Autism spectrum disorders (ASD) is a heterogeneous group of neurodevelopmental disorders characterized by synaptic dysfunction and defects in dendritic spine morphology. In the past decade, an extensive list of genes associated with ASD has been identified by genome-wide sequencing initiatives. Several of these genes functionally converge in the regulation of the Wnt/β-catenin signaling pathway, a conserved cascade essential for stem cell pluripotency and cell fate decisions during development. Here, we review current information regarding the transcriptional program of Wnt/β-catenin signaling in ASD. First, we discuss that Wnt/β-catenin gain and loss of function studies recapitulate brain developmental abnormalities associated with ASD. Second, transcriptomic approaches using patient-derived induced pluripotent stem cells (iPSC) cells, featuring mutations in high confidence ASD genes, reveal a significant dysregulation in the expression of Wnt signaling components. Finally, we focus on the activity of chromatin-remodeling proteins and transcription factors considered high confidence ASD genes, including CHD8, ARID1B, ADNP, and TBR1, that regulate Wnt/β-catenin-dependent transcriptional activity in multiple cell types, including pyramidal neurons, interneurons and oligodendrocytes, cells which are becoming increasingly relevant in the study of ASD. We conclude that the level of Wnt/β-catenin signaling activation could explain the high phenotypical heterogeneity of ASD and be instrumental in the development of new diagnostics tools and therapies. There apulation .The genetic etiology of autism was early recognized by Kanner, the moment he noted that autistic children came from highly intelligent families, though better at relating to concepts than to people . To datehttps://sfari.org/resources/sfari-gene). Nevertheless, most of the variants identified have a weak effect  have identified over 900 genes associated with ASDs  receptor and members of the single pass transmembrane low-density lipoprotein (LDL) receptor related proteins (LRP5/6), which acts as co-receptors . Wnt binA common neuroanatomical feature observed in ASD children is a premature brain overgrowth occurring during the first 3 years of life, which affects the cerebral cortex, limbic system, hippocampal formation, amygdala, and cerebellum . While tin vivo , which plays a central role in both canonical and non-canonical Wnt signaling , was oneelopment . SurprisWhile Dvl deficiency may point out to a loss of function in Wnt/β-catenin signaling in ASD, an APC conditional KO (cKO) mouse in excitatory pyramidal neurons showed increased β-catenin levels and target Wnt/β-catenin gene transcription, accompanied at the cellular level, with an increase in dendritic spine density in the cortex/hippocampus and spines with a stubby phenotype indicative of immature synapses; and at the behavioral level, by learning impairments, reduced social interest and repetitive behaviors . Conversin utero injection of XAV939 in the lateral ventricle at E14.5 leads to a transient increase in the number of intermediate progenitors, ultimately increasing the number of pyramidal neurons in the neocortex within layer 2/3, which was enough to trigger an enlarged neocortex and autistic behaviors in mice  box family, which activate gene expression by recruiting other DNA-binding factors and chromatin remodeling proteins to promoter sequences of target genes ; Table 17L2 gene , includiand ADHD . TCF7L2 and ADHD . TCF7L2 and ADHD , resembland ADHD . Finallyand ADHD  and choland ADHD , which mand ADHD .CHD8 +/-) affects the expression of thousands of genes in neural progenitors and early differentiating neurons (Recurrent mutations in the chromodomain helicase DNA binding protein 8 (CHD8) gene have been consistently identified in multiple individuals with ASD . Today,  neurons , which a neurons  and regu neurons . Altoget neurons .The AT-rich interactive domain-containing protein 1B (ARID1B) gene has also been consistently associated with ASD and syndromic and non-syndromic ID, and corpus callosum agenesis  and is sde novo variants in ADNP deficiency often show intellectual disability, facial dysmorphism, speech impediments, and motor dysfunctions  increased both binding with β-catenin and transcriptional activation of Wnt signaling in HEK293T cells, supporting the notion that that TBL1XR1 potentiation of Wnt signaling could be involved in ASD (in vitro and in vivo and is recruited to Wnt/β-catenin target genes in a β-catenin dependent manner (Several other products that modulate Wnt/β-catenin-dependent transcription have been genetically or functionally associated with ASD, including the activity dependent neuroprotective protein ADNP; , membersunctions . Interespression . Althougpression , little pression . Similarpression . Likewisd in ASD . Interesd in ASD . In the d in ASD  physicalt manner . MED13L t manner . Finallyt manner  is involt manner , a knownt manner  and potet manner  or GSK3βt manner . Altogett manner .In summary, deficiency in chromatin remodeling proteins and transcription factors associated with ASD widely recapitulate phenotypes resembled by β-catenin gain and loss of function experiments and highlights the importance of Wnt/β-catenin dependent transcription in ASD and related neurodevelopmental disorders.The role and regulatory mechanisms controlling the activation and inhibition of Wnt/β-catenin signaling have been extensively studied in human disease . While iIn the present work we reviewed the activity of Wnt/β-catenin signaling components in brain development and synaptic function in ASD that unequivocally points toward a dysregulation of Wnt/β-catenin associated targets. We further explore this observation by confirming that several transcriptional factors and chromatin remodeling proteins deemed ASD high confidence genes are also direct modulators of β-catenin mediated transcription. While we are reluctant to deem the levels of Wnt/β-catenin activation a “gray area,” we do think that both gain and loss of function of this pathway could explain the high heterogeneity of ASD. Furthermore, studies considering both temporal and spatial resolution of gene expression will be instrumental to fully comprehend this new “Wnt spectrum.”The data presented in this article also highlights the growing potential of different cell types in the neurobiology of ASD, in these contexts, cell type specific loss of function in interneurons and oligodendrocytes leads to ASD, giving a rising importance the Wnt pathway beyond the traditional excitatory pyramidal neuron and the development of the cerebral cortex.MC wrote the first draft and contributed to the overall structure of the manuscripts. MA revised the first draft, gave early contributions regarding content and designed and figures. FE-C and HL designed the figures and revised the final version. GU edited and contributed to the final manuscript version. GD wrote the manuscript, supervised overall writing and edited the final version. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Centipeda minima (L.) A. Braun & Asch is a well-studied plant in Chinese medicine that is used for the treatment of several diseases. A recent study has revealed the effects of extract of Cetipeda minima (CMX) standardized by brevilin A in inducing hair growth. However, the mechanism of action of CMX in human hair follicle dermal papilla cells (HFDPCs) has not yet been identified. We aimed to investigate the molecular basis underlying the effect of CMX on hair growth in HFDPCs. CMX induced the proliferation of HFDPCs, and the transcript-level expression of Wnt family member 5a (Wnt5a), frizzled receptor (FZDR), and vascular endothelial growth factor (VEGF) was upregulated. These results correlated with an increase in the expression of growth-related factors, such as VEGF and IGF-1. Immunoblotting and immunocytochemistry further revealed that the phosphorylation of ERK and JNK was enhanced by CMX in HFDPCs, and β-catenin accumulated significantly in a dose-dependent manner. Therefore, CMX substantially induced the expression of Wnt signaling-related proteins, such as GSK phosphorylation and β-catenin. This study supports the hypothesis that CMX promotes hair growth and secretion of growth factors via the Wnt/β-catenin, ERK, and JNK signaling pathways. In addition, computational predictions of drug-likeness, together with ADME property predictions, revealed the satisfactory bioavailability score of CMX compounds, exhibiting high gastrointestinal absorption. We suggest that CMX could be used as a promising treatment for hair regeneration and minimization of hair loss. Hair loss or alopecia is an incurable disease that affects the quality of life and is emotionally distressing, especially regarding self-confidence. Hair loss is caused by physiological situations related to hormonal imbalance, age, autoimmune conditions, medications, and genetics . AndrogeHair growth and hair loss in mammalian species are also controlled by a follicular cell cycle that includes periods of growth (anagen phase), regression (catagen phase), and rest (telogen phase) . These cCentipeda minima (L.) A. Braun & Asch. is plant in the Asteraceae family widely distributed in China, Korea, Japan, Australia, and India, and is commonly used Chinese herbal medicine. Aerial parts of C. minima have been used for centuries to treat diarrhea, asthma, nasal