BACKGROUND

Aging is accompanied by altered thinking (cognition) and feeling (mood), functions that depend on information processing by brain cortical cell microcircuits. We hypothesized that age-associated long-term functional and biological changes are mediated by gene transcriptomic changes within neuronal cell types forming cortical microcircuits, namely excitatory pyramidal cells (PYCs) and inhibitory gamma-aminobutyric acidergic neurons expressing vasoactive intestinal peptide (Vip), somatostatin (Sst), and parvalbumin (Pvalb).

METHODS

To test this hypothesis, we assessed locomotor, anxiety-like, and cognitive behavioral changes between young (2 months of age, n = 9) and old (22 months of age, n = 12) male C57BL/6 mice, and performed frontal cortex cell type-specific molecular profiling, using laser capture microscopy and RNA sequencing. Results were analyzed by neuroinformatics and validated by fluorescent in situ hybridization.

RESULTS

Old mice displayed increased anxiety and reduced working memory. The four cell types displayed distinct age-related transcriptomes and biological pathway profiles, affecting metabolic and cell signaling pathways, and selective markers of neuronal vulnerability (Ryr3), resilience (Oxr1), and mitochondrial dynamics (Opa1), suggesting high age-related vulnerability of PYCs, and variable degree of adaptation in gamma-aminobutyric acidergic neurons. Correlations between gene expression and behaviors suggest that changes in cognition and anxiety associated with age are partly mediated by normal age-related cell changes, and that additional age-independent decreases in synaptic and signaling pathways, notably in PYCs and somatostatin neurons, further contribute to behavioral changes.

CONCLUSIONS

Our study demonstrates cell-dependent differential vulnerability and coordinated cell-specific cortical microcircuit molecular changes with age. Collectively, the results suggest intrinsic molecular links among aging, cognition, and mood-related behaviors, with somatostatin neurons contributing evenly to both behavioral conditions.

BACKGROUND

Loss-of-function mutations in SLC12A3 coding for the thiazide-sensitive NaCl cotransporter (NCC) cause Gitelman's syndrome (GS), a recessively inherited salt-losing tubulopathy. Most GS patients are compound heterozygous. However, up to 30% of GS patients carry only a single mutant allele, and a normal SLC12A3 screening is also observed in a small subset of patients. Locus heterogeneity could explain the lack of detection of mutant SLC12A3 alleles in GS patients. The renal phenotype of the parvalbumin knockout mice pointed to PVALB as a candidate gene for GS for SLC12A3-negative cases.

METHODS

PCR and direct sequencing of PVALB was performed in 132 GS patients in whom only one or no (N = 79) mutant SLC12A3 allele was found. The possible interference of biallelic SNPs (single nucleotide polymorphisms) on normal transcription or normal splicing was investigated. Genotyping of 110 anonymous blood donors was performed to determine the allelic frequency in the normal population.

RESULTS

No sequence variants resulting in amino acid substitution or truncated protein within the PVALB gene were found in the 264 chromosomes tested. Ten biallelic SNPs, including six novel polymorphisms, were identified: five in the 5' UTR, none of them affecting predicted regulatory elements; three in the coding region, without alteration of the consensus splice sites, and two in the 3' UTR. The observed allelic frequencies did not differ significantly between GS patients and controls.

CONCLUSIONS

Our results strongly suggest that mutations in the PVALB gene are not involved in GS patients who harbour a single or no mutant SLC12A3 allele.

A deficit in parvalbumin neurons is found in schizophrenia and several animal models of the disease. In this preliminary study, we determined whether one such model, phencyclidine (PCP) administration, results in changes in DNA methylation in the rat Pvalb promoter.

DNA from hippocampus and prefrontal cortex from rats, which 6 weeks previously received either 2 mg/kg PCP or vehicle for 7 days, underwent bisulphite pyrosequencing to determine methylation.

PCP administration induced significantly greater methylation at one of two Pvalb CpG sites in both prefrontal cortex and hippocampus, while no significant difference was found in long interspersed nucleotide element-1, a global measure of DNA methylation.

Subchronic PCP administration results in a specific hypermethylation in the Pvalb promoter which may contribute to parvalbumin deficits in this animal model of psychosis.

