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Stereotaxic surgery has been an invaluable tool in systems neuroscience, applied in many experiments for the creation of site-targeted lesions, injection of anatomical tracers or implantation of electrodes or microdialysis probes. In this protocol, we describe stereotaxic surgery optimized for gene delivery by recombinant adeno-associated viruses and lentiviruses in mice and rats. This method allows the manipulation of gene expression in the rodent brain with excellent spatiotemporal control; essentially any brain region of choice can be targeted and cells (or a subpopulation of cells) in that region can be stably genetically altered at any postnatal developmental stage up to adulthood. Many aspects of the method, its versatility, ease of application and high reproducibility, make it an attractive approach for studying genetic, cellular and circuit functions in the brain. The entire protocol can be completed in 1-2 hours.

We have developed a quantitative method of measuring regional cerebral blood flow (rCBF) by using N-isopropyl-(iodine-123)p-iodoamphetamine and single-photon emission computed tomography (SPECT). Twenty-five dynamic SPECT images (24 sec/scan) were collected immediately after tracer injection using a ring-type SPECT system and the accumulation curve (C(t)) was obtained. The time-activity curve corresponding to the arterial blood activity curve was used as B(t). The latter curve was calculated from the lung time-activity curve monitored during scanning and corrected by the actual activity obtained by one-point blood sampling 5 min after tracer injection. The octanol extraction ratio during scanning was considered to be constant and taken as the value measured 5 min after tracer injection (E). The uptake constant (K) per pixel was calculated by the least squares fitting method as the slope of the linear relationship in which C(t)/E x B(t) was plotted against E x integral of t(o)B(tau)d tau/E x B(t). Functional maps of rCBF values were obtained on a 64 x 64 matrix by calculating the uptake constant per pixel and the cross calibration factor (CF) between the SPECT system and a well counter (rCBF = K.CF x 100).