Prenatal valproate exposure differentially affects parvalbumin-expressing neurons and related circuits in the cortex and striatum of mice

Autism spectrum disorders (ASD) comprise a number of heterogeneous neurodevelopmental diseases characterized by core behavioral symptoms in the domains of social interaction, language/communication and repetitive or stereotyped patterns of behavior. In utero exposure to valproic acid (VPA) has evolved as a highly recognized rodent ASD model due to the robust behavioral phenotype observed in the offspring and the proven construct-, face- and predictive validity of the model. The number of parvalbumin-immunoreactive (PV+) GABAergic interneurons has been consistently reported to be decreased in human ASD subjects and in ASD animal models. The presumed loss of this neuron subpopulation hereafter termed Pvalb neurons and/or PV deficits were proposed to result in an excitation/inhibition imbalance often observed in ASD. Importantly, loss of Pvalb neurons and decreased/absent PV protein levels have two fundamentally different consequences. Thus, Pvalb neurons were investigated in in utero VPA-exposed male (“VPA”) mice in the striatum, medial prefrontal cortex (mPFC) and somatosensory cortex (SSC), three ASD-associated brain regions. Unbiased stereology of PV+ neurons and Vicia Villosa Agglutinin-positive (VVA+) perineuronal nets, which specifically enwrap Pvalb neurons, was carried out. Analyses of PV protein expression and mRNA levels for Pvalb, Gad67, Kcnc1, Kcnc2, Kcns3, Hcn1, Hcn2, and Hcn4 were performed. We found a ∼15% reduction in the number of PV+ cells and decreased Pvalb mRNA and PV protein levels in the striatum of VPA mice compared to controls, while the number of VVA+ cells was unchanged, indicating that Pvalb neurons were affected at the level of the transcriptome. In selected cortical regions (mPFC, SSC) of VPA mice, no quantitative loss/decrease of PV+ cells was observed. However, expression of Kcnc1, coding for the voltage-gated potassium channel Kv3.1 specifically expressed in Pvalb neurons, was decreased by ∼40% in forebrain lysates of VPA mice. Moreover, hyperpolarization-activated cyclic nucleotide-gated channel (HCN) 1 expression was increased by ∼40% in the same samples from VPA mice. We conclude that VPA leads to alterations that are brain region- and gene-specific including Pvalb, Kcnc1, and Hcn1 possibly linked to homeostatic mechanisms. Striatal PV down-regulation appears as a common feature in a subset of genetic (Shank3B-/-) and environmental ASD models

17-β estradiol increases parvalbumin levels in Pvalb heterozygous mice and attenuates behavioral phenotypes with relevance to autism core symptoms

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by two core symptoms: impaired social interaction and communication, and restricted, repetitive behaviors and interests. The pathophysiology of ASD is not yet fully understood, due to a plethora of genetic and environmental risk factors that might be associated with or causal for ASD. Recent findings suggest that one putative convergent pathway for some forms of ASD might be the downregulation of the calcium-binding protein parvalbumin (PV). PV-deficient mice (PV−/−, PV+/−), as well as Shank1−/−, Shank3−/−, and VPA mice, which show behavioral deficits relevant to all human ASD core symptoms, are all characterized by lower PV expression levels.Methods: Based on the hypothesis that PV expression might be increased by 17-β estradiol (E2), PV+/− mice were treated with E2 from postnatal days 5–15 and ASD-related behavior was tested between postnatal days 25 and 31.Results: PV expression levels were significantly increased after E2 treatment and, concomitantly, sociability deficits in PV+/− mice in the direct reciprocal social interaction and the 3- chamber social approach assay, as well as repetitive behaviors, were attenuated. E2 treatment of PV+/+ mice did not increase PV levels and had detrimental effects on sociability and repetitive behavior. In PV−/− mice, E2 obviously did not affect PV levels; tested behaviors were not different from the ones in vehicle-treated PV−/− mice.Conclusion: Our results suggest that the E2-linked amelioration of ASD-like behaviors is specifically occurring in PV+/− mice, indicating that PV upregulation is required for the E2-mediated rescue of ASD-relevant behavioral impairments

Parvalbumin-expressing ependymal cells in rostral lateral ventricle wall adhesions contribute to aging-related ventricle stenosis in mice

Aging-associated ependymal-cell pathologies can manifest as ventricular gliosis, ventricle enlargement, or ventricle stenosis. Ventricle stenosis and fusion of the lateral ventricle (LV) walls is associated with a massive decline of the proliferative capacities of the stem cell niche in the affected subventricular zone (SVZ) in aging mice. We examined the brains of adult C57BL/6 mice and found that ependymal cells located in the adhesions of the medial and lateral walls of the rostral LVs upregulated parvalbumin (PV) and displayed reactive phenotype, similarly to injury-reactive ependymal cells. However, PV+ ependymal cells in the LV-wall adhesions, unlike injury-reactive ones, did not express glial fibrillary acidic protein. S100B+/PV+ ependymal cells found in younger mice diminished in the LV-wall adhesions throughout aging. We found that periventricular PV-immunofluorescence showed positive correlation to the grade of LV stenosis in nonaged mice (10-month-old) PV-knock out (PV-KO) mice. This suggests an involvement of PV+ ependymal cells in aging-associated ventricle stenosis. Additionally, we observed a time-shift in microglial activation in the LV-wall adhesions between age-grouped PV- KO and wild-type mice, suggesting a delay in microglial activation when PV is absent from ependymal cells. Our findings implicate that compromised ependymal cells of the adhering ependymal layers upregulate PV and display phenotype shift to “reactive” ependymal cells in aging-related ventricle stenosis; moreover, they also contribute to the progression of LV-wall fusion associated with a decline of the affected SVZ-stem cell niche in aged mice

