Neuron- and Pomc-specific TrpC5 deficiency blunts the acute anorexia by LepR and Ht2Cr activation

Wildtype, TrpC5KO (Riccio et al., 2009) and Pomc-creER^T2TrpC5^lox/Y mice were fasted overnight and intraperitoneally (i.p.) injected with either saline or leptin (5 mg/kg). Consistent with previous reports (Hill et al., 2008), i.p. injections of leptin reduced food intake in wildtype mice (at 1h) compared to the saline-injected group (Figures 2A and 2B). However, i.p. injections of leptin failed to decrease food intake in TrpC5KO and Pomc-creER^T2TrpC5^lox/Y mice (at 1h) versus the saline-injected group (Figures 2A and 2B).

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Figure 2

Leptin-, mCPP-, and lorcaserin-induced hypophagia is blunted in mice deficient for TrpC5 subunits. (A) and (B) Food consumption was measured 1 hour after leptin administration (5mg/kg, i.p.) and compared with food consumption in each animal following saline administration. (C) and (D) Cumulative food intake measured at 1, 4 and/or 6 hours after administration of Ht2Cr agonists mCPP (3mg/kg) or lorcaserin (1, 3, or 6mg/kg). (E) and (F) Food intake measured in response to lorcaserin (3mg/kg, i.p.). For (A)–(F), n = 9-14 per group; *p < 0.05.

We performed a similar series of experiments with both the non-selective Ht2Cr agonist (meta-Chlorophenylpiperazine – mCPP) (3 mg/kg) and lorcaserin (1, 3, and 6 mg/kg). As reported previously, the Ht2Cr agonists significantly reduced food intake in wildtype mice versus saline-injected groups (at 1h, 4h, and/or 6h Figures 2C-2F). However, i.p. injections of mCPP or lorcaserin failed to suppress food intake (at 1h, 4h, and/or 6h) in TrpC5KO, CamkIIαTrpC5^lox/Y and/or Pomc-creER^T2TrpC5^lox/Y mice compared to saline injections. Of note 3mg/kg of lorcaserin appears to be a threshold dose for the acute biological activity of food intake. Taken together, TrpC5 subunits are required in Pomc neurons for the acute anorexia induced by leptin receptor and Ht2Cr activation.

TrpC5 subunits are required for the acute activation of Pomc neurons by leptin

Whole-cell patch-clamp recordings were made in 58 arcuate Pomc neurons from Pomc-hrGFP mice (Parton et al., 2007; Ramadori et al., 2010; Sohn et al., 2011). 26 Pomc neurons were from wildtype and, 32 were in Pomc neurons deficient for TrpC5 subunits (Riccio et al., 2009). Pomc neurons from wildtype mice (n= 26) had a resting membrane potential of −41.5 ± 1.3 mV, a mean input resistance of 1260 ± 80 MΩ, and overshooting action potentials. When compared to values obtained from wildtype Pomc neurons, Pomc neurons deficient for TrpC 5 exhibited a hyperpolarized mean resting membrane potential (−46.3± 0.9 mV; 1085 ± 53 MΩ; n=32; p<0.05; supplemental figure 1A). Analogous results were obtained in Pomc neurons from Pomc-creER^T2 vs Pomc-creER^T2TrpC5^lox/Y mice (RMP = -44.3 ± 1.0mV from wildtype Pomc neurons, n=24; and RMP = -48.0 ± 1.4mV in Pomc neurons from Pomc-creER^T2Trpc5^lox/Y mice n = 22; p<0.05; supplemental figure 1A).

