The mGlu2/3 agonist LY379268 reduces sucrose taking, seeking, and motivation in male and female rats.

OBJECTIVES: The mGlu2/3 receptor agonist LY379268 reduces sucrose-seeking, but not sucrose-taking, in male rats. This study explored the generality of this effect across the sexes. In addition, the effect of the drug on motivation to receive sucrose was assessed. METHODS: Adult male and female Long-Evans rats ( N = 91) were challenged with LY379268 in three experiments: (1) a fixed ratio (FR) schedule of reinforcement (taking), (2) extinction of responding previously reinforced on the FR (seeking) or (3) responding reinforced on a progressive ratio (PR) schedule of reinforcement (motivation). For each experiment, rats first responded to 10% liquid sucrose on an FR in 10 daily 2-h sessions. For the PR study, this was followed by training on a PR for 7 daily 3-h sessions. Rats were then challenged in a counterbalanced order with LY379268 (0, 1.5, 3 and 6 mg/kg; IP; 30-min pretreatment) on test days, followed by either three reacquisition days of FR (experiments 1 and 2) or PR (experiment 3) responding. RESULTS: Female rats responded more to sucrose on the FR and PR. LY379268 reduced responding in all three experiments. LY379268 challenge to sucrose taking on the FR produced an inverted U-shaped function while extinction responding and responding for sucrose on the PR were decreased dose-dependently, with PR responding insensitive to the 1.5 mg/kg dose. There were no sex-dependent effects of the drug on sucrose-directed responding. CONCLUSIONS: The sucrose anti-taking, -seeking, and -motivation effects of LY379268 across male and female rats support further evaluation of glutamate modulation as an antiaddiction pharmacotherapy.

Human Stem Cell-derived Aggregates of Forebrain Astroglia Respond to Amyloid Beta Oligomers.

Astrocytes are vital components in neuronal circuitry and there is increasing evidence linking the dysfunction of these cells to a number of central nervous system diseases. Studying the role of these cells in human brain function in the past has been difficult due to limited access to the human brain. In this study, human induced pluripotent stem cells were differentiated into astrospheres using a hybrid plating method, with or without dual SMAD inhibition. The derived cells were assessed for astrocytic markers, brain regional identity, phagocytosis, calcium-transient signaling, reactive oxygen species production, and immune response. Neural degeneration was modeled by stimulation with amyloid-ß (Aß) 42 oligomers. Finally, co-culture was performed for the derived astrospheres with isogenic neurospheres. Results indicate that the derived astroglial cells express astrocyte markers with forebrain dorsal cortical identity, secrete extracellular matrix, and are capable of phagocytosing iron oxide particles and responding to Aß42 stimulation (higher oxidative stress, higher TNF-α, and IL-6 expression). RNA-sequencing results reveal the distinct transcriptome of the derived cells responding to Aß42 stimulation for astrocyte markers, chemokines, and brain regional identity. Co-culture experiments show the synaptic activities of neurons and the enhanced neural protection ability of the astroglial cells. This study provides knowledge about the roles of brain astroglial cells, heterotypic cell-cell interactions, and the formation of engineered neuronal synapses in vitro. The implications lie in neurological disease modeling, drug screening, and studying progression of neural degeneration and the role of stem cell microenvironment. Impact Statement Human pluripotent stem cell-derived astrocytes are a powerful tool for disease modeling and drug screening. However, the properties regarding brain regional identity and the immune response to neural degeneration stimulus have not been well characterized. Results of this study indicate that the derived astroglial cells express astrocyte markers with forebrain dorsal cortical identity, secrete extracellular matrix (ECM), and are capable of phagocytosing iron oxide particles and responding to amyloid-ß oligomers, showing the distinct transcriptome in astrocyte markers, chemokines, and brain regional identity. This study provides knowledge about the roles of brain astroglial cells, heterotypic cell-cell interactions, and engineering neural tissues in vitro.

Assembly of Human Stem Cell-Derived Cortical Spheroids and Vascular Spheroids to Model 3-D Brain-like Tissues.

Human cerebral organoids derived from induced pluripotent stem cells (iPSCs) provide novel tools for recapitulating the cytoarchitecture of human brain and for studying biological mechanisms of neurological disorders. However, the heterotypic interactions of neurovascular units, composed of neurons, pericytes, astrocytes, and brain microvascular endothelial cells, in brain-like tissues are less investigated. The objective of this study is to investigate the impacts of neural spheroids and vascular spheroids interactions on the regional brain-like tissue patterning in cortical spheroids derived from human iPSCs. Hybrid neurovascular spheroids were constructed by fusion of human iPSC-derived cortical neural progenitor cell (iNPC) spheroids, endothelial cell (iEC) spheroids, and the supporting human mesenchymal stem cells (MSCs). Single hybrid spheroids were constructed at different iNPC: iEC: MSC ratios of 4:2:0, 3:2:1 2:2:2, and 1:2:3 in low-attachment 96-well plates. The incorporation of MSCs upregulated the secretion levels of cytokines VEGF-A, PGE2, and TGF-ß1 in hybrid spheroid system. In addition, tri-cultured spheroids had high levels of TBR1 (deep cortical layer VI) and Nkx2.1 (ventral cells), and matrix remodeling genes, MMP2 and MMP3, as well as Notch-1, indicating the crucial role of matrix remodeling and cell-cell communications on cortical spheroid and organoid patterning. Moreover, tri-culture system elevated blood-brain barrier gene expression (e.g., GLUT-1), CD31, and tight junction protein ZO1 expression. Treatment with AMD3100, a CXCR4 antagonist, showed the immobilization of MSCs during spheroid fusion, indicating a CXCR4-dependent manner of hMSC migration and homing. This forebrain-like model has potential applications in understanding heterotypic cell-cell interactions and novel drug screening in diseased human brain.

Cell population balance of cardiovascular spheroids derived from human induced pluripotent stem cells.

Stem cell-derived cardiomyocytes and vascular cells can be used for a variety of applications such as studying human heart development and modelling human disease in culture. In particular, protocols based on modulation of Wnt signaling were able to produce high quality of cardiomyocytes or vascular cells from human pluripotent stem cells (hPSCs). However, the mechanism behind the development of 3D cardiovascular spheroids into either vascular or cardiac cells has not been well explored. Hippo/Yes-associated protein (YAP) signaling plays important roles in the regulation of organogenesis, but its impact on cardiovascular differentiation has been less evaluated. In this study, the effects of seeding density and a change in YAP signaling on 3D cardiovascular spheroids patterning from hPSCs were evaluated. Compared to 2D culture, 3D cardiovascular spheroids exhibited higher levels of sarcomeric striations and higher length-to-width ratios of α-actinin+ cells. The spheroids with high seeding density exhibited more α-actinin+ cells and less nuclear YAP expression. The 3D cardiovascular spheroids were also treated with different small molecules, including Rho kinase inhibitor (Y27632), Cytochalasin D, Dasatinib, and Lysophosphatidic acid to modulate YAP localization. Nuclear YAP inhibition resulted in lower expression of active ß-catenin, vascular marker, and MRTF, the transcription factor mediated by RhoGTPases. Y27632 also promoted the gene expression of MMP-2/-3 (matrix remodeling) and Notch-1 (Notch signaling). These results should help our understanding of the underlying effects for the efficient patterning of cardiovascular spheroids after mesoderm formation from hPSCs.