Mapping functional traces of opioid memories in the rat brain.

Addiction to psychoactive substances is a maladaptive learned behaviour. Contexts surrounding drug use integrate this aberrant mnemonic process and hold strong relapse-triggering ability. Here, we asked where context and salience might be concurrently represented in the brain during retrieval of drug-context paired associations. For this, we developed a morphine-conditioned place preference protocol that allows contextual stimuli presentation inside a magnetic resonance imaging scanner and investigated differences in activity and connectivity at context recall. We found context-specific responses to stimulus onset in multiple brain regions, namely, limbic, sensory and striatal. Differences in functional interconnectivity were found among amygdala, lateral habenula, and lateral septum. We also investigated alterations to resting-state functional connectivity and found increased centrality of the lateral septum in a proposed limbic network, as well as increased functional connectivity of the lateral habenula and hippocampal 'cornu ammonis' 1 region, after a protocol of associative drug-context. Finally, we found that pre- conditioned place preference resting-state connectivity of the lateral habenula and amygdala was predictive of inter-individual conditioned place preference score differences. Overall, our findings show that drug and saline-paired contexts establish distinct memory traces in overlapping functional brain microcircuits and that intrinsic connectivity of the habenula, septum, and amygdala likely underlies the individual maladaptive contextual learning to opioid exposure. We have identified functional maps of acquisition and retrieval of drug-related memory that may support the relapse-triggering ability of opioid-associated sensory and contextual cues. These findings may clarify the inter-individual sensitivity and vulnerability seen in addiction to opioids found in humans.

Aged Brain Metabolomics Study by Metabolic Profiling Analysis of Amino Acids, Organic Acids, and Fatty Acids in Cortex, Cerebellum, Hypothalamus, and Hippocampus of Rats.

BACKGROUND: Aging is a progressive process characterized by weakness in brain function. Although metabolomics studies on the brain related with aging have been conducted, it is not yet fully understood. A systematic metabolomics study was performed to search for biomarkers and monitor altered metabolism in various brain tissues of the cortex, cerebellum, hypothalamus, and hippocampus of young (8 months old) and old rats (22 months old). METHODS: Simultaneous profiling analysis of amino acids (AAs), organic acids (OAs), and fatty acids (FAs) in the brain tissues of young and old rats were performed by gas chromatography-tandem mass spectrometry. RESULTS: Under optimal conditions, AA, OA, and FA profiling methods showed good linearity (r ≥0.995) with limit of detection of ≤30 and 73.2 ng and limit of quantification of ≤90.1 and 219.5 ng, respectively. Repeatability varied from 0.4 to 10.4 and 0.8 to 14.8% relative standard deviation and accuracy varied from -11.3 to 10.3 and -12.8 to 14.1% relative error, respectively. In the profiling analysis, total 32, 43, 45, and 30 metabolites were determined in cortex, cerebellum, hypothalamus, and hippocampus, respectively. In statistical analysis, eight AAs (alanine, valine, leucine, isoleucine, threonine, serine, proline, and phenylalanine) in the cortex and four metabolites (alanine, phenylalanine, 3-hydoxypropionic acid, and eicosadienoic acid) in the cerebellum were significantly evaluated (Q-value <0.05, variable importance in projection scores ≥1.0). In all brain tissues, the score plots of orthogonal partial least square discriminant analysis were clearly separated between the young and old groups. CONCLUSIONS: Metabolomics results indicate that mechanistic targets of rapamycin complex 1, branched chain-amino acid, and energy metabolism are related to inflammation and mitochondrial dysfunction in the brain during aging. Thus, these results may explain the characteristic metabolism of brain aging.

Effects of Strawberry Tree Water Leaf Extract and Arbutin on Biochemical Markers and DNA Integrity in Brain Cells of Lewis Rats.

There is growing evidence that arbutin and plant extracts rich in arbutin, such as extracts of the strawberry tree (Arbutus unedo L.), exert a range of beneficial effects, including cyto- and genoprotective properties. This study evaluated the effects of strawberry tree water leaf extract (STE) and arbutin in the brain tissue of Lewis rats. STE or arbutin were administered per os to male and female rats at a dose of 200 mg/kg body weight/day for 14 or 28 days. Treatment outcomes were evaluated using biochemical markers (lipid peroxidation and the activities of the antioxidative enzymes catalase and superoxide dismutase). The effects of the tested substances on DNA integrity in brain cells were evaluated using the alkaline comet assay. The results suggest a high biocompatibility of both tested substances with rat brain tissue. No significant harmful disturbances were observed in the oxidative/antioxidative status or impairments of DNA integrity in the rat brain cells. Nearly all post-treatment values were within tolerable limits as compared to the matched control rats. Such encouraging findings support further research using other subtle biomarkers to clarify the safety aspects of arbutin and STE prior to the development of specific nutraceutical products.