allergies, and malaria. Recent pharmacological studies on an extract of C. minima have demonstrated that it has antioxidant, neuroprotective, antibacterial, and anticancer biological activities. Twelve major chemical constituents such as flavonoids, polyphenolic acid, and sesquiterpene lactones have been identified through HPLC-Q-TOF-MS [C. minima (CMX) by brevilin A on hair loss using a framework that integrated an in vitro investigation, a clinical study, and a network pharmacology-based analysis [Q-TOF-MS ,9. Previanalysis ,11. The Based on the connections between Wnt/β-catenin activation and hair development, researchers have identified the activators of this signaling pathway as potential interventions for hair therapy. Many natural and synthetic compounds have been reported to promote hair regeneration through the activation of Wnt/β-catenin signaling . HoweverAntibodies against β-catenin (D10A8), GSK3β (27C10), phospho-GSK3β (Ser 9), p38(A-12), phospho-p38 Thr180/Tyr182 (D3F9), ERK1/2 (p44/42), phospho-ERK (Thr202/Tyr204), SAPK/JNK, phospho-JNK (Thr183/Tyr185), AKT (5G3), phospho-AKT (Ser473), and GAPDH were purchased from Cell Signaling Technology . Additionally, antibodies against IGF (W18) and VEGF (C-1) were purchased from Santa Cruz Biotechnology . Goat anti-rabbit IgG cross-adsorbed secondary antibody, Texas Red-X, and SlowFade Gold antifade reagent with DAPI were obtained from Invitrogen . Cell culture slides for immunofluorescence were purchased from SPL .C. minima was purchased in December 2019 from Natural-herb . The material was identified by 1 of the authors (J.P.). A voucher specimen of the material (CM-2019-001) was deposited in the herbarium at Kyungsung University. CMX was prepared by D. Nature Co., Ltd.  using the efficient separation of brevilin A from C. minima by inducing phase separation in the emulsion as reported previously [eviously , and itsThe amounts of major compounds of the CMX were determined by HPLC-UV . For the HPLC conditions, refer to the previous study . Filtere2.Human hair follicle dermal papilla cells (HFDPCs) were purchased from PromoCell . The cells were maintained in a follicle dermal papilla cell basal medium (PromoCell) supplemented with 4% fetal calf serum, 0.4% bovine pituitary extract, 1 ng/mL basic fibroblast growth factor, and 5 μg/mL recombinant human insulin (PromoCell) at 37 °C in a humidified atmosphere of 5% CO4 cells per well. After 24 h of incubation, the cells were treated with CMX at various concentrations of 0.39, 0.78, 1.56, 3.13, 6.25, and 12.5 μg/mL for 24 h. The cells were incubated with water-soluble tetrazolium salt (WST-1) for 1 h. The absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader, and the results were expressed as percentages of the untreated controls.Cell viability was determined using an EZ-Cytox assay kit  . BrieflyTotal RNA was isolated from cells using the RNeasy Mini Kit  and quantified using the Nanodrop method. cDNA was synthesized using the AccuPower RocketScript Cycle RT Premix , and 1 µg of cDNA was used for PCR.−ΔΔCt method and normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the primers used in qRT-PCR are listed in qRT-PCR was performed using a PowerUP SYBR Green PCR Master Mix (Thermo Fisher Scientific) with a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) according to the manufacturer’s protocol . The mRNThe expression levels of KGF/FGF-7 (keratinocyte growth factor/fibroblast growth factor-7) and VEGF  in the culture medium were measured; 500 mL of the culture supernatant was harvested from the cultured cells. The levels of KGF/FGF-7 and VEGF were measured using an ELISA kit  according to the manufacturer’s instructions. Absorbance was determined at 450 nm using a microplate reader.HFDPCs were treated with the indicated concentrations of CMX for 24 h. Following treatment, the cells were washed with phosphate-buffered saline (PBS) twice and then lysed in a cold radioimmunoprecipitation assay (RIPA) buffer  containing 1 mM DTT , a phosphatase inhibitor cocktail , and a protease inhibitor cocktail  . The pro4 cells/well) and cultured in a growth medium containing the supplement mixture. Cells were fixed in 4% paraformaldehyde diluted in PBS for 15 min at room temperature. After blocking with a blocking reagent containing 5% BSA and 0.1% Triton X-100 in PBS for 60 min at room temperature, the cells were stained with β-catenin antibody (1:100)  overnight. The cells were rinsed 3 times with PBS and incubated with Texas Red-X goat anti-rabbit antibody for 2 h at room temperature. Cells were counterstained with DAPI and observed using a TH4-200 immunofluorescence microscopy system .HFDPCs were seeded in a 4-well chamber . The SwissADME computational tool allows the prediction of the following pharmacokinetic characteristics: gastrointestinal (GI) absorption, inhibition of some cytochrome P450 (CYP) enzymes regularly involved in interactions with xenobiotics , and skin permeability, with an accuracy of 71 to 89% [The pharmacokinetic and drug-likeness properties of CMX were predicted using the web-based tool SwissADME  from 3 independent experiments. Statistical analyses were performed with GraphPad Prism 5 . Statistical significance was presented as * The representative HPLC chromatograms of CMX are shown in We investigated the effect of CMX on the proliferation of HFDPCs. HFDPCs were treated with different concentrations  of CMX for 24 h. Cell viability was estimated using the EZ-Cytox assay. The viability of HFDPCs treated with CMX is shown in The Wnt pathway plays an essential role in induction of hair follicle growth . To deteHair regeneration is controlled by dermal papilla (DP) cells, which manage hair follicle cycling through secreted signaling factors . Thus, wWnt/β-catenin signaling plays a crucial role in the growth of DP cells ,22. The When cells are not stimulated by Wnt, cytoplasmic β-catenin is phosphorylated and degraded by kinases called casein kinase-I and GSK3 in the Axin and APC protein complexes. However, when Wnt is activated by external stimulation, the degradation of β-catenin is inhibited; therefore, the intracellular β-catenin level is increased and stabilized, and β-catenin translocates into the nucleus. Therefore, we performed an immunofluorescence assay to examine the accumulation and localization of β-catenin in HFDPCs after treatment with CMX for 24 h. CMX treatment resulted in a significant accumulation of β-catenin in the nucleus in a concentration-dependent manner compared with the DMSO control. This result correlated with the increase in β-catenin protein levels revealed by immunoblotting .Computational predictions of drug-likeness together with ADME property predictions can assess the possibility of potential lead compounds . We evalHair is one of the most unique features of mammals and is an indicator of individual health, as it serves multiple physiological functions, including protecting the body from environmental damage and maintaining body temperature . The haiC. minima is a well-known medicinal plant used for the treatment of whooping cough, cold, nasal allergy, asthma, diarrhea, and malaria in Chinese medicine. It is widely found in China, Korea, and Southeast Asia and is distributed over certain areas in Australia and India. Twelve major chemical constituents such as flavonoids, polyphenolic acid, and sesquiterpene lactones have been identified [C. minima [C. minima, was shown to induce hair regeneration, and increased hair count has been demonstrated by a previous study [entified ,9. Brevius study . The patus study ,31,32. Cus study . Therefous study ,34.Wnt/β-catenin signaling, among other signaling systems, has been implicated in the regulation of hair follicle morphogenesis and regeneration ,22,35. VWe observed that the levels of β-catenin and GSK3β, which are key intermediaries of the Wnt/β-catenin signaling pathway, were increased. Additionally, the MAPK and Akt signaling pathways are major regulators of cell proliferation. Our results showed that ERK and JNK were phosphorylated in CMX-treated cells in a dose-dependent manner. We demonstrated that these proteins are involved in the proliferation of HFDPCs. Follicle DP cells, located at the base of hair follicles, regulate the hair cycle by responding externally to stimuli and signals conveyed through cytokines and junctions .HDPFCs relay signals to epithelial cells through secreted molecules such as Wnt, Shh, and BMP during the hair cycle . Moreoveβ-catenin is an important regulator of Wnt-induced gene transcription. Using immunocytochemistry, we confirmed that β-catenin was significantly localized in the nucleus when the Wnt pathway was activated by CMX. Our results indicated that CMX upregulated the expression of growth factors such as VEGF and IGF via the Wnt/β-catenin, ERK, and JNK signaling pathways in HFDPCs. Taken together, our findings demonstrated that CMX can induce hair proliferation via the Wnt/β-catenin, ERK, and JNK signaling pathways, and secreted growth factors . In addiThis study showed that CMX regulates signaling in HFDPCs and stimulates hair regeneration through the Wnt/β-catenin, ERK, and JNK signaling pathways, resulting in the upregulation of VEGF and IGF factors. In conclusion, our study suggests the use of CMX, which has little toxicity, as an effective treatment for alopecia and highlights the possibility of cosmeceuticals in promoting hair regeneration.


