Cognitive dysfunction in early experimental metabolic dysfunction-associated steatotic liver disease is associated with systemic inflammation and neuroinflammation.

Background & Aims: Cognitive dysfunction is an increasingly recognised manifestation of metabolic dysfunction-associated steatotic liver disease (MASLD), but the mechanistic link remains unclear. The aim of this study was to investigate the hypothesis that experimental MASLD leads to cognitive dysfunction via systemic inflammation and neuroinflammation. Methods: Twenty male Sprague Dawley rats were randomised to a high-fat high-cholesterol (HFHC) diet to induce MASLD, or a standard diet (n = 10/group), for 16 weeks. Assessments included: MASLD severity (histology), neurobehaviour, inflammation (liver, plasma and cerebrospinal fluid), brain microglia and astrocyte activation, and synaptic density. Results: The HFHC diet induced MASLD with extensive steatosis and lobular inflammation without fibrosis. Several plasma cytokines were elevated (CXCL1, IL-6, IL-17, MIP-1α, MCP-1, IL-10; all p <0.05) and correlated with increases in hepatic chemokine gene expression. Cerebrospinal fluid concentrations of CXCL1 were elevated (p = 0.04). In the prefrontal brain cortex, we observed a 19% increase in microglial activation confirmed by Iba1 immunohistochemistry (p = 0.03) and 3H-PK11195 autoradiography (p <0.01). In parallel, synaptic density was reduced to 92%, assessed by 3H-UCB-J autoradiography (p <0.01). MASLD animals exhibited impaired memory to previously encountered objects in the novel object recognition test (p = 0.047) and showed depression-like behaviour evidenced by increased immobility time (p <0.01) and reduced swimming time (p = 0.03) in the forced swim test. Conclusions: Experimental non-fibrotic MASLD, as a model to reflect the early stage of human disease, results in cognitive impairment and depression-like behaviour. This is associated with an inflammatory phenotype not only in the liver but also in the plasma and brain, which together with diminished synaptic density, provides a pathophysiological link between liver disease and cognitive dysfunction in MASLD. Impact and implications: Cognitive dysfunction is an increasingly recognised comorbidity in patients with metabolic dysfunction-associated steatotic liver disease (MASLD), yet the underlying mechanisms remain unclear. This study provides evidence of impaired memory and depression-like symptoms in early experimental MASLD and indicates that hepatic inflammation may drive a systemic inflammatory response, resulting in neuroinflammation and reduced brain synaptic density. The evidence of impaired memory in MASLD and establishing its underlying pathophysiological link provides insights that could guide the development of potential new treatments for this increasingly common condition in people of working age. The study also emphasises the need to develop better tools for clinical cognitive testing, which will enable physicians to assess and manage brain dysfunction early in MASLD.

A single dose of cocaine raises SV2A density in hippocampus of adolescent rats.

OBJECTIVE: Cocaine is a highly addictive psychostimulant that affects synaptic activity with structural and functional adaptations of neurons. The transmembrane synaptic vesicle glycoprotein 2A (SV2A) of pre-synaptic vesicles is commonly used to measure synaptic density, as a novel approach to the detection of synaptic changes. We do not know if a single dose of cocaine suffices to affect pre-synaptic SV2A density, especially during adolescence when synapses undergo intense maturation. Here, we explored potential changes of pre-synaptic SV2A density in target brain areas associated with the cocaine-induced boost of dopaminergic neurotransmission, specifically testing if the effects would last after the return of dopamine levels to baseline. METHODS: We administered cocaine (20 mg/kg i.p.) or saline to rats in early adolescence, tested their activity levels and removed the brains 1 hour and 7 days after injection. To evaluate immediate and lasting effects, we did autoradiography with [3H]UCB-J, a specific tracer for SV2A, in medial prefrontal cortex, striatum, nucleus accumbens, amygdala, and dorsal and ventral areas of hippocampus. We also measured the striatal binding of [3H]GBR-12935 to test cocaine's occupancy of the dopamine transporter at both times of study. RESULTS: We found a significant increase of [3H]UCB-J binding in the dorsal and ventral sections of hippocampus 7 days after the cocaine administration compared to saline-injected rats, but no differences 1 hour after the injection. The [3H]GBR-12935 binding remained unchanged at both times. CONCLUSION: Cocaine provoked lasting changes of hippocampal synaptic SV2A density after a single exposure during adolescence.

