Locomotor behavior parameters in vehicle and shh pathway agonist (SAG) treated mice before and 7 days after stroke.

<p>*Difference between vehicle and SAG treated mice within pre-stroke or post-stroke condition (Student’s t-test).</p><p>HACTV (horizontal activity) = total number of beam interruptions that occurred in the horizontal sensors</p><p>TOTDIST (total distance travelled) = the total distance (cm) travelled.</p><p>MOVNO (number of movements) = the number of separate horizontal movements executed.</p><p>MOVTIME (movement time) = the amount of time (second) in ambulation.</p><p>RESTIME (rest time) = the amount of time (second) in rest.</p><p>VACTV (vertical activity) = total number of beam interruptions that occurred in the vertical sensors.</p><p>VMOVNO (vertical number of movements) = the number of separate vertical movements executed.</p><p>VTIME (vertical movement time) = the amount of time (second) in vertical activity</p><p>Locomotor behavior parameters in vehicle and shh pathway agonist (SAG) treated mice before and 7 days after stroke.</p

Nestin(+) cell-specific deletion of shh gene leads to more motor function deficits in shh iKO mice.

<p>I) strategy for nestin-expressing cell specific deletion of shh. II) in wt mice, shh expression is detected in many cells that are also nestin(+) in the SVZ (A) and in the ischemic site of the cortical stroke area (C). However, in shh iKO mice, only very few nestin positive cells express shh (panel B arrow). Similarly at the ischemic site in cortex (D), nestin (+) cells do not express shh while surrounding cells expressing shh are nestin negative. This confirms the deletion of the shh gene in both SVZ and cortical nestin-expressing cells near the ischemic site. III) Deletion of shh gene in nestin(+) cells in shh iKO mice leads to greater motor deficits in these ko mice compared to wt mice measured by total distance travelled and total movement number recorded in 24 hours at post-stroke day 7. Data normalized to pre-stroke value of each animal and presented as percentage of pre-stroke measurements. (** indicates p<0.01, * indicates p<0.05, n = 7–8, Student’s t-test). Scale bar = 50um.</p

Distal MCAo induces shh expression in the cortical ischemic site.

<p>A) and C) expression of shh and MAP2 in the contralateral side cortex. B) and D) shh expression in the ischemic site of the ipsilateral cortex. Arrow and inset showing a few shh+/MAP2+ cells. E) basal expression of shh and minimal or no expression of nestin in contralateral side of cortex and F) shh and nestin upregulation near the ischemic site in ipsilateral cortex. G and H) upregulation of GFAP in both cortex and striatum in the ipsilateral side. I and J) expression of shh and GFAP in the contralateral and ipsilateral sides of cortex at higher magnification. K/L/M) staining of nestin/GFAP/shh on the same section. N) Quantification of immunofluorescent intensity for shh, nestin and GFAP in contralateral and ipsilateral sides of the cortical area (n = 6). O, P, Q and R) colocalization analysis of shh/MAP2, shh/GFAP, shh/nestin and nesin/GFAP signals (protein on the y axis to the protein on x axis as labeled on the scatterplots. Pearson’s correlation coefficient (PCC) are marked on the bottom of the scatterplots. Scale bar = 100um. ** indicates p<0.01, Student’s t-test. Mouse brains were analyzed at 14 days after distal MCAo.</p

Using iPSC‐derived human DA neurons from opioid‐dependent subjects to study dopamine dynamics

Abstract Introduction: The dopaminergic (DA) system plays important roles in addiction. However, human DA neurons from drug‐dependent subjects were not available for study until recent development in inducible pluripotent stem cells (iPSCs) technology. Methods: In this study, we produced DA neurons differentiated using iPSCs derived from opioid‐dependent and control subjects carrying different 3′ VNTR (variable number tandem repeat) polymorphism in the human dopamine transporter (DAT or SLC6A3). In addition, the effects of valproic acid (VPA) exposures on iPSC‐derived human DA neurons are also examined. Results: We present the first evidence suggesting that the 3′ VNTR polymorphism in the hDAT gene affects DAT expression level in iPSC‐derived human DA neurons. In human DA neurons, which provide an appropriate cellular milieu, VPA treatment alters the expression of several genes important for dopaminergic neuron function including DAT, Nurr1, and TH; this might partly explain its action in regulating addictive behaviors. VPA treatment also significantly increased DA D2 receptor (Drd2) expression, especially in the opioid‐dependent iPSC cell lines. Conclusions: Our data suggest that human iPSC‐derived DA neurons may be useful in in vitro experimental model to examine the effects of genetic variation in gene regulation, to examine the underlying mechanisms in neurological disorders including drug addiction, and to serve as a platform for therapeutic development