In the present study, we found that about 25% of POMC neurons are depolarized by the 5-HT2CR agonist, mCPP via activation of TRPC channels. Additionally, these data suggest that the mCPP induced activation of POMC neurons is independent of GIRK channel activity. We also compared the activation of POMC neurons by mCPP and leptin, and found that mCPP-activated and leptin-activated POMC neurons comprised distinct populations. The segregation of mCPP- and leptin-activated POMC neurons was further confirmed by the use of a transgenic mouse model to identify the acute effects of serotonin and leptin on POMC neurons that either express or do not express leptin receptors. Our results demonstrate that serotonin and leptin, key anorexigenic signals, activate distinct subpopulations of POMC neurons via activation of TRPC channels.
The arcuate nucleus of the hypothalamus is one of the most studied regions in the brain as it relates to neuronal regulation of feeding and metabolism. Arcuate POMC neurons release α-MSH that activates downstream melanocortin receptors (MC3R/MC4R) resulting in decreased food intake. Arcuate neuropeptide Y (NPY) neurons release agouti-related peptide (AgRP) which antagonizes the action of α-MSH on MC3R/MC4R and increases food intake. Thus, α-MSH and AgRP reciprocally regulate the central melanocortin pathway to modulate energy balance and glucose homeostasis. The anorexigenic effect of d-Fen is mediated by the activation of POMC neurons through 5-HT2C
R and subsequent activation of melanocortin pathway (Heisler et al., 2002
; Xu et al., 2010b
). On the other hand, 5-HT1B
R agonists hyperpolarize NPY neurons which decrease the frequency of inhibitory postsynaptic currents (IPSCs) onto POMC neurons resulting in the activation of the central melanocortin pathway by indirectly increasing α-MSH release from POMC neurons and directly decreasing AgRP release (Heisler et al., 2006
). Of note, disturbances in the regulation of food intake and insulin sensitivity found in 5-HT2C
R null mice are normalized by the re-expression of 5-HT2C
R in POMC neurons (Xu et al., 2010a
; Xu et al., 2008
). Moreover, the activation of POMC neurons by 5-HT2C
Rs underlies these observations since mCPP did not depolarize POMC neurons from 5-HT2C
R null mice; rather depolarizing POMC neurons from mice which selectively expressed 5-HT2C
R in POMC neurons (Figure S1
; Xu et al., 2010a
). Therefore the melanocortin pathway is a key mediator through which serotonin regulates metabolism.
A recent study first suggested the role of hypothalamic GIRK channels in regulating food intake and body weight (Perry et al., 2008
). In the current study, we demonstrated a major role of GIRK1 subunits in both constitutively active and GABAB
-activated GIRK channel in POMC neurons. POMC neurons in GIRK1 knockout mice showed a significant (~6 mV) depolarization of the resting membrane potential and impaired hyperpolarizing response to baclofen. The GIRK1/2 heteromultimer is the neuronal GIRK channel prototype (Luscher and Slesinger, 2010
), and this would be the first example where an electrophysiological phenotype was observed in GIRK1 knockout but not GIRK2 knockout neurons. Since GIRK1 alone cannot form a functional channel (Hedin et al., 1996
; Kennedy et al., 1996
; Kennedy et al., 1999
; Krapivinsky et al., 1995
; Ma et al., 2002
), it suggests that GIRK1 is interacting with GIRK3 or GIRK4 to form the GIRK channel in POMC neurons. Although the physiological significance of GIRK1 subunit in POMC neurons remains to be determined, given the role of GABA release from NPY neurons in energy balance and the presence of GABAergic inputs to POMC neurons from adjacent NPY neurons (Cowley et al., 2001
; Tong et al., 2008
), GIRK channels may be the postsynaptic target to mediate the observed metabolic phenotypes.
