Atlas of exercise-induced brain activation in mice.

OBJECTIVES: There is significant interest in uncovering the mechanisms through which exercise enhances cognition, memory, and mood, and lowers the risk of neurodegenerative diseases. In this study, we utilize forced treadmill running and distance-matched voluntary wheel running, coupled with light sheet 3D brain imaging and c-Fos immunohistochemistry, to generate a comprehensive atlas of exercise-induced brain activation in mice. METHODS: To investigate the effects of exercise on brain activity, we compared whole-brain activation profiles of mice subjected to treadmill running with mice subjected to distance-matched wheel running. Male mice were assigned to one of four groups: a) an acute bout of voluntary wheel running, b) confinement to a cage with a locked running wheel, c) forced treadmill running, or d) placement on an inactive treadmill. Immediately following each exercise or control intervention, blood samples were collected for plasma analysis, and brains were collected for whole-brain c-Fos quantification. RESULTS: Our dataset reveals 255 brain regions activated by acute exercise in mice, the majority of which have not previously been linked to exercise. We find a broad response of 140 regulated brain regions that are shared between voluntary wheel running and treadmill running, while 32 brain regions are uniquely regulated by wheel running and 83 brain regions uniquely regulated by treadmill running. In contrast to voluntary wheel running, forced treadmill running triggers activity in brain regions associated with stress, fear, and pain. CONCLUSIONS: Our findings demonstrate a significant overlap in neuronal activation signatures between voluntary wheel running and distance-matched forced treadmill running. However, our analysis also reveals notable differences and subtle nuances between these two widely used paradigms. The comprehensive dataset is accessible online at www.neuropedia.dk, with the aim of enabling future research directed towards unraveling the neurobiological response to exercise.

TAAR1 agonists improve glycemic control, reduce body weight and modulate neurocircuits governing energy balance and feeding.

OBJECTIVE: Metabolic Syndrome, which can be induced or exacerbated by current antipsychotic drugs (APDs), is highly prevalent in schizophrenia patients. Recent preclinical and clinical evidence suggest that agonists at trace amine-associated receptor 1 (TAAR1) have potential as a new treatment option for schizophrenia. Intriguingly, preclinical tudies have also identified TAAR1 as a novel regulator of metabolic control. Here we evaluated the effects of three TAAR1 agonists, including the clinical development candidate ulotaront, on body weight, metabolic parameters and modulation of neurocircuits implicated in homeostatic and hedonic feeding. METHODS: Effects of TAAR1 agonists (ulotaront, RO5166017 and/or RO5263397) on body weight, food intake and/or metabolic parameters were investigated in rats fed a high-fat diet (HFD) and in a mouse model of diet-induced obesity (DIO). Body weight effects were also determined in a rat and mouse model of olanzapine-, and corticosterone-induced body weight gain, respectively. Glucose tolerance was assessed in lean and diabetic db/db mice and fasting plasma glucose and insulin examined in DIO mice. Effects on gastric emptying were evaluated in lean mice and rats. Drug-induced neurocircuit modulation was evaluated in mice using whole-brain imaging of c-fos protein expression. RESULTS: TAAR1 agonists improved oral glucose tolerance by inhibiting gastric emptying. Sub-chronic administration of ulotaront in rats fed a HFD produced a dose-dependent reduction in body weight, food intake and liver triglycerides compared to vehicle controls. In addition, a more rapid reversal of olanzapine-induced weight gain and food intake was observed in HFD rats switched to ulotaront or RO5263397 treatment compared to those switched to vehicle. Chronic ulotaront administration also reduced body weight and improved glycemic control in DIO mice, and normalized corticosterone-induced body weight gain in mice. TAAR1 activation increased neuronal activity in discrete homeostatic and hedonic feeding centers located in the dorsal vagal complex and hypothalamus with concurrent activation of several limbic structures. CONCLUSION: The current data demonstrate that TAAR1 agonists, as a class, not only lack APD-induced metabolic liabilities but can reduce body weight and improve glycemic control in rodent models. The underlying mechanisms likely include TAAR1-mediated peripheral effects on glucose homeostasis and gastric emptying as well as central regulation of energy balance and food intake.

