Fear factors

Credit: Neil Smith

The molecular events that underlie depression and anxiety are poorly understood but probably involve many factors. Corticotrophin-releasing factor (CRF) and serotonin (5-hydroxytryptamine (5-HT)) have been independently implicated in these disorders through their activation of CRF receptor 1 (CRFR1) and several 5-HT receptors, respectively. It was unknown whether these two systems interact, but now Magalhaes et al. show that CRFR1 activation increases the recruitment of 5-HT receptor 2A (5-HT_2A) to the cell surface, thereby modulating 5-HT_2A signalling.

CRFR1 and 5-HT₂ receptors are G protein-coupled receptors. Upon activation, 5-HT₂ receptors stimulate the phospholipase C signalling cascade, resulting in inositol phosphate production. The authors showed that CRF pretreatment of mouse cortical slices or cultured cells expressing both 5HT_2A and CRFR1 increased the 5-HT-induced formation of inositol phosphate. Inhibiting protein kinases downstream of either CRFR1 or 5-HT_2A activation did not change the CRF-mediated effect, indicating that it was not mediated by the signalling pathways associated with the receptors. The authors therefore examined whether CRFR1 activation directly influences 5-HT_2A receptors.

Immunofluorescence experiments showed that 5-HT_2A receptors were constitutively internalized, whereas CRFR1 only became internalized after CRF treatment. Blocking receptor internalization by expressing a dominant-negative endocytosis inhibitor eliminated the effect of CRF pretreatment on 5-HT_2A signalling. Thus, CRFR1 endocytosis is required for CRF-induced sensitization of the 5-HT_2A response to serotonin.

The authors further investigated the role of receptor trafficking in the interaction between CRF and 5-HT, and found that CRF pretreatment increased the cell surface expression of 5-HT_2A. Moreover, inhibiting receptor recycling by treating cells with monensin or by blocking fast recycling endosomes (by expressing a dominant-negative form of RAB4) respectively reduced and prevented the effect of CRF pretreatment on 5-HT_2A signalling. This suggests that CRF-induced sensitization of 5-HT_2A signalling involves receptor endocytosis and recycling, resulting in increased 5-HT_2A surface expression.

Receptor trafficking is regulated by PDZ domain-containing proteins, and both 5-HT_2A and CRFR1 contain interacting motifs for this domain. The authors showed that deleting these motifs reduced the effect of CRF pretreatment on 5-HT_2A surface expression and 5-HT_2A signalling.

The behavioural relevance of these findings was tested in mice in open field and elevated plus-maze tests for anxiety. The mice received a CRF infusion into the prefrontal cortex, followed by intraperitoneal administration of the 5-HT receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Although CRF or DOI treatment alone did not affect anxiety levels in these tests, the combined treatment increased anxiety-like behaviour. This synergistic effect was inhibited by pretreating mice intraperitoneally with a 5-HT_2A antagonist, indicating that an interaction between CRF and 5-HT_2A receptors increases anxiety-like behaviour.

Previous research had shown that CRF can stimulate the release of 5-HT. These new findings identify an additional way in which the two neurotransmitter systems interact to regulate anxiety-like behaviour, and hint at the potential of 5-HT_2A antagonists as anxiolytic therapeutics.