Drosophila melanogaster, it is now widely appreciated that active Wnt signaling in mammals is necessary for the development and growth of various tissues involved in whole-body metabolism, such as brain, liver, pancreas, muscle, and adipose. Moreover, elegant gain- and loss-of-function studies have dissected the tissue-specific roles of various downstream effector molecules in the regulation of energy homeostasis. This review attempts to highlight and summarize the contributions of the Wnt signaling pathway and its downstream effectors on whole-body metabolism and their influence on the development of metabolic diseases, such as diabetes and obesity. A better understanding of the Wnt signaling pathway in these tissues may aid in guiding the development of future therapeutics to treat metabolic diseases.The canonical Wnt signaling pathway is ubiquitous throughout the body and influences a diverse array of physiological processes. Following the initial discovery of the Wnt signaling pathway during wing development in  During embryogenesis, the Wnt signaling pathway has active and critical roles in tissue development, including, but not limited to, axon guidance, body segmentation, limb development, and stem cell differentiation . While iWhole-body metabolism is regulated by various tissues, including brain, liver, pancreas, muscle and adipose, and each contributes significantly to carbohydrate and/or lipid metabolism. The brain requires a constant uptake of oxygen and glucose, where astrocytes convert glucose to lactate and neurons use glucose and lactate in an oxidative manner . The livwingless (wg) in Drosophila melanogaster, where its mutation resulted in a wingless phenotype  receptor and lipoprotein-related proteins (LRP5/6). This leads to a cascade of downstream events, that culminate in the dissociation of the β-catenin “destruction complex” comprised of Glycogen Synthase Kinase 3 (GSK-3), Axin, Casein-kinase 1 α (CK1-α), and Adenomatosis Polypsis Coli (APC) . In the 2+ pathways  proteins and Wnt inhibitory factors, such as Dickkopf (DKK1), bind LRP5/6 directly to inhibit its association with the Fz receptor . On the Through the use of genetic approaches, the contributions of different cell types and tissues, such as adipose tissue, skeletal muscle, brain, pancreas, and liver, to overall metabolic homeostasis have been identified , and WntWnt1 deletion on brain development and viability   in mouse embryos, followed by their rapid downregulation by e16 . Wnt1 ovagenesis ; Table 3agenesis , showing-Fz8CRD) . Transge-Fz8CRD) .wnt2bb led to agenesis of hepatic tissue  and perivenous (PV) hepatocytes . PP hepaWls, the gene encoding the Wnt receptor GPR177, in murine endothelial cells (Glu1 (Glutamine synthase) and Rnase4 (Ribonuclease 4) were found to be upregulated at the neonatal stage, with no differences during the embryonic phase  are highly enriched within neuromuscular junctions , and oveβ-Catenin is a member of the Armadillo protein family and has been shown to have pleiotropic functions, including cell adhesion to gene transcription. One of the earliest, identified functions of β-catenin is its involvement in cell-cell adhesion via its association with E-cadherin to form adherens junctions . β-catenβ-catenin is characterized by the presence of 12 Armadillo repeats, which help to form a rigid center with flexibility at the C- and N- termini . ArmadilProteins related to β-catenin include plakoglobin (γ-catenin), δ-catenin, and α-catenin, and like β-catenin, they associate with E-cadherin . Among tDeletion of β-catenin in PDX1-positive cells in the pancreas revealed that β-catenin is necessary for acinar development, but not for the development of endocrine cells. Knockout mice exhibited early lethality and were smaller in size than wildtype mice, and they developed pancreatic hypoplasia . Histoloβ-catenin’s role in myogenic differentiation is not well-defined, as different groups have reported different effects of over-expression or depletion on the initiation of myogenesis . Retrovi+ muscle cell progenitors express low levels of nuclear β-catenin prior to the induction of myogenesis, followed by a marked increase post-differentiation  is an important component of the Wnt signaling pathway, as it binds to β-catenin to mediate the expression of critical Wnt-target genes. Genome-wide association studies have revealed a strong association with SNPs close to the TCF7L2 locus and the risk of developing of type 2 diabetes . These SGys2 (Glycogen Synthase 2) mRNA levels were decreased, while mRNA levels of Pck1, G6pc, and Fbp1 were increased, which may account for changes in glycogen synthesis and gluconeogenesis at birth (Systemic deletion of TCF7L2 (TCF7L2KO) in mice results in early lethality, as newborns were considerably hypoglycemic 3 h postpartum . Notablyat birth .Postnatal deletion of TCF7L2 in β-cells was not associated with any effects on body weight or glucose homeostasis in mice fed normal chow or high-fat diets . HistoloG6pc and Aldh3a2  resulted in mild fasting hypoglycemia, similar to what was observed with systemic TCF7L2KO mice . Microar Aldh3a2 . Opposit Aldh3a2 . Genes agulation . Moreovegulation .Myh7, a marker of myogenic maturation, in slow-muscle fibers. However, its expression was upregulated in the skeletal muscle of adult mice, with the exception of the soleus muscle, suggesting that TCF7L2 exerts developmental effects on myogenic maturation. Deletion of TCF7L2 in fetal myogenic cells using a Pax7-Cre driver was associated with reduced Myh7 and Myh2 gene expression in hind limb muscles and the diaphragm, supporting the hypothesis that TCF7L2 is important for myogenesis  has also been implicated in obesity in fruit flies. Abdominal fat body mass was increased following knockdown of wg using a Mef2-Gal4 driver  of obese individuals compared to subcutaneous adipose tissue (SCAT) , and Wntammation .LRP6 is essential for Wnt binding to Fz in the canonical pathway, but it can also inhibit the non-canonical Wnt signaling pathway . LRP6 muPck) and Glucose-6-phosphatase (G6p) in vivo, as well as in Hepa1-6 hepatoma cells, were decreased or increased following depletion or over-expression of β-catenin, respectively  due to negative effects on re-modeling of the actin cytoskeleton  in Drosophila and its replacement with a repetitive heterochromatin sequence resulted in a hyperactive Wnt signaling response, as Armadillo (B -catenin’s Drosophila ortholog) expression increased  exhibited decreased fed and fasting insulin levels, and when bred with mice heterozygous for Ir, haploinsufficiency of GSK3-β was able to inprove the metabolic phenotype of heterozygous Ir mice  in mice is known to cause perinatal lethality due to diabetic ketoacidosis, and mice heterozygous for ignaling . GSK3β iignaling . Mice he Ir mice . Previou Ir mice . When copoptosis . Expansiferation .Deletion of GSK-3β in β-cells is sufficient to partially rescue the diabetic phenotype of IRS2 knockout mice, as mice were normoglycemic despite being hyperinsulinemic . This waMlc1f-promoter driven Cre-mediated recombination results in mice with enhanced glucose tolerance. Morover, these mice had increased glycogen synthase, resulting in improved insulin signaling and actions, and this highlights GSK-3β’s importance in regulating insulin action in a tissue specific manner , which is derived from intestinal endocrine L cells, potentiates glucose-stimulated insulin secretion through actions on the GLP-1R on the surface of β-cells . In L ce β-cells . Indeed,ectively , 2015. Aectively . In adulectively .To examine TCF7L2’s effects on type 2 diabetes in zebrafish, heterozygous knockouts were generated by introducing a mutation in intron 1, which produced a truncated protein . Adult fSlc2a4 (GLUT4) mRNA. Adipocyte-specific TCF7L2 knockout mice were glucose intolerant and showed signs of hepatic insulin resistance that was associated with increased gluconeogenesis. Under high-fat diet conditions, knockout mice gained more weight and showed increased hypertrophy of inguinal white adipose tissue, and gene expression analysis of inguinal white adipose tissue displayed an increase in the Wnt target gene, Axin2, suggesting that the reduction of TCF7L2 activated the Wnt signaling pathway to initiate adipocyte hypertrophy.TCF7L2 has been demonstrated to have important roles in adipogenesis, but it is not without controversy . One stuin vitro and in vivo models. Mice fed a high fat diet exhibited an increased abundance of TCF7L2 and adiponectin expression in epididymal white adipose tissue, and this increase in TCF7L2 abundance could be recapitulated in adipose tissue from ob/ob and db/db mice  and Tgh2 gene expression, and adipocyte hypertrophy (In contrast, /db mice . Adipocyertrophy .Adipoq-Cre driver recapitulated the hyperglycemic phenotype seen by ex vivo, possibly due to the decreased islet expression of Glut2 mRNA. No differences in insulin secretion were seen in vivo under normal chow-fed conditions, possibly due to compensatory increases in circulating levels of NEFAs that can potentiate glucose-stimulated insulin secretion. Furthermore, under high-fat diet, no weight changes were seen but TCFL2 knockout diminished insulin secretion (Further studies done by ecretion .All three studies examining TCF7L2’s effects on adipocyte function and adipogenesis were performed on male mice using different Adipocyte-specific Cre driver mice. However, the exons targeted for recombination were also different for all three studies. Chen et al., floxed exon 11, known to encode the DNA binding HMG box . On the Although the Wnt signaling pathway is conserved across species and is necessary for embryogenesis and development, it is now clear that it also has essential metabolic roles in mammals. Moreover, aberrant expression or activity of various downstream effectors in the Wnt signaling pathway is associated with the development of chronic diseases, including diabetes and obesity. Given the pathway’s wide ranging effects and multiple downstream targets, more research is required to further our understanding of its involvement across different metabolic diseases.FAA wrote the manuscript. GEL edited the manuscript and the guarantor of this work. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This article presents the analysis of the deformability, structure and properties of the AZ61 cast magnesium alloy on the example of a new forging process of aircraft mount forgings. It was assumed that their production process would be based on drop forging on a die hammer. Two geometries of preforms, differing in forging degree, were used as the billet for the forging process. It was assumed that using a cast, unformed preform positively affects the deformability of hard-deformable magnesium alloys and flow kinematics during their forging and reduces the number of operations necessary to obtain the correct product. Numerical analysis of the proposed new technology was carried out using DEFORM 3D v.11, a commercial program dedicated to analyzing metal forming processes. The simulations were performed in the conditions of spatial strain, considering the full thermomechanical analysis. The obtained results of numerical tests confirmed the possibility of forming the forgings of aviation mounts from the AZ61 cast magnesium alloy with the proposed technology. They also allowed us to obtain information about the kinematics of the material flow during forming and process parameters, such as strain intensity distribution, temperatures, Cockcroft–Latham criterion and forming energy. The proposed forging process on a die hammer was verified in industrial conditions. The manufactured forgings of aircraft mounts made of AZ61 magnesium alloy were subjected to qualitative tests in terms of their structure, conductivity and mechanical properties. Lately, an increased interest in magnesium alloys has been noticeable ,3,4,5,6,However, magnesium is a reactive metal and very susceptible to corrosion, especially in environments containing chloride ions, limiting the application area of magnesium alloys. For this reason, it is necessary to protect the surface of magnesium components by applying additional paint coatings, conversion coatings or electrochemical coatings, or by using anodizing processes and vapor deposition of coatings ,12,13,14In aviation, a wide range of construction elements is used, e.g., gearbox, engine, wing, hull plating, door, wheels, landing gear, cockpit panels and seat elements . For exa3, allows to significantly decrease the product’s weight, even by 30%. The factors limiting the usage of magnesium alloys include low corrosion resistance, flammability, lower strength, specific forming conditions due to a narrow range of temperature parameters and sensitivity to strain, which results in high-cost of metal forming and mechanical machining [Magnesium alloys are also found in household items. Using magnesium, the density, of which is 1.74 g/cmachining ,20,21.The magnesium alloys are the most widely used as-cast alloys ,23,24,25The necessity of using castings is forced by the low availability of magnesium forgings, which would significantly increase the mechanical and functional properties of the final product and decrease the production cost. Despite the beneficial mechanical properties, using magnesium alloys for forming accounts for only 1% of the annual production of magnesium in the world, which is related to the limited plasticity of these magnesium alloys.Metal forming of magnesium alloys proves even more difficult due to the narrow range of temperature parameters and sensitivity to the strain. Therefore, forming magnesium alloys is carried out in high-temperature on forging machines with low operating speeds while maintaining isothermal temperature conditions during deformation. Currently, the few companies that have implemented the technology of forging magnesium alloys on specialized hydraulic presses with tool heating systems include Otto-Fuchs and Weisensee Warmpressteile from Germany and KUMZ from Russia.Some casting alloys are also formed, including by forging ,27. ThisThe die forging of magnesium alloys on forging equipment, such as forging hammers without specialized heating systems, would reduce production costs and increase productivity. Unfortunately, using standard shapes of castings for forging magnesium alloys, particularly hard-deforming, on traditional hammers does not guarantee correct forgings without cracks. Often the first cracks inside the material appear during the initial forging when forging is formed. Further processing of the forgings in the finishing impression reveals a defect in the form of cracks, which disqualifies the product. The solution to this problem could be using the ready-made preforms, which are cast to mirror the forgings as closely as possible. Theoretically, in terms of costs, using preforms obtained by the casting should be more advantageous than using billets, and consequently, should be an interesting alternative. In this case, the production process of the charge is shortened by the operations related to the