Uncovering CNS access of lipidated exendin-4 analogues by quantitative whole-brain 3D light sheet imaging.

Peptide-based drug development for CNS disorders is challenged by poor blood-brain barrier (BBB) penetrability of peptides. While acylation protractions (lipidation) have been successfully applied to increase circulating half-life of therapeutic peptides, little is known about the CNS accessibility of lipidated peptide drugs. Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful method to visualize whole-brain 3D distribution of fluorescently labelled therapeutic peptides at single-cell resolution. Here, we applied LSFM to map CNS distribution of the clinically relevant GLP-1 receptor agonist (GLP-1RA) exendin-4 (Ex4) and lipidated analogues following peripheral administration. Mice received an intravenous dose (100 nmol/kg) of IR800 fluorophore-labelled Ex4 (Ex4), Ex4 acylated with a C16-monoacid (Ex4_C16MA) or C18-diacid (Ex4_C18DA). Other mice were administered C16MA-acylated exendin 9-39 (Ex9-39_C16MA), a selective GLP-1R antagonist, serving as negative control for GLP-1R mediated agonist internalization. Two hours post-dosing, brain distribution of Ex4 and analogues was predominantly restricted to the circumventricular organs, notably area postrema and nucleus of the solitary tract. However, Ex4_C16MA and Ex9-39_C16MA also distributed to the paraventricular hypothalamic nucleus and medial habenula. Notably, Ex4_C18DA was detected in deeper-lying brain structures such as dorsomedial/ventromedial hypothalamic nuclei and the dentate gyrus. Similar CNS distribution maps of Ex4_C16MA and Ex9-39_C16MA suggest that brain access of lipidated Ex4 analogues is independent on GLP-1 receptor internalization. The cerebrovasculature was devoid of specific labelling, hence not supporting a direct role of GLP-1 RAs in BBB function. In conclusion, peptide lipidation increases CNS accessibility of Ex4. Our fully automated LSFM pipeline is suitable for mapping whole-brain distribution of fluorescently labelled drugs.

GLP-1 and nicotine combination therapy engages hypothalamic and mesolimbic pathways to reverse obesity.

Glucagon-like peptide-1 receptor (GLP-1R) agonists promote nicotine avoidance. Here, we show that the crosstalk between GLP-1 and nicotine extends beyond effects on nicotine self-administration and can be exploited pharmacologically to amplify the anti-obesity effects of both signals. Accordingly, combined treatment with nicotine and the GLP-1R agonist, liraglutide, inhibits food intake and increases energy expenditure to lower body weight in obese mice. Co-treatment with nicotine and liraglutide gives rise to neuronal activity in multiple brain regions, and we demonstrate that GLP-1R agonism increases excitability of hypothalamic proopiomelanocortin (POMC) neurons and dopaminergic neurons in the ventral tegmental area (VTA). Further, using a genetically encoded dopamine sensor, we reveal that liraglutide suppresses nicotine-induced dopamine release in the nucleus accumbens in freely behaving mice. These data support the pursuit of GLP-1R-based therapies for nicotine dependence and encourage further evaluation of combined treatment with GLP-1R agonists and nicotinic receptor agonists for weight loss.

Multimodal 3D Mouse Brain Atlas Framework with the Skull-Derived Coordinate System.

Magnetic resonance imaging (MRI) and light-sheet fluorescence microscopy (LSFM) are technologies that enable non-disruptive 3-dimensional imaging of whole mouse brains. A combination of complementary information from both modalities is desirable for studying neuroscience in general, disease progression and drug efficacy. Although both technologies rely on atlas mapping for quantitative analyses, the translation of LSFM recorded data to MRI templates has been complicated by the morphological changes inflicted by tissue clearing and the enormous size of the raw data sets. Consequently, there is an unmet need for tools that will facilitate fast and accurate translation of LSFM recorded brains to in vivo, non-distorted templates. In this study, we have developed a bidirectional multimodal atlas framework that includes brain templates based on both imaging modalities, region delineations from the Allen's Common Coordinate Framework, and a skull-derived stereotaxic coordinate system. The framework also provides algorithms for bidirectional transformation of results obtained using either MR or LSFM (iDISCO cleared) mouse brain imaging while the coordinate system enables users to easily assign in vivo coordinates across the different brain templates.