Similar to previous reports (Hill et al., 2008; Sohn et al., 2011), Pomc neurons that express LepRs were targeted from Pomc-hrGFPLepR-cre-tdtomato (PLT) mice (Sohn et al., 2011; Sun et al., 2016; Williams et al., 2014) to test the acute cellular effects of leptin (Figures 3A to 3E). Leptin (100 nM) depolarized 70.6 % (12 out of 17) of wildtype Pomc neurons that express LepRs by 6.7 ± 0.5 mV (n=12, Figures 3F, 3J and supplemental table 1). We found 1 cell (5.9 %) that was hyperpolarized by -10 mV, while the remaining 4 cells (23.5 %) were not responsive to leptin (0.3 ± 0.3 mV, n=4). Analysis of current-voltage relationships revealed that leptin decreased input resistance by 21.4 ± 3.0 % (n=12, from 1.2 ± 0.1 GΩ in control ACSF to 0.9 ± 0.1 GΩ in leptin) with a reversal potential of -26.0 ± 2.2 mV (n=12) (Figures 3G and 3H). These results confirmed that leptin activates a non-selective cation conductance to depolarize Pomc neurons.

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Figure 3

Trpc5 subunits are required for the acute leptin-induced depolarization of arcuate Pomc neurons. (A) Brightfield illumination of Pomc-hrGFPLepr-cretdtomato neuron from PLT mice. (B) and (C) The same neuron under FITC (hrGFP) and Alexafluor 594 (tdtomato) illumination. (D) Complete dialysis of Alexa Fluor 350 from the intracellular pipette. (E) Merge image illustrates colocalization of hr-GFP, tdtomato, and Alexa Fluor 350 indicative of a Pomc neuron which expresses Leprs. (F) Electrophysiological study demonstrates a Pomc-hrGFPLepr-cretdtomato (green/red) neuron that is depolarized in response to leptin (100nM). (G) Traces showing decreased voltage deflection and increased action potential frequency after leptin application. (H) Current versus voltage (I-V) plot from same WT neuron illustrating a characteristic decrease in input resistance subsequent to leptin application. Shown are responses before (control) and during leptin application. (I) demonstrates a current clamp recording of a Pomc-hrGFPLepr-cretdtomatoTrpC5KO (green/red) neuron in which leptin fails to induce a depolarization. (J) Histogram summarizing the acute effect of leptin on the membrane potential of Pomc neurons which express leptin receptors as well as express or do not express Trpc5 subunits (n= 12-14 per group). (K) Rostro-caudal and medio-lateral distribution of electrophysiological responses to leptin from Pomc neurons which express leptin receptors as well as express or do not express Trpc5 subunits.

In contrast, leptin failed to depolarize any of 15 Pomc neurons which express LepRs from TrpC5 knockout (PLT5KO) mice (Figures 3I, 3J and supplemental table 2): 14 cells (93.3 %) remained unresponsive to leptin (-0.1 ± 0.5 mV), while 1 cell (6.7 %) was hyperpolarized by -9 mV. Analogous results were obtained in recordings from Pomc neurons selectively deficient for TrpC5 subunits (from Pomc-creER^T2TrpC5 mice; supplemental figure 1B). These data suggest that TrpC5 subunits underlie the leptin-induced activation of a non-selective cation conductance resulting in the depolarization of Pomc neurons. Notably, the hyperpolarizing effects of leptin remained unchanged in Pomc neurons lacking TrpC5 subunits and these effects were accompanied by decreased input resistance with a reversal potential close to E_K. Similar results were obtained in ventral premammillary nucleus (PMv) neurons which express leptin receptors (supplemental material and supplemental figure 2). Thus, the null TrpC5 gene does not affect the functional expression of leptin receptors or other channels in our model.

After recordings, the sections were fixed in formalin to determine the location of recorded cells within the arcuate nucleus, as described previously (Sohn et al., 2011). Since acute leptin responses show a distinct distribution pattern (Williams et al., 2010), we targeted cells of similar region within the arcuate nucleus in both wildtype and TrpC5 knockout mice (Figure 3K).

