news and views
Inhaling: endocannabinoids and food intake Jaime G Maldonado-Avilés & Ralph J DiLeone
A commonly known effect of cannabinoid consumption is increased food craving and intake1. Starting with the identification of cannabinoid receptors2,3, many studies have established neurobiological mechanisms of cannabinoidinduced feeding. The cannabinoid receptor CB1 is extensively expressed in the CNS and regulates neurotransmission across brain regions that influence hunger, satiety and hedonic value of food. These known regions include the hypothalamus, nucleus accumbens and ventral tegmental area (mesolimbic system), and brainstem4. Thus, few would have expected to see a new region implicated in endocannabinoid regulation of feeding. Early work has demonstrated that removal of olfactory bulbs impairs calorie intake adjustment and meal pattern, while enhancing food intake in animals with ventromedial hypothalamic lesions5,6. These and subsequent studies began to illustrate the substantial influence that olfaction may have, not just on food perception, but on intake itself. Yet little is known regarding the specific molecular and cellular mechanisms by which olfactory bulb neurons influence feeding. In this issue of Nature Neuroscience, SoriaGómez et al.7 identify a surprising new neuronal pathway (Fig. 1) in which CB1 receptors in the main olfactory bulb influence food intake and scent detection. From its initial anatomical description, it became clear that the CB1 receptor was highly expressed in GABAergic interneurons8. It was later shown that lower levels of CB1 receptor are also found in many excitatory neurons9. Previous elegant work from Marsicano and colleagues used conditional mutants to show that deletion of CB1 receptor from glutamatergic neurons (Glu-CB1–/– mice) decreases fastinginduced food intake10. Because cannabinoid receptors modulate synaptic signaling by suppressing neurotransmitter release, reduced feeding in Glu-CB1–/– mice suggests that suppression of glutamate release by CB1 mediates this behavior. The brain region responsible for these effects remained unknown. Jaime G. Maldonado-Avilés and Ralph J. DiLeone are in the Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut, USA. e-mail: [email protected] or [email protected]
336
Soria-Gómez et al.7 analyzed CB1 expression across brain regions of the Glu-CB1–/– mice. Although the presence of cannabinoid receptors in the rodent olfactory bulb11 and their modulation of neuronal activity12 have been demonstrated, few studies have addressed the function of endocannabinoids in the main olfactory bulb (MOB), let alone any effect activation of cannabinoid receptors might have on feeding. The authors noted a clear reduction in CB1 protein in the granule cell layer (GCL) of the MOB of Glu-CB1–/– mice, particularly at putatively excitatory synapses, as determined by electron microscopy. To assess the potential role of olfactory bulb CB1 receptor on feeding, the authors first
measured levels of the endogenous endocannabinoids in the olfactory bulb after a 24-h fast. Compared with control mice provided with free access to food, fasted mice had substantially greater levels of the endocannabinoid anandamide in the MOB. These findings suggest that endocannabinoids are regulated in the olfactory bulb in metabolic states associated with changes in feeding. Furthermore, the authors showed that CB1 receptor antagonists delivered specifically to the MOB GCL reduced intake after fasting7. Thus, activation of CB1 receptors in the MOB GCL appears to influence normal feeding behavior. Given that CB1 is deleted from glutamatergic neurons in the Glu-CB1–/– mice, the observed
MOB
GCL AON/APC
Fasting
1
AEA
CB1
Glu
Glu
2 Glu
Granule cell
3 Food intake
Katie Vicari
npg
© 2014 Nature America, Inc. All rights reserved.
Feeding effects of CB1 receptors are commonly associated with exogenous cannabinoids, but a study now identifies a circuit by which endocannabinoid activation of CB1 receptors in the main olfactory bulb regulates normal food intake.