Modulation of the Blood-Brain Barrier by Sigma-1R Activation.

Sigma non-opioid intracellular receptor 1 (Sigma-1R) is an intracellular chaperone protein residing on the endoplasmic reticulum at the mitochondrial-associated membrane (MAM) region. Sigma-1R is abundant in the brain and is involved in several physiological processes as well as in various disease states. The role of Sigma-1R at the blood-brain barrier (BBB) is incompletely characterized. In this study, the effect of Sigma-1R activation was investigated in vitro on rat brain microvascular endothelial cells (RBMVEC), an important component of the blood-brain barrier (BBB), and in vivo on BBB permeability in rats. The Sigma-1R agonist PRE-084 produced a dose-dependent increase in mitochondrial calcium, and mitochondrial and cytosolic reactive oxygen species (ROS) in RBMVEC. PRE-084 decreased the electrical resistance of the RBMVEC monolayer, measured with the electric cell-substrate impedance sensing (ECIS) method, indicating barrier disruption. These effects were reduced by pretreatment with Sigma-1R antagonists, BD 1047 and NE 100. In vivo assessment of BBB permeability in rats indicates that PRE-084 produced a dose-dependent increase in brain extravasation of Evans Blue and sodium fluorescein brain; the effect was reduced by the Sigma-1R antagonists. Immunocytochemistry studies indicate that PRE-084 produced a disruption of tight and adherens junctions and actin cytoskeleton. The brain microcirculation was directly visualized in vivo in the prefrontal cortex of awake rats with a miniature integrated fluorescence microscope (aka, miniscope; Doric Lenses Inc.). Miniscope studies indicate that PRE-084 increased sodium fluorescein extravasation in vivo. Taken together, these results indicate that Sigma-1R activation promoted oxidative stress and increased BBB permeability.

Diffusion of brain metabolites highlights altered brain microstructure in type C hepatic encephalopathy: a 9.4 T preliminary study.

Introduction: Type C hepatic encephalopathy (HE) is a decompensating event of chronic liver disease leading to severe motor and cognitive impairment. The progression of type C HE is associated with changes in brain metabolite concentrations measured by 1H magnetic resonance spectroscopy (MRS), most noticeably a strong increase in glutamine to detoxify brain ammonia. In addition, alterations of brain cellular architecture have been measured ex vivo by histology in a rat model of type C HE. The aim of this study was to assess the potential of diffusion-weighted MRS (dMRS) for probing these cellular shape alterations in vivo by monitoring the diffusion properties of the major brain metabolites. Methods: The bile duct-ligated (BDL) rat model of type C HE was used. Five animals were scanned before surgery and 6- to 7-week post-BDL surgery, with each animal being used as its own control. 1H-MRS was performed in the hippocampus (SPECIAL, TE = 2.8 ms) and dMRS in a voxel encompassing the entire brain (DW-STEAM, TE = 15 ms, diffusion time = 120 ms, maximum b-value = 25 ms/µm2) on a 9.4 T scanner. The in vivo MRS acquisitions were further validated with histological measures (immunohistochemistry, Golgi-Cox, electron microscopy). Results: The characteristic 1H-MRS pattern of type C HE, i.e., a gradual increase of brain glutamine and a decrease of the main organic osmolytes, was observed in the hippocampus of BDL rats. Overall increased metabolite diffusivities (apparent diffusion coefficient and intra-stick diffusivity-Callaghan's model, significant for glutamine, myo-inositol, and taurine) and decreased kurtosis coefficients were observed in BDL rats compared to control, highlighting the presence of osmotic stress and possibly of astrocytic and neuronal alterations. These results were consistent with the microstructure depicted by histology and represented by a decline in dendritic spines density in neurons, a shortening and decreased number of astrocytic processes, and extracellular edema. Discussion: dMRS enables non-invasive and longitudinal monitoring of the diffusion behavior of brain metabolites, reflecting in the present study the globally altered brain microstructure in BDL rats, as confirmed ex vivo by histology. These findings give new insights into metabolic and microstructural abnormalities associated with high brain glutamine and its consequences in type C HE.

Repeated binge-like eating episodes in female rats alter adenosine A2A and dopamine D2 receptor genes regulation in the brain reward system.