Acute changes in systemic glycemia gate access and action of GLP-1R agonist on brain structures controlling energy homeostasis.

Therapies based on glucagon-like peptide-1 (GLP-1) long-acting analogs and insulin are often used in the treatment of metabolic diseases. Both insulin and GLP-1 receptors are expressed in metabolically relevant brain regions, suggesting a cooperative action. However, the mechanisms underlying the synergistic actions of insulin and GLP-1R agonists remain elusive. In this study, we show that insulin-induced hypoglycemia enhances GLP-1R agonists entry in hypothalamic and area, leading to enhanced whole-body fat oxidation. Mechanistically, this phenomenon relies on the release of tanycyctic vascular endothelial growth factor A, which is selectively impaired after calorie-rich diet exposure. In humans, low blood glucose also correlates with enhanced blood-to-brain passage of insulin, suggesting that blood glucose gates the passage other energy-related signals in the brain. This study implies that the preventing hyperglycemia is important to harnessing the full benefit of GLP-1R agonist entry in the brain and action onto lipid mobilization and body weight loss.

Digital Brain Maps and Virtual Neuroscience: An Emerging Role for Light-Sheet Fluorescence Microscopy in Drug Development.

The mammalian brain is by far the most advanced organ to have evolved and the underlying biology is extremely complex. However, with aging populations and sedentary lifestyles, the prevalence of neurological disorders is increasing around the world. Consequently, there is a dire need for technologies that can help researchers to better understand the complexity of the brain and thereby accelerate therapies for diseases with origin in the central nervous system. One such technology is light-sheet fluorescence microscopy (LSFM) which in combination with whole organ immunolabelling has made it possible to visualize an intact mouse brain with single cell resolution. However, the price for this level of detail comes in form of enormous datasets that often challenges extraction of quantitative information. One approach for analyzing whole brain data is to align the scanned brains to a reference brain atlas. Having a fixed spatial reference provides each voxel of the sample brains with x-, y-, z-coordinates from which it is possible to obtain anatomical information on the observed fluorescence signal. An additional and important benefit of aligning light sheet data to a reference brain is that the aligned data provides a digital map of gene expression or cell counts which can be deposited in databases or shared with other scientists. This review focuses on the emerging field of virtual neuroscience using digital brain maps and discusses some of challenges incurred when registering LSFM recorded data to a standardized brain template.

Peptide-YY3-36/glucagon-like peptide-1 combination treatment of obese diabetic mice improves insulin sensitivity associated with recovered pancreatic ß-cell function and synergistic activation of discrete hypothalamic and brainstem neuronal circuitries.

OBJECTIVE: Obesity-linked type 2 diabetes (T2D) is a worldwide health concern and many novel approaches are being considered for its treatment and subsequent prevention of serious comorbidities. Co-administration of glucagon like peptide 1 (GLP-1) and peptide YY3-36 (PYY3-36) renders a synergistic decrease in energy intake in obese men. However, mechanistic details of the synergy between these peptide agonists and their effects on metabolic homeostasis remain relatively scarce. METHODS: In this study, we utilized long-acting analogues of GLP-1 and PYY3-36 (via Fc-peptide conjugation) to better characterize the synergistic pharmacological benefits of their co-administration on body weight and glycaemic regulation in obese and diabetic mouse models. Hyperinsulinemic-euglycemic clamps were used to measure weight-independent effects of Fc-PYY3-36 + Fc-GLP-1 on insulin action. Fluorescent light sheet microscopy analysis of whole brain was performed to assess activation of brain regions. RESULTS: Co-administration of long-acting Fc-IgG/peptide conjugates of Fc-GLP-1 and Fc-PYY3-36 (specific for PYY receptor-2 (Y2R)) resulted in profound weight loss, restored glucose homeostasis, and recovered endogenous ß-cell function in two mouse models of obese T2D. Hyperinsulinemic-euglycemic clamps in C57BLKS/J db/db and diet-induced obese Y2R-deficient (Y2RKO) mice indicated Y2R is required for a weight-independent improvement in peripheral insulin sensitivity and enhanced hepatic glycogenesis. Brain cFos staining demonstrated distinct temporal activation of regions of the hypothalamus and hindbrain following Fc-PYY3-36 + Fc-GLP-1R agonist administration. CONCLUSIONS: These results reveal a therapeutic approach for obesity/T2D that improved insulin sensitivity and restored endogenous ß-cell function. These data also highlight the potential association between the gut-brain axis in control of metabolic homeostasis.