Figure 1 Metabolic state modulates feeding behaviors via cannabinoid activity in the main olfactory bulb (MOB). Soria-Gómez et al.7 show that fasting-induced feeding depends on MOB granule cell layer (GCL) CB1-mediated suppression of glutamate release by cortical neurons of the anterior olfactory nucleus or anterior piriform cortex (AON/APC). Fasting induces an increase in the endocannabinoid anandamide (AEA) in the MOB GCL (1), which activates CB1 receptors expressed on terminals from AON/APC neurons. This suppresses glutamate release (2). Lower glutamate release from AON/APC neurons underlies a subsequent increase in food intake (3). Anatomical diagrams represent coronal sections through the right hemisphere olfactory bulb (MOB) or the frontal pole (AON/APC) of an adult mouse brain.
volume 17 | number 3 | march 2014 nature neuroscience
npg
© 2014 Nature America, Inc. All rights reserved.
news and views reduction in fasting-induced feeding is likely to be a consequence of lower CB1-mediated suppression of glutamate release in the MOB GCL. The authors provided evidence consistent with this idea by infusing the NMDA-type glutamate receptor antagonist MK-801 or vehicle into the olfactory bulbs of wild-type or Glu-CB1–/– mice. Infusion of MK-801 into the olfactory bulbs of Glu-CB1–/– mice dose-dependently normalized the deficit in fasting-induced feeding, with mice infused with the highest dose exhibiting the same amount of fasting-induced feeding as wild-type mice. These findings suggest that the CB1-mediated suppression of glutamate release into the MOB GCL is an underlying mechanism of fasting-induced feeding. The authors noted that wild-type mice expressed almost no CB 1 (Cnr1) mRNA in the MOB GCL 7. What neurons, then, mediate the modulation of feeding by CB1 receptors in the MOB? MOB GCL neurons receive extensive feedback glutamatergic projections from cortical neurons located in the anterior olfactory nucleus (AON) and the anterior piriform cortex (APC)13. If CB1 receptors present in the MOB GCL suppress glutamate release from cortical AON or APC (AON/APC) neurons, and thereby increase food intake, then specific perturbations to the AON/APC-MOB pathway should influence CB 1-mediated fastinginduced feeding. Soria-Gómez et al. 7 performed several elegant experiments employing molecular, pharmacological and genetic tools to specifically address the role of cortical glutamatergic neurons in AON/ APC projecting to the MOB GCL. In the first set of experiments, the authors injected an adeno-associated virus encoding Cre recombinase (AAV-Cre) into the AON/ APC of mice with a loxP-flanked CB1 gene7. This manipulation eliminated CB1 expression exclusively from AON/APC neurons. AON/APC-specific CB1 deletion resulted in a substantial reduction of CB1 protein in the MOB GCL, consistent with the idea that AON/APC neurons are a major source of CB1 protein in the MOB GCL. Notably, the authors found that AON/APC-specific CB1 loss lowered food intake after a 24-h fast. Furthermore, they observed a strong correlation between loss of MOB CB1 immunoreactivity and increased food intake. These data argue for a model in which endocannabinoid activation of CB 1 receptors in the olfactory bulb suppresses glutamate release from AON/APC-projecting neurons. The authors further supported this idea with a conditional-rescue experiment. They generated a mutant, Stop-CB1,
by introducing a loxP-flanked stop cassette before the CB1 open reading frame7. These mice lack any CB 1 receptors. However, because the stop cassette in Stop-CB1 mice is flanked by loxP sites, expression of the CB1 receptor can be rescued by Cre recombinase. Like conventional CB 1–/– and GluCB1–/– mice, Stop-CB1 mice also exhibited a substantial decrease in fasting-induced food intake. Yet when CB 1 receptors were rescued in cortical glutamatergic neurons, the mice exhibited normal levels of fastinginduced feeding. Crucially, the authors also rescued CB1 expression exclusively in AON/ APC cortical neurons by locally infusing AAV-Cre into the AON/APC of Stop-CB1 mice. This restored levels of CB1 protein in the MOB GCL and corrected the deficit in fasting-induced feeding. Finally, the authors demonstrated that CB1 stimulation by the agonist WIN 55,212-2 in the MOB GCL suppresses excitatory postsynaptic