Parvalbumin (PV) is a classical member of the EF-hand protein superfamily that has been described as a Ca(2+) buffer and Ca(2+) transporter/shuttle protein and may also play an additional role in Mg(2+) handling. PV is exclusively expressed in the early part of the distal convoluted tubule in the human and mouse kidneys. Recent studies in Pvalb knockout mice revealed a role of PV in the distal handling of electrolytes: the lack of PV was associated with a mild salt-losing phenotype with secondary aldosteronism, salt craving and stronger bones compared with controls. A link between the Ca(2+)-buffering capacity of PV and the expression of the thiazide-sensitive Na(+)-Cl(-) cotransporter was established, which could be relevant to the regulation of sodium transport in the distal nephron. Variants in the PVALB gene that encodes PV have been described, but their relevance to kidney function has not been established. PV is also considered a reliable marker of chromophobe carcinoma and oncocytoma, two neoplasms deriving from the distal nephron. The putative role of PV in tumour genesis remains to be investigated.

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.

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.

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.

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.

KCNQ2/3 channels, ubiquitously expressed neuronal potassium channels, have emerged as indispensable regulators of brain network activity. Despite their critical role in brain homeostasis, the mechanisms by which KCNQ2/3 dysfunction lead to hypersychrony are not fully known. Here, we show that deletion of KCNQ2/3 channels changed PV+ interneurons', but not SST+ interneurons', firing properties. We also find that deletion of either KCNQ2/3 or KCNQ2 channels from PV+ interneurons led to elevated homeostatic potentiation of fast excitatory transmission in pyramidal neurons. Pvalb-Kcnq2 null-mice showed increased seizure susceptibility, suggesting that decreases in interneuron KCNQ2/3 activity remodels excitatory networks, providing a new function for these channels.

Excitation/inhibition imbalance has been proposed as a fundamental mechanism in the pathogenesis of neuropsychiatric and neurodevelopmental disorders, in which copy number variations of the Unc-51 like kinase 4 (ULK4) gene encoding a putative Serine/Threonine kinase have been reported in approximately 1/1000 of patients suffering pleiotropic clinical conditions of schizophrenia, depression, autistic spectrum disorder (ASD), developmental delay, language delay, intellectual disability, or behavioral disorder. The current study characterized behavior of heterozygous Ulk4 +/tm1a mice, demonstrating that Ulk4 +/tm1a mice displayed no schizophrenia-like behavior in acoustic startle reactivity and prepulse inhibition tests or depressive-like behavior in the Porsolt swim or tail suspension tests. However, Ulk4 +/tm1a mice exhibited an anxiety-like behavioral phenotype in several tests. Previously identified hypo-anxious (Atp1a2, Ptn, and Mdk) and hyper-anxious (Gria1, Syngap1, and Npy2r) genes were found to be dysregulated accordingly in Ulk4 mutants. Ulk4 was found to be expressed in GABAergic neurons and the Gad67+ interneurons were significantly reduced in the hippocampus and basolateral amygdala of Ulk4 +/tm1a mice. Transcriptome analyses revealed a marked reduction of GABAergic neuronal subtypes, including Pvalb, Sst, Cck, Npy, and Nos3, as well as significant upregulation of GABA receptors, including Gabra1, Gabra3, Gabra4, Gabra5, and Gabrb3. This is the first evidence that Ulk4 plays a major role in regulating GABAergic signaling and anxiety-like behavior, which may have implications for the development of novel anxiolytic treatments.

Background: In fast firing, parvalbumin (PV)-expressing (Pvalb) interneurons, PV acts as an intracellular Ca²⁺ signal modulator with slow-onset kinetics. In Purkinje cells of PV^-/- mice, adaptive/homeostatic mechanisms lead to an increase in mitochondria, organelles equally capable of delayed Ca²⁺ sequestering/buffering. An inverse regulation of PV and mitochondria likewise operates in cell model systems in vitro including myotubes, epithelial cells, and oligodendrocyte-like cells overexpressing PV. Whether such opposite regulation pertains to all Pvalb neurons is currently unknown. In oligodendrocyte-like cells, PV additionally decreases growth and branching of processes in a cell-autonomous manner.