An inhibitor-mediated beta-cell dedifferentiation model reveals distinct roles for FoxO1 in glucagon repression and insulin maturation.

OBJECTIVE: The loss of forkhead box protein O1 (FoxO1) signaling in response to metabolic stress contributes to the etiology of type II diabetes, causing the dedifferentiation of pancreatic beta cells to a cell type reminiscent of endocrine progenitors. Lack of methods to easily model this process in vitro, however, have hindered progress into the identification of key downstream targets and potential inhibitors. We therefore aimed to establish such an in vitro cellular dedifferentiation model and apply it to identify novel agents involved in the maintenance of beta-cell identity. METHODS: The murine beta-cell line, Min6, was used for primary experiments and high-content screening. Screens encompassed a library of small-molecule drugs representing the chemical and target space of all FDA-approved small molecules with an automated immunofluorescence readout. Validation experiments were performed in a murine alpha-cell line as well as in primary murine and human diabetic islets. Developmental effects were studied in zebrafish and C. elegans models, while diabetic db/db mouse models were used to elucidate global glucose metabolism outcomes. RESULTS: We show that short-term pharmacological FoxO1 inhibition can model beta-cell dedifferentiation by downregulating beta-cell-specific transcription factors, resulting in the aberrant expression of progenitor genes and the alpha-cell marker glucagon. From a high-content screen, we identified loperamide as a small molecule that can prevent FoxO inhibitor-induced glucagon expression and further stimulate insulin protein processing and secretion by altering calcium levels, intracellular pH, and FoxO1 localization. CONCLUSIONS: Our study provides novel models, molecular targets, and drug candidates for studying and preventing beta-cell dedifferentiation.

Whole-brain mapping of amylin-induced neuronal activity in receptor activity-modifying protein 1/3 knockout mice.

The pancreatic hormone amylin plays a central role in regulating energy homeostasis and glycaemic control by stimulating satiation and reducing food reward, making amylin receptor agonists attractive for the treatment of metabolic diseases. Amylin receptors consist of heterodimerized complexes of the calcitonin receptor and receptor-activity modifying proteins subtype 1-3 (RAMP1-3). Neuronal activation in response to amylin dosing has been well characterized, but only in selected regions expressing high levels of RAMPs. The current study identifies global brain-wide changes in response to amylin and by comparing wild type and RAMP1/3 knockout mice reveals the importance of RAMP1/3 in mediating this response. Amylin dosing resulted in neuronal activation as measured by an increase in c-Fos labelled cells in 20 brain regions, altogether making up the circuitry of neuronal appetite regulation (e.g., area postrema (AP), nucleus of the solitary tract (NTS), parabrachial nucleus (PB), and central amygdala (CEA)). c-Fos response was also detected in distinct nuclei across the brain that typically have not been linked with amylin signalling. In RAMP1/3 knockout amylin induced low-level neuronal activation in seven regions, including the AP, NTS and PB, indicating the existence of RAMP1/3-independent mechanisms of amylin response. Under basal conditions RAMP1/3 knockout mice show reduced neuronal activity in the hippocampal formation as well as reduced hippocampal volume, suggesting a role for RAMP1/3 in hippocampal physiology and maintenance. Altogether these data provide a global map of amylin response in the mouse brain and establishes the significance of RAMP1/3 receptors in relaying this response.

Effect of captopril on post-infarction remodelling visualized by light sheet microscopy and echocardiography.