potentials following optogenetic stimulation of AON/APC terminals. Together, these findings support the model that CB1 receptors on AON/APC terminals in the MOB GCL mediate fasting-induced feeding by suppressing glutamatergic transmission. Does the endocannabinoid signaling in the MOB also mediate the feeding effects of exogenous cannabinoids? To test this, the authors systemically administered a low dose of the CB1 receptor agonist ∆9-tetrahydrocannabinol (THC) (which substantially increases fastinginduced feeding in wild-type mice10) to mice in which the CB1 antagonist AM251 was infused into the MOB GCL. AM251 infusion into the MOB GCL blocked the effect of THC on feeding, suggesting that CB1 in the MOB GCL is needed for feeding responses to exogenous cannabinoids. Complementing this, the authors introduced Gq DREADDs (designer receptors exclusively activated by designer drugs14) into the AON region of wild-type mice to increase the activity of excitatory neurons (via the Camk2a promoter). Activation of AON/APC neurons blocked the increase in fasting-induced food intake induced by THC. Together, these findings indicate that, as with endogenous endocannabinoids, exogenous cannabinoids enhance food intake under certain metabolic conditions by activating CB1 receptors in the olfactory bulb and thereby suppressing glutamate release from cortical AON/APC neurons. Some interesting questions remain concerning the behavioral consequences of cannabinoid signaling in olfactory regions. It is not clear exactly how CB 1 activation in the MOB influences sensory perception and whether these changes converge on downstream pathways and signals known
nature neuroscience volume 17 | number 3 | march 2014
to regulate feeding, such as hypothalamic neurons. Soria-Gómez et al. 7 provide some initial clues, as they found that THC administration decreased the threshold for odor detection and impaired habituation to stimuli. However, as the authors note, although the effects on sensitivity and food intake were correlated, they could nonetheless be independent. Soria-Gómez et al.7 put forward a previously unappreciated neuronal circuit by which endocannabinoids can modulate normal feeding behavior by functioning in the olfactory bulb. Moreover, the same circuit can mediate the known feeding effects of exogenous cannabinoids. Interactions between metabolic state and olfaction have been known since early descriptions of how hunger enhances sensitivity to food odorants15. The present findings contribute to this knowledge by providing a cellular mechanism and neural circuit by which metabolic state alters responses to odors and food. The work is also likely to spur research on the role of this region in high-fat diet intake and the development of obesity. Soria-Gómez et al.’s results7 might suggest alternative strategies for targeting CB1 for therapeutic application in obesity. Finally, the study also serves to remind us that our models are never complete. In this case, given their central role as neuromodulators, it is likely that we will continue to be surprised by new substrates and mechanisms by which cannabinoids influence behavior. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Kirkham, T.C. Int. Rev. Psychiatry 21, 163–171 (2009). 2. Munro, S., Thomas, K.L. & Abu-Shaar, M. Nature 365, 61–65 (1993). 3. Matsuda, L.A., Lolait, S.J., Brownstein, M.J., Young, A.C. & Bonner, T.I. Nature 346, 561–564 (1990). 4. Bermudez-Silva, F.J., Cardinal, P. & Cota, D. J. Psychopharmacol. 26, 114–124 (2012). 5. Larue, C. & Le Magnen, J. Physiol. Behav. 5, 509–513 (1970). 6. Larue, C.G. & Le Magnen, J. Physiol. Behav. 9, 817–821 (1972). 7. Soria-Gómez, E. et al. Nat. Neurosci. 17, 407–415 (2014). 8. Katona, I. et al. J. Neurosci. 21, 9506–9518 (2001). 9. Marsicano, G. & Lutz, B. Eur. J. Neurosci. 11, 4213–4225 (1999). 10. Bellocchio, L. et al. Nat. Neurosci. 13, 281–283 (2010). 11. Herkenham, M. et al. J. Neurosci. 11, 563–583 (1991). 12. Wang, Z.-J., Sun, L. & Heinbockel, T. J. Neurosci. 32, 8475–8479 (2012). 13. Carson, K.A. Brain Res. Bull. 12, 629–634 (1984). 14. Lee, H.M., Giguere, P.M. & Roth, B.L. Drug Discov. Today published online, doi:10.1016/ j.drudis.2013.10.018 (1 November 2013). 15. Pager, J., Giachetti, I., Holley, A. & Le Magnen, J. Physiol. Behav. 9, 573–579 (1972).
