Diagram representing the mechanisms assumed to be involved in the (endo) cannabinoid effects on olfactory circuits inmice which have fasted. Credit: UPV/EHU
图示了被认为与大麻素对禁食小鼠嗅觉回路的影响有关的机制。来源:UPV / EHU
In a state of hunger or fasting, the increase in food intake that takes place after smelling food is linked to a type 1 cannabinoid receptor in the olfactory bulb, according to a study co-led by a UPV/EHU researcher and published today in the journal Nature Neuroscience.The work also shows that these receptors could also be a pharmacological target for the treatment of eating disorders, like those which lead to obesity or anorexia.
It is normal for the internal state of the organism to affect sensory perception, and therefore, to cause certain behaviour.One of the most well-known examples in these processes is the capacity of hunger to increase the sense of smell, and the search for and intake of food.However, until now the brain mechanism governing the connection between hunger, the sense of smell and food intake was unknown. A study conducted by an international group of researchers, including some from the UPV/EHU's department of Neurosciences, have unraveled the keys in this connection.
Before this study was carried out, the cannabinoid system was known to be linked to these phenomena."The level of endogenous cannabinoids in mammal brains is known to be increased by abstaining from food, or fasting, and the cannabinoid system is known to be an important component in regulating energy balance," explained PrdroGrandes, a researcher in the UPV/EHU's department of Neurosciences, and co-leader of the research together with Giovanni Marsicano, a researcher at the University of Bordeaux.
Endogenous cannabinoids are lipids produced on demand as a result of neuronal activity—in other words, in situations in which the system is specifically activated. The researchers who have conducted this study on mice have discovered the type of endogenous cannabinoid involved in these processes, the place where it acts, and the effect that is unleashed. Grandes explains it thus: "In situations of hunger, anandamide, which is a specific type of endogenous cannabinoid, is synthesised and acts on a specific receptor, CB1.These receptors are located at certain nerve endings in the olfactory bulb, whose function is to regulate excitatory synaptic transmission. When cannabinoids act on these CB1 receptors in the bulb, a reduction in the excitatory communication coming from olfactory areas in the cerebral cortex takes place, which ends in the innermost layer of the olfactory bulb. As a result, all the intrinsic functions that are on the level of the olfactory bulb are enhanced. So, for example, the cells that pick up smell transmit better, and therefore, the perception of smell is greater."
Supplementary Figure 1. Expression of CB₁ receptor mRNA in olfactory areas.(a) Representativecoronal pictures of fluorescent in situ hybridization (FISH) of CB1 receptor mRNA expression (red)in the MOB.(b-f) Detailed analysis of CB, receptor mRNA in different layers of the MOB. In wild-type mice (n=3), CB1 mRNA (green) is co-expressed with mRNA coding for tyrosine hydroxylase (TH, red, b) and GAD 65 (red, c) in the glomerular layer (GL, white arrows), but very sparse co-localization with GAD 65 was found in the granular cell layer (GCL, white arrows, d). No co-expression with the vesicular glutamate transporter 1(VGluT1) was detected in any layer (e,f).Note that CB₁ mRNA expression is not changed in Glu-CBi- mice (n=3) and it is absent in CBr-KO mice(n=3).
补充图1嗅觉区CB1受体mRNA的表达。(a) CB1受体mRNA在MOB中表达的荧光原位杂交(FISH)的代表性冠状图(红色)。(b-f) CB1受体mRNA在MOB不同层的详细分析。在野生型小鼠(n=3)中，CB1 mRNA(绿色)在肾小球层(GL，白色箭头)与酪氨酸羟化酶(TH，红色，b)和GAD 65(红色，c)编码的mRNA共表达，但在颗粒细胞层(GCL，白色箭头，d)与GAD 65的共定位非常稀疏。在任何层均未检测到与泡状谷氨酸转运蛋白1(VGluT1)共表达(e,f)。值得注意的是，在Glu-CBi-小鼠(n=3)中，CB₁mRNA的表达没有变化，而在CBr-KO小鼠(n=3)中则不存在。
Experiments with mutant mice
突变小鼠实验
This study has been conducted over a period of four years, so the discoveries have been gradually forthcoming. Initially, the researchers determined the distribution of the CB1 receptor in the brain. In this step, they were able to relate the location of the CB1 receptors in the excitatory endings.