Angiotensin converting enzyme inhibitors, among them captopril, improve survival following myocardial infarction (MI). The mechanisms of captopril action remain inadequately understood due to its diverse effects on multiple signalling pathways at different time periods following MI. Here we aimed to establish the role of captopril in late-stage post-MI remodelling. Left anterior descending artery (LAD) ligation or sham surgery was carried out in male C57BL/6J mice. Seven days post-surgery LAD ligated mice were allocated to daily vehicle or captopril treatment continued over four weeks. To provide comprehensive characterization of the changes in mouse heart following MI a 3D light sheet imaging method was established together with automated image analysis workflow. The combination of echocardiography and light sheet imaging enabled to assess cardiac function and the underlying morphological changes. We show that delayed captopril treatment does not affect infarct size but prevents left ventricle dilation and hypertrophy, resulting in improved ejection fraction. Quantification of lectin perfused blood vessels showed improved vascular density in the infarct border zone in captopril treated mice in comparison to vehicle dosed control mice. These results validate the applicability of combined echocardiographic and light sheet assessment of drug mode of action in preclinical cardiovascular research.

Whole-brain activation signatures of weight-lowering drugs.

OBJECTIVE: The development of effective anti-obesity therapeutics relies heavily on the ability to target specific brain homeostatic and hedonic mechanisms controlling body weight. To obtain further insight into neurocircuits recruited by anti-obesity drug treatment, the present study aimed to determine whole-brain activation signatures of six different weight-lowering drug classes. METHODS: Chow-fed C57BL/6J mice (n = 8 per group) received acute treatment with lorcaserin (7 mg/kg; i.p.), rimonabant (10 mg/kg; i.p.), bromocriptine (10 mg/kg; i.p.), sibutramine (10 mg/kg; p.o.), semaglutide (0.04 mg/kg; s.c.) or setmelanotide (4 mg/kg; s.c.). Brains were sampled two hours post-dosing and whole-brain neuronal activation patterns were analysed at single-cell resolution using c-Fos immunohistochemistry and automated quantitative three-dimensional (3D) imaging. RESULTS: The whole-brain analysis comprised 308 atlas-defined mouse brain areas. To enable fast and efficient data mining, a web-based 3D imaging data viewer was developed. All weight-lowering drugs demonstrated brain-wide responses with notable similarities in c-Fos expression signatures. Overlapping c-Fos responses were detected in discrete homeostatic and non-homeostatic feeding centres located in the dorsal vagal complex and hypothalamus with concurrent activation of several limbic structures as well as the dopaminergic system. CONCLUSIONS: Whole-brain c-Fos expression signatures of various weight-lowering drug classes point to a discrete set of brain regions and neurocircuits which could represent key neuroanatomical targets for future anti-obesity therapeutics.

An Optimized Mouse Brain Atlas for Automated Mapping and Quantification of Neuronal Activity Using iDISCO+ and Light Sheet Fluorescence Microscopy.

In recent years, the combination of whole-brain immunolabelling, light sheet fluorescence microscopy (LSFM) and subsequent registration of data with a common reference atlas, has enabled 3D visualization and quantification of fluorescent markers or tracers in the adult mouse brain. Today, the common coordinate framework version 3 developed by the Allen's Institute of Brain Science (AIBS CCFv3), is widely used as the standard brain atlas for registration of LSFM data. However, the AIBS CCFv3 is based on histological processing and imaging modalities different from those used for LSFM imaging and consequently, the data differ in both tissue contrast and morphology. To improve the accuracy and speed by which LSFM-imaged whole-brain data can be registered and quantified, we have created an optimized digital mouse brain atlas based on immunolabelled and solvent-cleared brains. Compared to the AIBS CCFv3 atlas, our atlas resulted in faster and more accurate mapping of neuronal activity as measured by c-Fos expression, especially in the hindbrain. We further demonstrated utility of the LSFM atlas by comparing whole-brain quantitative changes in c-Fos expression following acute administration of semaglutide in lean and diet-induced obese mice. In combination with an improved algorithm for c-Fos detection, the LSFM atlas enables unbiased and computationally efficient characterization of drug effects on whole-brain neuronal activity patterns. In conclusion, we established an optimized reference atlas for more precise mapping of fluorescent markers, including c-Fos, in mouse brains processed for LSFM.