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Inhaling: endocannabinoids and food intake Jaime G Maldonado-Avilés & Ralph J DiLeone
A commonly known effect of cannabinoid consumption is increased food craving and intake1. Starting with the identification of cannabinoid receptors2,3, many studies have established neurobiological mechanisms of cannabinoidinduced feeding. The cannabinoid receptor CB1 is extensively expressed in the CNS and regulates neurotransmission across brain regions that influence hunger, satiety and hedonic value of food. These known regions include the hypothalamus, nucleus accumbens and ventral tegmental area (mesolimbic system), and brainstem4. Thus, few would have expected to see a new region implicated in endocannabinoid regulation of feeding. Early work has demonstrated that removal of olfactory bulbs impairs calorie intake adjustment and meal pattern, while enhancing food intake in animals with ventromedial hypothalamic lesions5,6. These and subsequent studies began to illustrate the substantial influence that olfaction may have, not just on food perception, but on intake itself. Yet little is known regarding the specific molecular and cellular mechanisms by which olfactory bulb neurons influence feeding. In this issue of Nature Neuroscience, SoriaGómez et al.7 identify a surprising new neuronal pathway (Fig. 1) in which CB1 receptors in the main olfactory bulb influence food intake and scent detection. From its initial anatomical description, it became clear that the CB1 receptor was highly expressed in GABAergic interneurons8. It was later shown that lower levels of CB1 receptor are also found in many excitatory neurons9. Previous elegant work from Marsicano and colleagues used conditional mutants to show that deletion of CB1 receptor from glutamatergic neurons (Glu-CB1–/– mice) decreases fastinginduced food intake10. Because cannabinoid receptors modulate synaptic signaling by suppressing neurotransmitter release, reduced feeding in Glu-CB1–/– mice suggests that suppression of glutamate release by CB1 mediates this behavior. The brain region responsible for these effects remained unknown. Jaime G. Maldonado-Avilés and Ralph J. DiLeone are in the Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut, USA. e-mail: [email protected] or [email protected]
336
Soria-Gómez et al.7 analyzed CB1 expression across brain regions of the Glu-CB1–/– mice. Although the presence of cannabinoid receptors in the rodent olfactory bulb11 and their modulation of neuronal activity12 have been demonstrated, few studies have addressed the function of endocannabinoids in the main olfactory bulb (MOB), let alone any effect activation of cannabinoid receptors might have on feeding. The authors noted a clear reduction in CB1 protein in the granule cell layer (GCL) of the MOB of Glu-CB1–/– mice, particularly at putatively excitatory synapses, as determined by electron microscopy. To assess the potential role of olfactory bulb CB1 receptor on feeding, the authors first
measured levels of the endogenous endocannabinoids in the olfactory bulb after a 24-h fast. Compared with control mice provided with free access to food, fasted mice had substantially greater levels of the endocannabinoid anandamide in the MOB. These findings suggest that endocannabinoids are regulated in the olfactory bulb in metabolic states associated with changes in feeding. Furthermore, the authors showed that CB1 receptor antagonists delivered specifically to the MOB GCL reduced intake after fasting7. Thus, activation of CB1 receptors in the MOB GCL appears to influence normal feeding behavior. Given that CB1 is deleted from glutamatergic neurons in the Glu-CB1–/– mice, the observed
MOB
GCL AON/APC
Fasting
1
AEA
CB1
Glu
Glu
2 Glu
Granule cell
3 Food intake
Katie Vicari
npg
© 2014 Nature America, Inc. All rights reserved.
Feeding effects of CB1 receptors are commonly associated with exogenous cannabinoids, but a study now identifies a circuit by which endocannabinoid activation of CB1 receptors in the main olfactory bulb regulates normal food intake.