Methods: The in vivo effects of absence of PV were investigated in inhibitory Pvalb neurons expressing EGFP, present in the somatosensory and medial prefrontal cortex, striatum, thalamic reticular nucleus, hippocampal regions DG, CA3, and CA1 and cerebellum of mice either wild-type or knockout (PV^-/-) for the Pvalb gene. Changes in Pvalb neuron morphology and PV concentrations were determined using immunofluorescence, followed by 3D-reconstruction and quantitative image analyses.

Results: PV deficiency led to an increase in mitochondria volume and density in the soma; the magnitude of the effect was positively correlated with the estimated PV concentrations in the various Pvalb neuron subpopulations in wild-type neurons. The increase in dendrite length and branching, as well as thickness of proximal dendrites of selected PV^-/- Pvalb neurons is likely the result of the observed increased density and length of mitochondria in these PV^-/- Pvalb neuron dendrites. The increased branching and soma size directly linked to the absence of PV is assumed to contribute to the increased volume of the neocortex present in juvenile PV^-/- mice. The extended dendritic branching is in line with the hypothesis of local hyperconnectivity in autism spectrum disorder (ASD) and ASD mouse models including PV^-/- mice, which display all ASD core symptoms and several comorbidities including cortical macrocephaly at juvenile age.

Conclusion: PV is involved in most proposed mechanisms implicated in ASD etiology: alterations in Ca²⁺ signaling affecting E/I balance, changes in mitochondria structure/function, and increased dendritic length and branching, possibly resulting in local hyperconnectivity, all in a likely cell autonomous way.

Keywords: Autism spectrum disorder; Calcium homeostasis; Calcium-binding protein; Mitochondria; Parvalbumin; Pvalb neurons.

Epilepsy is one of the most common neurological disorders, yet its pathophysiology is poorly understood due to the high complexity of affected neuronal circuits. To identify dysfunctional neuronal subtypes underlying seizure activity in the human brain, we have performed single-nucleus transcriptomics analysis of >110,000 neuronal transcriptomes derived from temporal cortex samples of multiple temporal lobe epilepsy and non-epileptic subjects. We found that the largest transcriptomic changes occur in distinct neuronal subtypes from several families of principal neurons (L5-6_Fezf2 and L2-3_Cux2) and GABAergic interneurons (Sst and Pvalb), whereas other subtypes in the same families were less affected. Furthermore, the subtypes with the largest epilepsy-related transcriptomic changes may belong to the same circuit, since we observed coordinated transcriptomic shifts across these subtypes. Glutamate signaling exhibited one of the strongest dysregulations in epilepsy, highlighted by layer-wise transcriptional changes in multiple glutamate receptor genes and strong upregulation of genes coding for AMPA receptor auxiliary subunits. Overall, our data reveal a neuronal subtype-specific molecular phenotype of epilepsy.

OBJECTIVE

The parvalbumin (PV)-containing subgroup of GABAergic neurons is particularly affected in schizophrenia and animal models of psychosis, including after methamphetamine (METH) administration. We investigated whether METH dependence and METH-induced psychosis may involve an effect on DNA methylation of the PVALB promoter.

METHODS

The methylation of a PVALB promoter sequence was determined in 100 METH-dependent and 102 control subjects using pyrosequencing.

RESULTS

A significant increase in PVALB methylation was observed in METH dependence and METH-induced psychosis. No significant effect on long interspersed nucleotide element-1 methylation, a measure of global DNA methylation, was observed.

CONCLUSIONS

These results demonstrate a specific association between elevated PVALB methylation and METH-induced psychosis. This finding may contribute to the GABAergic deficits associated with METH dependence.

