2.17. Intranasal drug administration

For the intranasal dose–response studies, rats received different concentrations of H3 relaxin (2.75, 27.5, 275, or 2,750 μg·kg⁻¹), 14s21 peptide (0.015, 0.15, 1.5, or 15 mg·kg⁻¹), or sterile isotonic saline. For the highest doses (2,750 μg·kg⁻¹ H3 relaxin and 15 mg·kg⁻¹ 14s21 peptide), the peptides were dissolved in 20% DMSO and sterile saline. Dose solution aliquots for each experiment were stored at −80°C until the last day of the experiment. Intranasal delivery in SD rats was carried out manually without anaesthesia as previously described (Lukas & Neumann, 2012; Thorne, Pronk, Padmanabhan, & Frey, 2004) with slight modifications. To minimize non‐specific stress during the application procedure, each rat was handled (including adapting the animal to the supine position) by the handler for 10 min daily for at least 5 days prior the day of the experiment. Intranasal administration of vehicle was performed in the same way as drug treatment, that is, holding the animal in the supine position. In detail, the head of a conscious rat was restrained in a supine position, and two pads were placed under the dorsal neck to extend the head back towards the supporting surface. All rats were treated with relaxin‐3, 14s21 peptide, or saline/vehicle delivered over both nares alternatively using a 100‐μl pipette. An average of 50‐μl total peptide dose solution divided into 8‐ to 10‐μl drops was administered every 1–2 min by alternating between each naris, over a total of 6.5 to 7 min, until the drugs were naturally sniffed in by the rat (Migliore et al., 2014). The rat was held for an additional 30–60 s to ensure the fluid was inhaled. Rats were then returned to their home cage until behavioural testing started 25–35 min later.