Figure 1 Metabolic state modulates feeding behaviors via cannabinoid activity in the main olfactory bulb (MOB). Soria-Gómez et al.7 show that fasting-induced feeding depends on MOB granule cell layer (GCL) CB1-mediated suppression of glutamate release by cortical neurons of the anterior olfactory nucleus or anterior piriform cortex (AON/APC). Fasting induces an increase in the endocannabinoid anandamide (AEA) in the MOB GCL (1), which activates CB1 receptors expressed on terminals from AON/APC neurons. This suppresses glutamate release (2). Lower glutamate release from AON/APC neurons underlies a subsequent increase in food intake (3). Anatomical diagrams represent coronal sections through the right hemisphere olfactory bulb (MOB) or the frontal pole (AON/APC) of an adult mouse brain.
volume 17 | number 3 | march 2014 nature neuroscience
npg
© 2014 Nature America, Inc. All rights reserved.
news and views reduction in fasting-induced feeding is likely to be a consequence of lower CB1-mediated suppression of glutamate release in the MOB GCL. The authors provided evidence consistent with this idea by infusing the NMDA-type glutamate receptor antagonist MK-801 or vehicle into the olfactory bulbs of wild-type or Glu-CB1–/– mice. Infusion of MK-801 into the olfactory bulbs of Glu-CB1–/– mice dose-dependently normalized the deficit in fasting-induced feeding, with mice infused with the highest dose exhibiting the same amount of fasting-induced feeding as wild-type mice. These findings suggest that the CB1-mediated suppression of glutamate release into the MOB GCL is an underlying mechanism of fasting-induced feeding. The authors noted that wild-type mice expressed almost no CB 1 (Cnr1) mRNA in the MOB GCL 7. What neurons, then, mediate the modulation of feeding by CB1 receptors in the MOB? MOB GCL neurons receive extensive feedback glutamatergic projections from cortical neurons located in the anterior olfactory nucleus (AON) and the anterior piriform cortex (APC)13. If CB1 receptors present in the MOB GCL suppress glutamate release from cortical AON or APC (AON/APC) neurons, and thereby increase food intake, then specific perturbations to the AON/APC-MOB pathway should influence CB 1-mediated fastinginduced feeding. Soria-Gómez et al. 7 performed several elegant experiments employing molecular, pharmacological and genetic tools to specifically address the role of cortical glutamatergic neurons in AON/ APC projecting to the MOB GCL. In the first set of experiments, the authors injected an adeno-associated virus encoding Cre recombinase (AAV-Cre) into the AON/ APC of mice with a loxP-flanked CB1 gene7. This manipulation eliminated CB1 expression exclusively from AON/APC neurons. AON/APC-specific CB1 deletion resulted in a substantial reduction of CB1 protein in the MOB GCL, consistent with the idea that AON/APC neurons are a major source of CB1 protein in the MOB GCL. Notably, the authors found that AON/APC-specific CB1 loss lowered food intake after a 24-h fast. Furthermore, they observed a strong correlation between loss of MOB CB1 immunoreactivity and increased food intake. These data argue for a model in which endocannabinoid activation of CB 1 receptors in the olfactory bulb suppresses glutamate release from AON/APC-projecting neurons. The authors further supported this idea with a conditional-rescue experiment. They generated a mutant, Stop-CB1,
by introducing a loxP-flanked stop cassette before the CB1 open reading frame7. These mice lack any CB 1 receptors. However, because the stop cassette in Stop-CB1 mice is flanked by loxP sites, expression of the CB1 receptor can be rescued by Cre recombinase. Like conventional CB 1–/– and GluCB1–/– mice, Stop-CB1 mice also exhibited a substantial decrease in fasting-induced food intake. Yet when CB 1 receptors were rescued in cortical glutamatergic neurons, the mice exhibited normal levels of fastinginduced feeding. Crucially, the authors also rescued CB1 expression exclusively in AON/ APC cortical neurons by locally infusing AAV-Cre into the AON/APC of Stop-CB1 mice. This restored levels of CB1 protein in the MOB GCL and corrected the deficit in fasting-induced feeding. Finally, the authors demonstrated that CB1 stimulation by the agonist WIN 55,212-2 in the MOB GCL suppresses excitatory postsynaptic