OBJECTIVE: Binge-eating disorder is an eating disorder characterized by recurrent binge-eating episodes, during which individuals consume excessive amounts of highly palatable food (HPF) in a short time. This study investigates the intricate relationship between repeated binge-eating episode and the transcriptional regulation of two key genes, adenosine A2A receptor (A2AAR) and dopamine D2 receptor (D2R), in selected brain regions of rats. METHOD: Binge-like eating behavior on HPF was induced through the combination of food restrictions and frustration stress (15 min exposure to HPF without access to it) in female rats, compared to control rats subjected to only restriction or only stress or none of these two conditions. After chronic binge-eating episodes, nucleic acids were extracted from different brain regions, and gene expression levels were assessed through real-time quantitative PCR. The methylation pattern on genes' promoters was investigated using pyrosequencing. RESULTS: The analysis revealed A2AAR upregulation in the amygdala and in the ventral tegmental area (VTA), and D2R downregulation in the nucleus accumbens in binge-eating rats. Concurrently, site-specific DNA methylation alterations at gene promoters were identified in the VTA for A2AAR and in the amygdala and caudate putamen for D2R. DISCUSSION: The alterations on A2AAR and D2R genes regulation highlight the significance of epigenetic mechanisms in the etiology of binge-eating behavior, and underscore the potential for targeted therapeutic interventions, to prevent the development of this maladaptive feeding behavior. These findings provide valuable insights for future research in the field of eating disorders. PUBLIC SIGNIFICANCE: Using an animal model with face, construct, and predictive validity, in which cycles of food restriction and frustration stress evoke binge-eating behavior, we highlight the significance of epigenetic mechanisms on adenosine A2A receptor (A2AAR) and dopamine D2 receptor (D2R) genes regulation. They could represent new potential targets for the pharmacological management of eating disorders characterized by this maladaptive feeding behavior.

Ammoniagenic Action of Valproate without Signs of Hepatic Dysfunction in Rats: Possible Causes and Supporting Evidence.

(1) Background: Valproic acid (VPA) is one of the frequently prescribed antiepileptic drugs and is generally considered well tolerated. However, VPA neurologic adverse effects in the absence of liver failure are fairly common, suggesting that in the mechanism for the development of VPA-induced encephalopathy, much more is involved than merely the exposure to hyperammonemia (HA) caused by liver insufficiency to perform detoxification. Taking into account the importance of the relationship between an impaired brain energy metabolism and elevated ammonia production, and based on the ability of VPA to interfere with neuronal oxidative pathways, the current study intended to investigate a potential regional ammoniagenic effect of VPA on rats' brains by determining activities of the enzymes responsible for ammonia production and neutralization. (2) Methods: Rats received a single intraperitoneal injection of VPA (50, 100, 250, 500 mg/kg). Plasma, the neocortex, the cerebellum, and the hippocampus were collected at 30 min after injection. The levels of ammonia, urea, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured in blood plasma. The activities of glutaminase and glutamate dehydrogenase (GDH) in mitochondria and the activities of AMP deaminase (AMPD), adenosine deaminase (ADA), and glutamine synthetase (GS) in cytosolic fractions isolated from rat brain regions were measured. Ammonia, ALT, and AST values were determined in the mitochondrial and cytosolic fractions. (3) Results: Multi-dose VPA treatment did not significantly affect the plasma levels of ammonia and urea or the ALT and AST liver enzymes. Significant dose-independent increases in the accumulation of ammonia were found only in the cytosol from the cerebellum and there was a strong correlation between the ammonia level and the ADA activity in this brain structure. A significant decrease in the AMPD and AST activities was observed, while the ALT activity was unaffected. Only the highest VPA dose (500 mg/kg) was associated with significantly less activity of GS compared to the control in all studied brain structures. In the mitochondria of all studied brain structures, VPA caused a dose-independent increases in ammonia levels, a high concentration of which was strongly and positively correlated with the increased GDH and ALT activity, while glutaminase activity remained unchanged, and AST activity significantly decreased compared to the control in all studied brain structures. (4) Conclusions: This study highlights the rat brain region-specific ammoniagenic effects of VPA, which may manifest themselves in the absence of hyperammonemia. Further research should analyze how the responsiveness of the different brain regions may vary in VPA-treated animals that exhibit compromised energy metabolism, leading to increased ammoniagenesis.

Probes for the heterogeneity of muscimol binding sites in rat brain.