The acute mCPP-induced activation of Pomc neurons requires TrpC5 subunits

To test the acute cellular effects of Ht2Crs, we targeted Pomc neurons that do not express LepRs from PLT mice (Figures 4A to 4E), as previously described (Sohn et al., 2011). We confirmed that mCPP (4 μM) depolarized 55 % (5 out of 9) of wildtype Pomc neurons that do not express LepRs by 6 ± 0.6 mV (n=5, Figures 4F, 4J, and supplemental table 1). The remaining 4 cells (45 %) were not responsive to mCPP (0.4 ± 0.2 mV, n=4). Application of current steps revealed that mCPP decreases input resistance by 14.8 ± 4.8 % (n=5, from 1.5 ± 0.1 GΩ in control ACSF to 1.3 ± 0.1 GΩ in mCPP) with a reversal potential of -14 ± 4.8 mV (n=5) (Figures 4G and 4H). In contrast, mCPP failed to depolarize all Pomc neurons tested from Trpc5 knockout (PLT5KO) mice (0.1 ± 0.2 mV, n=19, Figures 4I, 4J, and supplemental table 2). Similar to the acute leptin effects in the current study, we targeted cells in similar region and responses were mapped within the arcuate nucleus (Figure 4K).

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Figure 4

Trpc5 subunits are required for the acute mCPP-induced depolarization of arcuate Pomc neurons. (A) Brightfield illumination of Pomc-hrGFP neuron from PLT mice. (B) and (C) The same neuron under FITC (hrGFP) and Alexafluor 594 (tdtomato) illumination. (D) Complete dialysis of Alexa Fluor 350 from the intracellular pipette. (E) Merge image illustrates colocalization of hr-GFP and Alexa Fluor 350 indicative of a Pomc neuron which does not express Leprs. (F) Electrophysiological study demonstrates a Pomc-hrGFP (green) neuron from PLT mice that depolarized in response to mCPP (4μM). Traces showing decreased voltage deflection and increased action potential frequency after mCPP application. (H) Current versus voltage (I-V) plot from same WT neuron illustrating a characteristic decrease in input resistance subsequent to mCPP application. Shown are responses before (control) and during mCPP application. (I) demonstrates a current clamp recording of a Pomc-hrGFPTrcp5 ^-/Y (green) neuron in which mCPP fails to induce a depolarization. (J) Histogram summarizing the acute effect of mCPP on the membrane potential of Pomc neurons which do not express leptin receptors as well as express or do not express Trpc5 subunits (n= 13-19 per group). (K) Rostro-caudal and medio-lateral distribution of electrophysiological responses to mCPP from Pomc neurons which do not express leptin receptors as well as express or do not express Trpc5 subunits.

Generation of a cre-conditional TrpC5 allele (TrpC5^fl/y)

The TrpC5 targeted clone obtained from EUCOMM was injected into blastocysts to obtain highly chimeric mice. These mice were then bred to obtain germline transmission of the targeted TrpC5 allele. The TrpC5^lox/Y mice were generated following flp-mediated removal of the neo cassette leaving two loxP sequences flanking exons 5 of the TrpC5 gene. Deletion of exon 5 creates a frameshift mutation.

Tamoxifen treatment to induce adult-onset ablation of TrpC5 in Pomc neurons

Tamoxifen (0.15mg/g; Sigma-Aldrich) dissolved in corn oil (Sigma-Aldrich) was administered i.p. for 5 consecutive days to 6-weekold male TrpC5^fl/y mice (controls) and TrpC5^fl/y × Pomc-cre:ERT2 littermate mice. Corn oil was used as a vehicle control.

Electrophysiology

Whole-cell patch-clamp recordings from Pomc-hrGFP neurons with or without leptin receptors were maintained in hypothalamic slice preparations and data analysis were performed as previously described (Hill et al., 2008). Briefly, 4- to 16-week-old male mice were anesthetized and transcardially perfused with a modified ice-cold artificial CSF (ACSF) (described below), in which an equiosmolar amount of sucrose was substituted for NaCl. The mice were then decapitated, and the entire brain was removed, and immediately submerged in ice-cold, carbogen-saturated (95% O2 and 5% CO2) ACSF (126 mM NaCl, 2.8 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 5 mM glucose). Coronal sections (250 μm) were cut with a Leica VT1000S Vibratome and then incubated in oxygenated ACSF at room temperature for at least 1 hr before recording. Slices were transferred to the recording chamber and allowed to equilibrate for 10–20 min before recording. The slices were bathed in oxygenated ACSF (32°C–34°C) at a flow rate of ~2 ml/min.