In neurodevelopmental disorders (NDDs) including autism spectrum disorder (ASD) and schizophrenia, impairment/malfunctioning of a subpopulation of interneurons expressing the calcium-binding protein parvalbumin (PV) -here termed Pvalb neurons- has gradually emerged as a possible cause. These neurons may represent a hub or point-of-convergence in the etiology of NDD. Increased oxidative stress associated with mitochondria impairment in Pvalb neurons is discussed as an essential step in schizophrenia etiology. Since PV downregulation is a common finding in ASD and schizophrenia individuals and PV-deficient (PV-/-) mice show a strong ASD-like behavior phenotype, we investigated the putative link between PV expression, alterations in mitochondria and oxidative stress. In a longitudinal study with 1, 3, and 6-months old PV-/- and wild type mice, oxidative stress was investigated in 9 Pvalb neuron subpopulations in the hippocampus, striatum, somatosensory cortex, medial prefrontal cortex, thalamic reticular nucleus (TRN) and cerebellum. In Pvalb neuron somata in the striatum and TRN, we additionally determined mitochondria volume and distribution at these three time points. In all Pvalb neuron subpopulations, we observed an age-dependent increase in oxidative stress and the increase strongly correlated with PV expression levels, but not with mitochondria density in these Pvalb neurons. Moreover, oxidative stress was elevated in Pvalb neurons of PV-/- mice and the magnitude of the effect was again correlated with PV expression levels in the corresponding wild type Pvalb neuron subpopulations. The PV-dependent effect was insignificant at 1 month and relative differences between WT and PV-/- Pvalb neurons were largest at 3 months. Besides the increase in mitochondria volume in PV's absence in TRN and striatal PV-/- Pvalb neurons fully present already at 1 month, we observed a redistribution of mitochondria from the perinuclear region toward the plasma membrane at all time points. We suggest that in absence of PV, slow Ca²⁺ buffering normally exerted by PV is compensated by a (mal)adaptive, mostly sub-plasmalemmal increase in mitochondria resulting in increased oxidative stress observed in 3- and 6-months old mice. Since PV-/- mice display core ASD-like symptoms already at 1 month, oxidative stress in Pvalb neurons is not a likely cause for their ASD-related behavior observed at this age.

Keywords: ROS; autism; mitochondria; parvalbumin; schizophrenia.

The PKD1 gene encodes polycystin-1 (PC1), a mechanosensor triggering intracellular responses upon urinary flow sensing in kidney tubular cells. Mutations in PKD1 lead to autosomal dominant polycystic kidney disease (ADPKD). The involvement of PC1 in renal electrolyte handling remains unknown since renal electrolyte physiology in ADPKD patients has only been characterized in cystic ADPKD. We thus studied the renal electrolyte handling in inducible kidney-specific Pkd1 knockout (iKsp- Pkd1-/-) mice manifesting a precystic phenotype. Serum and urinary electrolyte determinations indicated that iKsp- Pkd1-/- mice display reduced serum levels of magnesium (Mg2+), calcium (Ca2+), sodium (Na+), and phosphate (Pi) compared with control ( Pkd1+/+) mice and renal Mg2+, Ca2+, and Pi wasting. In agreement with these electrolyte disturbances, downregulation of key genes for electrolyte reabsorption in the thick ascending limb of Henle's loop (TA;, Cldn16, Kcnj1, and Slc12a1), distal convoluted tubule (DCT; Trpm6 and Slc12a3) and connecting tubule (CNT; Calb1, Slc8a1, and Atp2b4) was observed in kidneys of iKsp- Pkd1-/- mice compared with controls. Similarly, decreased renal gene expression of markers for TAL ( Umod) and DCT ( Pvalb) was observed in iKsp- Pkd1-/- mice. Conversely, mRNA expression levels in kidney of genes encoding solute and water transporters in the proximal tubule ( Abcg2 and Slc34a1) and collecting duct ( Aqp2, Scnn1a, and Scnn1b) remained comparable between control and iKsp- Pkd1-/- mice, although a water reabsorption defect was observed in iKsp- Pkd1-/- mice. In conclusion, our data indicate that PC1 is involved in renal Mg2+, Ca2+, and water handling and its dysfunction, resulting in a systemic electrolyte imbalance characterized by low serum electrolyte concentrations.

Tonotopic differences in the structure and physiological function, e.g., synapse number, membrane properties, Ca²⁺ channels, Ca²⁺ dependence of exocytosis and vesicle pool replenishment of inner hair cells (IHCs) along the longitudinal cochlear axis have recently been discovered, suggesting different gene expression patterns of IHCs. To determine whether IHCs present different gene expression patterns along the longitudinal cochlear axis, apical and basal IHCs were collected separately using the suction pipette technique from adult mouse cochleae for RNA-seq and genome-wide transcriptome analysis. We found 689 annotated genes showed more than 2-fold increase in expression. Interestingly, 93.4% of the differentially expressed genes (DEGs) was upregulated in apical IHCs. Although a subset of genes that related to IHC machinery and deafness were found to be differentially expressed, a gradient of gene expression was indeed detected in Ocm, Pvalb, Prkd1, Fbxo32, Nme2, and Sncg, which may play putative roles in the Ca²⁺ buffering and survival regulation. The expression of these genes was validated by real-time quantitative PCR (RT-qPCR) or immunostaining. We conclude that IHCs from different mouse cochlear longitudinal position have different gene expression profiles. Our data might serve as a valuable resource for exploring the molecular mechanisms underlying different biological properties as well as the survival regulation of IHCs.