potentials following optogenetic stimulation of AON/APC terminals. Together, these findings support the model that CB1 receptors on AON/APC terminals in the MOB GCL mediate fasting-induced feeding by suppressing glutamatergic transmission. Does the endocannabinoid signaling in the MOB also mediate the feeding effects of exogenous cannabinoids? To test this, the authors systemically administered a low dose of the CB1 receptor agonist ∆9-tetrahydrocannabinol (THC) (which substantially increases fastinginduced feeding in wild-type mice10) to mice in which the CB1 antagonist AM251 was infused into the MOB GCL. AM251 infusion into the MOB GCL blocked the effect of THC on feeding, suggesting that CB1 in the MOB GCL is needed for feeding responses to exogenous cannabinoids. Complementing this, the authors introduced Gq DREADDs (designer receptors exclusively activated by designer drugs14) into the AON region of wild-type mice to increase the activity of excitatory neurons (via the Camk2a promoter). Activation of AON/APC neurons blocked the increase in fasting-induced food intake induced by THC. Together, these findings indicate that, as with endogenous endocannabinoids, exogenous cannabinoids enhance food intake under certain metabolic conditions by activating CB1 receptors in the olfactory bulb and thereby suppressing glutamate release from cortical AON/APC neurons. Some interesting questions remain concerning the behavioral consequences of cannabinoid signaling in olfactory regions. It is not clear exactly how CB 1 activation in the MOB influences sensory perception and whether these changes converge on downstream pathways and signals known
nature neuroscience volume 17 | number 3 | march 2014
to regulate feeding, such as hypothalamic neurons. Soria-Gómez et al. 7 provide some initial clues, as they found that THC administration decreased the threshold for odor detection and impaired habituation to stimuli. However, as the authors note, although the effects on sensitivity and food intake were correlated, they could nonetheless be independent. Soria-Gómez et al.7 put forward a previously unappreciated neuronal circuit by which endocannabinoids can modulate normal feeding behavior by functioning in the olfactory bulb. Moreover, the same circuit can mediate the known feeding effects of exogenous cannabinoids. Interactions between metabolic state and olfaction have been known since early descriptions of how hunger enhances sensitivity to food odorants15. The present findings contribute to this knowledge by providing a cellular mechanism and neural circuit by which metabolic state alters responses to odors and food. The work is also likely to spur research on the role of this region in high-fat diet intake and the development of obesity. Soria-Gómez et al.’s results7 might suggest alternative strategies for targeting CB1 for therapeutic application in obesity. Finally, the study also serves to remind us that our models are never complete. In this case, given their central role as neuromodulators, it is likely that we will continue to be surprised by new substrates and mechanisms by which cannabinoids influence behavior. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Kirkham, T.C. Int. Rev. Psychiatry 21, 163–171 (2009). 2. Munro, S., Thomas, K.L. & Abu-Shaar, M. Nature 365, 61–65 (1993). 3. Matsuda, L.A., Lolait, S.J., Brownstein, M.J., Young, A.C. & Bonner, T.I. Nature 346, 561–564 (1990). 4. Bermudez-Silva, F.J., Cardinal, P. & Cota, D. J. Psychopharmacol. 26, 114–124 (2012). 5. Larue, C. & Le Magnen, J. Physiol. Behav. 5, 509–513 (1970). 6. Larue, C.G. & Le Magnen, J. Physiol. Behav. 9, 817–821 (1972). 7. Soria-Gómez, E. et al. Nat. Neurosci. 17, 407–415 (2014). 8. Katona, I. et al. J. Neurosci. 21, 9506–9518 (2001). 9. Marsicano, G. & Lutz, B. Eur. J. Neurosci. 11, 4213–4225 (1999). 10. Bellocchio, L. et al. Nat. Neurosci. 13, 281–283 (2010). 11. Herkenham, M. et al. J. Neurosci. 11, 563–583 (1991). 12. Wang, Z.-J., Sun, L. & Heinbockel, T. J. Neurosci. 32, 8475–8479 (2012). 13. Carson, K.A. Brain Res. Bull. 12, 629–634 (1984). 14. Lee, H.M., Giguere, P.M. & Roth, B.L. Drug Discov. Today published online, doi:10.1016/ j.drudis.2013.10.018 (1 November 2013). 15. Pager, J., Giachetti, I., Holley, A. & Le Magnen, J. Physiol. Behav. 9, 573–579 (1972).
337