Preclinical quality, safety, and efficacy of a human embryonic stem cell-derived product for the treatment of Parkinson's disease, STEM-PD.

Cell replacement therapies for Parkinson's disease (PD) based on transplantation of pluripotent stem cell-derived dopaminergic neurons are now entering clinical trials. Here, we present quality, safety, and efficacy data supporting the first-in-human STEM-PD phase I/IIa clinical trial along with the trial design. The STEM-PD product was manufactured under GMP and quality tested in vitro and in vivo to meet regulatory requirements. Importantly, no adverse effects were observed upon testing of the product in a 39-week rat GLP safety study for toxicity, tumorigenicity, and biodistribution, and a non-GLP efficacy study confirmed that the transplanted cells mediated full functional recovery in a pre-clinical rat model of PD. We further observed highly comparable efficacy results between two different GMP batches, verifying that the product can be serially manufactured. A fully in vivo-tested batch of STEM-PD is now being used in a clinical trial of 8 patients with moderate PD, initiated in 2022.

Peering into the Brain's Estrogen Receptors: PET Tracers for Visualization of Nuclear and Extranuclear Estrogen Receptors in Brain Disorders.

Estrogen receptors (ERs) play a multitude of roles in brain function and are implicated in various brain disorders. The use of positron emission tomography (PET) tracers for the visualization of ERs' intricate landscape has shown promise in oncology but remains limited in the context of brain disorders. Despite recent progress in the identification and development of more selective ligands for various ERs subtypes, further optimization is necessary to enable the reliable and efficient imaging of these receptors. In this perspective, we briefly touch upon the significance of estrogen signaling in the brain and raise the setbacks associated with the development of PET tracers for identification of specific ERs subtypes in the brain. We then propose avenues for developing efficient PET tracers to non-invasively study the dynamics of ERs in the brain, as well as neuropsychiatric diseases associated with their malfunction in a longitudinal manner. This perspective puts several potential candidates on the table and highlights the unmet needs and areas requiring further research to unlock the full potential of PET tracers for ERs imaging, ultimately aiding in deepening our understanding of ERs and forging new avenues for potential therapeutic strategies.

Changes in hypothalamic mu-opioid receptor expression following acute olanzapine treatment in female rats: Implications for feeding behavior.

Advances have been made in recent years in using opioid receptor antagonists as an adjunct therapy to psychotropic medication to reduce debilitating weight gain and metabolic adverse effects associated with in particular second generation antipsychotics. However, it is unknown whether second generation antipsychotics produce a change in opioid receptor expression in the brain. The present study investigated early changes in opioid receptor expression in the female rat hypothalamus, a master controller of hunger and metabolic regulation, after acute treatment with olanzapine, a commonly used second generation antipsychotic. Using quantitative spatial in situ hybridization and receptor autoradiography, expression levels of the three opioid receptors; kappa, mu and delta, were determined at mRNA and protein level, respectively, in the five hypothalamic areas: paraventricular nucleus, arcuate nucleus, ventromedial nucleus, dorsomedial nucleus and lateral hypothalamus. After 48 h of olanzapine treatment at clinically relevant plasma concentration weight gain and food intake changes, and increased plasma glucose were observed in female rats. Olanzapine treatment also led to a significant increase in mu opioid receptor availability in the arcuate nucleus, which contains both satiety and hunger controlling neurons. No other areas showed any opioid receptor expressional changes with olanzapine treatment on neither at mRNA nor protein level. Technical difficulties made it impossible to analyze mRNA levels in the lateral hypothalamus and overall binding of delta opioid receptors. Thus, the present study provided insights in to how olanzapine at clinically relevant plasma levels already at an early stage modulated the opioid system in the hypothalamus.

