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. Author manuscript; available in PMC: 2025 Feb 1.
Published in final edited form as: J Cell Physiol. 2023 Sep 8;239(2):e31117. doi: 10.1002/jcp.31117

Leptin Excites Basolateral Amygdala Principal Neurons and Reduces Food Intake by LepRb-JAK2-PI3K-dependent Depression of GIRK Channels

Cody A Boyle 1, Phani K Kola 1, Chidiebele S Oraegbuna 1, Saobo Lei 1,#
PMCID: PMC10920395  NIHMSID: NIHMS1928325  PMID: 37683049

Abstract

Leptin is an adipocyte-derived hormone that modulates food intake, energy balance, neuroendocrine status, thermogenesis and cognition. Whereas a high density of leptin receptors has been detected in the basolateral amygdala (BLA) neurons, the physiological functions of leptin in the BLA have not been determined yet. We found that application of leptin excited BLA principal neurons by activation of the long form leptin receptor, LepRb. The LepRb-elicited excitation of BLA neurons was mediated by depression of the G protein-activated inwardly rectifying potassium (GIRK) channels. Janus Kinase 2 (JAK2) and phosphoinositide 3-kinase (PI3K) were required for leptin-induced excitation of BLA neurons and depression of GIRK channels. Microinjection of leptin into the BLA reduced food intake via activation of LepRb, JAK2 and PI3K. Our results may provide a cellular and molecular mechanism to explain the physiological roles of leptin in vivo.

Keywords: excitability, action potential, K+ channels, amygdala, peptide, synapse, appetite

Graphical Abstract

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1. Introduction

Leptin, the protein product of the ob gene, is an adipocyte-derived hormone that plays a key role in the regulation of food intake, energy balance, neuroendocrine status, thermogenesis and cognition (Park & Ahima, 2014, 2015; Tartaglia, 1997). Leptin exerts its effects by interacting with specific membrane receptors (LepRs). The LepRs have at least five splice variants, but the long form leptin receptor (LepRb) is the major form capable of signal transduction (Allison & Myers, 2014; Park & Ahima, 2014; Tartaglia, 1997). The short forms may serve as a serum binding protein that functions in leptin stabilization or sequestration or as a leptin transporter (Allison & Myers, 2014; Tartaglia, 1997). Leptin binds to LepRb, activating the associated Janus Kinase 2 (JAK2) tyrosine kinase (Allison & Myers, 2014; Bjorbaek & Kahn, 2004; Chua, 2009; Gorska et al., 2010; Park & Ahima, 2014). Activated JAK2 phosphorylates the intracellular tail of LepRb on three tyrosine residues (Tyr985, Tyr1077, Tyr1138). Tyr985 recruits the Src homology region 2-containing protein tyrosine phosphatase (SHP2) to participate in extracellular signal-regulated kinase (ERK) signaling and serves as a binding site for the negative feedback regulator, suppressor of cytokine signaling 3 (SOCS3). STAT5 and STAT3 binds to Tyr1077 and Tyr1138, respectively. Leptin also recruits the insulin receptor substrate 2 (IRS2)/phosphoinositide 3-kinase (PI3K) and SH2B1 pathways, although the mechanism of their recruitment to LepRb remains unclear. LepRb mRNA is expressed in the hypothalamus, cerebral cortex, thalamus, cerebellum, area postrema, anterior pituitary (Lin et al., 2000) and amygdala (Burguera et al., 2000; Hikita et al., 2000; Lin et al., 2000; Udagawa, Hatta, Naora, & Otani, 2000). In the amygdala, LepRb mRNA is located in the basolateral amygdala (BLA) (Han, Yan, Luo, Liu, & Wang, 2003; Udagawa et al., 2000; W. Wang et al., 2015) and lateral amygdala (LA) (Udagawa et al., 2000; W. Wang et al., 2015). Whereas the central amygdala (CeA) is innervated by the LepRb-expressing projections from the neurons in the ventral tegmental area in mice, the CeA neurons do not expressed LepRb (Leshan et al., 2010; Udagawa et al., 2000). However, the physiological functions of leptin in the amygdala have not been determined.