extrusion of the billets themselves and the initial forging of the preforms. It seems that such a solution may also have several valuable advantages. The mechanical properties of the forgings obtained from the billets are comparable for both technologies, and in many cases, favor those cast due to their low anisotropy. There are, however, differences in the structure of the forgings made of ingot compared to using wrought billet forging. The forging made of an extruded billet receives in some sections a fibrous structure, which results in different values of strength properties depending on the direction of the fibers in the forging. In some cases, this may be disadvantageous from the point of view of the distribution of the mechanical properties in the product. A more fragmented and homogeneous structure in the entire volume of the product favor forgings made of cast materials.It was assumed that using a preform cast as close as possible to the shape of the forging in the plane of the die division would have a positive effect on the kinematics of metal flow, especially when deforming less deformable grades of magnesium alloys, which would allow obtaining a correct product without defects with a more precise shape and dimensions. By limiting the number of operations needed to produce the forging, greater material and energy savings can be achieved.Therefore, it was considered advisable to test this concept and develop new technology for forging magnesium alloys from cast preforms. The AZ61 magnesium alloy with good strength properties, which is of interest to the aerospace and automotive industries, was selected for the study. The article analyses the deformability, structure and properties of the AZ61 cast magnesium alloy in the form of preforms used in the new hammer forging process of the forgings of the aircraft mounts.The research concentrated on a new forming process of forging an aircraft mount a manufacIt was assumed that in the new process of forging an aircraft mount from AZ61 grade alloy, the geometry of the preform would be similar to the forging, especially in terms of the outline in the area of die division. Using a not-wrought preform positively influences the flow kinematics and deformability of the material during forging. Using a billet with precise/accurate dimensions in the form of cast preform allows reducing material waste compared to the process currently used in the industry, that is, forging from an extruded billet. The process is realized in one forging operation in a finishing die, using typical forging machines, die hammers and inexpensive tool heating methods . It is also worth mentioning that using a ready-made cast forging for the forging process limits the number and duration of operations required for the forging to be obtained, which influences the effectiveness and decreases the labor consumption of the process. Elements subjected to metal forming following the new technology are of better quality, resulting from a better macro- and microstructure, whereas its surfaces are smoother, which, in turn, enhance the functional and mechanical properties of the product compared to the ones made from cast only.0 to the height of the deformed forging. The forgings differ mainly in height and thus in the degree of forging and the cross-sectional dimensions in the division plane. The degree of forging, i.e., the ratio of the height of the forging preform to the height of the forging product for the first variant was hI/h0 = 38.5/28.5 = 1.35, and for the second variant, hII/h0 = 43.5/28.5 = 1.52. The scheme of the geometry of the preforms and the forging is shown in −1 to 10 s−1 and strain values in the range 0–1.The numerical analysis of forging the aircraft mount in a hammer from AZ61 grade alloy was performed using the finite elements method (FEM) in Deform 3D ver. 11.0 software . FEM simulations were conducted with the spatial strain state and applying the full thermomechanical model. For the new process, two preforms geometries were designed, with a more and less significant forging degree, being the ratio of the height of the billet h2K and between workpiece and tools 4.5 kW/m2K [The forging process for manufacturing magnesium alloy aircraft bracket is performed with a hammer described by the impact energy of 36 kJ and the weight of the dropping part of 1200 kg. The heat transfer coefficient between the workpiece and the environment was assumed to be 0.03 kW/m5 kW/m2K . The fri5 kW/m2K .AZ61 magnesium alloy cast into sand molds was used for the experimental tests. The chemical composition of the AZ61 alloy is shown in The experimental tests of the new process of forming forgings of aircraft mounts from AZ61 alloy castings were carried out in industrial conditions at ZOP Co. Ltd. FORGING PLANT . All forging tests were performed on an MPM 3150 die hammer  a using aTo assess the quality of the formed AZ61 alloy forgings of aircraft mounts obtained during industrial tests, the structure, electrical conductivity and mechanical properties were tested. The macrostructure tests were carried out in the cross-section of the mounting rib  for cast6 crystal source and an SDD-type EDS detector.Microstructural examinations were carried out using the NIKON MA200 optical microscope . Quantitative microstructure analysis was performed using Image-Pro 10 software. The analysis was carried out both in the central and near-surface areas of the samples, as shown in The next performed qualitative tests were tests of specific electrical conductivity. They were carried out to evaluate the structure of the material after forging and after heat treatment. Conductivity measurements were made on the metallographic polished sections analyzed during the microstructure tests. The conductivity of the samples was measured with a SIGMATEST model 2.069 . The measurements were made at an ambient temperature of 21 °C.Strength tests were carried out on samples made of heat-treated cast preforms and on forgings of mounts. The geometry of the samples and the test procedure were following ISO 6892-1. A Shimadzu AG-X plus 20KN testing machine  was used for the tests, equipped with a longitudinal extensometer to measure deformations and to control the test speed in the feedback loop. The tests were carried out under controlled conditions at 21 °C. The speed of the test was variable. In the elastic range, the strain was controlled using the signal from the extensometer, and the speed was 0.025%/s. In the range of plastic flow, the traverse displacement speed was 0.9 mm/min.Hardness measurements were made using the Vickers method with the Future-tech FM800 hardness tester . Measurements were made on the HV0.5 scale following PN-EN ISO 6507-1: 2006The theoretical analysis results confirmed the possibility of forming the forging of the aircraft mount from AZ61 magnesium alloy from cast preforms of an assumed geometry. The numerical analysis results also provided information on the important parameters of the process, such as strain intensity, distribution of temperature and Cockcroft–Latham damage criterion and the forming energy. The distribution of the effective strain in the formed forging of the aircraft mount is shown in Additionally, the distribution of temperature in the formed forgings  was analmax-maximum principal stress, σH-equivalent stress according to Huber’s hypothesis, 𝜀-strain intensity, C1-integral value.Along with the analysis of the parameters discussed above, the risk of cracking in the forging was researched. For the theoretical analysis of this phenomenon, the Cockroft–Latham (C–L) failure criterion  in a mod1 = CCL, plastic fracture of the material occurs. The results are shown in The C-L criterion assumes that when the work done by tensile stresses in uniform tension reaches a certain critical value CFEM analysis allowed to determine the forming energy  occurrinThe results of experimental tests carried out in industrial conditions confirm the good deformability of the AZ61cast magnesium alloy and the possibility of producing aircraft mounts from both geometry variants of the cast-shaped preforms. The microanalysis results indicated a significant heterogeneity of the chemical composition of the preform in the as-cast state, which was characteristic of this type of alloy. Three basic regions were distinguished, regions of different composition: the zone of Mg solid solution with low content of alloying additives, precipitations of the intermetallic phase (most likely Mg17Al12) and eutectic regions. As a result of the homogenization process, the chemical composition became homogeneous and was close to the nominal one over the entire tested cross-section.Based on studying the microstructure of the cast preforms in their raw state, the structures shown in All raw castings had a homogeneous structure throughout the tested cross-section. This structure was characteristic of this as-cast alloy and was dendritic. It consisted of chain precipitates of intermetallic phased, most of which were β phase (Mg17Al12) surrounded by eutectic precipitated. The alloy matrix was a solid solution of αMg. After homogenization of the castings, grain growth was visible, with the dissolution of most of the precipitates in the alloy matrix.After the preform of the first variant was hammered, the microstructure had a significantly finer grain both in the central zone and near the surface. In the third area, with the highest intensity of deformation, the grain was the finest. The grain size was relatively uniform . The twin deformations were visible.In the case of forging a higher preform (second variant), the situation was analogous. The greatest grain refinement occurs in the third area, and in areas 1 and 2, the grain sizes were less homogeneous compared to the lower preform. However, the difference was not significant. The grain size differences were acceptable. The shape of the grains in all studied areas indicated that the processes of dynamic recovery and recrystallization were significantly advanced. When comparing the structural homogeneity obtained in the considered variants of the preforms, it should be noted that, although the most favorable microstructure was obtained for area 3 of the preform II, the differences in individual areas were smaller in the case of the I variant preform. This may indicate that a high degree of forging promotes fragmentation and homogeneity. However, the differences in the individual areas indicate that the distribution of deformations in the II variant forging was less homogeneous.After heat treatment, the grain regrowth was noticed. However, it was not as significant as after homogenization. Lenticular secondary precipitated were visible (discontinuous). In all areas, the grain size was similar, and its shape was regular.A quantitative assessment of the microstructure was carried out in the areas analyzed on the surface of preforms and forgings. The as-cast grain size was significant, mainly due to undeveloped grain boundaries in the dendritic structure. The grain was still relatively large after homogenization. As a result of forging, the grain broke down, and due to recrystallization, the average grain size dropped significantly, especially in areas with high deformation value (3 area), as indicated by the results of numerical simulations. For the variant with a higher degree of deformation, the average grain size was smaller in all areas.The use of heat treatment increased the grain size in all areas, particularly for variant I. The obtained results indicate that using high deformation values had a positive effect on the microstructure obtained at the end of the process.2, and for area 3, below 60 μm2. For variant II in areas 1 and 2, the values were analogous, and in area 3, most of the grains were smaller than 40 μm2. After the heat treatment, the grain size increased and the differences in grain size in the individual areas reduced. The analysis of the grain size distribution in individual areas showed that in most cases, the smallest grains were the most numerous group and that there were single grains of a much larger size. In the case of forged samples, for variant I in areas 1 and 2, most of the grains had a surface area below 100 μmThe objective of this study was to investigate the formability of AZ61 cast magnesium alloy using the example of an innovative technique for producing forging of aircraft mounts by hammer forging from cast preforms. The numerical and experimental results demonstrate that AZ61 cast magnesium alloy in sand molds has good formability in the hammer die forging process, and the proposed new forging method is a viable way of producing forgings of aircraft mounts with the required shape. The new method ensures considerable material and energy savings and higher properties of the product than previously applied techniques, consisting of forming aircraft mounts only from casts by machining operations.Based on the analyses conducted, the following conclusions were formulated:The developed new technology of forging from preforms assumes greater production efficiency due to the shortening of the time of forging in one forging operation compared to the multi-stage forging from the extruded rod used in the industry.Structural studies have shown that the material’s structure undergoes substantial evolution at different stages of the process. Homogenization results in obtaining a consistent initial structure for the die forging process. After forging, significant grain refinement was observed, which is not uniform throughout the forging volume, but the grain size differences are perfectly acceptable. Further heat treatment makes the grain sizes and shapes uniform and allows the alloy to be strengthened by secondary precipitation. The conducted tests and conductivity analysis confirm the observed evolution of the material’s microstructure during the process. The detected decrease in hardness promotes forming, while its significant increase after heat treatment has a positive effect on the performance of forgings. Small differences in the values obtained for variants I and II may result, first of all, from the different cooling kinetics of castings with different volumes and the different degree of forging. Finally, variant II has higher mechanical properties and conductivity, but the differences are small. Comparing the maximum values of the strength and hardness properties of the new forging technology from forgings to the traditional rod forging technology, they are as follows: new technology-Rm = 250 MPa, HV = 75.5; bar forging-Rm = 292 MPa, HV = 65.7 .Lightweight magnesium alloys belong to the group of construction materials considered key importance for the future. Therefore, it is necessary to conduct further research developing the technique for manufacturing magnesium alloy products using forging hammers that have significantly higher output capacity than hydraulic presses. The positive results of previous studies on the hammer forging process from AZ61 magnesium alloy-shaped cast preforms prove that the research should be continued for other workable magnesium alloys.