Introduction: The plant-based alkaloid muscimol is a potent agonist of inhibitory GABAA-neurotransmitter receptors. GABAA receptors are a heterogeneous family of pentameric complexes, with 5 out of 19 subunits assembling around the central anion pore. Muscimol is considered to bind to all receptor subtypes at the orthosteric drug binding site at the ß+/α- interface. Recently, we observed that the antipsychotic drugs clozapine (CLZ), loxapine (LOX) and chlorpromazine (CPZ) although exerting functional inhibition on multiple GABAA receptor subtypes showed diverging results in displacing 3H-muscimol. While a complete displacement could be observed in hippocampal membranes by bicuculline (BIC), and no displacement with CPZ, the compounds CLZ and LOX competed partially. Non-sigmoidal, complex dose response curves were indicative of multiple sites. In the current study we now aimed to investigate more extensively this heterogeneity of bicuculline sensitive muscimol sites in rat brain. Methods: We tested membranes from four different brain regions (hippocampus, cerebellum, thalamus and striatum) and selected recombinantly expressed subunit combinations with displacement assays. 3H-muscimol displacement was tested with BIC, LOX, CLZ and CPZ. In silico ligand structural analysis and computational docking was performed. Results: We observed a unique pharmacology of each tested compound in the studied brain regions. Combining two of the tested ligands suggests that in striatum all CLZ sites are contained in the pool of LOX sites, while the CPZ sites may in part be non-overlapping with LOX sites. Experiments on recombinantly expressed receptors indicate, that BIC can displace 3H-muscimol from all tested receptors, while LOX and CLZ display different and variable competition indicative of multiple sites. Molecular docking produced structural correlates of the observed diversity of muscimol sites on the basis of bicuculline bound experimental structures. Discussion: These findings indicate that 3H-muscimol binding sites in rat brain are heterogeneous, with different populations of receptors, which are CPZ, LOX or CLZ sensitive or insensitive. These binding sites show a varying distribution in different rat brain regions. Molecular docking suggests that the so-called loop F region of α subunits drives the observed differences.

Disposable paper electrodes for detection of changes in dopamine concentrations in rat brain homogenates.

Dopamine, the main catecholamine neurotransmitter plays an important role in renal, cardiovascular, central nervous systems, and pathophysiological processes. The abnormal dopamine levels can result in neurological disorders such as Parkinson's, Alzheimer's, schizophrenia, acute anxiety, neuroblastoma and also contribute to cognitive dysfunctions. Given the widespread importance of dopamine concentration levels, it is imperative to develop sensors that are able to monitor dopamine. Herein, we have developed pre-anodized disposable paper electrode modified with 1-pyrenebutyric acid, for the selective and sensitive determination of dopamine. The sensor was characterized with Fourier transform infrared spectroscopy, Energy dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and electrochemical techniques for addressing the robust formation and electrochemical activity. The modified electrode exhibited excellent electrocatalytic activity towards dopamine without the common interference from ascorbic acid. The calibration plot for the dopamine sensor resulted linear range from 0.003 µM to 0.5 µM with a detection limit of 0.11 nM. The sensor's potential utility was tested by monitoring dopamine concentration changes in rat brain homogenates when subjected to neurotoxicity. The developed sensor was validated with gold-standard UV-Vis spectroscopy studies and computational studies were performed to understand the interaction between 1-pyrenebutyric acid and dopamine.

[Mechanobiological Mechanisms Involved in the Regualation of the Blood-Brain Barrier by Fluid Shear Force]. / æµä½“å‰ªåˆ‡åŠ›è°ƒæŽ§è¡€è„‘å±éšœçš„åŠ›å­¦ç”Ÿç‰©å­¦æœºåˆ¶ç ”ç©¶.