Dysregulation of parvalbumin expression in the Cntnap2−/− mouse model of autism spectrum disorder

Due to the complex and heterogeneous etiology of autism spectrum disorder (ASD), identification of convergent pathways and/or common molecular endpoints in the pathophysiological processes of ASD development are highly needed in order to facilitate treatment approaches targeted at the core symptoms. We recently reported on decreased expression of the Ca2+-binding protein parvalbumin (PV) in three well- characterized ASD mouse models, Shank1−/−, Shank3B−/− and in utero VPA- exposed mice. Moreover, PV-deficient mice (PV+/− and PV−/−) were found to show behavioral impairments and neuroanatomical changes closely resembling those frequently found in human ASD individuals. Here, we combined a stereology-based approach with molecular biology methods to assess changes in the subpopulation of PV-expressing (Pvalb) interneurons in the recently characterized contactin-associated protein-like 2 (Cntnap2−/−) knockout mouse model of ASD. The CNTNAP2 gene codes for a synaptic cell adhesion molecule involved in neurodevelopmental processes; mutations affecting the human CNTNAP2 locus are associated with human ASD core symptoms, in particular speech and language problems. We demonstrate that in Cntnap2−/− mice, no loss of Pvalb neurons is evident in ASD- associated brain regions including the striatum, somatosensory cortex (SSC) and medial prefrontal cortex (mPFC), shown by the unaltered number of Pvalb neurons ensheathed by VVA-positive perineuronal nets. However, the number of PV- immunoreactive (PV+) neurons and also PV protein levels were decreased in the striatum of Cntnap2−/− mice indicating that PV expression levels in some striatal Pvalb neurons dropped below the detection limit, yet without a loss of Pvalb neurons. No changes in PV+ neuron numbers were detected in the cortical regions investigated and also cortical PV expression levels were unaltered. Considering that Cntnap2 shows high expression levels in the striatum during human and mouse embryonic development and that the cortico-striato-thalamic circuitry is important for speech and language development, alterations in striatal PV expression and associated (homeostatic) adaptations are likely to play an important role in Cntnap2−/− mice and, assumingly, in human ASD patients with known Cntnap2 mutations

Reduction in parvalbumin expression not loss of the parvalbumin-expressing GABA interneuron subpopulation in genetic parvalbumin and shank mouse models of autism

Background A reduction of the number of parvalbumin (PV)-immunoreactive (PV⁺) GABAergic interneurons or a decrease in PV immunoreactivity was reported in several mouse models of autism spectrum disorders (ASD). This includes Shank mutant mice, with SHANK being one of the most important gene families mutated in human ASD. Similar findings were obtained in heterozygous (PV+/-) mice for the Pvalb gene, which display a robust ASD-like phenotype. Here, we addressed the question whether the observed reduction in PV immunoreactivity was the result of a decrease in PV expression levels and/or loss of the PV-expressing GABA interneuron subpopulation hereafter called “Pvalb neurons”. The two alternatives have important implications as they likely result in opposing effects on the excitation/inhibition balance, with decreased PV expression resulting in enhanced inhibition, but loss of the Pvalb neuron subpopulation in reduced inhibition.Methods Stereology was used to determine the number of Pvalb neurons in ASD-associated brain regions including the medial prefrontal cortex, somatosensory cortex and striatum of PV-/-, PV+/-, Shank1-/- and Shank3B-/- mice. As a second marker for the identification of Pvalb neurons, we used Vicia Villosa Agglutinin (VVA), a lectin recognizing the specific extracellular matrix enwrapping Pvalb neurons. PV protein and Pvalb mRNA levels were determined quantitatively by Western blot analyses and qRT-PCR, respectively.Results Our analyses of total cell numbers in different brain regions indicated that the observed “reduction of PV⁺ neurons” was in all cases, i.e., in PV+/-, Shank1-/- and Shank3B-/- mice, due to a reduction in Pvalb mRNA and PV protein, without any indication of neuronal cell decrease/loss of Pvalb neurons evidenced by the unaltered numbers of VVA⁺ neurons.ConclusionsOur findings suggest that the PV system might represent a convergent downstream endpoint for some forms of ASD, with the excitation/inhibition balance shifted towards enhanced inhibition due to the down-regulation of PV being a promising target for future pharmacological interventions. Testing whether approaches aimed at restoring normal PV protein expression levels and/or Pvalb neuron function might reverse ASD-relevant phenotypes in mice appears therefore warranted and may pave the way for novel therapeutic treatment strategies

