Golgi staining and dendritic spine counting

Golgi staining of brains was performed as per manufacturer’s instructions using FD Rapid GolgiStain Kit (FD Neurotechnologies) as previously described (Sanderson et al., 2012). Briefly, Golgi-stained pyramidal neuron apical dendrites in CA1 in stratum radiatum were imaged by transmitted light on a Zeiss Axiovert 200M microscope, using a 63x Plan-Apo/1.4 NA objective and a Coolsnap CCD camera (Photometrics) operated by Slidebook 5.0 software (Intelligent Imaging Innovations). Spines were counted from image stacks using ImageJ for 20–105 μm segments of secondary or higher-order dendrites > 40 μm from the cell body. Counts are expressed as spines/μm for three mice per genotype with 19 to 21 neurons from different sections along the rostral-caudal axis analyzed per mouse and between one and four dendritic segments counted and averaged per neuron (n = # neurons).