Intranasal application of the neuropeptide alarin leads to c-fos activation

Background Neuropeptide alarin is a 25 amino acid peptide derived by alternative splicing of the GALP gene. It regulates several physiological functions; feeding behavior, energy and glucose homeostasis, body temperature and reproduction. Accumulating evidence indicates that alarin is involved in onset of obesity. For instance, alarin alters acute food intake in lean rats and mice upon intracerebroventricular injection. Here, we investigated the effects of intranasal administration of alarin as a minimal invasive route of peptide delivery. Our aim was to elucidate if the blood-brain barrier can be bypassed and to determine the effects on activation of the immediate early gene c-fos in various brain regions. Furthermore, we investigated whether long-term treatment is safe without major side effects.
Methods Lean mice were treated with a single intranasal dose of 10, 20 and 30 nmol alarin in 10 µl 5% alpha cyclodextrine or vehicle and results were compared to untreated naïve counterparts (short-term model). Two hours post-injection, transcardial perfusion with paraformaldehyde was performed, brains were excised, post-fixed, cryoprotected in 30% sucrose and sagitally sliced. Immunofluorescence staining was performed on free-floating tissue sections using an anti-c-fos antibody as indicator of neuronal activation. Images were acquired using the Olympus slide scanner VS120. To examine whether long-term application of alarin is safe and whether it affects food intake and body weight in lean and obese mice, mice were treated over 13 weeks with either a 60% fat diet to induce obesity or a 10% fat control diet. After 2 weeks of habituation, 10 nmol alarin or vehicle was intranasally applied on a daily basis for 4 consecutive weeks. Changes in behavior, appearance, social interaction of mice and alterations in body weight, composition of adipose tissue and other toxic effects were monitored.
Results Here we demonstrate that intranasally applied alarin can bypass the blood-brain as short-term application of alarin resulted in dose-dependent c-fos activation in various brain regions, particularly the hypothalamus, the central control of feeding and energy homeostasis. Low-dose intranasal alarin (10 nmol) did not alter body weight, appearance or social behavior of mice neither in the short- nor in the long-term model. Moreover, in the long-term model body weight and adipose tissue composition was unaltered in obese and normal weight mice compared to vehicle-treated mice.
Discussion Short- and long-term intranasal application of alarin was proven to be safe without major side effects on appearance and behavior of mice. Intranasal application of alarin represents a minimal invasive route of application and was found to bypass the blood-brain barrier as shown by c-fos activation of various brain regions. Further studies will be performed to explore the modes of action of intranasally applied alarin.