Regulation of Corticotropin-Releasing Factor (CRF) Messenger Ribonucleic Acid and CRF Peptide in the Amygdala: Studies in Primary Amygdalar Cultures

Abstract

Amygdalar CRF has been implicated in the mediation of stress behaviors. The signal transduction pathways that regulate amygdalar CRF are not well understood. In this report, we have examined the effect of protein kinase A and C activators, dexamethasone, and interleukin 6 on CRF messenger RNA (mRNA) and CRF peptide expression in dissociated amygdalar cultures. The amygdala from E19 rat pups was dissected out bilaterally and dissociated in 0.25% trypsin for 10-15 min and plated. On day 17 in culture, CRF mRNA and peptide were measured following treatment with the following agents: forskolin, the phorbol ester-phorbol 12 myristate 13-acetate (TPA), dexamethasone, and interleukin-6 (IL6). Both forskolin and IL6, but not TPA, increased CRF mRNA in a time- and dose-dependent manner. Secretion and intracellular content of the CRF peptide also increased with both forskolin and IL6 treatment but not with TPA. Dexamethasone treatment did not alter the expression of CRF message or peptide. Transfection of the primary cultures with a rat CRF promoter-luciferase reporter construct followed by treatment with all four agents produced alterations in luciferase expression that were consistent with changes observed at the level of CRF mRNA and peptide. The results suggest that CRF regulation in the amygdala differs from that known to occur in the hypothalamus, and that elevation of IL6 levels within the central nervous system may directly act to stimulate CRF production and secretion from limbic structures such as the amygdala, to promote subsequent behavioral changes.

Coordinate and Divergent Regulation of Corticotropin-Releasing Factor (CRF) and CRF-Binding Protein Expression in an Immortalized Amygdalar Neuronal Cell Line**Portions of this report were presented in abstract form at the 1997 and 1998 annual meetings of The Endocrine Society.

“…We conclude, therefore, that the immortalized cells are refractory to the effects of dex to inhibit CRF production, both in basal and stimulated states. This is consistent with our report that dex is ineffective at altering basal CRF production in primary amygdalar cultures (18), although both observations contrast with reports for hypothalamic CRF systems, where dex inhibits both basal and stimulated CRF production (19,20,29). This differential effect of glucocorticoids on CRF regulation in various brain regions may be caused by the differential expression of glucocorticoid receptors in the various experimental systems; this is an area requiring experimental investigation in our cells.…”

“…Cells were grown in 8-well Lab-Tek II Chamber Slide Systems (Nalge Nunc International, Naperville, IL) and fixed, using 4% paraformaldehyde (pH 7.4), as described previously (18). Cells were incubated overnight at room temperature in a 1:2000 dilution of rabbit polyclonal anti-CRF antiserum (rc70, Dr. W. Vale) in PBS containing 0.2% Triton X-100.…”

“…Using primary cultures of dispersed amygdalar neurons, we observed that, like hypothalamic CRF neurons, CRF in amygdalar neurons is positively regulated by cytokines and forskolin (18). Unlike hypothalamic CRF neurons (19,20), however, primary amygdalar CRF neurons seem to be refractory to the negative feedback effects of glucocorticoids (18). The use of primary cultures of amygdalar neurons presents several challenges.…”

Depressão, antidepressivos e sistema imune: um novo olhar sobre um velho problema

“…Paralelamente, a exposição prolongada aos hormônios glicocorticóides também seria capaz de reduzir os níveis de noradrenalina no locus coeruleus, correlacionando, dessa forma, o estresse à depressão 23 . Há uma série de evidências sobre o papel do CRH liberado pela amígdala na mediação das respostas comportamentais ao estresse, e a elevação nos níveis de Il-6 dentro do sistema nervoso central pode estimular diretamente a produção e secreção de CRH por estruturas límbicas como a amígdala, promovendo alterações comportamentais 24 . O CRH, por sua vez, ativa macrófa-gos, monócitos, micróglia e astrócitos para liberarem citocinas pró-inflamatórias 15 .…”

Geschlechtsspezifische Unterschiede der schlafendokrinen Regulation und deren Bedeutung für die Pathophysiologie der Major Depression

“…Glukokortikoide in der Amygdala gibt (Beyer et al 1988;Kasckow et al 1997;Makino et al 1994). Da die Amygdala eine besondere Bedeutung bei den Gedächtnisfunktionen für aversive Reize einnimmt Roozendaal 2000), und in der Depression eine verstärkte Aktivierung der Amygdala beschrieben wurde (Drevets 1999;Frodl et al 2002), könnte dieses Kerngebiet eine zentrale Rolle für die neuroendokrinen Veränderungen bei Patienten mit Major Depression spielen.…”