Aquatic macro-invertebrates play a vital role in the food chain of river ecosystem at several trophic guilds and consumer levels, and are used as biomonitoring tools for aquatic ecosystem health. However, hydrologic conditions of these ecosystems have been severely altered because of the increase in urban development and agricultural expansion. This study examined benthic invertebrate response to processes that structure their community in the Wewe River, segmented into intact, medium, and severe condition zones. We sampled in 100 stations in a period of 4 months in the wet  and 3 months in the dry  seasons. Geometric series, rarefaction, and Hill numbers models were used to quantify invertebrate assemblages, while ordination technique, canonical correspondence analysis, was used to evaluate the influence of predictive factors on their assemblages. A total of 2,075 individuals belonging to 20 family taxa were registered. There was no significant difference in benthic assemblages between the dry and wet seasons. Predictive factors accounted for 47.04 and 50.84% variances, respectively. Taxa distribution patterns differed significantly only in the severely disturbed zone during the wet season. Neptidae, Libellulidae, and Chironomidae were the most abundant taxa, indicating their broad range habitat preference and their ability to adapt to seasonal changes. Asellidae and Perlidae were the least detected, suggesting their sensitivity to elevated levels of some water quality parameters. The findings highlight the threats to the benthic community and overall functional state of the Wewe River, with the need to consider the proposed conservation interventions indicated in this study. Within Changes in benthic invertebrate distributions caused by river regulation may occur because of altered habitat, flow patterns, sediment input, water quality, and thermal regimes ,6. ApartStreams and rivers in urban centres worldwide have been severely polluted because of increase in urban development ,20. In GGiven the lack of scientific information about benthic invertebrates’ status in the Kumasi Metropolitan Area, it is not clear how these disturbance-related drivers have directly influenced physicochemical parameters and the consequent effect on benthic invertebrates. Furthermore, because of their sensitivity to aquatic environments, benthic invertebrates are widely considered as good indicators of water quality , by aidiIn this paper, we assessed seasonal response of benthic invertebrate to physicochemical parameters in the Wewe River. To achieve this broad aim, we sought to answer the following objectives: (1) Are there variations in the abundance and distribution patterns along the three condition zones of the Wewe River? (2) Are there differences in benthic diversity among the three condition zones of the Wewe River? (3) What processes structured benthic assemblages among the three condition zones of the Wewe River? We hypothesized that (a) benthic invertebrate assemblages will differ between seasons, in terms of both taxonomic diversity and number of individuals, because benthic invertebrates have the capacity to recover rapidly from extreme drought periods ; (b) phy22.1The study area is a suburban forest reserve in the Kumasi Metropolis of Ghana and surrounded by communities like Ayigya, Weweso, Bomso, Gyenyasi, Kentikrono, and Ahinsan. Farming along the fringes of the river occurs year round. Waste water from nearby settlements and farm waste equally drains into the river course through direct channel connectivity. Wewe River is a typical unregulated system and takes its source from mountains near Aboabo Nkwanta and flows for about 8.125 km southwest towards Abirem and Weweso . The riv2.2intact condition class (10–15): areas dominated by different vegetation strata that cover the full length of the segment, which is linked to natural fluvial forms and slightly fragmented; moderately disturbed condition class (7–9): areas with vegetation cover nearly half of the study zone being disturbed; 1–3 m active channel width and about 10–30% exotic and ruderal species present; severely disturbed condition class (1–6): areas where 60% of the riparian corridor is reduced by human-led activities; vegetation covering <30% (mainly grasses/herbs and isolated woody species), with channel banks connected to agricultural fields. The end-to-end distance within each condition zone included the following: 180 m , 937 m , and 533 m   methods). The river course was classified into three condition zones, because it is surrounded by a forest reserve, with some disturbance along the riparian zone, which could have a direct influence on the water quality and consequently on benthic community structure. The index ranges from 1 and 15. Thus, of ±5 m) , Plate 12.32 mouth area, and 1 m length dimensions). The D-frame sweep net was driven deep into the sediment and against the flow direction, to trap all organisms inhabiting the sediment column beneath surface water. The D-frame sweep nets are best used as qualitative or semi-quantitative analyses where a diversity of specimens (e.g. IBI or other indices) is more important than density estimates sampling.Benthic invertebrates were collected in biweekly intervals for a 7-month period – 4 months in the wet season  and 3 months in the dry season . There is no rainfall in the dry season, and this reflects in the low flow rate. A total of 100 samples were randomly collected across the three segments , with six replicates per sample point. We sampled each condition zone once per week. Invertebrates were collected using a D-frame sweep net . Physicochemical drivers were log transformed where appropriate to achieve normality.Water quality variables, DO, TDS, EC, surface water temperature, salinity, mercury content (mm Hg), and pH, were measured 2.5Objective 1: Are there variations in the abundance and distribution patterns of benthic taxa along the three condition zones of the Wewe River?2.5.1Ho) that invertebrate abundance distribution and richness did not differ in each of the three segments or condition zones classes of the Wewe River.Both benthic invertebrates and physicochemical drivers’ dataset were initially subjected to square root transformation to homogenize variances and achieve normality . Invertex-axis and the abundance plotted on the y-axis. Analysis of covariance was used to test for the significant difference of the equality of the slope of invertebrate abundance distributions among the three segments of the Wewe River.All registered invertebrate order in each of the three segments of the river were ranked from the most to the least abundant on the rank abundant curve , with eaObjective 2: Are there differences in benthic richness and diversity among the three condition zones of the Wewe River?2.5.2n” less than Individual-based rarefaction model  was perfocument} . Rarefacocument}  and haveocument} ,47.S is the number of species in the assemblage, and the ith species has relative abundance pi, i = 1, 2,..., S.Finally, invertebrate diversity was quantified using Hill numbers ,50,51,52Objective 3: What processes structured benthic assemblages among the three condition zones of the Wewe River?2.5.3t-test was performed to determine seasonal variability among invertebrates’ families and physicochemical drivers. Where significant difference was detected, we further used Tukey’s HSD post hoc test to determine the habitats that differed. Spearman’s rank correlation test was performed to evaluate the significant relationship among physicochemical drivers. All the analyses were performed using PAST ver. 3.18 Package [Canonical correspondence analysis (CCA) was used to evaluate the influence of predictive factors on benthic assemblages . CCA is  Package .33.1n = 693) and wet  seasons and among the habitat condition zones  and wet  seasons  and intact and severe condition zones (P < 0.0001), whereas during the wet season, it was between the moderate and severe condition zones (P < 0.0007) and intact and severe condition zones (P < 0.0006). Although we detected more family taxa in the wet season (n = 17) than the dry season (n = 12), mean seasonal variations were not substantially significant   . From in = 0.75) .3.2Z = −0.517, P = 0.61, Fligner Kileen test for equal C.V.) , intact , and severely disturbed  condition zones in the dry season (P < 0.901) and intact (P < 0.99), with the exception of the severely disturbed condition zone   . Individy season , Table 1 0.0004) , Table 1n = 78), Odontopygidae (n = 73), and Baetidae (n = 72) were the highest ranked on the abundance distribution curve and were widely distributed across the three condition zones, during the dry season , Dytiscidae , and Perlidae  were the least ranked in the moderate and intact condition zone, reflecting their sensitivity to habitat perturbation. Overall, the severely disturbed condition zone supported the most abundant family taxa.Of the 20 benthic families registered, Libellulidae , Libellulidae , and Chironomidae  were the highest ranked invertebrate families in all three condition zones, while Asellidae (n = 17) and termitidae (n = 45) were the least ranked and infrequent taxa, whose distribution occurred in narrow ranges in the three condition zones , although the number of benthic families appeared more in the wet season (n = 17) than in the dry season (n = 12)  . General(n = 12) . Observe(n = 12) . ConditiF2,6 = 0.0461, P = 0.65) and wet  seasons, and ranged as qD = 2.974–2.996 in the dry and wet seasons (qD = 2.996) and the severe condition zone  (qD = 2.991) to be the most diverse in the dry and wet seasons, respectively (qD = 2.974) and wet (qD = 2.854) seasons. The low diversity in the moderate condition zone appears to reflect in its lowest abundance of individuals in both seasons as well  and surface water temperature  on Axis I, and total dissolved solids  and EC  on Axis II, as the major physicochemical drivers of benthic invertebrate assemblages among the three condition zones in the dry season ), TDS , and SS  on Axis I and Mercury  and pH  on Axis II were found to influence the community structure of benthic invertebrate  and wet  seasons. Furthermore, seasonal variability in physicochemical drivers was equally not significant .The matrices of the invertebrate-site biplot generated by CCA showed DO  and EC  in the severe condition zone (DOW = downstream), along Axes I and II (upper left of the CCA diagram). On the lower left of the ordination diagram, Libellulidae, Odontopygidae, and Gerridae were negatively associated with total dissolved solids  and DO  on Axes I and II  may be an effect of the high flow rate, causing oxygen to dissolve in the water, and simultaneously carrying floating debris  and Termitidae  in the moderate (MID – midstream) and severely disturbed (DOW – downstream) condition zones. The least abundance of Notonemouridae (n = 9) and Corydalidae (n = 9) in these two zones was largely linked to the influence of EC on Axis II  and EC  on Axes I and II contributed to the low abundance of Asellidae, Perlidae, and Platycnemididae, located in the upper right hand of the ordination diagram. Similarly, the influence of weak to near-neutral pH levels  partly contributed to high abundance of Dytiscidae, Ephemeridae, and Lestidae. However, abundance of Termitidae (n = 3) was affected by the near-neutral levels of pH, especially in the moderately disturbed MID – midstream, condition zones  (n = 112) and Chironomidae (n = 109) was found to strongly correlate with total dissolved solids  and SS , on Axis 1. Other taxa, namely Gerridae, Gomphidae, and Hydropsychidae, on the upper left of the CCA diagram, were within the tolerable range of surface water temperature in the severe condition zone (downstream).In the wet season, we observed that increased level of mercury  , Table 644.1Studies of seasonal dynamics in benthic invertebrate communities among smaller lotic systems have been reported by a number of ecologists ,59,60,61Studies in an intermittent river in North Africa found similar variability in diversity of benthic invertebrate community during the dry and wet seasons . HoweverHigher taxa abundance in the wet season was thought to coincide with the larval developmental stages and differing responses of taxa to changes in water quality in the dry and wet seasons’ shift, which is largely driven by seasonal-specific disturbances (i.e. intensity of farming practices mostly in the wet season) and changes in the physical environment of the Wewe River. Norris and Thoms  argued tLow abundance of taxa, especially in the moderate and intact condition zones during the dry season, may occur due to the low levels of concentrated DO, related to low water volume and flow rate . ExtremeChanges in stream conditions as a result of seasonality and other direct anthropogenic drivers such as farming, dumping of solid waste in streams, and burning can affect some benthic community structure that are sensitive extreme habitat perturbation, and this probably explained why Corydalidae, Notonemouridae, and Protoneuridae were only found during the dry season, whereas Belostomatidae, Gomphidae, and Neptidae thrived only in wet season . The dom4.2Benthic habitats are complex, and a variety of environmental variables acting at multiple spatial scales regulate the composition and distribution patterns of stream macro-invertebrate assemblages in a synergistic fashion ,75. For In a similar study on seasonal variations in benthic invertebrates, Dudgeon  found hyImpact of higher levels of mercury concentration and SS on the low abundance and distribution of Asellidae and Perlidae especially in the moderate condition zone was probably a result of intensive use of agrochemicals on nearby farmlands, deposition of solid waste on the riparian zone, and the flow of liquid waste from surrounding human settlements, during the wet season. An increase in mercury concentration might have contributed to oxygen depletion, which consequently reduced Asellidae population. Such negative impacts on the functional role of invertebrates as litter decomposers could be impaired and consequently have long-term effects on the ecosystem health of the Wewe River, leading to loss of biodiversity. Hellawell  revealed5(a)farming activities within the riparian zone should be banned and farmers relocated in places further away from the riparian zones;(b)diversion of liquid sewage spillway emptying into the river course; and(c)dumping of solid refuse along the riparian zone must be banned by authorities and managers of the Wewe River.This study assessed the seasonal response of benthic invertebrate to physicochemical drivers in the Wewe River. Our findings showed that benthic invertebrate assemblages did not differ in the dry and wet seasons. Nonetheless, there was a slight increase among individuals, taxa richness, and diversity in the wet season compared with the dry season. The most severely disturbed segment (downstream) of the Wewe River registered the highest number of individuals, suggesting the role of habitat perturbation and sample size in influencing macro-invertebrate heterogeneity and abundance. Dominance of Chironomidae in the dry and wet seasons was attributed to their broad range habitat preferences and their ability to adapt to seasonal changes in physicochemical driver conditions, while the lowest abundance of Asellidae and Perlidae was probably due to their sensitivity to elevated levels of some water quality parameters, namely mercury and low dissolve oxygen concentration, which were characteristics of the moderately disturbed zone (upstream) of the Wewe River. Thus, for effective management of the Wewe River, we recommend that Asellidae and Perlidae be considered as a suit of indicator benthic orders to monitor the water quality health. Other conservation measures that should be considered in protecting the overall ecosystem of the Wewe River include the following:In terms of the setbacks that arose from this study, we noted the difficulty in sampling with less than four persons per segment of the river per day. The extreme stress or tiredness observed among the researchers may have resulted in the lack of detection or loss of some benthic invertebrate samples, as we could not sample every section of the different condition zones of the riverine continuum. We suggest that future studies should involve not less than seven persons, sampling in each segment of a riverine continuum.


Loss of GIRK1 did not alter motivated behavior for an appetitive reward or impair overall performance in an operant-based attention set-shifting model of cognitive flexibility; however it did alter types of errors committed during the visual cue test. Unexpectedly, baseline sex differences were also identified in these tasks, with females exhibiting overall poorer performance compared to males and distinct types of errors, highlighting potential differences in task-related problem-solving. Interestingly, reductions in PVI GIRK signaling did not correspond to changes in membrane excitability but did increase action potential (AP) firing at higher current injections in PVI of males, but not females. This is the first investigation on the role that PVI GIRK-signaling has on membrane excitability, AP firing, and their role on affect and cognition together increasing the understanding of PVI cellular mechanisms and function.Pathological impairments in the regulation of affect  and flexible decision-making are commonly observed across numerous neuropsychiatric disorders and are thought to reflect dysfunction of cortical and subcortical circuits that arise in part from imbalances in excitation and inhibition within these structures. Disruptions in GABA transmission, in particular, that from parvalbumin-expressing interneurons (PVI), has been highlighted as a likely mechanism by which this imbalance arises, as they regulate excitation and synchronization of principle output neurons. G protein-gated inwardly rectifying potassium ion (GIRK/Kir3) channels are known to modulate excitability and output of pyramidal neurons in areas like the medial prefrontal cortex and hippocampus; however, the role GIRK plays in PVI excitability and behavior is unknown. Male and female mice lacking GIRK1 in PVI (Girk1 Cognitive flexibility is the ability to adapt behavior in response to changing environmental contingencies and is a critical component of everyday life. As such, impairments in flexibility increase susceptibility to negative life events , reduce emotional control, and promote the development of maladaptive behaviors that can disrupt the capacity of individuals to engage in their lives effectively  and flexible decision-making require a balance of excitation and inhibition (E/I) in numerous cortical and subcortical circuits including the medial prefrontal cortex  channels produce a slow hyperpolarizing current which modulates neuron excitability and spike firing Arbr/J; Stock No:017320) then backcrossed to create flox/floxGirk1. Female flox/floxGirk1 mice positive for PVcre were then bred with male mice purchased from Jackson Laboratories expressing tdtomato in parvalbumin-positive neurons 15Gfng/J; Stock No: 027395) and backcrossed to generate experimental flox/floxGirk1 mice hemizygous for PVcre and PVtdtomato. For all experiments, flox/floxGirk1 without cre recombinase present were used as controls whereas flox/floxGirk1 with cre recombinase was used as the experimental group. For behavioral experiments, a subset of control and experimental mice also expressed tdtomato in parvalbumin-positive neurons. For electrophysiology experiments, mice also expressed tdtomato in parvalbumin-positive neurons. Mice were housed in a temperature and humidity-controlled room with a 12/12 h light/dark cycle with food and water available ad libitum except throughout attention set-shifting and the progressive ratio test. Male and female experimental mice were PD78 ± 2 days at the start of visual cue testing. Behavioral procedures were conducted in the light phase. Experiments were approved by the Institutional Animal Care and Use Committee at Marquette University.Mice were handled for 3 days then tested in the elevated plus-maze (EPM), followed by attention set-shifting training and testing, progressive ratio, and forced swim test FST; . The batMice were tested for anxiety-like behaviors using the EPM as previously described  and were modified and based on methods previously described , with each trial followed by a 20 s time out. Mice were required to reach criterion of ≤5 omissions total on two consecutive days to move forward. Once the lever training criterion was reached, lever bias was assessed during which both the right and left lever were presented and reinforced on a fixed ratio of one for a total of seven trials. If a mouse did not have ≤5 omissions for two consecutive days within 15 days of lever training, training was ceased and the mouse did not progress through testing (N = 12).During visual cue testing was conducted until 150 trials or 10 consecutive correct responses were reached with a minimum of at least 30 trials. If the criterion was not reached on the first day of testing, a second or third day of testing was conducted. During visual cue testing, an illuminated cue light was presented above either the left or right lever in a pseudorandomized order. A response on the lever underneath the illuminated cue light resulted in reward delivery and a 20 s time out; incorrect responses resulted in only the time