Objective: To explore the mechanobiological mechanism of fluid shear force (FSF) on the protection, injury, and destruction of the structure and function of the blood-brain barrier (BBB) under normal physiological conditions, ischemic hypoperfusion, and postoperative hyperperfusion conditions. BBB is mainly composed of brain microvascular endothelial cells. Rat brain microvascular endothelial cells (rBMECs) were used as model cells to conduct the investigation. Methods: rBMECs were seeded at a density of 1×105 cells/cm2 and incubated for 48 h. FSF was applied to the rBMECs at 0.5, 2, and 20 dyn/cm2, respectively, simulating the stress BBB incurs under low perfusion, normal physiological conditions, and high FSF after bypass grafting when there is cerebral vascular stenosis. In addition, a rBMECs static culture group was set up as the control (no force was applied). Light microscope, scanning electron microscope (SEM), and laser confocal microscope (LSCM) were used to observe the changes in cell morphology and cytoskeleton. Transmission electron microscope (TEM) was used to observe the tight junctions. Immunofluorescence assay was performed to determine changes in the distribution of tight junction-associated proteins claudin-5, occludin, and ZO-1 and adherens junction-associated proteins VE-cadherin and PECAM-1. Western blot was performed to determine the expression levels of tight junction-associated proteins claudin-5, ZO-1, and JAM4, adherens junction-associated protein VE-cadherin, and key proteins in Rho GTPases signaling (Rac1, Cdc42, and RhoA) under FSF at different intensities. Results: Microscopic observation showed that the cytoskeleton exhibited disorderly arrangement and irregular orientation under static culture and low shear force (0.5 dyn/cm2). Under normal physiological shear force (2 dyn/cm2), the cytoskeleton was rearranged in the orientation of the FSF and an effective tight junction structure was observed between cells. Under high shear force (20 dyn/cm2), the intercellular space was enlarged and no effective tight junction structure was observed. Immunofluorescence results showed that, under low shear force, the gap between the cells decreased, but there was also decreased distribution of tight junction-associated proteins and adherens junction-associated proteins at the intercellular junctions. Under normal physiological conditions, the cells were tightly connected and most of the tight junction-associated proteins were concentrated at the intercellular junctions. Under high shear force, the gap between the cells increased significantly and the tight junction and adherens junction structures were disrupted. According to the Western blot results, under low shear force, the expression levels of claudin-5, ZO-1, and VE-cadherin were significantly up-regulated compared with those of the control group (P<0.05). Under normal physiological shear force, claudin-5, ZO-1, JAM4, and VE-cadherin were highly expressed compared with those of the control group (P<0.05). Under high shear force, the expressions of claudin-5, ZO-1, JAM4, and VE-cadherin were significantly down-regulated compared with those of the normal physiological shear force group (P<0.05). Under normal physiological shear force, intercellular expressions of Rho GTPases proteins (Rac1, Cdc42, and RhoA) were up-regulated and were higher than those of the other experimental groups (P<0.05). The expressions of Rho GTPases under low and high shear forces were down-regulated compared with that of the normal physiological shear force group (P<0.05). Conclusion: Under normal physiological conditions, FSF helps maintain the integrity of the BBB structure, while low or high shear force can damage or destroy the BBB structure. The regulation of BBB by FSF is closely related to the expression and distribution of tight junction-associated proteins and adherens junction-associated proteins.

Spiral laser scanning photoacoustic microscopy for functional brain imaging in rats.

Significance: There are many neuroscience questions that can be answered by a high-resolution functional brain imaging system. Such a system would require the capability to visualize vasculature and measure neural activity by imaging the entire brain continually and in rapid succession in order to capture hemodynamic changes. Utilizing optical excitation and acoustic detection, photoacoustic technology enables label-free quantification of changes in endogenous chromophores, such as oxyhemoglobin, deoxyhemoglobin, and total hemoglobin. Aim: Our aim was to develop a sufficiently high-resolution, fast frame-rate, and wide field-of-view (FOV) photoacoustic microscopy (PAM) system for the purpose of imaging vasculature and hemodynamics in a rat brain. Approach: Although the most PA microscopy systems use raster scanning (or less commonly Lissajous scanning), we have developed a simple-to-implement laser scanning optical resolution PAM system with spiral scanning (which we have named "spiral laser scanning photoacoustic microscopy" or sLS-PAM) to acquire an 18 mm diameter image at fast frame rate (more than 1 fps). Such a system is designed to permit continuous rat brain imaging without the introduction of photobleaching artifacts. Conclusion: We demonstrated the functional imaging capability of the sLS-PAM system by imaging cerebral hemodynamics in response to whisker and electrical stimulation and used it for vascular imaging of a modeled brain injury. We believe that we have demonstrated the development of a simple-to-implement PAM system, which could become an affordable functional neuroimaging tool for researchers.

The impact of multifunctional enkephalin analogs and morphine on the protein changes in crude membrane fractions isolated from the rat brain cortex and hippocampus.

Endogenous opioid peptides serve as potent analgesics through the opioid receptor (OR) activation. However, they often suffer from poor metabolic stability, low lipophilicity, and low blood-brain barrier permeability. Researchers have developed many strategies to overcome the drawbacks of current pain medications and unwanted biological effects produced by the interaction with opioid receptors. Here, we tested multifunctional enkephalin analogs LYS739 (MOR/DOR agonist and KOR partial antagonist) and LYS744 (MOR/DOR agonist and KOR full antagonist) under in vivo conditions in comparison with MOR agonist, morphine. We applied 2D electrophoretic resolution to investigate differences in proteome profiles of crude membrane (CM) fractions isolated from the rat brain cortex and hippocampus exposed to the drugs (10 mg/kg, seven days). Our results have shown that treatment with analog LYS739 induced the most protein changes in cortical and hippocampal samples. The identified proteins were mainly associated with energy metabolism, cell shape and movement, apoptosis, protein folding, regulation of redox homeostasis, and signal transduction. Among these, the isoform of mitochondrial ATP synthase subunit beta (ATP5F1B) was the only protein upregulation in the hippocampus but not in the brain cortex. Contrarily, the administration of analog LYS744 caused a small number of protein alterations in both brain parts. Our results indicate that the KOR full antagonism, together with MOR/DOR agonism of multifunctional opioid ligands, can be beneficial in treating chronic pain states by reducing changes in protein expression levels but retaining analgesic efficacy.