Maternal immune activation (MIA) increases risk for neuropsychiatric disorders such as autism spectrum disorder (ASD) in offspring later in life through unknown causal mechanisms. Growing evidence implicates parvalbumin-containing GABAergic interneurons as a key target in rodent MIA models. We targeted a specific neurodevelopmental window of parvalbumin interneurons in a mouse MIA model to examine effects on spatial working memory, a key domain in ASD that can manifest as either impairments and improvements both clinically and in animal models. Pregnant dams received three consecutive intraperitoneal injections of Polyinosinic:polycytidylic acid (poly(I:C), 5mg/kg) at gestational days 13, 14 and 15. Spatial working memory was assessed in young adult offspring using touchscreen operant chambers and the Trial-Unique Non-matching to Location (TUNL) task. Anxiety, novelty seeking and short-term memory were assessed using Elevated Plus Maze (EPM) and Y-maze novelty preference tasks. Fluorescent immunohistochemistry was used to assess hippocampal parvalbumin cell density, intensity and co-expression with perineuronal nets. qPCR was used to assess the expression of putatively implicated gene pathways. MIA targeting a window of parvalbumin interneuron development increased spatial working memory performance on the TUNL touchscreen task which was not influenced by anxiety or novelty seeking behaviour. The model reduced fetal mRNA levels of Gad1 and adult hippocampal mRNA levels of Pvalb and the distribution of low intensity parvalbumin interneurons was altered. We speculate a specific timing window for parvalbumin interneuron development underpins the apparently paradoxical improved spatial working memory phenotype found both across several rodent models of autism and clinically in ASD.

Keywords: Immune activation; TUNL; autism; parvalbumin; poly(I:C); spatial working memory; touchscreen.

Nervous necrosis virus (NNV) infects a wide range of larval and juvenile fish species, thereby causing enormous economic losses in the aquaculture industry. Possible solutions to this problem include the use of antimicrobial peptides (AMPs), which directly inhibit bacterial growth, and also modulate host signaling mechanisms. The AMPs epinecidin (Epi)-1 and Tilapia hepcidin (TH) 1-5 have been demonstrated to be effective against Nervous necrosis virus infection in medaka (Oryzias latipes). However, the underlying molecular mechanisms are yet to be explored. Here, microarray analyses were performed to examine how NNV infection and/or epinecidin-1 or TH1-5 treatment affects gene expression in medaka; such analyses enabled the prediction of host signaling pathways affected by virus infection and/or regulated by epinecidin-1 and TH1-5. Transcriptome analysis revealed altered expression of genes involved in B cell activation, T cell activation, adipocytokine signaling, and mast cell activation. We subsequently used real-time PCR to analyze expression of key genes involved in these signaling mechanisms. Medaka infected with NNV exhibited up-regulation of PVALB, CEBPA, IFIM, IFN, IL-6ST, NF-kB2, SOC3, SP1, and TGFB1, and such increases were prevented by pre-treatment with epinecidin-1 or TH1-5. Immunohistochemistry using the anti-NNV antibody to stain brain and eye sections revealed that epinecidin-1 treatment during or after infection clears viral load, while TH1-5 treatment only reduces viral numbers if applied during infection. These observations demonstrate that epinecidin-1 and TH1-5 modulate NNV-induced host signaling mechanisms, thereby preventing viral multiplication in host organisms.