TRPC channels are a family of the larger TRP channels, and further classified into 4 subfamilies (TRPC1, TRPC4/5, TRPC3/6/7, and TRPC2) (Clapham et al., 2001
). TRPC channels are known to have numerous physiological functions (Clapham et al., 2001
; Freichel et al., 2005
), including a role of TRPC3 in motor coordination (Hartmann et al., 2008
) and TRPC5 in fear conditioning (Riccio et al., 2009
) in brain. Interestingly, recent evidence suggests an emerging role for TRPC channels in the regulation of energy homeostasis by leptin (Qiu et al., 2010
) and now serotonin. We found an involvement of PLC in 5-HT2C
R depolarization of POMC neurons. It has been shown that TRPC3/6/7 are activated by diacylglycerol (DAG) (Hofmann et al., 1999
; Trebak et al., 2003
), but it is not clear if these channels are also activated by DAG generated from PLC-protein kinase C (PKC) signaling pathway in native systems. On the other hand, TRPC4/5 has been shown to be activated by PLC and Gq protein-coupled receptors (GqPCRs) (Strubing et al., 2001
). Thus TRPC4/5 are potential molecular candidates mediating the mCPP-induced depolarization of POMC neurons. This hypothesis is supported by the single cell reverse transcription polymerase chain reaction (RT-PCR) data performed in mouse POMC neurons suggesting that the most prevalent subunit in POMC neurons was TRPC5, and this was followed by TRPC1, 4 and 7 (Qiu et al., 2010
). Moreover, the leptin-mediated inward currents were shown to be dependent on PLCγ which is a possible downstream signaling molecule of PI3K pathway. Although these data suggest potential compositions of the TRPC channel involved in the 5-HT2C
R acute activation of POMC neurons, the identity of the TRPC channel remains undefined.
The current study suggests that TRPC channels may be a common ion channel mediating the acute effects of the two potent anorexigenic signals, leptin (Qiu et al., 2010
) and serotonin. Although leptin and serotonin share a common target of cellular activation, TRPC channels, it was unclear if the acute effects of serotonin and leptin are observed in a similar subpopulation of arcuate POMC neurons. It is possible that 5-HT2C
R and leptin receptor activate different intracellular signaling pathways within the same neuron. For instance, 5-HT2C
R has been shown to activate PLC-PKC-IP3
-dependent signaling pathways while leptin receptor activates PI3K-dependent downstream pathways both resulting in activation of TRPC channels. An alternative possibility is that POMC neurons activated by 5-HT2C
R and leptin receptor are anatomically segregated in the arcuate nucleus. This possibility was recently demonstrated for the acute effects of leptin and insulin, as at least 2 functionally heterogeneous groups of arcuate POMC neurons (Williams et al., 2010
). We found in the present study that mCPP and leptin activate distinct subpopulations of POMC neurons ( and ). Our results support the model of a diversity of POMC neuronal populations suggesting that there are at least 3 functionally heterogeneous groups of POMC neurons.
Intriguingly, deletion of leptin receptors selectively in POMC neurons does not significantly alter food intake (Balthasar et al., 2005
; Hill et al., 2010
). However recent evidence suggests reactivation of 5-HT2C
R selectively in POMC neurons blunts the hyperphagia characterisctic of 5-HT2C
R null mouse (Xu et al., 2008
). Together with the current study suggesting that 5-HT2C
R and LepRs both activate POMC neurons via a TRPC conductance (Qiu et al., 2010
), these data suggest a segregation of the metabolic effects of leptin and serotonin in arcuate POMC neurons. In support of these data, we now demonstrate via the use of a novel transgenic line (PLT mice) that the acute effects of leptin and serotonin are segregated in POMC neurons.
Our results also indicate that mCPP-activated and leptin-activated POMC neuronal subpopulations may modify the activity of POMC neurons which project to different brain regions and activate melanocortin pathways of distinct functions. We previously reported a divergence of melanocortin pathways in controlling food intake and energy expenditure (Balthasar et al., 2005
). MC4Rs in paraventricular hypothalamus and amygdala were responsible for the regulation of food intake while those in other unidentified brain regions were responsible for energy expenditure. It is currently unclear which areas each subpopulation of POMC neurons projects to, but the possibility of differential projection by mCPP- or leptin-activated POMC neurons will be an exciting focus of future studies.
In conclusion, our results provide a cellular mechanism for the ability of 5-HT to activate POMC neurons. This may prove to be useful in the design of rational pharmaceutical strategies to combat obesity and diabetes. We also provided evidence that POMC neurons are activated by either 5-HT2CR or leptin receptor alone, but not by both. Our results further highlight the functional heterogeneity of POMC neurons regulating energy balance.