Radioligand Binding Assays

To determine H₁R density at E12 (three litters per experiment) and E14 (one litter per experiment), a total membrane preparation for five independent experiments per group were obtained combining the telencephalon and mesencephalon. The tissue was homogenized in a hypotonic solution (10 mM Tris–HCI, 1 mM EGTA, pH 7.4) and centrifuged (20,000 ×g at 4°C for 20 min). The resulting pellet (membranes) was re-suspended in 450 μl incubation buffer (50 mM Tris–HCI, pH 7.4) and sonicated (3×, 5 s). Binding assays were performed as previously in detail elsewhere (Soria-Jasso et al., 1997). Briefly, membrane aliquots were incubated for 60 min at 30°C in 100 μl incubation buffer containing a nearly saturating concentration (10 nM) of the selective H₁R antagonist [³H]-mepyramine (PerkinElmer, Waltham, MA, United States). Specific binding was defined as that insensitive to 10 μM unlabelled mepyramine and was normalized to the amount of protein per sample determined by the BCA method (E12 ∼2 μg; E14 ∼22 μg).

The density of dopamine D₂ receptors in striatal nerve terminals was evaluated in a synaptosomal membrane preparation obtained as described in detail elsewhere (Márquez-Gómez et al., 2018) from a pool of P21 males or females (four pups per determination for a total of three experiments per group). Synaptosomes were lysed in a hypotonic solution (10 mM Tris–HCI, 1 mM EGTA, pH 7.4), the suspension was centrifuged (20,000 ×g, 4°C, 20 min) and the pellet (membranes) was re-suspended in 0.5 ml incubation buffer (50 mM Tris–HCI, 10 mM KCl, 4 mM MgCl₂, 1 mM EDTA, 100 μM ketanserin, pH 7.4). Aliquots were incubated with 2 nM [³H]-spiperone (PerkinElmer) in a final 100 μl volume at 25°C for 90 min. Non-specific binding was defined in the presence of 100 μM (±)-butaclamol (Sigma-Aldrich).

The incubation was stopped by filtration through Whatman GF/B glass microfiber filters (GE Healthcare Life Science, Marlborough, MA, United States), pre-soaked for 2 h in 0.3% polyethylenimine and the radioactivity retained in the filters was determined by scintillation counting. Data were normalized to the amount of protein per sample (∼72 μg).