Autoradiographic characterization of [18 F]PSMA-1007 binding in rat brain.

Carboxypeptidase II (CBPII) in brain metabolizes the neuroactive substance N-acetyl-L-aspartyl-L-glutamate (NAGG) to yield the elements of glutamate and N-acetyl-aspartate (NAA). In peripheral organs, CBPII is known as prostrate specific membrane antigen (PSMA), which presents an important target for nuclear medicine imaging in prostate cancer. Available PSMA ligands for PET imaging do not cross the blood-brain barrier, and there is scant knowledge of the neurobiology of CBPII, despite its implication in the regulation of glutamatergic neurotransmission. In this study we used the clinical PET tracer [18 F]-PSMA-1007 ([18 F]PSMA) for an autoradiographic characterization of CGPII in rat brain. Ligand binding and displacement curves indicated a single site in brain, with KD of about 0.5 nM, and Bmax ranging from 9 nM in cortex to 19 nM in white matter (corpus callosum and fimbria) and 24 nM in hypothalamus. The binding properties of [18 F]PSMA in vitro should enable its use for autoradiographic investigations of CBPII expression in animal models of human neuropsychiatric conditions.

Treadmill exercise modulates nigral and hippocampal cannabinoid receptor type 1 in the 6-OHDA model of Parkinson's disease.

Physical exercise benefits Parkinson's disease (PD) patients but the mechanism is unclear. Cannabinoid receptor type 1 (CB1R) is known to be reduced in PD patients and animal models. We test the hypothesis that binding of the CB1R inverse agonist, [3H]SR141716A, is normalized by treadmill exercise in the toxin-induced 6-hydroxydopamine (6-OHDA) model of PD. Male rats had unilateral striatal injections of 6-OHDA or saline. After 15 days, half were submitted to treadmill exercise and half remained sedentary. [3H]SR141716A autoradiography was performed in postmortem tissue from striatum, substantia nigra (SN) and hippocampus. There was a 41% decrease of [3H]SR141716A specific binding in the ipsilateral SN of 6-OHDA-injected sedentary animals which was attenuated to 15% by exercise, when compared to saline-injected animals. No striatal differences were observed. A 30% bilateral hippocampal increase was observed in both healthy and 6-OHDA exercised groups. In addition, a positive correlation between nigral [3H]SR141716A binding and nociceptive threshold was observed in PD-exercised animals (p = 0.0008), suggesting a beneficial effect of exercise in the pain associated with the model. Chronic exercise can reduce the detrimental effects of PD on nigral [3H]SR141716A binding, similar to the reported reduction after dopamine replacement therapy, so should be considered as an adjunct therapy for PD.

Sex and Estrous Cycle Are Not Mediators of S-Ketamine's Rapid-Antidepressant Behavioral Effects in a Genetic Rat Model of Depression.

BACKGROUND: Recent preclinical and clinical studies have shed light on the possible impact of sex and estrous/menstrual cycle on ketamine's antidepressant action but with incongruous results. The preclinical studies that have shown the effects of ovarian sex hormones have not done so in animal models of depression. Thus, the aim of the present study is to scrutinize the acute behavioral responses to a subanesthetic dose of S-ketamine in males vs females and in different estrous phases in free-cycling females in a well-powered translational approach. METHODS: We evaluated the behavioral sensitivity to 20 mg/kg S-ketamine (i.p.) in male and female Flinders Sensitive Line rats (FSLs) and their counterpart Flinders Resistant Line rats (FRLs) subjected to the open field and forced swim tests. Female rats were disaggregated into different estrous phases, and the behavioral outcomes were compared. RESULTS: Acute administration of S-ketamine had robust antidepressant-like effects in FSLs. Within our study power, we could not detect sex- or estrous cycle-specific different antidepressant-like responses to S-ketamine in FSLs. Fluctuations in the levels of ovarian sex hormones across different estrous cycles did not behaviorally affect S-ketamine's rapid-acting antidepressant mode of action. No sex-related or estrous cycle-related impact on behavioral despair was observed even among FRLs and saline-treated FSLs. CONCLUSIONS: We conclude that physiological oscillations of estrogen and progesterone levels neither amplify nor diminish the behavioral antidepressant-like effect of S-ketamine. In addition, fluctuations of ovarian sex hormones do not predispose female animals to exhibit enhanced or reduced depressive-like and anxiety-like behaviors.