Development of an innovative turn-on fluorescent probe for targeted in-vivo detection of nitric oxide in rat brain extracts as a biomarker for migraine disease.

Nitric oxide (NO) is one of the reactive nitrogen species (RNS) that has been proposed to be a key signaling molecule in migraine. Migraine is a neurological disorder that is linked to irregular NO levels, which necessitates precise NO quantification for effective diagnosis and treatment. This work introduces a novel fluorescent probe, 2,3-diaminonaphthelene-1,4-dione (DAND), which was designed and synthesized to selectively detect NO in-vitro and in-vivo as a migraine biomarker. DAND boasts high aqueous solubility, biocompatibility, and facile synthesis, which enable highly selective and sensitive detection of NO under physiological conditions. NO reacts with diamine moieties (recognition sites) of DAND, results in the formation of a highly fluorescent product (DAND-NO) known as 1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione at λem 450 nm. The fluorescence turn-on sensing mechanism operates through an intramolecular charge transfer (ICT) mechanism. To maximize fluorescence signal intensity, parameters including DAND concentration, reaction temperature, reaction time and pH were systematically optimized for sensitive and precise NO determination. The enhanced detection capability (LOD = 0.08 µmol L-1) and high selectivity of the probe make it a promising tool for NO detection in brain tissue homogenates. This demonstrates the potential diagnostic value of the probe for individuals suffering from migraine. Furthermore, this study sheds light on the potential role of zolmitriptan (ZOLM), an antimigraine medication, in modulating NO levels in the brain of rats with nitroglycerin-induced migraine, emphasizing its significant impact on reducing NO levels. The obtained results could have significant implications for understanding how ZOLM affects NO levels and may aid in the development of more targeted and effective migraine treatment strategies.

The Locare workflow: representing neuroscience data locations as geometric objects in 3D brain atlases.

Neuroscientists employ a range of methods and generate increasing amounts of data describing brain structure and function. The anatomical locations from which observations or measurements originate represent a common context for data interpretation, and a starting point for identifying data of interest. However, the multimodality and abundance of brain data pose a challenge for efforts to organize, integrate, and analyze data based on anatomical locations. While structured metadata allow faceted data queries, different types of data are not easily represented in a standardized and machine-readable way that allow comparison, analysis, and queries related to anatomical relevance. To this end, three-dimensional (3D) digital brain atlases provide frameworks in which disparate multimodal and multilevel neuroscience data can be spatially represented. We propose to represent the locations of different neuroscience data as geometric objects in 3D brain atlases. Such geometric objects can be specified in a standardized file format and stored as location metadata for use with different computational tools. We here present the Locare workflow developed for defining the anatomical location of data elements from rodent brains as geometric objects. We demonstrate how the workflow can be used to define geometric objects representing multimodal and multilevel experimental neuroscience in rat or mouse brain atlases. We further propose a collection of JSON schemas (LocareJSON) for specifying geometric objects by atlas coordinates, suitable as a starting point for co-visualization of different data in an anatomical context and for enabling spatial data queries.

Reference Gene Validation in the Embryonic and Postnatal Brain in the Rat Hyperhomocysteinemia Model.

Maternal hyperhomocysteinemia (HCY) induced by genetic defects in methionine cycle enzymes or vitamin imbalance is known to be a pathologic factor that can impair embryonal brain development and cause long-term consequences in the postnatal brain development as well as changes in the expression of neuronal genes. Studies of the gene expression on this model requires the selection of optimal housekeeping genes. This work aimed to analyze the expression stability of housekeeping genes in offspring brain. Pregnant female Wistar rats were treated daily with a 0.15% L-methionine solution in the period starting on the 4th day of pregnancy until delivery, to cause the increase in the homocysteine level in fetus blood and brain. Housekeeping gene expression was assessed by RT-qPCR on whole embryonic brain and selected rat brain areas at P20 and P90. The amplification curves were analyzed, and raw means Cq data were imported to the RefFinder online tool to assess the reference genes stability. Most of the analyzed genes showed high stability of mRNA expression in the fetal brain at both periods of analysis (E14 and E20). However, the most stably expressed genes at different age points differed. Actb, Ppia, Rpl13a are the most stably expressed on E14, Ywhaz, Pgk1, Hprt1 - on E20 and P20, Hprt1, Actb, and Pgk1 - on P90. Gapdh gene used as a reference in various studies demonstrates high stability only in the hippocampus and cannot be recommended as the optimal reference gene on HCY model. Hprt1 and Pgk1 genes were found to be the most stably expressed in the brain of rat subjected to HCY. These two genes showed high stability in the brain on E20 and in various areas of the brain on the P20 and P90. On E14, the preferred genes for normalization are Actb, Ppia, Rpl13a.