这项研究已经进行了四年多，所以发现是逐渐出现的。最初，研究人员确定了CB1受体在大脑中的分布。在这一步中，他们能够将CB1受体在兴奋性末梢中的位置联系起来。
Then they characterised the function of the CB1 receptor in the mechanisms described: during a state of hunger; in sense of smell perception; and in food intake."We saw that the CB1 receptor is necessary in these mechanisms, since if it was pharmacologically blocked, or if it was eliminated genetically (by producing mice lacking it), the mice ate less when they were in a state of hunger," says Grandes.
然后，他们描述了CB1受体在以下机制中的功能：在饥饿状态下；嗅觉：在嗅觉感知方面；还有食物摄入。“我们发现CB1受体在这些机制中是必要的，因为如果它被药理学阻断，或者如果它被基因消除(通过产生缺乏它的老鼠)，老鼠在饥饿状态下吃得更少，”格兰德斯说。
Supplementary Figure 5.(a,b) Expression of the CB1 receptor protein in the AON (a) and thehippocampus (b) of wild-type (WT, n=3), Stop-CB₁(n=3),CB₁-RS(n=3)and Glu-CB₁-RS mice (n=3).Note the absence of CB₁ receptor protein in Stop-CB, mice and its complete rescue in global CB₁-RSmice. According to the low levels of CB₁ receptors on cortical glutamatergic neurons, Glu-CB₁-RSmice display only slightly above-background staining. The presence of abundant CB₁ receptor proteinin the inner molecular layer of the dentate gyrus (b) and in the GCL/MOB (compare with Figure 4a ofmain text) confirms the presence of abundant receptors at terminals of hippocampal mossy cells,and at terminals of centrifugal feedback projections of olfactory cortical areas.(c) Percentage ofincrease in food intake of CB₁-RS, Glu-CB₁-RS and AON-CB₁-RS mice as compared to respective Stop-CB₁ mice.
图5 (a,b)野生型(WT, n=3)、Stop-CB₁(n=3)、CB₁-RS(n=3)和Glu-CB₁-RS小鼠(n=3)的AON (a)和海马(b)中CB1受体蛋白的表达。请注意，在Stop-CB小鼠中缺乏CB₁受体蛋白，并且在全球CB₁-RSmice中完全恢复。根据皮质谷氨酸神经元上CB₁受体的低水平，Glu-CB₁-RSmice仅显示略高于背景的染色。在齿状回的内分子层(b)和GCL/MOB中(与主要文本的图4a相比)存在丰富的CB₁受体蛋白，证实了在海马苔藓状细胞的末端和嗅觉皮质区离心反馈投影的末端存在丰富的受体。(c)与各自的停止CB₁小鼠相比，CB₁-RS, Glu-CB₁-RS和AON-CB₁-RS小鼠的食物摄入量增加百分比。
Later on, they corroborated this function of the CB1 receptor in mice that did not have this receptor, but which had been made to express it "by means of various gene manipulations," explains the researcher. "In these experiments the mice were found to eat more again when they displayed CB1 receptors in the neuronal pathway described, which projects from the cerebral cortex to the olfactory bulb." At the same time, by means of in vivo studies, the role of the CB1 receptor was confirmed in the mechanism that leads to increased food intake in the mouse.