Exploration of possible sex bias in acute social stress research: a semi-systematic review.

Stress can have a significant impact on the daily lives of individuals and can increase vulnerability to a number of medical conditions. This study aims to estimate the ratio of male to female participants in acute social stress research in healthy individuals. We examined original research articles published over the last 20 years. Each article was screened to determine the total number of female and male participants. We extracted data from 124 articles involving a total of 9539 participants. A total of 4221 (44.2%) participants were female, 5056 (53.0%) were male and 262 (2.7%) were unreported. Articles incorporating only females were significantly underrepresented compared to articles incorporating only males. Forty articles (63.5%) which presented data from both females and males, failed to analyse and interpret the results by sex, a significant methodological limitation. In conclusion, in the literature published over the last 20 years, female participants are significantly underrepresented. In the studies where females are represented, severe methodological limitations are apparent. Researchers should be conscious of sexual dimorphism, menstrual phase and use of hormonal contraception, which may impact the interpretation of their results.

Development and validation of the Male Post-coital Affect Scale for heterosexual men.

OBJECTIVE: The aim of this study was to examine the reliability and validity of the Male Post-coital Affect Scale (MPAS), which was developed to assess positive post-coital feelings in men. METHODS: After a pilot study, we validated our scale on a sample of American heterosexual men, who answered our questionnaire on the internet through Amazon Mechanical Turk. We tested the reliability using internal consistency. The validity was examined by assessing content, face and construct validity by testing the association between our scale, the Experience in Close Relationships Scale and other instruments. RESULTS: A total of 484 volunteers were included in the study. Cronbach's α for the scale was 0.83. Our scale was negatively correlated with attachment avoidance, r(482) = -0.36, p < 0.001) and Perceived Stress Scale, r(482) = -0.18, p < 0.001, and positively correlated with sexual satisfaction, r(482) = 0.18, p < 0.001. CONCLUSION: The MPAS is a reliable and valid tool to assess positive post-coital feelings in men.

Spatial quantification of single cell mRNA and ligand binding of the kappa opioid receptor in the rat hypothalamus.

Detailed quantification of brain tissue provides a deeper understanding of changes in expression and function. We have created a pipeline to study the detailed expression patterns of the kappa opioid receptor in the rat hypothalamus using high resolution fluorescence microscopy and receptor autoradiography. The workflow involved structured serial sampling of rat hypothalamic nuclei, in situ detection of mRNA and receptor expression, and advanced image analysis. Our results demonstrate how maintaining spatial information can lead to increased understanding of RNA and protein expression. In addition, we show the detailed expression patterns of the kappa opioid receptor in the rat hypothalamus.

A genetically modified minipig model for Alzheimer's disease with SORL1 haploinsufficiency.

The established causal genes in Alzheimer's disease (AD), APP, PSEN1, and PSEN2, are functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease's initial preclinical phase. Mutations in SORL1, encoding the endosome recycling receptor SORLA, are found in 2%-3% of individuals with early-onset AD, and SORL1 haploinsufficiency appears to be causal for AD. To test whether SORL1 can function as an AD causal gene, we use CRISPR-Cas9-based gene editing to develop a model of SORL1 haploinsufficiency in Göttingen minipigs, taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young adult minipigs is found to phenocopy the preclinical in vivo profile of AD observed with APP, PSEN1, and PSEN2, resulting in elevated levels of ß-amyloid (Aß) and tau preceding amyloid plaque formation and neurodegeneration, as observed in humans. Our study provides functional support for the theory that SORL1 haploinsufficiency leads to endosome cytopathology with biofluid hallmarks of autosomal dominant AD.