The gene expression of parvalbumin (Pvalb), a high-affinity calcium-binding protein and the major fish allergen, was significantly increased in the tilapia fry treated with methyltestosterone (MT) as examined using a subtractive hybridization assay. Using the real-time quantitative PCR, we further confirmed the increased Pvalb expression in the MT-treated tilapia fry. The 568 base pairs (bp) tilapia Pvalb (tPvalb) cDNA clone was fully sequenced and found to contain a coding region of 330 bp, which encodes a 108 amino acids protein with a molecular weight of 11,370.5 and an calculated isoelectric point of 4.56. The predicted secondary structure of tPvalb is comprised of seven alpha helices. It contains two characteristic EF-hand calcium-binding motifs, one PKC and five casein kinase II consensus phosphorylation sites. The tPvalb is highly homologous to the selected fish Pvalbs at a similarity ranging from 53% to 80%. The phylogenetic tree analysis showed that the tPvalb is closest to the Scomber japonicus Pvalb. The tPvalb was found to express in the heart, muscle, gill, kidney, brain and ovary of adult fish by RT-PCR analysis. In situ hybridization also revealed that the tPvalb was highly expressed in the hypothalamus and sarcoplasmic reticulum. A tPvalb glutathione S-transferase (GST) fusion protein was generated and digested by thrombin to remove the GST moiety. Further Western analysis showed that the tPvalb protein was cross-reacted to an anti-rat Pvalb antibody. Those results suggest that Pvalb is evolutionally conserved in tilapia.

Deficits of brain parvalbumin (PV) are a consistent finding in schizophrenia and models of psychosis. We investigated whether this is associated with abnormal PV gene (PVALB) methylation in the brain in schizophrenia. Bisulfite pyrosequencing was used to determine cytosine (CpG) methylation in a PVALB promoter sequence. Greater PVALB methylation was found in schizophrenia hippocampus, while no differences were observed in prefrontal cortex. LINE-1 methylation, a measure of global methylation, was also elevated in both regions in schizophrenia, although the PVALB change was independent of this effect. These results provide the first evidence that PVALB promoter methylation is abnormal in schizophrenia and suggest that this epigenetic finding may relate to the reduction of PV expression seen in the disease.

Autism spectrum disorders (ASD) are persistent conditions resulting from disrupted/altered neurodevelopment. ASD multifactorial etiology-and its numerous comorbid conditions-heightens the difficulty in identifying its underlying causes, thus obstructing the development of effective therapies. Increasing evidence from both animal and human studies suggests an altered functioning of the parvalbumin (PV)-expressing inhibitory interneurons as a common and possibly unifying pathway for some forms of ASD. PV-expressing interneurons (short: PVALB neurons) are critically implicated in the regulation of cortical networks' activity. Their particular connectivity patterns, i.e., their preferential targeting of perisomatic regions and axon initial segments of pyramidal cells, as well as their reciprocal connections, enable PVALB neurons to exert a fine-tuned control of, e.g., spike timing, resulting in the generation and modulation of rhythms in the gamma range, which are important for sensory perception and attention.New methodologies such as induced pluripotent stem cells (iPSC) and genome-editing techniques (CRISPR/Cas9) have proven to be valuable tools to get mechanistic insight in neurodevelopmental and/or neurodegenerative and neuropsychiatric diseases. Such technological advances have enabled the generation of PVALB neurons from iPSC. Tagging of these neurons would allow following their fate during the development, from precursor cells to differentiated (and functional) PVALB neurons. Also, it would enable a better understanding of PVALB neuron function, using either iPSC from healthy donors or ASD patients with known mutations in ASD risk genes. In this concept paper, the strategies hopefully leading to a better understanding of PVALB neuron function(s) are briefly discussed. We envision that such an iPSC-based approach combined with emerging (genetic) technologies may offer the opportunity to investigate in detail the role of PVALB neurons and PV during "neurodevelopment ex vivo."

EF-hand Ca²⁺-binding protein family members, α- and β-parvalbumins have been studied for decades. Yet, considerable information is lacking distinguishing functional differences between mammalian α-parvalbumin (PVALB) and oncomodulin (OCM), a branded β-parvalbumin. Herein, we provide an overview detailing the current body of work centered around OCM as an EF-Hand Ca²⁺-binding protein and describe potential mechanisms of OCM function within the inner ear and immune cells. Additionally, we posit that OCM is evolutionarily distinct from PVALB and most other β-parvalbumins. This review summarizes recent studies pertaining to the function of OCM and emphasizes OCM as a parvalbumin possessing a unique cell and tissue distribution, Ca²⁺ buffering capacity and phylogenetic origin.

