Mice lacking Ptprd exhibit deficits in goal-directed behavior and female-specific impairments in sensorimotor gating.

Protein Tyrosine Phosphatase receptor type D (PTPRD) is a member of the protein tyrosine phosphatase family that mediates cell adhesion and synaptic specification. Genetic studies have linked Ptprd to several neuropsychiatric phenotypes, including Restless Leg Syndrome (RLS), opioid abuse disorder, and antipsychotic-induced weight gain. Genome-wide association studies (GWAS) of either pediatric obsessive-compulsive traits, or Obsessive-Compulsive Disorder (OCD), have identified loci near PTPRD as genome-wide significant, or strongly suggestive for this trait. We assessed Ptprd wild-type (WT), heterozygous (HT), and knockout (KO) mice for behavioral dimensions that are altered in OCD, including anxiety and exploration (open field test, dig test), perseverative behavior (splash-induced grooming, spatial d), sensorimotor gating (prepulse inhibition), and home cage goal-directed behavior (nest building). No effect of genotype was observed in any measure of the open field test, dig test, or splash test. However, Ptprd KO mice of both sexes showed impairments in nest building behavior. Finally, female, but not male, Ptprd KO mice showed deficits in prepulse inhibition, an operational measure of sensorimotor gating that is reduced in female, but not male, OCD patients. Our results indicate that constitutive lack of Ptprd may contribute to the development of certain domains that are altered OCD, including goal-directed behavior, and reduced sensorimotor gating specifically in females.

Aldosterone: Renal Action and Physiological Effects.

Aldosterone exerts profound effects on renal and cardiovascular physiology. In the kidney, aldosterone acts to preserve electrolyte and acid-base balance in response to changes in dietary sodium (Na+ ) or potassium (K+ ) intake. These physiological actions, principally through activation of mineralocorticoid receptors (MRs), have important effects particularly in patients with renal and cardiovascular disease as demonstrated by multiple clinical trials. Multiple factors, be they genetic, humoral, dietary, or otherwise, can play a role in influencing the rate of aldosterone synthesis and secretion from the adrenal cortex. Normally, aldosterone secretion and action respond to dietary Na+ intake. In the kidney, the distal nephron and collecting duct are the main targets of aldosterone and MR action, which stimulates Na+ absorption in part via the epithelial Na+ channel (ENaC), the principal channel responsible for the fine-tuning of Na+ balance. Our understanding of the regulatory factors that allow aldosterone, via multiple signaling pathways, to function properly clearly implicates this hormone as central to many pathophysiological effects that become dysfunctional in disease states. Numerous pathologies that affect blood pressure (BP), electrolyte balance, and overall cardiovascular health are due to abnormal secretion of aldosterone, mutations in MR, ENaC, or effectors and modulators of their action. Study of the mechanisms of these pathologies has allowed researchers and clinicians to create novel dietary and pharmacological targets to improve human health. This article covers the regulation of aldosterone synthesis and secretion, receptors, effector molecules, and signaling pathways that modulate its action in the kidney. We also consider the role of aldosterone in disease and the benefit of mineralocorticoid antagonists. © 2023 American Physiological Society. Compr Physiol 13:4409-4491, 2023.

Dopamine D2 receptor overexpression in the nucleus accumbens core induces robust weight loss during scheduled fasting selectively in female mice.

Anorexia nervosa (AN) is an eating disorder observed predominantly in women and girls that is characterized by a low body-mass index, hypophagia, and hyperactivity. Activity-based anorexia (ABA), which refers to the weight loss, hypophagia, and hyperactivity exhibited by rodents exposed to both running wheels and scheduled fasting, provides a model for aspects of AN. Increased dopamine D2/D3 receptor binding in the anteroventral striatum has been reported in AN patients. We virally overexpressed D2Rs on nucleus accumbens core (D2R-OENAc) neurons that endogenously express D2Rs, and tested mice of both sexes in the open field test, ABA paradigm, and intraperitoneal glucose tolerance test (IGTT). D2R-OENAc did not alter baseline body weight, but increased locomotor activity in the open field across both sexes. During constant access to food and running wheels, D2R-OENAc mice of both sexes increased food intake and ran more than controls. However, when food was available only 7 h a day, only female D2R-OENAc mice rapidly lost 25% of their initial body weight, reduced food intake, and substantially increased wheel running. Surprisingly, female D2R-OENAc mice also rapidly lost 25% of their initial body weight during scheduled fasting without wheel access and showed no changes in food intake. In contrast, male D2R-OENAc mice maintained body weight during scheduled fasting. D2R-OENAc mice of both sexes also showed glucose intolerance in the IGTT. In conclusion, D2R-OENAc alters glucose metabolism in both sexes but drives robust weight loss only in females during scheduled fasting, implicating metabolic mechanisms in this sexually dimorphic effect.