The pipette solution for whole-cell recording was modified to include an intracellular dye (Alexa Fluor 594 or Alexa Fluor 350) for whole-cell recording: 120 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 5 mM EGTA, 1 mM CaCl2, 1 mM MgCl2, and 2 mM MgATP, 0.03 mM Alexa Fluor 594 or Alexa Fluor 350 hydrazide dye (pH 7.3). Epifluorescence was briefly used to target fluorescent cells, at which time the light source was switched to infrared differential interference contrast imaging to obtain the whole-cell recording (Zeiss Axioskop FS2 Plus or Nikon FN1 equipped with a fixed stage and a QuantEM:512SC electron-multiplying charge-coupled device camera). Electrophysiological signals were recorded using an Axopatch 700B amplifier (Molecular Devices), low-pass filtered at 2–5 kHz, and analyzed offline on a PC with pCLAMP programs (Molecular Devices). Recording electrodes had resistances of 2.5–5 MΩ when filled with the K-gluconate internal solution. Input resistance was assessed by measuring voltage deflection at the end of the response to a hyperpolarizing rectangular current pulse steps (500 ms of −10 to −50 pA).

Leptin (100 nM; provided by A.F. Parlow, through the National Hormone and Peptide Program), mCPP (4 μM, Sigma Aldrich), and Lorcaserin (4 μM, provided by Kathryn Cunningham), were added to the ACSF for specific experiments. Solutions containing leptin, mCPP, or lorcaserin were typically perfused for 2–4 min. A drug effect was required to be associated temporally with peptide application, and the response had to be stable within a few minutes. A neuron was considered depolarized or hyperpolarized if a change in membrane potential was at least 2 mV in amplitude.

Analyses of leptin-, mCPP- and lorcaserin-induced hypophagia

Leptin (5 mg/kg; provided by A.F. Parlow, through the National Hormone and Peptide Program)), mCPP (3 mg/kg; Sigma-Aldrich), lorcaserin (1, 3, and 6 mg/kg; proved by Kathryn Cunningham), and vehicle (sterile saline) were administered i.p. in a counterbalanced manner to chow-fed 18-hour overnight-fasted mice as previously described (Berglund et al., 2013; Williams et al., 2014; Xu et al., 2010b). Food intake was measured hourly for 6 hours and then a single measurement at 24 hours.

Analyses of lorcaserin-induced alterations in glucose tolerance test (GTT) and insulin tolerance test (ITT)

For GTTs mice were intraperitoneally injected with 1.5 g/kg body weight of dextrose after an overnight fast. Mice were injected with lorcaserin (1.5 mg/kg) or vehicle 45 min prior to glucose injection. We drew blood samples at 0, 15, 30, 60, 90, and 120 min time points and measured glucose levels with a Bayer contour glucometer.

For ITTs, mice were fasted for 3 hours with water ad libitum. Mice were injected with lorcaserin (1.5 mg/kg) or vehicle 45 min prior to insulin injection. After measurement of basal levels of glucose, insulin (1.2 U/kg, Eli Lilly and Company) was administrated i.p. Blood glucose levels were monitored at given time points after insulin injection.

We thank Dr. Joel K. Elmquist (The University of Texas Southwestern Medical Center, Dallas, Texas, USA) for kindly providing us with the LepR-cre and Pomc-hrGFP mice. We thank Dr. David E. Clapham (Howard Hughes Medical Institute, Boston Children's Hospital, Boston, Massachusetts, USA) for generously providing us with the TrpC5KO mice (Riccio et al., 2009). This work was supported by grants to T.Y. (China Scholarship Council 201406280111), J-W.S (The National Research Foundation of Korea NRF-2015M3A9E7029177 and NRF-2016R1C1B2006614 and from the Korean Ministry of Health and Welfare HI14C1946), J.S (National Natural Science Foundation of China No.81300689 & No.81670783), X.K. (K99 DK106550), K.A.C (K05 DA020087), Y.C. (The National Natural Science Foundation of China #81471049) T.L. (AHA 14SDG20370016), and K.W.W. (R01 DK100699).