后来，他们在没有这种受体的老鼠身上证实了CB1受体的这种功能，但这些老鼠“通过各种基因操作”来表达它，研究人员解释说。“在这些实验中，当小鼠在描述的从大脑皮层到嗅球的神经元通路中显示CB1受体时，它们会再次吃得更多。”同时，通过体内研究，证实了CB1受体在小鼠食物摄入增加机制中的作用。
Supplementary Figure 6. Activation of centrifugal glutamatergic transmission to the GCL/MOB by the Ga-DREADD approach. (a) Representative coronal pictures of the anterior olfactory nucleus (AON)and the main olfactory bulb(MOB) from mice injected in the AON with rAAV CaMK-DREAAD-mCherry. Due to the expression of DREADD-mCherry exclusively at somatic level, the fluorescentsignal is detected only in the AON and not in the MOB, where infected neurons project (comparewith Figure 5c of main text).(b) Phospo-CREB immunohistochemistry in mice injected with rAAVCaMK-DREAAD in the AON and injected with saline (veh) or 1 mg/kg of CNO 30 minutes beforesacrifice. Note the activation of both MOB and AON (dotted lines) following DREADD stimulation withCNO.(c) Food intake in control mice injected with rAAV CaMK-mCherry in the AON (AON-mCherry)after administration of saline (VEH) or 1mg/kg CNO.
补充图6 通过Ga-DREADD方法激活GCL/MOB的离心谷氨酸能传递。(a)小鼠前嗅核注射rAAV camk - dread - mcherry后，前嗅核(AON)和主嗅球(MOB)的代表性冠状图。(b)在AON中注射rAAVCaMK-DREAAD的小鼠，在献祭前30分钟注射生理盐水(veh)或1 mg/kg CNO的小鼠的Phospo-CREB免疫组化。注意CNO刺激DREADD后MOB和AON的激活(虚线)。(c)在给予生理盐水(VEH)或1mg/kg CNO后，在AON (AON- mcherry)注射rAAV CaMK-mCherry的对照小鼠的食物摄入量。
Finally, the researchers were able to observe that these phenomena are not unleashed by the endogenous cannabinoids alone—in other words, those produced by the organism itself. "One type of exogenous cannabinoid, THC, which is the psychoactive component of cannabis, also led to the increase in the perception of the sense of smell and appetite when injected into these mice. Of course this effect always takes place in fasting conditions; in the cases in which the mice had eaten their fill, this mechanism did not kick in," says the UPV/EHU researcher.
Translating this study to the clinical sphere could lead to the production of new drugs that could be useful for treating certain eating disorders. "In people with anorexia, we could stimulate intake by enhancing these mechanisms. And by contrast, in cases of obesity, the aim would be to try and reduce the function of these CB1 receptors to reduce the perception of smell and thus get these individuals to eat less when they are hungry."
Supplementary Figure 8.(a) Food intake and AUC of odor detection threshold values afterdifferent doses of THC or (b) URB597. Note that positive correlations between food intakeand olfactory detection were found only with the hyperphagic doses of THC(1mg/kg) andURB597(10mg/kg).
图8 (a)不同剂量四氢大麻酚后的食物摄入量和气味检测阈值的AUC或(b) URB597。注意，食物摄入量与嗅觉检测之间的正相关关系仅与四氢大麻酚(1mg/kg)和urb597 (10mg/kg)的过量剂量有关。
More information: E. Soria-Gómez, L. Bellocchio, L. Reguero, G. Lepousez, C. Martin, M. Bendahmane, S. Ruehle, F. Remmers, T. Desprez, I. Matias, T. Wiesner, A. Cannich, A. Nissant, A. Wadleigh, H. C. Pape, A. P. Chiarlone, C. Quarta, D. Verrier, P. Vincent, F. Massa, B. Lutz, M. Guzmán, H. Gurden, G. Ferreira, P. M Lledo, P. Grandes, G. Marsicano. 2014. "The endocannabinoid system controls food intake via olfactory processes".Nature Neuroscience.DOI: 10.1038/nn.3647
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