Inducible and reversible silencing of the Pvalb gene in mice: An in vitro and in vivo study

Inducible and reversible regulation of gene expression is a powerful approach for unraveling gene functions. Here, we describe the generation of a system to efficiently downregulate in a reversible and inducible manner the Pvalb gene coding for the calcium‐binding protein parvalbumin (PV) in mice. We made use of an IPTG‐inducible short hairpin RNA to activate Pvalb transcript knockdown and subsequently downregulate PV. The downregulation was rapidly reversed after withdrawal of IPTG. In vitro and in vivo experiments revealed a decrease in PV expression of ≥50% in the presence of IPTG and full reversibility after IPTG removal. We foresee that the tightly regulated and reversible PV downregulation in mice in vivo will provide a new tool for the control of Pvalb transcript expression in a temporal manner. Because PV protein and PVALB transcript levels were found to be lower in the brain of patients with autism spectrum disorder and schizophrenia, the novel transgenic mouse line might serve as a model to investigate the putative role of PV in these neurodevelopmental disorders

Autophagy-related protein LC3 and Beclin-1 in the first trimester of pregnancy

Autophagy is a degradation process that acts in response to environmental stressors. Recently, autophagy has been detected in normal term, preeclamptic and intrauterine growth-restricted placentas. The object of this work was to investigate the presence of autophagy in first trimester voluntary interruption of pregnancy placental villi by the expression of autophagy-related proteins, light chain 3 (LC3), and Beclin-1. In first trimester placental villi laser scanning confocal microscopy (LSCM) analysis revealed LC3 and Beclin-1 immunoreactivity prevalently located in villous cytotrophoblasts. Using LSCM, LC3, and Beclin-1 were localized to the cytoplasm of the trophoblast layer in human full-term placentas. Beclin-1 expression and LC3 activation were confirmed by western blotting. These data emphasize that autophagy activation is different among cytotrophoblasts and syncytiotrophoblasts depending on the gestational age and thus we speculate that autophagy might play a prosurvival role throughout human pregnancy

Effect of image registration on 3D absorbed dose calculations in 177 Lu-DOTATOC Peptide Receptor Radionuclide Therapy

Peptide receptor radionuclide therapy (PRRT) is an effective MRT (molecular radiotherapy) treatment, which consists of multiple administrations of a radiopharmaceutical labelled with 177Lu or 90Y. Through sequential functional imaging a patient specific 3D dosimetry can be derived. Multiple scans should be previously co-registered to allow accurate absorbed dose calculations. The purpose of this study is to evaluate the impact of image registration algorithms on 3D absorbed dose calculation. A cohort of patients was extracted from the database of a clinical trial in PRRT. They were administered with a single administration of 177Lu-DOTATOC. All patients underwent 5 SPECT/CT sequential scans at 1 h, 4 h, 24 h, 40 h, 70 h post-injection that were subsequently registered using rigid and deformable algorithms. A similarity index was calculated to compare rigid and deformable registration algorithms. 3D absorbed dose calculation was carried out with the Raydose Monte Carlo code. The similarity analysis demonstrated the superiority of the deformable registrations (p < .001). Average absorbed dose to the kidneys calculated using rigid image registration was consistently lower than the average absorbed dose calculated using the deformable algorithm (90% of cases), with percentage differences in the range [−19; +4]%. Absorbed dose to lesions were also consistently lower (90% of cases) when calculated with rigid image registration with absorbed dose differences in the range [−67.2; 100.7]%. Deformable image registration had a significant role in calculating 3D absorbed dose to organs or lesions with volumes smaller than 100 mL. Image based 3D dosimetry for 177Lu-DOTATOC PRRT is significantly affected by the type of algorithm used to register sequential SPECT/CT scans

Early administration of the phytoestrogen genistein induces sex specific permanent alterations of nitrergic and vasopressinergic systems

Soy foods contain phytoestrogens as genistein (GEN) which may interfere with endocrine system and, during developmental critical periods, lead to permanent alterations of estrogen sensitive hypothalamic circuits. In a previous study, we demonstrated that GEN exposure through mothers resulted in an anxiolytic effect and a concurrent decrease of neural NO synthase (nNOS)+ cells in amygdale of male offspring [1]. This was consistent with both the role of NOS system in anxiety regulation and its sensitivity to gonadal hormones. In the present experiment, we analyzed anxiety levels and changes of neuronal circuits in mice directly fed with vehicle, Estradiol (E2) or GEN from birth (postnatal day 0, PND0) to PND8. Behavioral tests were conducted at PND60 and the mice were sacrificed at PND90. Coronal serial sections were processed for immunohistochemistry against nNOS and vasopressin (AVP). The GEN treatment had a dichotomic behavioral effects on sexes: anxiolitic on females while anxiogenic on males. Concurrently nNOS+ and AVP+ cell density in some hypothalamic nuclei was affected. Interestingly only a few of those effects were mimicked by E2 treatment suggesting that GEN may act trough different intracellular pathways.These results raise concerns about the possible long-term effects of soy-based food in livestock that largely use soy-based supplements and show hypo-fertility problems, as pigs. Similar concerns could involve the long-term use of soy-based formulas for babies