out. Omissions were counted as stated above and were not counted towards a trial to criterion  when data was analyzed but counted towards the daily 150 trials. The next day, extradimensional shift testing was conducted, during which the reinforced lever was always the opposite lever of the previously assessed lever bias. The cue light was presented in a manner/order similar to that during the visual cue test; however, it was not associated with the correct response. Mice were run in the extradimensional shift test until 10 consecutive responses or 150 trials were conducted with a minimum of 30 trials. The day after the extradimensional shift test criterion was reached , reversal testing was conducted during which the reinforced lever was always that of the previously assessed lever bias; however, the cue light was presented identically to that used during the visual cue test. For set-shifting experiments, all tests for a mouse were excluded if >15 omissions in a given test were recorded as this may reflect mechanical issues, motoric, or motivational issues and may influence responding on subsequent tests.The following day, initial errors were errors that were made within each block until there were less than six in a single block. Once there were less than six errors in a single block, errors in all subsequent blocks were characterized as regressive errors. For the extradimensional set shift test, tests were divided into bins of 16 trials. Errors that were made based on the previous visual cue test rule used, such that the response was made on the lever under the illuminated cue light, were considered perseverative until less than six errors in a single bin were made. Errors in the next bin and subsequent bins were considered regressive errors. Never reinforced errors were those that were incorrect but the response was not on the lever underneath the illuminated cue light. For the reversal test, perseverative and regressive errors were assessed as described above; however, errors were considered perseverative until less than 10 errors in a bin were made. Separately, errors were also divided into errors that were made towards the cue light distractor and away from the cue light distractor. Response latencies were measured as the amount of time from the extension of the lever until a response was made.For further behavioral assessment, the types of errors were analyzed based on previously published methods -5 . Coronal slices (300 μm) containing the mPFC were collected using a Leica VT1000S vibratome. Slices were immediately incubated at 31°C for 10 min in a solution containing 119 mM NaCl, 2.5 mM KCl, 1 mM NaH2PO4, 26.2mM NaHCO3, 11 mM glucose, 0.4 mM ascorbic acid, 4 mM MgCl2, and 1 mM CaCl2. Slices were then removed, allowed to cool to room temperature, and incubated further for a minimum of 35 min.Mice were anesthetized with isoflurane (Henry Schein), decapitated, and the brain removed and put in ice-cold 95% O3, 10 mM glucose, 0.4 mM ascorbic acid, 1.3 mM MgCl2, and 2 mM CaCl2) was gravity perfused at a temperature of 29–33°C at a flow rate of ~1.5–2.5 ml/min. Sutter Integrated Patch Amplifier (IPA) with Igor Pro  was used for the data acquisition software. Recordings were filtered at 2 kHz and sampled at 5 kHz. Layer 5/6 PVI were identified based on the presence of td-tomato fluorescence. For all recordings, adequate whole-cell access (Ra < 40 MΩ) was maintained. Borosilicate glass pipettes were filled with 140 mM K-Gluconate, 5.0 mM HEPES, 1.1 mM EGTA, 2.0 mM MgCl2, 2.0 mM Na2-ATP, 0.3 mM Na-GTP, and 5.0 mM phosphocreatine . For ML297 recordings, a baseline with <20% fluctuation current was obtained, followed by bath application of 10 μM ML297  in 0.04% DMSO (Sigma–Aldrich). Evoked currents were reversed using bath application of 0.30 mM barium chloride (Thermo Fisher Scientific). For rheobase and action potential (AP) frequency, a 20 pA current-step injection was used . Capacitance, membrane resistance, and resting membrane potential (RMP) were taken as a simultaneous value after obtaining whole-cell access. AP duration was measured as the time to reach half the amplitude for the first action potential. Afterhyperpolarization (AHP) amplitude was measured from the spike threshold equipotential point to the maximum amplitude of the first action potential hyperpolarization. Sample sizes are denoted as n for the number of recordings/cells and N for the number of mice.Whole-cell recordings were performed as previously described  from behavioral tests were excluded from analyses . For electrophysiology data, cell-based and animal-based analyses were conducted during which each recording was a single data point or recordings from each animal were averaged and counted as a single data point, respectively.Data are presented as mean ± SEM. SigmaPlot 11.0 was used to perform statistical analyses. A 2  × 2  analysis of variance was used for all comparisons except assessment of progressive ratio breakpoint which violates parametric assumptions, and therefore a Kruskal–Wallis test was used. The Student–Newman–Keuls method for multiple Constitutive knockout of GIRK signaling, including channels expressing the GIRK1 subunit, has been shown to alter anxiety- and depression-like behavior, learning, and memory, as well as motivation for appetitive rewards  = 5.91, p = 0.018). There was also a main effect of condition, with cre-positive mice having increased percent open arm time compared to control mice  = 7.05, p = 0.010); however no sex by condition interaction was observed  = 0.07, p = 0.789; To examine the behavioral relevance of PVI GIRK1 signaling, we assessed affect-related behavior using the EPM and FST. Individual differences in locomotor activity were controlled for by assessing the percent of time spent in the open arm divided by the total time assessed. A main effect of sex was identified, with males having increased percent time in the open arm compared to females  = 0.87, p = 0.356; condition: F = 1.91, p = 0.174; interaction: F = 0.20, p = 0.657; F = 4.78, p = 0.034), with male cre-positive mice having significantly more immobile episodes than control counterparts (p < 0.001), whereas there were no differences between conditions in female mice (p = 0.200). Female controls also had increased immobile episodes compared to the male controls (p < 0.001), whereas male and female cre-positive mice did not differ  using a progressive ratio model. Responses on the left reinforced lever, the breaking point at which the mouse would no longer respond, and the right nonreinforced lever were recorded. Males had an overall greater number of left lever presses compared to females, regardless of condition, whereas controls and cre-positive mice had similar number of responses  = 18.50, p < 0.001; condition: F = 0.22, p = 0.640; interaction: F = 2.43, p = 0.128; H(3) = 10.76, p = 0.013), with breakpoints in cre-positive males greater than cre-positive female mice, and no other significant post-hoc comparisons  = 5.44, p = 0.026). In this case, females displayed a higher number of presses; however no effect of condition or interaction was observed  = 0.15, p = 0.702; interaction: F = 0.26, p = 0.614; To examine if the loss of GIRK1 in PVI impacts motivation and to control for any potential differences observed in our set-shifting experiments (see below), we assessed responding for an appetitive liquid reward  = 0.94, p = 0.340), condition  = 1.64, p = 0.210), nor an interaction  = 1.59, p = 0.216; data not shown) on the number of days to reach this criterion. Once mice passed lever training criterion and were assessed for a side bias, testing in the visual cue was conducted. There was an effect of sex on the total trials to reach criterion during the visual cue test  = 8.32, p = 0.007) but no effect of condition  = 0.42, p = 0.522) or a sex by condition interaction  = 0.41, p = 0.527; F = 8.32, p = 0.007) but no effect of condition  = 0.31, p = 0.583) nor a sex by condition interaction  = 0.41, p = 0.710; Our recent work has shown that disruption of GIRK1 signaling in mPFC prelimbic pyramidal neurons impairs cognitive performance in an attentional set-shifting model of cognitive flexibility  = 7.72, p = 0.009; post-hoc comparisons indicating no significant difference between male control and cre-positive mice (p = 0.154), whereas female cre-positive mice had fewer initial errors compared to control mice (p = 0.012). Notably, control females had significantly more initial errors compared to control males (p = 0.027), whereas male and female cre-positive mice did not differ (p = 0.116). Examination of regressive errors showed no main effect of condition  = 0.61, p = 0.441) or a condition by sex interaction  = 1.98, p = 0.169); however females had significantly more regressive errors compared to males  = 9.09, p = 0.005; In a more refined investigation into errors, we divided the error type based on errors made before the mice received less than six errors in one 16 trial bin  and those after (regressive errors), the latter of which reflects an inability to maintain the rule strategy. For initial errors, there was significant sex by condition interaction  = 76.88, p < 0.001; F = 6.01, p = 0.017) with post-hoc comparisons indicating that male cre-positive mice took less time to respond compared to male controls (p = 0.006), while females, regardless of condition, had similar response latencies (p = 0.758). There were no other significant interactions  = 1.06, p = 0.307; condition by response type: F = 2.09, p = 0.153; sex by condition by response type: F = 3.06, p = 0.085).The speed of processing and general motor function of each mouse can be measured by taking the average latency to respond after the lever was presented  = 0.84, p = 0.365), condition  = 0.21, p = 0.653), or sex by condition  = 0.00, p = 0.978; F = 2.22, p = 0.145), condition  = 0.79, p = 0.381), or sex by condition  = 0.37, p = 0.551; F = 4.34, p = 0.045), while there were no differences of condition on number of perseverative errors  = 0.71, p = 0.404) or a sex by condition interaction  = 0.03, p = 0.882; F = 0.72, p = 0.402), main effect of condition  = 0.01, p = 0.910), nor a sex by condition interaction  = 0.01, p = 0.094) on regressive errors during the extradimensional shift test  = 2.27, p = 0.141; condition: F = 0.03, p = 0.857; interaction: F = 1.28, p = 0.265; F = 7.39, p = 0.008; F = 0.21, p = 0.645; response type: F = 0.08, p = 0.772; sex by condition: F = 0.00, p = 0.974; sex by response type: F = 0.00, p = 0.973; condition by response type: F = 0.64, p = 0.427; sex by condition by response type: F = 0.26, p = 0.614).During the extradimensional shift test, there were no differences in trials to reach criterion comparing sex  = 8.03, p = 0.008), but there were no