Rodent brain extraction and dissection: A comprehensive approach.

The neuroscience is continuously expanding field, and conducting experiments serves as one of the most effective approaches to enhance and broad our understanding of this fascinating field. Most of the lab work in neuroscience involves the use of animal models such as rats and mice for experiments dedicated to monitoring cerebral changes. The study:•Introduces a practical method for brain extraction without perfusion with paraformaldehyde prioritizing brain integrity and avoiding damage.•Offers a detailed, step-by-step dissection guide for different brain regions, including the hippocampus, cerebral cortex, corpus striatum, thalamus, cerebellum, and medial prefrontal cortex, from rodent brains, accompanied by high-resolution images that provide anatomical clarity.•Presents enhanced reliability, precision, and detailed anatomical descriptions.Conclusion: This study has introduced a reliable technique for brain extraction that eliminates the need for paraformaldehyde perfusion. Furthermore, a comprehensive methodology has been presented for extracting different brain regions from rodent brains.

Effect of Jianpi Bushen Huoxue Prescription on Rat Brain Microvascular Endothelial Cells Based on HIF-1α/VEGF Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effects of Jianpi Bushen Huoxue prescription （JPBSHX） on rat brain microvascular endothelial cells （RBMECs） based on hypoxia-inducible factor-1α （HIF-1α）/vascular endothelial growth factor （VEGF） signaling pathway， aiming to provide a theoretical basis for the treatment of ischemic stroke. MethodTwelve 8-week-old male SPF-grade SD rats were selected. Eight of them were randomly chosen and given 3.25 g·mL-1 JPBSHX solution by gavage at a dose of 10 mL·kg-1 for 5 consecutive days to prepare the medicated serum， which was then preserved for later use. The remaining four rats were given the same volume of normal saline. Follow-up operations were the same as those of the above eight rats. Normal rat serum was collected and stored for later use. RBMECs were revived， cultured， passaged， and randomly divided into five groups： normal group （20% normal rat serum+80% high glucose DMEM）， model group （hypoxia-reoxygenation injury） （20% normal rat serum+80% glucose-free DMEM）， medicated serum group （20% JPBSHX-medicated serum+80% glucose-free DMEM）， medicated serum+HIF-1α inhibitor group （20% JPBSHX-medicated serum+HIF-1α inhibitor 1 mg +80% glucose-free DMEM）， and medicated serum+VEGF inhibitor group （20% JPBSHX-medicated serum +VEGF inhibitor 1 mg+80% glucose-free DMEM）. The relative protein expression levels of Claudin-1 and Claudin-5 in RBMECs， the expression levels of HIF-1α and VEGF in RBMEC culture supernatants， the repair ability of RBMECs， and the number of nodes， microvessels， and their lengths after 72 h of culture were observed in each group. ResultAfter 24 h of reoxygenation， the scratch healing rate in the model group was significantly lower than in the normal group （P<0.01）. Compared with the result in the model group， the scratch healing rates significantly improved in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group （P<0.05）. However， the healing rates in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group were significantly lower than that in the medicated serum group （P<0.05）. The number of nodes， microvessels， and total length of microvessels in the model group were significantly lower than those in the normal group （P<0.01）. These indicators significantly improved in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group compared with those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group compared with those in medicated serum group （P<0.05）. The relative expression levels of Claudin-1 and Claudin-5 proteins were significantly lower in the model group than in the normal group （P<0.01）. These levels were significantly higher in medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group than those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group than those in the medicated serum group （P<0.05）. The expression levels of HIF-1α and VEGF in the RBMEC culture supernatants were significantly lower in the model group than those in the normal group （P<0.01）. These levels were significantly higher in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group than those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group than those in the medicated serum group （P<0.05）. ConclusionJPBSHX can promote the proliferation， migration， and angiogenesis， such as tubule formation， of RBMECs damaged by hypoxia-reoxygenation injury， and this effect may be achieved through the regulation of the HIF-1α/VEGF signaling pathway.

The Development and Full Validation of a Novel Liquid Chromatography Electrochemical Detection Method for Simultaneous Determination of Nine Catecholamines in Rat Brain.

