ABSTRACT
Neurones in the centrally projecting Edinger-Westphal nucleus (EWcp) are the main site of urocortin 1 (Ucn1) synthesis in the mammalian brain, and are assumed to play a role in the stress response of the animal. Because endocannabinoid signalling has also been strongly implicated in stress, we hypothesised that endocannabinoids may modulate the functioning of the urocortinergic EWcp. First, using in situ hybridisation, we demonstrated cannabinoid receptor 1 (CB1R) mRNA expression in mouse EWcp-neurones that were Ucn1-negative. Dual- and triple-label immunocytochemistry revealed the presence of CB1R in several GABA-immunopositive fibres juxtaposed to EWcp-Ucn1 neurones. To test functional aspects of such an anatomical constellation, we compared acute (1 h of restraint) and chronic (14 days of chronic mild stress) stress-induced changes in wild-type (WT) and CB1R knockout (CB1R-KO) mice. Acute and especially chronic stress resulted in an increase in Ucn1 content of the EWcp, which was attenuated in CB1R-KO mice. CB1R-KO mice had higher basal and chronic stress-induced adrenocorticotrophin and corticosterone levels and were more anxious on the elevated plus-maze versus WT. Collectively, our results show for the first time EWcp-Ucn1 neurones are putatively innervated by endocannabinoid sensitive, inhibitory, GABAergic afferents. In addition, we provide novel evidence that the absence of the CB1 receptor alters the Ucn1 mRNA and peptide levels in EWcp neurones, concomitant with an augmented stress response and increased anxiety-like behaviour.
Subject(s)
Endocannabinoids/pharmacology , Mesencephalon/drug effects , Neurons/drug effects , Stress, Psychological/pathology , Urocortins/metabolism , Acute Disease , Animals , Anxiety/etiology , Anxiety/genetics , Anxiety/metabolism , Behavior, Animal/drug effects , Behavior, Animal/physiology , Chronic Disease , Hypothalamo-Hypophyseal System/drug effects , Hypothalamo-Hypophyseal System/metabolism , Hypothalamo-Hypophyseal System/physiology , Male , Mesencephalon/metabolism , Mesencephalon/pathology , Mice , Mice, Knockout , Neurons/metabolism , Neurons/physiology , Pituitary-Adrenal System/drug effects , Pituitary-Adrenal System/metabolism , Pituitary-Adrenal System/physiology , Receptor, Cannabinoid, CB1/genetics , Receptor, Cannabinoid, CB1/metabolism , Stress, Psychological/genetics , Stress, Psychological/metabolism , Urocortins/genetics , gamma-Aminobutyric Acid/genetics , gamma-Aminobutyric Acid/metabolismABSTRACT
Housekeeping gene (HKG) mRNAs are used to normalize expression data of genes of interest in quantitative reverse transcriptase polymerase chain reaction studies. Such normalization assumes constant HKG gene expression under all circumstances. Although sporadic evidence suggests that HKG expression may not always fulfill this requirement and, therefore, such normalization may lead readily to erroneous results, this fact is generally not sufficiently appreciated by investigators. Here, we have systematically analyzed the expression of three common HKGs, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunit 18S and beta-actin, in two different stress paradigms, in various brain areas, in male and in female rats. HKG expressions differed considerably with respect to brain area, type of stressor and gender, in an HKG-specific manner. Therefore, we conclude that before final experimentation, pilot expression studies are necessary to select an HKG which expression is unaffected by the experimental factor(s), allowing reliable interpretation of expression data of genes of interest.
