarizing to hyperpolarizing in CA3 pyramidal neurons, and this is restored by the NKCC1 chloride importer antagonist bumetanide. Furthermore, MIA hippocampal pyramidal neurons at birth have a more exuberant apical arbor organization and increased apical dendritic length than age-matched controls. The frequency of spontaneous glutamatergic postsynaptic currents is also increased in MIA offspring, as well as the pairwise correlation of the synchronized firing of active cells in CA3. These alterations produced by MIA persist, since at P14-15 GABA action remains depolarizing, produces excitatory action, and network activity remains elevated with a higher frequency of spontaneous glutamatergic postsynaptic currents. Therefore, the pathogenic actions of MIA lead to important morphophysiological and network alterations in the hippocampus already at birth.

Enhanced Glutamatergic Currents at Birth in Shank3 KO Mice.

Autism spectrum disorders (ASD) are neurodevelopmental disorders induced by genetic and environmental factors. In our recent studies, we showed that the GABA developmental shifts during delivery and the second postnatal week are abolished in two rodent models of ASD. Maternal treatment around birth with bumetanide restored the GABA developmental sequence and attenuated the autism pathogenesis in offspring. Clinical trials conducted in parallel confirmed the usefulness of bumetanide treatment to attenuate the symptoms in children with ASD. Collectively, these observations suggest that an alteration of the GABA developmental sequence is a hallmark of ASD. Here, we investigated whether similar alterations occur in the Shank3 mouse model of ASD. We report that in CA3 pyramidal neurons, the driving force and inhibitory action of GABA are not different in naïve and Shank3-mutant age-matched animals at birth and during the second postnatal week. In contrast, the frequency of spontaneous excitatory postsynaptic currents is already enhanced at birth and persists through postnatal day 15. Therefore, in CA3 pyramidal neurons of Shank3-mutant mice, glutamatergic but not GABAergic activity is affected at early developmental stages, hence reflecting the heterogeneity of mechanisms underlying the pathogenesis of ASD.

Alteration in the time and/or mode of delivery differentially modulates early development in mice.

Delivery is a complex biological process involving hormonal and mechanical stimuli that together condition the survival and development of the fetus out of the womb. Accordingly, changes in the time or way of being born are associated with an alteration of fundamental biological functions and hypothesized to promote the emergence of neurodevelopmental disorders. Hence, the steadily rise in preterm birth and cesarean section (CS) delivery rates over the past years has become a worldwide health concern. In our previous work, we reported that even though no long-term autistic-like deficits were observed, mice born preterm by CS presented early transient neuronal and communicative defects. However, understanding if these alterations were due to an early birth combined with CS delivery, or if prematurity solely could lead to a similar outcome remained to be evaluated. Using mice born either at term or preterm by vaginal or CS delivery, we assessed early life ultrasonic vocalizations and the onset of eye opening. We report that alterations in communicative behaviors are finely attuned and specifically affected either by preterm birth or by the association between CS delivery and preterm birth in mice, while delayed onset of eye opening is due to prematurity. Moreover, our work further underlies a gender-dependent vulnerability to changes in the time and/or way of being born with distinct outcomes observed in males and females. Thus, our results shed light on the intricacy of birth alterations and might further explain the disparities reported in epidemiological studies.

Abnormal Development of Glutamatergic Synapses Afferent to Dopaminergic Neurons of the Pink1(-/-) Mouse Model of Parkinson's Disease.

In a preceding study, we showed that in adult pink1(-/-) mice, a monogenic animal model of Parkinson's disease (PD), striatal neurons display aberrant electrical activities that precede the onset of overt clinical manifestations. Here, we tested the hypothesis that the maturation of dopaminergic (DA) neurons of the pink1(-/-) substantia nigra compacta (SNc) follows, from early stages on, a different developmental trajectory from age-matched wild type (wt) SNc DA neurons. We used immature (postnatal days P2-P10) and young adult (P30-P90) midbrain slices of pink1(-/-) mice expressing the green fluorescent protein in tyrosine hydroxylase (TH)-positive neurons. We report that the developmental sequence of N-Methyl-D-aspartic acid (NMDA) spontaneous excitatory postsynaptic currents (sEPSCs) is altered in pink1(-/-) SNc DA neurons, starting from shortly after birth. They lack the transient episode of high NMDA receptor-mediated neuronal activity characteristic of the immature stage of wt SNc DA neurons. The maturation of the membrane resistance of pink1(-/-) SNc DA neurons is also altered. Collectively, these observations suggest that electrical manifestations occurring shortly after birth in SNc DA neurons might lead to dysfunction in dopamine release and constitute an early pathogenic mechanism of PD.

Response to Comment on "Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring".

Bambini-Junior et al. questioned whether our treatment in two rodent models of autism has a long-lasting effect into adulthood. In response, we show that bumetanide treatment around delivery attenuates autistic behavioral features in adult offspring. Therefore, the polarity of γ-aminobutyric acid (GABA) actions during delivery exerts long-lasting priming actions after birth.

Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring.

We report that the oxytocin-mediated neuroprotective γ-aminobutyric acid (GABA) excitatory-inhibitory shift during delivery is abolished in the valproate and fragile X rodent models of autism. During delivery and subsequently, hippocampal neurons in these models have elevated intracellular chloride levels, increased excitatory GABA, enhanced glutamatergic activity, and elevated gamma oscillations. Maternal pretreatment with bumetanide restored in offspring control electrophysiological and behavioral phenotypes. Conversely, blocking oxytocin signaling in naïve mothers produced offspring having electrophysiological and behavioral autistic-like features. Our results suggest a chronic deficient chloride regulation in these rodent models of autism and stress the importance of oxytocin-mediated GABAergic inhibition during the delivery process. Our data validate the amelioration observed with bumetanide and oxytocin and point to common pathways in a drug-induced and a genetic rodent model of autism.

Mechanisms and effects of seizures in the immature brain.

The developing immature brain is not simply a small adult brain but rather possesses unique physiological properties. These include neuronal ionic currents that differ markedly from those in the adult brain, typically being longer-lasting and less selective. This enables immature heterogeneous neurons to connect and fire together but at the same time, along with other features may contribute to the enhanced propensity of the developing brain to become epileptic. Indeed, immature neurons tend to readily synchronize and thus generate seizures. Here, we review the differences between the immature and adult brain, with particular focus on the developmental sequence of γ-aminobutyric acid that excites immature neurons while being inhibitory in the normal adult brain. We review the mechanisms underlying the developmental changes to intracellular chloride levels, as well as how epileptiform activity can drive pathologic changes to chloride balance in the brain. We show that regulation of intracellular chloride is one important factor that underlies both the ease with which seizures can be generated and the facilitation of further seizures. We stress in particular the importance of understanding normal developmental sequences and how they are interrupted by seizures and other insults, and how this knowledge has led to the identification of potential novel treatments for conditions such as neonatal seizures.

Midbrain dopaminergic neurons generate calcium and sodium currents and release dopamine in the striatum of pups.

Midbrain dopaminergic neurons (mDA neurons) are essential for the control of diverse motor and cognitive behaviors. However, our understanding of the activity of immature mDA neurons is rudimentary. Rodent mDA neurons migrate and differentiate early in embryonic life and dopaminergic axons enter the striatum and contact striatal neurons a few days before birth, but when these are functional is not known. Here, we recorded Ca(2+) transients and Na(+) spikes from embryonic (E16-E18) and early postnatal (P0-P7) mDA neurons with dynamic two-photon imaging and patch clamp techniques in slices from tyrosine hydroxylase-GFP mice, and measured evoked dopamine release in the striatum with amperometry. We show that half of identified E16-P0 mDA neurons spontaneously generate non-synaptic, intrinsically driven Ca(2+) spikes and Ca(2+) plateaus mediated by N- and L-type voltage-gated Ca(2+) channels. Starting from E18-P0, half of the mDA neurons also reliably generate overshooting Na(+) spikes with an abrupt maturation at birth (P0 = E19). At that stage (E18-P0), dopaminergic terminals release dopamine in a calcium-dependent manner in the striatum in response to local stimulation. This suggests that mouse striatal dopaminergic synapses are functional at birth.

Differential regulation of BACE1 promoter activity by nuclear factor-kappaB in neurons and glia upon exposure to beta-amyloid peptides.

The brains of Alzheimer's disease (AD) patients display cerebrovascular and parenchymal deposits of beta-amyloid (A beta) peptides, which are derived by proteolytic processing by the beta-site APP-cleaving enzyme 1 (BACE1) of the amyloid precursor protein (APP). The rat BACE1 promoter has a nuclear factor-kappaB (NF-kappaB) binding site. Deletion studies with a BACE1 promoter/luciferase reporter suggest that the NF-kappaB binding DNA consensus sequence plays a suppressor role, when occupied by NF-kappaB, in the regulation of neuronal brain BACE1 expression. Here we characterize a signal transduction pathway that may be responsible for the increases in A beta associated with AD. We propose that the transcription factor NF-kappaB acts as a repressor in neurons but as an activator of BACE1 transcription in activated astrocytes present in the CNS under chronic stress, a feature present in the AD brain. The activated astrocytic stimulation of BACE1 may in part account for increased BACE1 transcription and subsequent processing of Ab eta in a cell-specific manner in the aged and AD brain. As measured by reporter gene promoter constructs and endogenous BACE1 protein expression, a functional NF-kappaB site was stimulatory in activated astrocytes and A beta-exposed neuronal cells and repressive in neuronal and nonactivated astrocytic cells. Given the evidence for increased levels of activated astrocytes in the aged brain, the age- and AD-associated increases in NF-kappaB in brain may be significant contributors to increases in A beta, acting as a positive feedback loop of chronic inflammation, astrocyte activation, increased p65/p50 activation of BACE1 transcription, and further inflammation.