Elsevier

Neuroscience

Volume 204, 1 March 2012, Pages 230-244
Neuroscience

Endocannabinoids, Emotional Behavior and Psychiatric Illness
Cannabinoid type 1 receptors located on single-minded 1–expressing neurons control emotional behaviors

https://doi.org/10.1016/j.neuroscience.2011.08.049Get rights and content

Abstract

This study has investigated the role of hypothalamic and amygdalar type-1 cannabinoid (CB1) receptors in the emotional and neuroendocrine responses to stress. To do so, we used the Cre/loxP system to generate conditional mutant mice lacking the CB1 gene in neurons expressing the transcription factor single-minded 1 (Sim1). This choice was dictated by former evidence for Sim1-Cre transgenic mice bearing Cre activity in all areas expressing Sim1, which chiefly includes the hypothalamus (especially the paraventricular nucleus, the supraoptic nucleus, and the posterior hypothalamus) and the mediobasal amygdala. Genomic DNA analyses in Sim1-CB1−/− mice indicated that the CB1 allele was excised from the hypothalamus and the amygdala, but not from the cortex, the striatum, the thalamus, the nucleus accumbens, the brainstem, the hippocampus, the pituitary gland, and the spinal cord. Double-fluorescent in situ hybridization experiments further indicated that Sim1-CB1−/− mice displayed a weaker CB1 receptor mRNA expression in the paraventricular nucleus of the hypothalamus and the mediobasal part of the amygdala, compared to wild-type animals. Individually housed Sim1-CB1−/− mice and their Sim1-CB1+/+ littermates were exposed to anxiety and fear memory tests under basal conditions as well as after acute/repeated social stress. A principal component analysis of the behaviors of Sim1-CB1−/− and Sim1-CB1+/+ mice in anxiety tests (open field, elevated plus-maze, and light/dark box) revealed that CB1 receptors from Sim1-expressing neurons exert tonic, albeit opposite, controls of locomotor and anxiety reactivity to novel environments. No difference between genotypes was observed during the recall of contextual fear conditioning or during active avoidance learning. Sim1-CB1−/−, but not Sim1-CB1+/+, mice proved sensitive to an acute social stress as this procedure reverted the increased ambulation in the center of the open field. The stimulatory influence of repeated social stress on body and adrenal weights, water intake, and sucrose preference was similar in the two genotypes. On the other hand, repeated social stress abolished the decrease in cued-fear conditioned expression that was observed in Sim1-CB1−/− mice, compared to Sim1-CB1+/+ mice. This study suggests that CB1 receptors located on Sim1-expressing neurons exert a tonic control on locomotor reactivity, unconditioned anxiety, and cued-fear expression under basal conditions as well as after acute or repeated stress.

This article is part of a Special Issue entitled: Stress, Emotional Behavior and the Endocannabinoid System.

Highlights

▶CB1 receptors on Sim1-expressing neurons control locomotor reactivity and anxiety. ▶Cued-fear expression is decreased in Sim-CB1−/− mutants. ▶Repeated stress reverses the decreased cued-fear memory observed in Sim-CB1−/− mutants. ▶Stress-induced adrenal hyperplasia is similar in Sim-CB1+/+ and Sim-CB1−/− mice.

Section snippets

Animals

Sim1-Cre expressing mice (The Jackson Laboratory, Bar Harbor, ME, USA) were crossed with homozygous CB1-flox mice (CB1f/f) (Marsicano et al., 2003) to obtain heterozygous Cre-expressing/CB1-flox mice (CB1Sim1-Cre;f/+, step 1). These mice were again crossed with CB1f/f to obtain homozygous CB1Sim1-Cre;f/f mice (step 2). Male mice from step 2 were finally bred with CB1f/f females to generate littermate experimental animals (CB1Sim1-Cre;f/f and CB1f/f, called thereafter Sim1-CB1−/− and Sim1-CB1+/+

Focal excision of the CB1 allele in the CNS of Sim1-CB1−/− mice

The PCR analysis of genomic DNA extracted from brain regions of wild-type and mutant mice revealed an effective deletion of the CB1 allele in the hypothalamus and the amygdala of Sim1-CB1−/− mice (Fig. 2). On the other hand, the CB1 allele was found to be present in the mutant mouse cortex, striatum, thalamus, nucleus accumbens, brainstem, hippocampus, pituitary gland, and spinal cord (Fig. 2). In keeping with the role of the PVN in the regulation of the HPA axis, we also checked whether the

Discussion

A previous study has reported that Sim1-Cre mice effectively express Cre activity in all brain regions/nuclei that express Sim1 (Balthasar et al., 2005). In turn, in situ hybridization experiments aimed at tagging areas expressing Sim1 mRNAs have shown strong expression in the hypothalamus—especially in PVN/SON—and in the nucleus of the lateral olfactory tract (Balthasar et al., 2005). Lower expression levels of Sim1 expression were observed in the MBA, in the LH and PH, in the BNST, and in the

Conclusion

This study reveals that CB1 receptors located on Sim1-expressing neurons exert a tonic inhibitory control over locomotor reactivity and a tonic stimulatory control on anxiety behavior when confronted acutely with novel environments. On the other hand, following acute and repeated stress, these receptors exert an inhibitory control over anxiety and cued-conditioned fear, respectively. In keeping with the high concentration of Sim1-expressing neurons in the PVN and the reduction of CB1 receptor

Acknowledgments

We are grateful to colleagues from the laboratory for fruitful discussions, to Delphine Gonzales and Claire Lordan and the Genotyping Platform of the NeuroCentre Magendie for mouse genotyping, to Magali Billy, Magalie Soares, Fiona Corailler, Hélène Doat, Jean-Pierre Villars, and the Animalerie Centrale of the NeuroCentre Magendie for taking care of the animals. This study was supported by grants from la Délégation Générale à l'Armement (to S.D.), EU-FP7 (Reprobesity, Health-F2-2008-22371 to

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