The contraction and relaxation events of the muscle is mediated by the coordination of many important calcium cycling proteins of ryanodine receptor (RYR), troponin C (TNNC), parvalbumin (PVALB), sarcoendoplasmic reticulum calcium transport ATPase (SERCA) and calsequestrin (CASQ). In higher vertebrates, the expression level of calcium cycling proteins are positively correlated to the muscle contraction/relaxation ability of the cell. In this study, we used RNAseq to explore the expression profile of calcium cycling genes between two marine fish of Bombay duck (Harpadon nehereus) and beltfish (Trichiurus lepturus) with poor and robust swimming activities, respectively. We have studied the hypothesis whether the expression level of calcium cycling proteins are also positive correlated to swimming ability in fish. We used Illumina sequencing technology (NextSeq500) to sequence, assemble and annotate the muscle transcriptome of Bombay duck for the first time. A total of 47,752,240 cleaned reads (deposited in NCBI SRA database with accession number of SRX1706379) were obtained from RNA sequencing and 26,288 unigenes (with N50 of 486 bp) were obtained after de novo assembling with Trinity software. BLASTX against NR, GO, KEGG and eggNOG databases show 100%, 65%, 26%, 94% and 88% annotation rate, respectively. Comparison of the dominantly expressed unigenes in fish muscle shows calcium cycling gene expression in beltfish (SRX1674471) is 1.4- to 51.6-fold higher than Bombay duck. Among five calcium cycling genes, the fold change results are very significant in CASQ (51.6 fold) and PVALB (9.1 fold) and both of them are responsive for calcium binding to reduce free calcium concentration in the sarcoendoplasmic reticulum and cytoplasm. In conclusion, we confirmed that the high abundant expression rate of calcium cycling genes in robust swimming fish species. The current muscle transcriptome and identified calcium cycling gene data can provide more insights into the muscle physiology of fish.

Developmental exposure to valproic acid (VPA), a model compound for experimental autism, has shown to primarily target GABAergic interneuron subpopulations in hippocampal neurogenesis of rat offspring. The VPA-exposed animals had revealed late effects on granule cell lineages, involving progenitor cell proliferation and synaptic plasticity. To investigate the possibility whether hippocampal neurogenesis in postpubertal rats in a protocol of 28-day repeated exposure is affected in relation with the property of a developmental neurotoxicant by developmental exposure, VPA was orally administered to 5-week-old male rats at 0, 200, 800 and 900 mg/kg body weight/day for 28 days. At 900 mg/kg, GFAP⁺ cells increased in number, but DCX⁺ cells decreased in number in the granule cell lineages. Moreover, CHRNB2⁺ cells and NeuN⁺ postmitotic neurons decreased in number in the hilus of the dentate gyrus. Transcript level examined at 900 mg/kg in the dentate gyrus was increased with Kit, but decreased with Dpsyl3, Btg2, Pvalb and Chrnb2. These results suggest that VPA increased type-1 stem cells in relation to the activation of SCF-KIT signaling and suppression of BTG2-mediated antiproliferative effect on stem cells. VPA also decreased type-3 progenitor cells and immature granule cells probably in relation with PVALB⁺ interneuron hypofunction and reduced CHRNB2⁺ interneuron subpopulation in the hilus, as well as with suppression of BTG2-mediated terminal differentiation of progenitor cells. Thus, the disruption pattern of VPA by postpubertal exposure was different from developmental exposure. However, disruption itself can be detected, suggesting availability of hippocampal neurogenesis in detecting developmental neurotoxicants in a 28-day toxicity study.

