ABSTRACT
The adipocyte-derived hormone leptin plays a critical role in the control of reproduction via signalling in hypothalamic neurones. The drivers of the hypothalamic-pituitary-gonadal axis, the gonadotrophin-releasing hormone (GnRH) neurones, do not have the receptors for leptin. Therefore, intermediate leptin responsive neurones must provide leptin-to-GnRH signalling. We investigated the populations of leptin responsive neurones that provide input to the rostral preoptic area (rPOA) where GnRH cell bodies reside. Fluorescent retrograde tracer beads (RetroBeads; Lumafluor Inc., Naples, FL, USA) were injected into the rPOA of transgenic leptin receptor enhanced green fluorescent protein (Lepr-eGFP) reporter mice. Uptake of the RetroBeads by Lepr-eGFP neurones was assessed throughout the hypothalamus. RetroBead uptake was most evident in the medial arcuate nucleus (ARC), the dorsomedial nucleus (DMN) and the ventral premammillary nucleus (PMV) of the hypothalamus. The uptake of RetroBeads specifically by Lepr-eGFP neurones was highest in the medial ARC (18% of tracer-labelled neurones Lepr-eGFP-positive). Because neurones that are both leptin responsive and GABAergic play a critical role in the regulation of fertility by leptin, we next focussed on the location of these populations. To address whether GABAergic neurones in leptin-responsive hypothalamic regions project to the rPOA, the experiment was repeated in GABA neurone reporter mice (Vgat-tdTomato). Between 10% and 45% of RetroBead-labelled neurones in the ARC were GABAergic, whereas uptake of tracer by GABAergic neurones in the DMN and PMV was very low (< 5%). These results show that both leptin responsive and GABAergic neurones from the ARC project to the region of the GnRH cell bodies. Our findings suggest that LEPR-expressing GABA neurones from the ARC may be mediators of leptin-to-GnRH signalling.
Subject(s)
GABAergic Neurons/drug effects , Leptin/pharmacology , Preoptic Area/drug effects , Animals , Arcuate Nucleus of Hypothalamus/drug effects , Arcuate Nucleus of Hypothalamus/metabolism , Cell Tracking/methods , Female , GABAergic Neurons/physiology , Gonadotropin-Releasing Hormone/metabolism , Hypothalamus/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Preoptic Area/cytology , Receptors, Leptin/geneticsABSTRACT
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.