Df(h22q11)/+ mouse model exhibits reduced binding levels of GABAA receptors and structural and functional dysregulation in the inhibitory and excitatory networks of hippocampus.

The 22q11.2 hemizygous deletion confers high risk for multiple neurodevelopmental disorders. Inhibitory signaling, largely regulated through GABAA receptors, is suggested to serve a multitude of brain functions that are disrupted in the 22q11.2 deletion syndrome. We investigated the putative deficit of GABAA receptors and the potential substrates contributing to the inhibitory and excitatory dysregulations in hippocampal networks of the Df(h22q11)/+ mouse model of the 22q11.2 hemizygous deletion. The Df(h22q11)/+ mice exhibited impairments in several hippocampus-related functional domains, represented by impaired spatial memory and sensory gating functions. Autoradiography using the [3H]muscimol tracer revealed a significant reduction in GABAA receptor binding in the CA1 and CA3 subregions, together with a loss of GAD67+ interneurons in CA1 of Df(h22q11)/+ mice. Furthermore, electrophysiology recordings exhibited significantly higher neuronal activity in CA3, in response to the GABAA receptor antagonist, bicuculline, as compared with wild type mice. Density and volume of dendritic spines in pyramidal neurons were reduced and Sholl analysis also showed a reduction in the complexity of basal dendritic tree in CA1 and CA3 subregions of Df(h22q11)/+ mice. Overall, our findings demonstrate that hemizygous deletion in the 22q11.2 locus leads to dysregulations in the inhibitory circuits, involving reduced binding levels of GABAA receptors, in addition to functional and structural modulations of the excitatory networks of hippocampus.

Synaptic Vesicle Glycoprotein 2A: Features and Functions.

In recent years, the field of neuroimaging dramatically moved forward by means of the expeditious development of specific radioligands of novel targets. Among these targets, the synaptic vesicle glycoprotein 2A (SV2A) is a transmembrane protein of synaptic vesicles, present in all synaptic terminals, irrespective of neurotransmitter content. It is involved in key functions of neurons, focused on the regulation of neurotransmitter release. The ubiquitous expression in gray matter regions of the brain is the basis of its candidacy as a marker of synaptic density. Following the development of molecules derived from the structure of the anti-epileptic drug levetiracetam, which selectively binds to SV2A, several radiolabeled markers have been synthetized to allow the study of SV2A distribution with positron emission tomography (PET). These radioligands permit the evaluation of in vivo changes of SV2A distribution held to be a potential measure of synaptic density in physiological and pathological conditions. The use of SV2A as a biomarker of synaptic density raises important questions. Despite numerous studies over the last decades, the biological function and the expressional properties of SV2A remain poorly understood. Some functions of SV2A were claimed, but have not been fully elucidated. While the expression of SV2A is ubiquitous, stronger associations between SV2A and Υ amino butyric acid (GABA)-ergic rather than glutamatergic synapses were observed in some brain structures. A further issue is the unclear interaction between SV2A and its tracers, which reflects a need to clarify what really is detected with neuroimaging tools. Here, we summarize the current knowledge of the SV2A protein and we discuss uncertain aspects of SV2A biology and physiology. As SV2A expression is ubiquitous, but likely more strongly related to a certain type of neurotransmission in particular circumstances, a more extensive knowledge of the protein would greatly facilitate the analysis and interpretation of neuroimaging results by allowing the evaluation not only of an increase or decrease of the protein level, but also of the type of neurotransmission involved.

The intersection of astrocytes and the endocannabinoid system in the lateral habenula: on the fast-track to novel rapid-acting antidepressants.

