Modulation of Cerebral Activity Induced by α‐casozepine, a Benzodiazepine‐like Peptide Derived from Bovine Casein

Abstract

The tryptic hydrolysate of bovine αs1‐casein (CH) displays anxiolytic properties highlighted in several animal species and in humans. Unlike benzodiazepines (BZD), the most prescribed anxiolytic drugs, CH shows neither addiction, dependence, sedation nor toxicity. The search for a carrier bioactive molecule within CH led to the α‐casozepine (α‐CZP), a decapeptide which anxiolytic properties were confirmed in rats. Its affinity for the benzodiazepine site of the GABAA receptor has helped getting the α‐CZP closer to the BZD family, despite a much lower affinity than the BZD reference diazepam. The aim of this study was to characterise the changes in the activity of the brain areas involved in the reduction of anxiety after intraperitoneal administration of α‐CZP by labelling neuronal activity in mice brain, in order to characterise its mechanism of action.Swiss mice were fed with a soy‐protein based diet containing no caseins, in order to prevent the presence or potential release of bioactive peptides from these milk proteins. Animals (8 per group) were placed in an anxiety‐producing situation (light‐dark box) 30 minutes after an intraperitoneal injection of α‐CZP (1 mg/kg), diazepam (1 mg/kg) or of the vehicle used to solubilize the molecules. The molecules were then perfused with formalin 1h30 after this stimulus. Brain expression of c‐Fos (a marker of neuronal activity) was measured by automatic counting with immunofluorescence on sagittal and coronal brains sections.The anxiolytic effects of α‐CZP on mice were confirmed using the light‐dark box (significant augmentation of the time spent in the aversive lit box). Immunofluorescence analysis showed a significant lower expression of c‐Fos in the prefrontal cortex (−60%), hippocampus (−40%), nucleus accumbens (−50%) and hypothalamus (−60%) after administration of the α‐CZP compared to the vehicle. The same profiles were observed after diazepam injection. A significant increase in the expression of c‐Fos in the amygdala (+300%), observed only after the administration of α‐CZP, indicates a different mechanism of action compared to diazepam. Results were confirmed on coronal sections.In conclusion, this study showed that an intraperitoneal administration of α‐CZP, a bioactive peptide resulting of a food protein hydrolysis, allows a modulation of neuronal activity in different brain regions involved in the regulation of anxiety and thereby can partly explain the anxiolytic activity of the peptide. A binding of α‐CZP on BZD receptors could explain the diminution of neuronal activity in different brain regions associated with the anxiolytic effects of the peptide. Moreover, a different mechanism of action of that of diazepam could also account for the absence of side effects observed with CH and this could be explained by the differences observed in the amygdala activation.