Vt 1200 semi automatic vibrating blade microtome

To further understand the neuron distribution and development of DA, GABA, and glutamate neurons following prenatal nicotine exposure in each sub-region of the VTA, samples were taken 7 (P7), 14 (P14) and 21 (P21) days after birth and were compared with saline groups of the same age. On the appropriate postnatal day 7 (nicotine: n = 7; saline: n = 7), day 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. Brains were removed rapidly and sectioned using a VT1200 semiautomatic vibrating blade microtome (Leica, Nussloch, Eisfeld, Germany) at a speed of 0.5 mm/s. Horizontal brain slices of the VTA were obtained by cutting from the ventral side of the brain (1500 µm deep) for ages P7, P14, and P21 before the VTA sub-region samples were collected. For the P7, P14, and P21 groups, 300 µm thick slices were collected for each of the PN, PIF, and PBP sub-regions of the VTA. Two 1 mm biopsy punches (Integra Miltex, VWR, Radnor, PA, USA) were used to collect VTA brain punches bilaterally. Tissue samples were kept fresh in RNAlater (Invitrogen, Thermo Fisher Scientific, USA). Brain slices from each sub-region was taken and samples were collected. Their relative gene expression was analyzed using the primers for qPCR listed in Table 1.

14 animals were randomly pooled together from different pregnant rats (n = 3). They were aged approximately 4 weeks (male and female), previously exposed to nicotine (n = 7) or saline (n = 7) through the placenta and after birth through breast milk were anesthetized with isoflurane before decapitation using a guillotine. Brains were rapidly removed and sectioned on a VT1200 semiautomatic vibrating blade microtome (Leica, Nussloch, Eisfeld, Germany) into 250 μm thick horizontal slices containing the top (PBP), middle (PIF), and bottom (PN) sub-regions of the VTA. Bilateral brain punches containing the VTA were collected under negative pressure using a 1 mm biopsy punch (Integra Miltex, VWR, Radnor, PA, USA) and deposited in 500 μL of ice-cold RNAlater (Invitrogen, Thermo Fisher Scientific, USA). Separate biopsy punches were used for each sub-region to prevent cross-contamination. Total RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Next, cDNA was prepared using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific, Carlsbad, CA, USA) according to manufacturer’s instructions and reverse transcription (RT) was performed on a T100 thermal cycler (Bio-Rad, Hercules, CA, USA).