Deep learning reveals 3D atherosclerotic plaque distribution and composition.

Complications of atherosclerosis are the leading cause of morbidity and mortality worldwide. Various genetically modified mouse models are used to investigate disease trajectory with classical histology, currently the preferred methodology to elucidate plaque composition. Here, we show the strength of light-sheet fluorescence microscopy combined with deep learning image analysis for characterising and quantifying plaque burden and composition in whole aorta specimens. 3D imaging is a non-destructive method that requires minimal ex vivo handling and can be up-scaled to large sample sizes. Combined with deep learning, atherosclerotic plaque in mice can be identified without any ex vivo staining due to the autofluorescent nature of the tissue. The aorta and its branches can subsequently be segmented to determine how anatomical position affects plaque composition and progression. Here, we find the highest plaque accumulation in the aortic arch and brachiocephalic artery. Simultaneously, aortas can be stained for markers of interest (for example the pan immune cell marker CD45) and quantified. In ApoE-/- mice we observe that levels of CD45 reach a plateau after which increases in plaque volume no longer correlate to immune cell infiltration. All underlying code is made publicly available to ease adaption of the method.

3D quantification of changes in pancreatic islets in mouse models of diabetes type I and II.

Diabetes is characterized by rising levels of blood glucose and is often associated with a progressive loss of insulin-producing beta cells. Recent studies have demonstrated that it is possible to regenerate new beta cells through proliferation of existing beta cells or trans-differentiation of other cell types into beta cells, raising hope that diabetes can be cured through restoration of functional beta cell mass. Efficient quantification of beta cell mass and islet characteristics is needed to enhance drug discovery for diabetes. Here, we report a 3D quantitative imaging platform for unbiased evaluation of changes in islets in mouse models of type I and II diabetes. To determine whether the method can detect pharmacologically induced changes in beta cell volume, mice were treated for 14 days with either vehicle or the insulin receptor antagonist S961 (2.4 nmol/day) using osmotic minipumps. Mice treated with S961 displayed increased blood glucose and insulin levels. Light-sheet imaging of insulin and Ki67 (also known as Mki67)-immunostained pancreata revealed a 43% increase in beta cell volume and 21% increase in islet number. S961 treatment resulted in an increase in islets positive for the cell proliferation marker Ki67, suggesting that proliferation of existing beta cells underlies the expansion of total beta cell volume. Using light-sheet imaging of a non-obese diabetic mouse model of type I diabetes, we also characterized the infiltration of CD45 (also known as PTPRC)-labeled leukocytes in islets. At 14 weeks, 40% of the small islets, but more than 80% of large islets, showed leukocyte infiltration. These results demonstrate how quantitative light-sheet imaging can capture changes in individual islets to help pharmacological research in diabetes.

Circulating Triglycerides Gate Dopamine-Associated Behaviors through DRD2-Expressing Neurons.

Energy-dense food alters dopaminergic (DA) transmission in the mesocorticolimbic (MCL) system and can promote reward dysfunctions, compulsive feeding, and weight gain. Yet the mechanisms by which nutrients influence the MCL circuitry remain elusive. Here, we show that nutritional triglycerides (TGs), a conserved post-prandial metabolic signature among mammals, can be metabolized within the MCL system and modulate DA-associated behaviors by gating the activity of dopamine receptor subtype 2 (DRD2)-expressing neurons through a mechanism that involves the action of the lipoprotein lipase (LPL). Further, we show that in humans, post-prandial TG excursions modulate brain responses to food cues in individuals carrying a genetic risk for reduced DRD2 signaling. Collectively, these findings unveil a novel mechanism by which dietary TGs directly alter signaling in the reward circuit to regulate behavior, thereby providing a new mechanistic basis by which energy-rich diets may lead to (mal)adaptations in DA signaling that underlie reward deficit and compulsive behavior.

Automated Image Analyses of Glomerular Hypertrophy in a Mouse Model of Diabetic Nephropathy.