Ketamine induces immediate and delayed alterations of OCD-like behavior.

RATIONALE: Obsessive-compulsive disorder (OCD) is a psychiatric disorder characterized by intrusive obsessive thoughts and/or compulsive behaviors. Currently, serotonin reuptake inhibitors (SRIs) provide the only pharmacological monotherapy for OCD, but response rates are insufficient. Ketamine, a noncompetitive NMDA receptor antagonist, was reported to have rapid, sustained therapeutic effects in OCD patients. However, the mechanisms remain unknown. OBJECTIVES: Here, we aimed to provide a platform for investigating mechanisms underlying anti-OCD effects of ketamine treatment by assessing whether ketamine pretreatment could alleviate 5-HT1B receptor (5-HT1BR)-induced OCD-like behavior in mice. METHODS: We assessed whether acute ketamine (0, 3, 10, 30 mg/kg), administered at two pretreatment time points (30 min, 24 h), would modulate 5-HT1BR-induced OCD-like behavior in mice. Behavioral measures were perseverative hyperlocomotion in the open field and deficits in prepulse inhibition (PPI) induced by acute pharmacological 5-HT1BR challenge. RESULTS: Three milligrams per kilogram of ketamine reduced 5-HT1BR-induced perseverative hyperlocomotion, but not PPI deficits, 24 h postinjection. In contrast, higher doses of ketamine were either ineffective (10 mg/kg) or exacerbated (30 mg/kg) 5-HT1BR-induced perseverative hyperlocomotion 30 min postinjection. At 24 h postinjection, 30 mg/kg ketamine reduced perseverative hyperlocomotion across all groups. CONCLUSIONS: Our results suggest that the 5-HT1BR-induced model of OCD-like behavior is sensitive to a low dose of ketamine, a potential fast-acting anti-OCD treatment, and may provide a tool for studying mechanisms underlying the rapid therapeutic effects of ketamine in OCD patients.

Btbd3 expression regulates compulsive-like and exploratory behaviors in mice.

BTB/POZ domain-containing 3 (BTBD3) was identified as a potential risk gene in the first genome-wide association study of obsessive-compulsive disorder (OCD). BTBD3 is a putative transcription factor implicated in dendritic pruning in developing primary sensory cortices. We assessed whether BTBD3 also regulates neural circuit formation within limbic cortico-striato-thalamo-cortical circuits and behaviors related to OCD in mice. Behavioral phenotypes associated with OCD that are measurable in animals include compulsive-like behaviors and reduced exploration. We tested Btbd3 wild-type, heterozygous, and knockout mice for compulsive-like behaviors including cage-mate barbering, excessive wheel-running, repetitive locomotor patterns, and reduced goal-directed behavior in the probabilistic learning task (PLT), and for exploratory behavior in the open field, digging, and marble-burying tests. Btbd3 heterozygous and knockout mice showed excessive barbering, wheel-running, impaired goal-directed behavior in the PLT, and reduced exploration. Further, chronic treatment with fluoxetine, but not desipramine, reduced barbering in Btbd3 wild-type and heterozygous, but not knockout mice. In contrast, Btbd3 expression did not alter anxiety-like, depression-like, or sensorimotor behaviors. We also quantified dendritic morphology within anterior cingulate cortex, mediodorsal thalamus, and hippocampus, regions of high Btbd3 expression. Surprisingly, Btbd3 knockout mice only showed modest increases in spine density in the anterior cingulate, while dendritic morphology was unaltered elsewhere. Finally, we virally knocked down Btbd3 expression in whole, or just dorsal, hippocampus during neonatal development and assessed behavior during adulthood. Whole, but not dorsal, hippocampal Btbd3 knockdown recapitulated Btbd3 knockout phenotypes. Our findings reveal that hippocampal Btbd3 expression selectively modulates compulsive-like and exploratory behavior.

