Spine
volumes, densities, and turnover rates were analyzed in 3D using custom software. Because spine addition rates can vary between cultures and experimental Doxorubicin price days, spine addition rates are reported as a percent of matched controls (calculated using all spines gained in the three posttreatment time points). Absolute spine addition and loss rates for all experiments are documented in Table S1. We prepared 1,000× stocks by dissolving MG132 (A.G. Scientific) and KN-93 (Tocris) in DMSO and bicuculline (Tocris), lactacystin (EMD Biochemicals), myristoylated PKI 14–22 amide (Tocris), Rp-cAMPS (Tocris), and CPP (Sigma) in water. Vehicle controls were matched in identity and volume to that in which the inhibitor was dissolved. When two drugs were applied, the vehicle consisted of the sum of the vehicles for both drugs. Slices were imaged at 30°C in magnesium-free ACSF containing 5 mM MNI-caged-glutamate. Image stacks were acquired immediately before and after the uncaging stimulus, which consisted of 50 pulses (720 nm, ∼12 mW at the sample) of 4 ms duration delivered at 5 Hz by parking
the beam at a point ∼0.5 μm from the edge of a secondary or tertiary apical dendrite. No more than four uncaging trials were performed on the same neuron. The success rate of de novo spine outgrowth was determined by two blind evaluators. Comparison of success rate across conditions was made by Fisher’s selleck exact test. Error bars represent standard error of the mean and significance was set at p = 0.05 (two-tailed t test, unless otherwise noted). All statistics were calculated across cells. ∗p < 0.05 and ∗∗p <
0.001. We thank Judy Callis, Aldrin Gomes, and Jim Trimmer for advice and reagents; Lauren Boudewyn, Julie Heiner, and Sarah Mikula for help with experiments and analysis; and Elva Diaz, Jim Trimmer, and Georgia Woods for critical reading of the manuscript. This work was supported by a Burroughs Wellcome Career Award in the Biomedical Sciences (K.Z.), an NSF CAREER Award (0845285 K.Z. and H.V.R.), and the NIH (T32GM007377 A.M.H.; MARC-GM083894 H.V.R.; NS062736 K.Z., A.M.H., Sitaxentan and H.V.R.; NS054732 G.N.P.; AG017502 J.W.H. and I.S.S.). H.V.R. was a participant in the BUSP Program (supported by NIH-IMSD GM056765, HHMI 52005892). “
“Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG expansion in exon 1 of the huntingtin gene (Huntington’s Disease Collaborative Research Group, 1993). This mutation translates into an elongated glutamine tract in the N terminus of the huntingtin protein. Patients with HD display progressive movement dysfunction, including hyperkinetic involuntary movements, chorea, and dystonia, as well as cognitive impairments. Presently, there is no effective treatment for HD. The majority of potential therapies now under development are aimed at ameliorating symptoms of one of several proposed molecular consequences of mutant huntingtin, i.e.