Despite attaining significant advances toward better management of depressive disorders, we are still facing several setbacks. Developing rapid-acting antidepressants with sustained effects is an aspiration that requires thinking anew to explore possible novel targets. Recently, the lateral habenula (LHb), the brain's "anti-reward system", has been shown to go awry in depression in terms of various molecular and electrophysiological signatures. Some of the presumed contributors to such observed aberrations are astrocytes. These star-shaped cells of the brain can alter the firing pattern of the LHb, which keeps the activity of the midbrain's aminergic centers under tight control. Astrocytes are also integral parts of the tripartite synapses, and can therefore modulate synaptic plasticity and leave long-lasting changes in the brain. On the other hand, it was discovered that astrocytes express cannabinoid type 1 receptors (CB1R), which can also take part in long-term plasticity. Herein, we recount how the LHb of a depressed brain deviates from the "normal" one from a molecular perspective. We then try to touch upon the alterations of the endocannabinoid system in the LHb, and cast the idea that modulation of astroglial CB1R may help regulate habenular neuronal activity and synaptogenesis, thereby acting as a new pharmacological tool for regulation of mood and amelioration of depressive symptoms.

Spontaneous partial recovery of striatal dopaminergic uptake despite nigral cell loss in asymptomatic MPTP-lesioned female minipigs.

The Göttingen minipig is a large animal with a gyrencephalic brain that expresses -complex behavior, making it an attractive model for Parkinson's disease research. Here, we investigate the temporal evolution of presynaptic dopaminergic function for 14 months after injections of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into the minipig using a multi-tracer longitudinal positron emission tomography (PET) design. We injected seven sedated minipigs with 1-2 mg/kg of MPTP, and two with saline, three times a week over four weeks. We monitored behavioral deficits using a validated motor scale and walking mat. Brains were imaged with (+)-⍺-[11C]-dihydrotetrabenazine ([11C]-DTBZ) and [18F]-dihydroxyphenylalanine ([18F]-FDOPA) PET at baseline and 1, 3, 10 and 14 months after MPTP injection, and immunohistochemistry was used to assess nigral cell loss. The minipigs showed mild bradykinesia and impaired coordination at early timepoints after MPTP. PET revealed decreases of striatal [11C]-DTBZ and [18F]-FDOPA uptake post-MPTP with partial spontaneous recovery of [18F]-FDOPA after 10 months. Postmortem analysis estimated an MPTP-induced nigral loss of 57% tyrosine hydroxylase+ and 43% Nissl-stained cells. Normal motor function despite substantial damage to the dopaminergic system is consistent with prodromal Parkinson's disease, and offers an opportunity for testing disease-modifying therapies. However, partial spontaneous recovery of dopamine terminal function must be taken into account in future studies.

Tips and traps for behavioural animal experimentation.

Behavioural animal experimentation is an inseparable part of research trying to understand the biological underpinnings of human behaviour, diseases and disorders. Working with animals comes with great responsibility to achieve reliable and reproducible results of highest scientific quality. In a simple step-by-step fashion, we highlight some common issues that may occur along the path to conducting behavioural animal experimentations and posit some solutions and grounds to ensure the excellence of work done in this research area while aspiring to improve conditions for laboratory animals. It entails topics of study design, animal and experimenter welfare, experimental considerations and frequentist biostatistics. At the end, we direct to some guidelines and manuals that may prove valuable to researchers in this field. Our ten simple tips and traps are meant for students who are learning about important concepts for the first time; graduates whose statistics training all too often has neglected the concept of power in experimental design; and researches who would like a light-hearted refresher on these topics. With this perspective, we hope that you will avoid falling into traps and find answers to what you always wanted to know about conducting behavioural animal experimentation.

Validation and optimisation of an automatic blood sampler for preclinical positron emission tomography research in domestic pigs.

In preclinical positron emission tomography animal studies, continuous blood sampling is used to measure the time course of the activity concentration in arterial blood. However, pigs have hypercoagulable blood that tends to clot inside plastic tubes. We tested several tube materials and lengths and the use of three-way connectors. We validated set-ups for automated blood sampling with and without blood recirculation that could run for 90 minutes without problematic clots and without any evidence of emboli formation during necropsy.