skf 81297 hydrobromide  (Tocris)

Journal: iScience

Article Title: Modulation of striatal cAMP levels: A key pathway in the treatment of hyperkinetic movement disorders

doi: 10.1016/j.isci.2025.114457

Figure Lengend Snippet: Mutant AC5 has a reduced expression level but an increased specific activity in response to Golf-mediated stimulation (A) AC5 protein levels, quantified by immunoblot in striatal homogenates, are decreased in Adcy5 R419W/R419W . GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is used as housekeeping protein. (B) cAMP production in the presence of various concentrations of D1R ( SKF81297 ) or A 2A R ( CGS21680 ) agonists or AC5 activator Forskolin in fresh striatal homogenates containing neuronal membranes. cAMP production was quantified by an HTRF cAMP assay, normalized to total protein levels, expressed as percentage of unstimulated basal cAMP production in Adcy5 +/+ striata, and normalized to relative AC5 protein levels in Adcy5 +/+ and Adcy5 R419W/R419W to estimate AC5 activity (%). cAMP production levels suggest that mutant AC5 activity was significantly increased in response to SKF81297 or CGS21680 (D1R or A 2A R agonists) as compared to wild-type, whereas response to forskolin (AC5 activator) remained similar for mutant and wild-type AC5s. Statistics: data are presented as mean ± SEM. Student test (A) and three-parameter logistic regression (B). ∗∗∗ p < 0.001 (see for detailed statistics). Fresh striatal homogenates from Adcy5 R419W/R419W are in light blue ( n = 3–5) and from Adcy5 +/+ littermates in black ( n = 2–4).

Article Snippet: SKF81297 , Tocris , 1447/10.

Techniques: Mutagenesis, Expressing, Activity Assay, Western Blot, cAMP Assay

Journal: Brain Sciences

Article Title: Dopaminergic Inhibition of the Inwardly Rectifying Potassium Current in Direct Pathway Medium Spiny Neurons in Normal and Parkinsonian Striatum

doi: 10.3390/brainsci15090979

Figure Lengend Snippet: Inhibition of Kir occludes the motor stimulation of D1 agonism. SKF81297 (0.2 μg in 0.2 μL saline) unilateral microinjection into the DS induced contralateral rotations in 5 TH-KO mice, see . BaCl 2 (0.2 µg in 0.2 μL) unilateral microinjection into the DS induced robust contralateral rotations in 6 TH KO mice, see . This Ba effect had a fast onset and also decayed quickly, likely because Ba can diffuse away quickly. Concurrent microinjection of BaCl 2 (0.2 µg) and SKF81297 (0.2 µg in the same 0.2 µL together with BaCl 2 in 7 TH KO mice) triggered only 58 rotations, similar to the 52 rotations triggered by BaCl 2 microinjection alone. The data points at 10 min after injection are most pertinent to our question on Kir and motor stimulation and were analyzed with one-way ANOVA; data points at and after 30 min after drug injection likely involve differential diffusion of Ba and SKF81297 besides their respective pharmacological effects and thus were not analyzed statistically. The insert on the right shows our histological verification of the injection site. The chronically implanted guide cannula was 26 Ga (outer dia. 0.41 mm), and the injection needle was 33 Ga (outer dia. 0.18 mm) that was removed after injection for minimizing tissue damage; the guide cannula tip was above the intended injection site also for minimizing tissue damage. M1, primary motor cortex; S1, primary somatosensory cortex; NAc, nucleus accumbens; OT, olfactory tubercle.

Article Snippet: SKF81297 -HBr was purchased from Tocris (cat. # 1447).

Techniques: Inhibition, Saline, Microinjection, Injection, Diffusion-based Assay

Journal: Brain Sciences

Article Title: Dopaminergic Inhibition of the Inwardly Rectifying Potassium Current in Direct Pathway Medium Spiny Neurons in Normal and Parkinsonian Striatum

doi: 10.3390/brainsci15090979

Figure Lengend Snippet: Integration of our present findings into the functional synaptic circuits of the cortico-basal ganglia-thalamo-cortical loop ( A ) that control motoric and cognitive behaviors. Striatal D1-MSNs (also known as dMSNs) selectively project to the GABAergic substantia nigra pars reticulata (SNr), a key output nucleus of the basal ganglia ( A1 ). MSNs are so named because of their medium-sized cell body and numerous dendritic spines where synaptic inputs are received ( A2 ). Our present findings fill a key knowledge gap. D1Rs are highly and selectively expressed in dMSNs—these neurons project precisely to SNr. D1R agonism inhibits the tonically active inwardly rectifying K current (Kir) and thus increases D1-MSN intrinsic excitability, spike firing ( B , B1 ), and inhibitory synaptic GABA output, inhibiting GABA neurons in the SNr, disinhibiting the glutamatergic thalamus, and consequently promoting behavior ( C ); the 4 Pitx3Null male mice were treated with 10 mg/kg L-dopa and 5 mg/kg benserazide; 1 mg/kg D1 agonist SKF81297 had similar effects; similar motor stimulation was triggered in TH KO mice; no sex difference was observed). Not depicted here and not the topic of our paper, dopamine inhibits D2-MSNs, further stimulating behavior, i.e., D1 agonism and D2 agonism can stimulate behavior independently, but they normally work together synergistically . Additionally, D1R activation is established to stimulate G olf and then adenylyl cyclase (AC) and cAMP production. Our prior work using cAMP-producing Gs-GREADD also supports the mechanism depicted here . Our present work fills an important knowledge gap about the key ion channel mediating the profound DA/D1 behavior stimulation. D2R agonism also stimulates behavior (synergistically with D1R agonism , unpublished data of Zhou lab). AON, anterior olfactory nucleus; MOB, main olfactory bulb; Glu, glutamate; Hippo, hippocampus; IC, inferior colliculus; SC, superior colliculus; NAc, nucleus accumbens; ORB, orbital cortical area; OT, olfactory tubercle; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; ZI, zona incerta.

Article Snippet: SKF81297 -HBr was purchased from Tocris (cat. # 1447).

Techniques: Functional Assay, Control, Activation Assay