differences comparing conditions  = 1.59, p = 0.216) or a sex by condition interaction  = 0.04, p = 0.849; F = 4.54, p = 0.041); however there was no difference comparing the conditions  = 0.24, p = 0.630) or a sex by condition interaction  = 0.07, p = 0.793; F = 0.16, p = 0.695; condition: F = 0.02, p = 0.882; sex by condition: F = 0.00, p = 0.969; F = 3.94, p = 0.056; condition: F = 0.12, p = 0.727; sex by condition: F = 0.14, p = 0.709; F = 5.30, p = 0.028) with females making more errors towards the distractor  = 1.11, p = 0.299) or a sex by condition interaction  = 0.00, p = 0.988). There were also no differences in number of errors made away from the distractor  = 1.43, p = 0.241; condition: F = 0.24, p = 0.629; interaction: F = 0.31, p = 0.582; F = 2.17, p = 0.146; condition: F = 1.81, p = 0.184; response type: F = 0.44, p = 0.510; sex by condition: F = 1.00, p = 0.322; sex by response type: F = 0.45, p = 0.506; condition by response type: F = 0.00, p = 0.944; sex by condition by response type: F = 0.79, p = 0.377; During the reversal test, females took more trials to reach criterion compared to males  that correlated with a decrease in input resistance (not shown) and was blocked by subsequent application of barium chloride (0.3 mM). Comparison across sex and condition (con vs. cre) showed that ML297-mediated currents were not different in PVI from males vs. females  = 0.26, p = 0.616) nor was there a sex by condition interaction  = 0.00, p = 0.972). PVI recorded from cre-positive mice showed a significant reduction in ML297-induced current compared to PVI from control mice  = 10.23, p = 0.004; n = 6/N = 4; male cre n = 8/N = 4; female control n = 6/N = 5; female cre n = 5/N = 3). Similarly, animal-based analysis during which data points were averaged for each animal show no significant effect of sex  = 0.89, p = 0.364) nor a sex by condition interaction  = 0.00, p = 0.951); however there was a main effect of condition  = 4.83, p = 0.048). Similar to initial findings, recordings from cells in cre-positive mice had a significant reduction in ML297-induced current compared to cells from cre-negative mice indicating a reduction in GIRK1 signaling.Past work has identified an important role for GIRK1 signaling in hippocampal PVI  = 0.22, p = 0.641; condition: F = 0.05, p = 0.826; interaction: F = 2.10, p = 0.157; F = 0.43, p = 0.518; condition: F = 0.05, p = 0.821; interaction: F = 0.00, p = 0.980; n = 9/N = 6; male cre n = 8/N = 4; female control n = 11/N = 5; female cre n = 11/N = 5). RMP was more negative in females compared to males  = 5.13, p = 0.032) however there was no main effect of condition  = 0.02, p = 0.898) nor a sex by condition interaction  = 0.61, p = 0.442; n = 9/N = 6; male cre n = 8/N = 4; female control n = 6/N = 4; female cre n = 7/N = 4). The threshold to fire an action potential was also more negative in females compared to males  = 13.46, p = 0.001) with no main effect of condition  = 3.86, p = 0.060) nor a sex by condition interaction  = 0.09, p = 0.773; n = 8/N = 6; male cre n = 8/N = 4; female control n = 7/N = 4; female cre n = 7/N = 4). Rheobase  did not differ based on sex or condition  = 0.21, p = 0.651; condition: F = 1.02, p = 0.322; interaction: F = 0.59, p = 0.449; n = 9/N = 6; male cre n = 8/N = 4; female control n = 7/N = 4; female cre n = 7/N = 4). The action potential amplitude  = 3.51, p = 0.072; condition: F = 4.16, p = 0.052; interaction: F = 0.88, p = 0.356; F = 0.07, p = 0.797; condition: F = 0.00, p = 0.988; interaction: F = 2.75, p = 0.109; n = 8/N = 6; male cre n = 8/N = 4; female control n = 7/N = 4; female cre n = 7/N = 4). There was a significant effect of condition on the afterhyperpolarization amplitude, with cre-positive mice having greater afterhyperpolarizations compared to control mice  = 7.23, p = 0.203). However, there were no differences in sex  = 1.36, p = 0.255) nor a sex by condition interaction  = 1.71, p = 0.203; n = 8/N = 6; male cre n = 8/N = 4; female control n = 6/N = 3; female cre n = 7/N = 4). Lastly, during the current-step injection in PVI from males, there was a significant current by condition interaction  = 2.01, p = 0.016) with cre-positive cells firing significantly more action potentials at higher currents (240–300 pA) compared to controls  = 0.69, p = 0.423) or a condition by current interaction  = 0.75, p = 0.735; n = 9/N = 6; male cre n = 8/N = 4; female control n = 7/N = 4; female cre n = 7/N = 4). Together, these data indicate that loss of GIRK1 does not impact activation threshold but does increase neuronal firing of male PVI.To determine how loss of GIRK1 signaling impacts membrane properties we next accessed capacitance, membrane resistance, RMP, threshold to fire an action potential, rheobase, amplitude and duration of initial action potential, AHP amplitude, and spike firing frequency in response to increasing current injections. For cell-based analyses, there were no differences in capacitance  = 0.03, p = 0.867; condition: F = 0.19, p = 0.669; interaction: F = 2.72, p = 0.119) or resistance  = 0.13, p = 0.728; condition: F = 0.04, p = 0.836; interaction: F = 0.01, p = 0.937). Similar to the cell-based statistics, RMP was more negative in females compared to males  = 4.59, p = 0.050) however there was no main effect of condition  = 0.09, p = 0.775) nor a sex by condition interaction  = 0.52, p = 0.484). The threshold to fire an action potential was also more negative in females compared to males  = 13.19, p = 0.003) with no main effect of condition  = 3.82, p = 0.071) nor a sex by condition interaction  = 0.73, p = 0.406). Rheobase did not differ based on sex or condition  = 1.06, p = 0.321; condition: F = 1.71, p = 0.212; interaction: F = 1.06, p = 0.321). Unlike cell-based statistics which showed no difference in action potential amplitude, animal-based statistics revealed a significant difference with females having significantly greater amplitude compared to males  = 5.94, p = 0.029; condition: F = 2.79, p = 0.117; interaction: F = 1.18, p = 0.296). The duration at 50% the amplitude did not differ between groups  = 0.07, p = 0.794; condition: F = 0.15, p = 0.706; interaction: F = 2.63, p = 0.127). Unlike cell-based statistics, there was no significant effect on the afterhyperpolarization amplitude  = 1.30, p = 0.275; condition: F = 4.50, p = 0.054; interaction: F = 1.25, p = 0.284). Lastly, during the current-step injection in PVI, there were no significant effects of condition or condition by current interactions in males  = 1.80, p = 0.217; interaction: F = 1.24, p = 0.255) or females  = 1.83, p = 0.225; interaction: F = 1.39, p = 0.174). Similar to cell-based statistics, these data indicate that loss of GIRK1 does not impact activation threshold however also does not alter neuronal firing in males or females, despite significantly reducing GIRK1 signaling.Animal-based statistics, during which recordings from each animal were averaged, show similar findings to the cell-based statistics. There were no differences in capacitance , and known differences in the electrophysiological characteristics of each cell-type, taking the average of cells from each mouse may increase the risk of a Type 1 error by reducing variability in the statistical analysis. Frequently, each animal contributes one to three recordings within a dataset, therefore, the use of animal-based statistics to average recordings also significantly reduced the sample size which may result in a Type 2 error. Although cell-based statistics revealed a significantly greater afterhyperpolarization amplitude in cre-positive compared to controls, there were no differences when analyzed using animal-based statistics. Similarly, while cell-based statistics revealed greater action potential firing from cre-positive mice compared to controls in males, there were also no differences when analyzed using animal-based statistics. The lack of findings using animal-based statistics may be the result of decreased power due to reductions in sample size from averaging all recordings within each animal.The lack of a consistent effect on traditional measures of membrane excitability , as well as lack of a robust behavioral phenotype, may reflect a variety of factors. First, the knockout of PVI GIRK-signaling was already in effect during critical development periods which may have led to compensatory changes in non-GIRK effectors more readily modulating membrane excitability. Although a viral vector to specifically target PVI GIRK-signaling is not readily available, it would be beneficial for future research to target PVI GIRK channels specifically during adulthood to reduce the potential for compensation of other signaling during development. Further, neuronal GIRK channels can be homo- and heterotetrameric complexes formed primarily by the assembly of GIRK1, GIRK2, and GIRK3 subunits  and take longer to respond compared to males. Females also require more trials to reach criterion in a test of reversal learning. Further investigation of the error types shows a greater number of errors on the lever underneath the cue  indicating that they are still perseverating on the visual cue rule, despite learning a new rule during the extradimensional shift. To our knowledge, this is the first study to investigate sex differences in an operant based attentional set-shifting model of cognitive flexibility. It should be noted that although gonadal hormones influence cognition (Luine and Frankfurt, Findings from the current study suggest that PVI GIRK1 signaling does not mediate membrane excitability to the extent that it does in principle pyramidal neurons and may differentially impact firing frequency in males and females. Further, while reductions in PVI GIRK1 signaling influenced anxiety-like behaviors, effects on cognitive performance were more nuanced. While outside the scope of this study, the demonstration that GIRK channels are present in prelimbic PVI requires further investigation into their function, as past work has shown that differences in subunit expression and coupling dictate responsivity to various Gi-coupled GPCR agonists, and thus may inform future drug therapies targeting GIRKs or Gi-coupled GPCRs. Further, the current findings have provided unexpected insight into how biological sex impacts cognitive processing associated with an operant-based model of cognitive flexibility which may have important implications for treating pathological deficits in cognitive control.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Marquette University Instituational Animal Care and Use Committee.EA and MH designed, discussed, planned all experiments, and wrote the manuscript. EA, AE, SD, HD’A, and MH performed experiments and analyzed data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.