Fig 2: FuG-B lentiviral labelling of ‘classical’ dSPNs in rat and mouse. (A) Overview image of a sagittal rat brain section. GFP-positive cells are found throughout the striatum, but to lesser extent also in the cortex. Scale bar: 0.5 cm (B) Low magnification confocal image of a coronal brain section shows FuG-B-GFP-labeled cells throughout striatum. Scale bar: 500 μm. (C) Co-labeling shows that these FuG-B-GFP positive cells in the rat striatum are positive for DARPP-32, a marker of SPNs. Scale bar: 25 μm. (D) We found 98.7% of co-labelling of FuG-B-GFP and strong tdTomato fluorescence after injecting the same vector in transgenic BAC-drd1a-tdTomato mice (n = 152 cells in 6 mice). Scale bars: 25 μm.

Fig 3: AAV-mediated expression of AKAR in rat striatum and photometry recording in anesthetized and freely moving rats. (a) Representative image showing expression of AKAR and staining for the neuronal marker NeuN in a coronal section containing the dorsal striatum (i.e. caudate-putamen) of an adult rat that was sacrificed 5 weeks following stereotaxic injection of AAV-AKAR. (b) Representative high-magnification image showing fluorescent DARPP-32 immunolabelling of fixed striatal tissue, together with AKAR expression. (c) Schematic representation of the ratiometric photometry system. (d) Exemplar trace showing CFP and YFP channels, and calculated FRET ratio, recorded for 30 s from AKAR-transduced rat dorsal striatum at 100 Hz sampling rate. The lower panels show photometry recordings obtained in rats maintained either under light isoflurane anesthesia (e,f) or freely moving (g), with FRET values averaged within consecutive 30-s time bins. (e) After 15 min of recording, rats were injected s.c. (vertical dotted line) with vehicle or the D1R agonist SKF 81297, with or without 30-min pre-treatment with the D1R antagonist SCH 23390. (f) Rats were injected with vehicle or the dopamine D2R agonist quinpirole. Responses were recorded for 30 s every 2 min. (g) Photometry recording performed in freely-moving rats with either AKAR or AKARctl, the latter being a negative control biosensor lacking a phosphorylation site. Rats were recorded at 2-min intervals, and were challenged with either vehicle or SKF 81297 (5 mg/kg s.c.), 15 min after the start of recording. For all experiments, individual rats were recorded under vehicle and drug conditions on consecutive days in a counterbalanced design. (e–g) These panels show the mean ± SEM (n = 3 rats), where the value for each rat is the mean FRET over a 30 s long 100-Hz recording such as is shown in (d). Refer to “Materials and methods” for full description of data processing and representation.

Fig 4: Bcl11b and Ppp1r1b/DARPP-32 in human fibroblasts.(A) Bcl11b expression in individual fibroblast samples from five control (normal) subjects (C1–C5) and from five patients with LNS (L1–L5). ?ct values are normalized to control mRNA (GAPDH). (B) Normalized Bcl11b expression in controls (CTL) and LNS patients. (C) Ppp1r1b/DARPP-32 expression in individual fibroblast samples from five control (normal) subjects (C1–C5) and from five patients with LNS (L1–L5). ?ct values are normalized to control mRNA (GAPDH). (D) Normalized Ppp1r1b/DARPP-32 expression in controls (CTL) and LNS patients (*P<0.05, t-test).

Fig 5: Results from the experiment on medium-spiny neurons (MSNs) in the caudate-putamen (CPu). Pups were exposed to one of the following conditions: hypoxia-ischemia (HI) and diluent (HI + DIL), single treatment with adipose-derived-mesenchymal stem cells (HI + AD-MSCs×1), double treatment with AD-MSCs (HI + AD-MSCs×2) or no HI and diluent (i.e., an uninjured control condition). (A,B) Effect of delayed treatment with either AD-MSCs, or diluent, on postnatal day (PN) 14 and PN16, after perinatal hypoxia-ischemia (HI) at PN7, on (A) the average body weight from PN7 to PN21, (B) the average right cerebral hemisphere brain weight at PN21, and (C–E) the stereological parameters relevant to the measurement of all MSNs within the CPu, including (C) the total reference volume (Vref) of the CPu, (D) the corrected density (Nv) of dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32)-positive/bromodeoxyuridine (BrdU)-negative and DARPP-32-positive/BrdU-positive MSNs in the CPu, and (E) the absolute number (N) of DARPP-32-positive/BrdU-negative and DARPP-32-positive/BrdU-positive for each group. All data in C-E are from the right cerebral hemisphere at PN21. (A–E) n = 6/7 per group. In a subset of these animals (F–H, n = 2/3 per group), the effect on progenitor DARPP-32-positive/BrdU-positive MSNs was also investigated. The stereological results relevant to this measurement of MSNs are indicated for (F) the total reference volume (Vref) for the CPu, (G) corrected density (Nv) of DARPP-32-positive/BrdU-positive MSNs in the CPu and (H) the absolute number (N) of DARPP-32-positive/BrdU-positive MSNs for each group. Note that three BrdU injections were administered daily, starting at 7 days after the induction of HI, to permit tracking of the differentiation of neuroblasts. The body weight data were statistically analyzed using used repeated measures ANOVA. The brain weight and stereological data were statistically analyzed used one-way ANOVA and Holm–Šídák’s post-hoc test corrected for multiple comparisons. * p < 0.05, ** p < 0.01. ns, not statistically significant. See also the text in the Results for more details on the statistical outcomes.