animal preparation
All protocols and surgical procedures were approved by the Institutional Animal Care and Use Committee (IACUC), the University of Houston Animal Care and Operation (ACO) and approved guidelines and regulations (protocol code 16-017 and date of approval: July 10, 2019), conducted according to ARRIVE guidelines. A pregnant female Sprague-Dawley rat (Charles River, Wilmington, MA, USA) was ordered to arrive on day 4 of gestation and delivered to the University of Houston Animal Care Operations. On day 7 of gestation, 72 hours after arrival, subcutaneously osmotic pumps (Alzet, Cupertino, CA, USA) containing nicotine bitartrate (Sigma-Aldrich, St. Louis, MO, USA) released at a rate of 6 mg. Embed. /kg/day or an equivalent volume of saline vehicle is administered for controls. The nicotine concentration infused into the osmotic pump is calculated based on the body weight of adult rats. The nicotine is then released via osmotic pumps and passed to puppies throughout pregnancy and through breastfeeding from day 7 of gestation to day 14 of life. . Animals are observed and weighed daily while kept in her ACO facility on a 12 h light/12 h dark schedule at a temperature of 22 ± 2 °C and 65% humidity. Access to standard food and water was provided.
Preparing slices
To further understand the neuronal distribution and development of DA, GABA, and glutamate neurons after prenatal nicotine exposure in each subregion of the VTA, postnatal day 7 (P7), 14 (P14) and 21 (P21) Samples were collected at 12:00 and compared with an age-matched saline group. appropriate postnatal day 7 (nicotine: n = 7; saline: n = 7), 14 (nicotine: n = 20; saline: n = 14), or 21 (nicotine: n = 18). , saline: n = 14) ), pups were randomly pooled and anesthetized with isoflurane before decapitation. The brain was quickly removed and he sectioned at a speed of 0.5 mm/s using a VT1200 semi-automatic vibrating blade microtome (Leica, Nussloch, Eisfeld, Germany). Horizontal brain slices of the VTA were obtained by cutting from the ventral side (1500 µm depth) of age P7, P14, and P21 brains before VTA subregion samples were collected. For the P7, P14, and P21 groups, 300-μm-thick slices were collected for each of the PN, PIF, and PBP subregions of the VTA. VTA brain punches were collected bilaterally using two 1 mm biopsy punches (Integra Miltex, VWR, Radnor, PA, USA). Tissue samples were kept fresh with RNAlater (Invitrogen, Thermo Fisher Scientific, USA). Brain slices were taken from each subregion and samples were collected. Their relative gene expression was analyzed using the qPCR primers listed in Table 1.
RNA extraction and cDNA preparation
Tissue samples were pessed with RLT buffer and beta-mercaptoethanol solution (Sigma Life Science, Darmstadt, Germany), and samples were passed through a 20-gauge syringe (Air-Tite Products Co., Inc., Virginia Beach, VA) 10 times. cDNA was then prepared using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer’s instructions and placed in a T100 thermal cycler (Bio-Rad, Hercules, CA, USA). ). Final volume of 20 µL: 25 °C for 10 minutes, 37 °C for 2 hours, 85 °C for 5 minutes. All reactions were run in triplicate using a no-template control (NTC).
qPCR
We designed qPCR studies according to MIQE guidelines. Taqman Gene Expression Assay primers, as shown in Table 1, for GAD1 (assay ID: Rn00690300_m1) and GAD2 (assay ID: Rn00561244_m1) for GABA, Slc6a3 (assay ID: Rn00562224_m1) and Th (assay ID: Rn00562500_m1) for dopamine, and Slc17a6 (assay ID: Rn00584780_m1) dopamine and glutamate are run in triplicate within each subregion sample. GAPDH (Assay ID: Rn01775763_g1) served as a reference gene and was also run in triplicate for each sample. TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, Carlsbad, CA) is used in assays involving the following parameters: 50 °C for 2 minutes, 95 °C for 2 minutes, 95 °C for 1 second. 40 cycles, 20 s at 60 °C on a StepOnePlus real-time PCR system (Applied Biosystems, Thermo Fisher Scientific, Carlsbad, CA). A comparative Ct method (ΔΔCt) was calculated using the StepOnePlus Real-Time PCR System and used to compare saline GAPDH samples to determine relative expression values. Three RT-qPCR reactions were performed for all validation experiments.
statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA), including degrees of freedom for the F-test. Statistical significance was assessed using repeated measures two-way analysis of variance (ANOVA) followed by his Tukey’s post hoc analysis. Unpaired t-tests with Welch’s correction were used when comparing gene expression profiles between nicotine- and saline-treated groups for subregions. Statistics were given as t = t-value and p = p-value. Benjamini–Hochberg False Discovery Rate (FDR) p-values were calculated to correct for multiple tests.29Genes adjusted for p < 0.05 in FDR were considered significantly differentially expressed. Sample sizes were calculated using standard power calculations, requiring an effect size of 30% at 80% power. Values are expressed as the arithmetic mean ± standard error of the mean (SEM). Throughout the manuscript, independent biological replicates are defined as experiments performed independently on material derived from different animals.
Animal research protocol
The animal research protocol was approved by the Institutional Animal Care and Use Committee (IACUC) and the University of Houston Animal Care and Operation (ACO) (protocol code 16-017 and approval date: July 10, 2019).
