Computational protocol: Top-down descending facilitation of spinal sensory excitatory transmission from the anterior cingulate cortex

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Protocol publication

[…] The gene encoding GCaMP6s (addgene #40753) were subcloned into the pENTR plasmid. The GCaMP6s cassette was transferred into the AAV shuttle vector (pZac2.1, provided by the Vector Core of the University of Pennsylvania) with ESYN, an enhanced human synapsin promoter (addgene #32581), and the woodchuck post-regulatory element (WPRE) sequence (pZac2.1-ESYN-GCaMP6s-WPRE). AAV vectors were co-transfected with AAV serotype 9 helper plasmid into human embryonic kidney 293 (HEK293) cells and were purified by two rounds cesium chloride density gradient purification steps. The vector was dialyzed against phosphate-buffered saline (PBS) containing 0.001% (v/v) Pluronic-F68 using Amicon Ultra 100 K filter units (Millipore, Darmstadt, Germany). Viral titer was determined by Pico Green fluorometric reagent (Molecular Probes, OR, USA). Viral aliquots were stored at −80 °C until use. Under anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg), 500 nl of AAV (1 × 1013 viral genomic copies/ml) was delivered with a glass microcapillary into the left side of SDH between Th13 and L1 vertebrae (200 μm in depth from the dura).SDH neurons were affected with AAV9-ESYN-GCaMP6s-WPRE in one side of SDH (L4-5) in adult mice. Three to four weeks after GCaMP6s transduction with AAV, mice were deeply anesthetized by subcutaneous injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and custom made spinal cord imaging chamber was attached to vertebrae at T12-L1 as previously reported with minor modifications. The skin was incised at Th11-L2. The paravertebral muscle overlying Th12-L1 was removed and tendons are severed from the transverse processes. The laminectomy was performed at Th13, and a custom-made spinal chamber was attached on the laminectomy. Imaging window was covered with Kwik Sil elastomer, and a glass coverslip was placed to seal the window. For ACC stimulation, a craniotomy (1 mm in diameter) centered 1.5 mm anterior to the bregma and 0.3 mm lateral to the midline was made using a dental drill.Two-photon Ca2+ imaging was performed under isoflurane anesthesia by using an Olympus FV1000 with a 25 × 1.05 NA water-immersion lens (Olympus) and Spectra Physics Mai-Tai IR laser tuned at 900 nm for two-photon excitation for GCaMP6s. During two-photon imaging, mice were placed on a heating pad. Ca2+ imaging was performed on the neurons that located at a depth of 25–70 μm of one side of lumber spinal cord. The electrode for ACC stimulation was inserted through craniotomy to place electrode tip in the ACC area contralateral to the spinal imaging side (stereotaxic coordinates: 1.5 mm anterior to the bregma and 0.3 mm lateral, 1.0 mm ventral to the dura). After 10 min with confirmation of no bleeding from electrode insertion site, peripheral sensory stimuli were applied to the hindpaw using brush and subsequently forceps. An electrical stimulation delivered to the ACC was performed with rectangular pulses and lasted for 300 ms (pulse duration, 100 µs; intensity, 100 µA; frequency, 100 Hz), and after 2 min, peripheral sensory stimulus (3 s) were applied to the left hindpaw again. Obtained image data (512 × 512 pixels, 3.5–5 s/frame) were analyzed with Image J (http://imagej.nih.gov/ij/). The fluorescent signals were quantified by measuring the mean pixel intensities of the cell body of each neurons. The fluorescent change was defined as ΔF/F0 = (Ft−F0)/F0. Ft was the fluorescent intensity at time t, and F0 was the baseline intensity obtained by first value of pre-stimulus. The fluorescence intensity changed less than 20% from initial response is considered no change.The noxious and innocuous mechanical stimuli were applied to the receptive field of the hindlimb ipsilateral to the Ca2+ responses recording neurons by using toothed forceps, brush or air puffs (Pressure system IIe, Toohey Company, Fairfield, NJ, USA), respectively. The brush, air puff, and pinch stimuli were applied at least three times before and after ACC stimulation and the duration for each stimulation lasted for 5 s. To keep a fixed strength noxious stimulation, the toothed forceps was clamped during skin pinching. [...] All experiments were carried out as blind to genotype and the conditions of the experiments, unless indicated in naïve animals. Data were collected and processed randomly, and no data points were excluded. No statistical methods were used to predetermine sample sizes, but our sample sizes were similar to those reported in previous publications. Statistical comparisons were made using the unpaired, paired t-test, and one way repeated measures ANOVA (Student–Newman–Keuls test was used for post hoc comparison). The normal distribution and the variation within each group of data was verified by using Sigmaplot 11.0 software before applying statistical comparison. Analyzed numbers (n) for each set of experiments are indicated in the corresponding figure legends or main text sections. The examples shown in each figure are representative and were reproducible at least three times for each set of experiments. All data were presented as the mean ± S.E.M. In all cases, p < 0.05 was considered statistically significant. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Diseases Peripheral Nervous System Diseases, Cranial Nerve Injuries