Assessing Activity-based Anorexia in Mice.

Rodents develop activity-based anorexia (ABA) when exposed to a restricted feeding schedule and allowed free access to a running wheel. These conditions lead to a life-threatening reduction in body weight. However, rodents exposed to only one of these conditions ultimately adapt to re-establish normal body weight. Although increased running coupled with reduction in voluntary food intake appear paradoxical under ABA conditions, ABA behavior is observed across numerous mammalian species. The ABA paradigm provides an animal model for anorexia nervosa (AN), an eating disorder with severe dysregulation of appetite-behavior. Subjects are singly housed with free access to a running wheel. Each day, the subject is offered food for a limited amount of time. During the course of the experiment, a subject's body weight decreases from high activity and low caloric intake. The duration of the study varies based on how long food is offered daily, the type of food offered, the strain of mouse, if drugs are being tested, and environmental factors. A lack of effective pharmacological treatments for AN patients, their low quality of life, high cost of treatment, and their high mortality rate indicate the urgency to further research AN. We provide a basic outline for performing ABA experiments with mice, offering a method to investigate AN-like behavior in order to develop novel therapies. This protocol is optimized for use in Balb/cJ mice, but can easily be manipulated for other strains, providing great flexibility in working with different questions, especially related to genetic factors of ABA.

Aldosterone alters the chromatin structure of the murine endothelin-1 gene.

UNLABELLED: Aldosterone increases sodium reabsorption in the renal collecting duct and systemic blood pressure. Paradoxically, aldosterone also induces transcription of the endothelin-1 (Edn1) gene to increase protein (ET-1) levels, which inhibits sodium reabsorption. AIMS: Here we investigated changes in the chromatin structure of the Edn1 gene of collecting duct cell lines in response to aldosterone treatment. The Edn1 gene has a CpG island that encompasses the transcription start site and four sites in the 5' regulatory region previously linked to transcriptional regulation. MATERIALS AND METHODS: The chromatin structure of the Edn1 gene was investigated using a quantitative PCR-based DNaseI hypersensitivity assay in murine hepatocyte (AML12), renal cortical collecting duct (mpkCCDC14), outer medullary collecting duct1 (OMCD1), and inner medullary collecting duct-3 (IMCD-3) cell lines. KEY FINDINGS: The CpG island was uniformly accessible. One calcium-responsive NFAT element remained at low chromatin accessibility in all cell lines under all conditions tested. However, the second calcium responsive NFAT element located at -1563bp upstream became markedly more accessible in IMCD-3 cells exposed to aldosterone. Importantly, one established aldosterone hormone response element HRE at -671bp relative to the transcription start site was highly accessible, and another HRE (-551bp) became more accessible in aldosterone-treated IMCD-3 and OMCD1 cells. SIGNIFICANCE: The evidence supports a model in which aldosterone activation of the mineralocorticoid receptor (MR) results in the MR-hormone complex binding at HRE at -671bp to open chromatin structure around other regulatory elements in the Edn1 gene.

Effect of mineralocorticoid treatment in mice with collecting duct-specific knockout of endothelin-1.

Aldosterone increases blood pressure (BP) by stimulating sodium (Na) reabsorption within the distal nephron and collecting duct (CD). Aldosterone also stimulates endothelin-1 (ET-1) production that acts within the CD to inhibit Na reabsorption via a negative feedback mechanism. We tested the hypothesis that this renal aldosterone-endothelin feedback system regulates electrolyte balance and BP by comparing the effect of a high-salt (NaCl) diet and mineralocorticoid stimulation in control and CD-specific ET-1 knockout (CD ET-1 KO) mice. Metabolic balance and radiotelemetric BP were measured before and after treatment with desoxycorticosterone pivalate (DOCP) in mice fed a high-salt diet with saline to drink. CD ET-1 KO mice consumed more high-salt diet and saline and had greater urine output than controls. CD ET-1 KO mice exhibited increased BP and greater fluid retention and body weight than controls on a high-salt diet. DOCP with high-salt feeding further increased BP in CD ET-1 KO mice, and by the end of the study the CD ET-1 KO mice were substantially hypernatremic. Unlike controls, CD ET-1 KO mice failed to respond acutely or escape from DOCP treatment. We conclude that local ET-1 production in the CD is required for the appropriate renal response to Na loading and that lack of local ET-1 results in abnormal fluid and electrolyte handling when challenged with a high-salt diet and with DOCP treatment. Additionally, local ET-1 production is necessary, under these experimental conditions, for renal compensation to and escape from the chronic effects of mineralocorticoids.