Background: Glomerular hypertrophy is a hallmark of kidney injury in metabolically induced renal diseases such as obesity-associated glomerulopathies and diabetic nephropathy (DN). Methods: Using light sheet fluorescent microscopy (LSFM) and 3D image analysis, we tested algorithms for automated and unbiased quantification of total glomerular numbers and individual glomerular volume in the uninephrectomized (UNx) db/db mouse model of DN. Results: At 6 weeks after surgery, db/db and UNx db/db mice showed increased urine albumin-to-creatinine ratio (ACR) compared with db/+ control mice. Before euthanasia, glomeruli were labeled in vivo by injecting tomato lectin. Whole-kidney LSFM 3D image analysis revealed that mean glomerular volume was significantly increased in UNx db/db mice compared with db/+ mice. Moreover, analysis of individual glomerular volume showed a shift in volume distribution toward larger glomeruli and thereby demonstrated additive effects of diabetes and UNx on induction of glomerular hypertrophy. The automatized quantification showed no significant differences in glomerular numbers among db/+, db/db, and UNx db/db mice. These data correlated with glomerular numbers as quantified by subsequent stereologic quantification. Conclusions: Overall, LSFM coupled with automated 3D histomorphometric analysis was demonstrated to be advantageous for unbiased assessment of glomerular volume and numbers in mouse whole-kidney samples. Furthermore, we showed that injection of fluorescently labeled lectin and albumin can be used as markers of nephron segments in the mouse kidneys, thus enabling functional assessment of kidney physiology, pathology, and pharmacology in preclinical rodent models of kidney disease.

Quantitative whole-brain 3D imaging of tyrosine hydroxylase-labeled neuron architecture in the mouse MPTP model of Parkinson's disease.

Parkinson's disease (PD) is a basal ganglia movement disorder characterized by progressive degeneration of the nigrostriatal dopaminergic system. Immunohistochemical methods have been widely used for characterization of dopaminergic neuronal injury in animal models of PD, including the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model. However, conventional immunohistochemical techniques applied to tissue sections have inherent limitations with respect to loss of 3D resolution, yielding insufficient information on the architecture of the dopaminergic system. To provide a more comprehensive and non-biased map of MPTP-induced changes in central dopaminergic pathways, we used iDISCO immunolabeling, light-sheet fluorescence microscopy (LSFM) and deep-learning computational methods for whole-brain three-dimensional visualization and automated quantitation of tyrosine hydroxylase (TH)-positive neurons in the adult mouse brain. Mice terminated 7 days after acute MPTP administration demonstrated widespread alterations in TH expression. Compared to vehicle controls, MPTP-dosed mice showed a significant loss of TH-positive neurons in the substantia nigra pars compacta and ventral tegmental area. Also, MPTP dosing reduced overall TH signal intensity in basal ganglia nuclei, i.e. the substantia nigra, caudate-putamen, globus pallidus and subthalamic nucleus. In contrast, increased TH signal intensity was predominantly observed in limbic regions, including several subdivisions of the amygdala and hypothalamus. In conclusion, mouse whole-brain 3D imaging is ideal for unbiased automated counting and densitometric analysis of TH-positive cells. The LSFM-deep learning pipeline tracked brain-wide changes in catecholaminergic pathways in the MPTP mouse model of PD, and may be applied for preclinical characterization of compounds targeting dopaminergic neurotransmission.

Integrated Brain Atlas for Unbiased Mapping of Nervous System Effects Following Liraglutide Treatment.

Light Sheet Fluorescence Microscopy (LSFM) of whole organs, in particular the brain, offers a plethora of biological data imaged in 3D. This technique is however often hindered by cumbersome non-automated analysis methods. Here we describe an approach to fully automate the analysis by integrating with data from the Allen Institute of Brain Science (AIBS), to provide precise assessment of the distribution and action of peptide-based pharmaceuticals in the brain. To illustrate this approach, we examined the acute central nervous system effects of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide. Peripherally administered liraglutide accessed the hypothalamus and brainstem, and led to activation in several brain regions of which most were intersected by projections from neurons in the lateral parabrachial nucleus. Collectively, we provide a rapid and unbiased analytical framework for LSFM data which enables quantification and exploration based on data from AIBS to support basic and translational discovery.