Objectives: Chemical neurotransmission, managed by neurotransmitters, has a crucial role in brain processes such as fear, memory, learning, and pain, or neuropathology such as schizophrenia, epilepsy, anxiety/depression, and Parkinson's disease. The measurement of these compounds is used to elucidate the disease mechanisms and evaluate the outcomes of therapeutic interventions. However, this can be quite difficult because of various matrix effects and the problems of chromatographic separation of analysts. In the current study; for the first time, an optimized and fully validated high-performance liquid chromatography-electrochemical detection (HPLC-EC) method according to Food and Drug Administration and European Medicines Agency Bioanalytical Validation Guidance was developed for the simultaneous analysis of nine neurotransmitter compounds, namely dopamine, homovanilic acid, vanilmandelic acid, serotonin (SER), 5-hydroxyindole-3-acetic acid, 4-hydroxy-3-methoxyphenylglycol, norepinephrine, 3,4 dihydroxyphenylacetic acid, and 3-methoxytyramine and simultaneously determined in rat brain samples. Materials and Methods: Separation was achieved with 150 mm x 4.6 mm, 2.6 µm Kinetex F5 (Phenomenex, USA) column isocratically, and analysis was carried out by HPLC equipped with a DECADE II EC detector. Results: The method exhibited good selectivity, and the correlation coefficient values for each analyte's calibration curves were > 0.99. The detection and quantification limits ranged from 0.01 to 0.03 ng/mL and 3.04 to 9.13 ng/mL, respectively. The stability of the analyses and method robustness were also examined in detail in the study, and the obtained results are presented statistically. Conclusion: The developed and fully validated method has been successfully applied to actual rat brain samples, and important results have been obtained. In the rat brain sample analysis, the lowest number of SER and the highest amount of noradrenaline were found.

Automatic brain extraction for rat magnetic resonance imaging data using U2-Net.

Objective.Skull stripping is a key step in the pre-processing of rodent brain magnetic resonance images (MRI). This study aimed to develop a new skull stripping method via U2-Net, a neural network model based on deep learning method, for rat brain MRI.Approach.In this study, 599 rats were enrolled and U2-Net was applied to segment MRI images of rat brain. The intercranial tissue of each rat was manually labeled. 476 rats (approximate 80%) were used for training set while 123 rats (approximate 20%) were used to test the performance of the trained U2-Net model. For evaluation, the segmentation result by the U2-Net model is compared with the manual label, and traditional segment methods. Quantitative evaluation, including Dice coefficient, Jaccard coefficient, Sensitivity, Specificity, Pixel accuracy, Hausdorff coefficient, True positive rate, False positive rate and the volumes of whole brain, were calculated to compare the segmentation results among different models.Main results.The U2-Net model was performed better than the software of RATS and BrainSuite, in which the quantitative values of training U2-Net model were 0.9907 ± 0.0016 (Dice coefficient), 0.9816 ± 0.0032 (Jaccard coefficient), 0.9912 ± 0.0020 (Sensitivity), 0.9989 ± 0.0002 (Specificity), 0.9982 ± 0.0003 (Pixel accuracy), 5.2390 ± 2.5334 (Hausdorff coefficient), 0.9902 ± 0.0025 (True positive rate), 0.0009 ± 0.0002(False positive rate) respectively.Significance.This study provides a new method that achieves reliable performance in rat brain skull stripping of MRI images, which could contribute to the processing of rat brain MRI.

Increased Apoptosis in Subcortical Regions of The Visual Pathway in Offspring Born to Diabetic Rats.

OBJECTIVE: Diabetes in pregnancy is a prevalent disease that can affect the central nervous system of the fetus by hyperglycemia. This study aimed to investigate the impact of maternal diabetes on neuronal apoptosis in the superior colliculus (SC) and the lateral geniculate nucleus (LGN) in male neonates born to diabetic mothers. MATERIALS AND METHODS: In this experimental study, female adult rats were separated into three groups: control, diabetic (induced using an intraperitoneal injection of streptozotocin), and insulin-treated diabetic [diabetes controlled by subcutaneous neutral protamine hagedorn (NPH)-insulin injection]. Male neonates from each group were euthanized on 0, 7, and 14 postnatal days (P0, P7, and P14, respectively), and apoptotic cells were identified using TUNEL staining. RESULTS: The numerical density per unit area (NA) of apoptotic cells was significantly higher in SC and the dorsal LGN (dLGN) in neonates born to the diabetic rats compared to the control group at P0, P7, and P14. However, insulin treatment normalized the number of apoptotic cells. CONCLUSION: This study demonstrated that maternal diabetes increased apoptosis in dLGN and SC of male neonates at P0, P7, and P14.