Tissue-specific and time-dependent regulation of the endothelin axis by the circadian clock protein Per1.

AIMS: The present study is designed to consider a role for the circadian clock protein Per1 in the regulation of the endothelin axis in mouse kidney, lung, liver and heart. Renal endothelin-1 (ET-1) is a regulator of the epithelial sodium channel (ENaC) and blood pressure (BP), via activation of both endothelin receptors, ETA and ETB. However, ET-1 mediates many complex events in other tissues. MAIN METHODS: Tissues were collected in the middle of murine rest and active phases, at noon and midnight, respectively. ET-1, ETA and ETB mRNA expressions were measured in the lung, heart, liver, renal inner medulla and renal cortex of wild type and Per1 heterozygous mice using real-time quantitative RT-PCR. KEY FINDINGS: The effect of reduced Per1 expression on levels of mRNAs and the time-dependent regulation of expression of the endothelin axis genes appeared to be tissue-specific. In the renal inner medulla and the liver, ETA and ETB exhibited peaks of expression in opposite circadian phases. In contrast, expressions of ET-1, ETA and ETB in the lung did not appear to vary with time, but ET-1 expression was dramatically decreased in this tissue in Per1 heterozygous mice. Interestingly, ET-1 and ETA, but not ETB, were expressed in a time-dependent manner in the heart. SIGNIFICANCE: Per1 appears to regulate expression of the endothelin axis genes in a tissue-specific and time-dependent manner. These observations have important implications for our understanding of the best time of day to deliver endothelin receptor antagonists.

MicroRNA regulation of endothelin-1 mRNA in renal collecting duct cells.

AIMS: Recently, microRNAs (miRNAs) have been implicated in control of Edn1 mRNA in several tissues. Here we examined the role of miRNA action on Edn1 mRNA expression in renal distal collecting duct cells. MAIN METHODS: A microarray study was conducted to provide a comprehensive assessment of miRNAs present in a murine inner medullary collecting duct (mIMCD-3) cell line. The experiment was designed as a comparison between mIMCD-3 cells grown in the presence and absence of aldosterone. Argonaute (Ago) immunoprecipitation experiments were used to investigate binding of the RNA induced silencing complex (RISC) to Edn1 mRNA. KEY FINDINGS: Thirty-four miRNAs were detected in very high abundance in mIMCD-3 cells, and a large number of others were present at lower levels. The microarray experiments were validated by quantitative PCR analysis of selected miRNAs. The microarray data, in combination with in silico examination of the Edn1 3' UTR provided a panel of candidate miRNAs that could act upon the Edn1 expression. Edn1 mRNA was co-immunoprecipitated with an Argonaute protein antibody, and this interaction was blocked by anti-miR-709 oligonucleotides. SIGNIFICANCE: These results define the miRNA landscape of the mIMCD-3 cell line. Moreover, Edn1 was shown to interact with Argonaute protein suggesting that it is a target of the RNA induced silencing complex (RISC).

Endothelin-1 inhibits sodium reabsorption by ET(A) and ET(B) receptors in the mouse cortical collecting duct.

The collecting duct (CD) is a major renal site for the hormonal regulation of Na homeostasis and is critical for systemic arterial blood pressure control. Our previous studies demonstrated that the endothelin-1 gene (edn1) is an early response gene to the action of aldosterone. Because aldosterone and endothelin-1 (ET-1) have opposing actions on Na reabsorption (JNa) in the kidney, we postulated that stimulation of ET-1 by aldosterone acts as a negative feedback mechanism, acting locally within the CD. Aldosterone is known to increase JNa in the CD, in part, by stimulating the epithelial Na channel (ENaC). In contrast, ET-1 increases Na and water excretion through its binding to receptors in the CD. To date, direct measurement of the quantitative effect of ET-1 on transepithelial JNa in the isolated in vitro microperfused mouse CD has not been determined. We observed that the CD exhibits substantial JNa in male and female mice that is regulated, in part, by a benzamil-sensitive pathway, presumably ENaC. ENaC-mediated JNa is greater in the cortical CD (CCD) than in the outer medullary CD (OMCD); however, benzamil-insensitive JNa is present in the CCD and not in the OMCD. In the presence of ET-1, ENaC-mediated JNa is significantly inhibited. Blockade of either ETA or ETB receptor restored JNa to control rates; however, only ETA receptor blockade restored a benzamil-sensitive component of JNa. We conclude 1) Na reabsorption is mediated by ENaC in the CCD and OMCD and also by an ENaC-independent mechanism in the CCD; and 2) ET-1 inhibits JNa in the CCD through both ETA and ETB receptor-mediated pathways.