The prevalence of autism spectrum disorder (ASD)-a type of neurodevelopmental disorder-is increasing and is around 2% in North America, Asia, and Europe. Besides the known genetic link, environmental, epigenetic, and metabolic factors have been implicated in ASD etiology. Although highly heterogeneous at the behavioral level, ASD comprises a set of core symptoms including impaired communication and social interaction skills as well as stereotyped and repetitive behaviors. This has led to the suggestion that a large part of the ASD phenotype is caused by changes in a few and common set of signaling pathways, the identification of which is a fundamental aim of autism research. Using advanced bioinformatics tools and the abundantly available genetic data, it is possible to classify the large number of ASD-associated genes according to cellular function and pathways. Cellular processes known to be impaired in ASD include gene regulation, synaptic transmission affecting the excitation/inhibition balance, neuronal Ca²⁺ signaling, development of short-/long-range connectivity (circuits and networks), and mitochondrial function. Such alterations often occur during early postnatal neurodevelopment. Among the neurons most affected in ASD as well as in schizophrenia are those expressing the Ca²⁺-binding protein parvalbumin (PV). These mainly inhibitory interneurons present in many different brain regions in humans and rodents are characterized by rapid, non-adaptive firing and have a high energy requirement. PV expression is often reduced at both messenger RNA (mRNA) and protein levels in human ASD brain samples and mouse ASD (and schizophrenia) models. Although the human PVALB gene is not a high-ranking susceptibility/risk gene for either disorder and is currently only listed in the SFARI Gene Archive, we propose and present supporting evidence for the Parvalbumin Hypothesis, which posits that decreased PV level is causally related to the etiology of ASD (and possibly schizophrenia).

Keywords: E/I balance; GABAergic neurons; ROS; autism (ASD); calcium signal modulator; mitochondria; parvalbumin; schizophrenia.

Neuronal cell identity is established during development and must be maintained throughout an animal's life (Fishell G, Heintz N. Neuron 80: 602-612, 2013). Transcription factors critical for establishing neuronal identity can be required for maintaining it (Deneris ES, Hobert O. Nat Neurosci 17: 899-907, 2014). Posttranscriptional regulation also plays an important role in neuronal differentiation (Bian S, Sun T. Mol Neurobiol 44: 359-373, 2011), but its role in maintaining cell identity is less established. To better understand how posttranscriptional regulation might contribute to cell identity, we examined the proprioceptive neurons in the dorsal root ganglion (DRG), a highly specialized sensory neuron class, with well-established properties that distinguish them from other neurons in the ganglion. By conditionally ablating Dicer in mice, using parvalbumin (Pvalb)-driven Cre recombinase, we impaired posttranscriptional regulation in the proprioceptive sensory neuron population. Knockout (KO) animals display a progressive form of ataxia at the beginning of the fourth postnatal week that is accompanied by a cell death within the DRG. Before cell loss, expression profiling shows a reduction of proprioceptor specific genes and an increased expression of nonproprioceptive genes normally enriched in other ganglion neurons. Furthermore, although central connections of these neurons are intact, the peripheral connections to the muscle are functionally impaired. Posttranscriptional regulation is therefore necessary to retain the transcriptional identity and support functional specialization of the proprioceptive sensory neurons.NEW & NOTEWORTHY We have demonstrated that selectively impairing Dicer in parvalbumin-positive neurons, which include the proprioceptors, triggers behavioral changes, a lack of muscle connectivity, and a loss of transcriptional identity as observed through RNA sequencing. These results suggest that Dicer and, most likely by extension, microRNAs are crucially important for maintaining proprioception. Additionally, this study hints at the larger question of how neurons maintain their functional and molecular specificity.

Ovary development appears to be under polygenic control, and is influenced by multiple genetic factors that may vary from organism to organism. To gain a better insight into the molecular mechanisms of carp ovary development, Suppression Subtractive Hybridization (SSH) DNA libraries in two species of Yellow River carp were analyzed. Primordial gonads and stage II ovaries were used as testers, and adult ovaries as drivers. One hundred and fifty differentially-expressed candidate genes were examined by Southern blot microarray hybridization. We identified 41 differentially-expressed genes in the PG (Primordial gonad) library and 37 in the stage II ovary library. Gene Ontology Biological Pathway analysis showed the genes were involved in signal transduction, proteolysis process, cell differentiation, TGF-β signal and other biological responses. Twenty-two candidate genes were selected and further characterized using qRT-PCR. Pvalb, epd, and MYH were found specifically expressed in PG, while bmp2b, desmin and fp1 were specifically expressed in stage II ovary. Our results indicate that these genes could be used as biomarkers of the early development of carp ovary. This finding will provide a basis for further understanding of the complex gonad developmental molecular mechanisms in Yellow River carp.