Manipulations in the peripheral stalk of the Saccharomyces cerevisiae F1F0-ATP synthase.

The Saccharomyces cerevisiae F(1)F(0)-ATP synthase peripheral stalk is composed of the OSCP, h, d, and b subunits. The b subunit has two membrane-spanning domains and a large hydrophilic domain that extends along one side of the enzyme to the top of F(1). In contrast, the Escherichia coli peripheral stalk has two identical b subunits, and subunits with substantially altered lengths can be incorporated into a functional F(1)F(0)-ATP synthase. The differences in subunit structure between the eukaryotic and prokaryotic peripheral stalks raised a question about whether the two stalks have similar physical and functional properties. In the present work, the length of the S. cerevisiae b subunit has been manipulated to determine whether the F(1)F(0)-ATP synthase exhibited the same tolerances as in the bacterial enzyme. Plasmid shuffling was used for ectopic expression of altered b subunits in a strain carrying a chromosomal disruption of the ATP4 gene. Wild type growth phenotypes were observed for insertions of up to 11 and a deletion of four amino acids on a nonfermentable carbon source. In mitochondria-enriched fractions, abundant ATP hydrolysis activity was seen for the insertion mutants. ATPase activity was largely oligomycin-insensitive in these mitochondrial fractions. In addition, very poor complementation was seen in a mutant with an insertion of 14 amino acids. Lengthier deletions yielded a defective enzyme. The results suggest that although the eukaryotic peripheral stalk is near its minimum length, the b subunit can be extended a considerable distance.

The b (arg36) contributes to efficient coupling in F(1)F (O) ATP synthase in Escherichia coli.

In Escherichia coli, the F(1)F(O) ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been constructed in an attempt to achieve an intragenic suppressor of the b (arg36-->ile) and the b (arg36-->glu) mutations. The b (arg36-->ile) mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b (arg36-->glu) mutation was largely suppressed in the b (arg36-->glu,glu39-->arg) double mutant. E. coli expressing the b (arg36-->glu,glu39-->arg) subunit grew well on succinate-based medium. F(1)F(O) ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence suggests that efficient coupling in F(1)F(O) ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk.

Closing the gap: collaborative learning as a strategy to embed evidence within occupational therapy practice.

RATIONALE: The principles of clinical governance apply as guidelines for good practice to all practitioners. However, evidence-based practice (EBP) is proving a challenge for practitioners who lack the confidence to consume published research. For therapists not wishing to undertake formal study there is a risk of becoming disempowered within a culture of EBP. Opportunities to develop skills in consuming research have focused on the information dissemination model that has limited effect. Mutual reflective learning processes are recommended to empower practitioners to bridge the theory-practice gap. AIM: An action research approach investigated practice based collaborative learning as a catalyst to increase therapist's competence and confidence in consuming research and to explore the transition toward EB practitioner. METHOD AND RESULTS: A diagnostic survey reaffirmed therapist's lack of confidence in EBP. Formative interviews (n = 5) found an over reliance on professional craft and personal knowledge. Research knowledge was not included in participants' construct of a good practitioner and engagement in higher order critical reflection was limited. Collaborative learning groups (n = 6) embedded in practice integrated research, theory, practice and critical reflection. Supported by the collegial learning environment, a learning package developed participants' confidence and competence in consuming published research. Summative interviews (n = 5) evaluated the group and found that therapists were empowered to incorporate propositional knowledge into their clinical reasoning, engage in critical reflection and challenge their practice. They felt confident to incorporate EBP into their continuing professional development plans. Sustainability of these changes requires commitment from the therapists and the workplace.