The amygdala is involved in modulating emotion (Dejean et al., 2015; Janak & Tye, 2015; Ressler, 2010; Tye et al., 2011), pain (Neugebauer, 2015; Neugebauer, Li, Bird, & Han, 2004; Veinante, Yalcin, & Barrot, 2013), alcohol use disorders (Gilpin, Herman, & Roberto, 2015; Silberman, Shi, Brunso-Bechtold, & Weiner, 2008) and appetite (Petrovich, 2011, 2013; Smith & Lawrence, 2018; Zanchi et al., 2017). The LA and BLA mostly contain glutamatergic pyramidal neurons, but the CeA is composed of distinct GABAergic neurons. The CeA contains 3 subnuclei named as capsular, lateral, and medial nucleus of CeA (LeDoux, 2000) (abbreviated as CeC, CeL and CeM, respectively). The LA receives multisensory information from the thalamus (LeDoux, Cicchetti, Xagoraris, & Romanski, 1990; Tully, Li, Tsvetkov, & Bolshakov, 2007), integrated sensory information from the cortex (McDonald, 1998), and noxious stimulus information from the brainstem regions (Johansen, Cain, Ostroff, & LeDoux, 2011). Information flows from the LA and BLA into the CeA, which sends out information through amygdala efferents (Duvarci & Pare, 2014), although the CeA also receives noxious stimulus information from the brainstem regions (Bernard, Huang, & Besson, 1992; Neugebauer, Galhardo, Maione, & Mackey, 2009). In this study, we probed the effects of leptin on the activity of BLA projection neurons based on the following rationales: 1) animals with lesions of BLA showed altered feeding behaviors (Holland & Petrovich, 2005; Petrovich & Gallagher, 2003; Y. Wang, Fontanini, & Katz, 2006); 2) leptin is well-known to modulate appetite (Park & Ahima, 2015); 3) LepRb are expressed in the BLA (Han et al., 2003; Udagawa et al., 2000; W. Wang et al., 2015). Our results indicate that LepRb activation excites BLA projection neurons by JAK2 and PI3K-mediated suppression of the G protein-activated inwardly rectifying potassium (GIRK) channels and these intracellular signaling molecules are involved in leptin-elicited reduction of food intake. Our results may provide a cellular and molecular mechanism to explain the physiological roles of leptin in the amygdala.

2. Material and Methods

2.1. Preparation of amygdala slices

Coronal brain slices (300 μm) were prepared from virgin male and female Sprague-Dawley (SD) rats (30–45 days old) purchased from Envigo RMS, INC. (Indianapolis, IN). The animals were housed in the Center for Biomedical Research in the University of North Dakota with food and water available ad libitum. The animal rooms were maintained on a 14/10 h light–dark cycle (lights on at 7:00 a.m.), with a room temperature of 22°C. After being deeply anaesthetized with isoflurane, animals were decapitated and their brains were dissected out. The cerebellum was trimmed and the caudal pole of the brain was glued to the plate of a vibrotome (Leica VT1200S). The cutting solution contained (in mM) 50 sucrose, 104 NaCl, 24.4 NaHCO3, 3.3 KCl, 1.24 NaH2PO4, 2.5 CaCl2, 1.5 MgCl2 and 10.2 glucose, saturated with 95% O2 and 5% CO2. Cuttings were made from the rostral pole of the brain and slices were collected from both hemispheres when the structure of amygdala appeared. Slices were incubated at 35°C for 30 min in the solution containing (mM) 12 sucrose, 124 NaCl, 24 NaHCO3, 3.4 KCl, 1.25 NaH2PO4, 2.5 CaCl2, 1.5 MgCl2, and 10 glucose and then kept at room temperature until use. All animal procedures conformed to the guidelines approved by the University of North Dakota Animal Care and Use Committee.

2.2. Recordings of resting membrane potentials, action potentials and holding currents

Whole-cell patch-clamp recordings using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA) in current- or voltage-clamp mode were made from the principal neurons in the BLA visually identified with infrared video microscopy (Olympus BX51WI) and differential interference contrast optics. The bath was maintained between 33°C and 34°C by an in-line heater and an automatic temperature controller (TC-324C, Warner Instruments). The extracellular solution contained (in mM) 130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 2.5 CaCl2, 1.5 MgCl2 and 10 glucose, saturated with 95% O2 and 5% CO2. Kynurenic acid (1 mM) and picrotoxin (100 μM) were supplemented in the extracellular solution to block potential indirect actions from synaptic transmission. The recording electrodes were filled with (in mM) 120 K+-gluconate, 10 KCl, 5 NaCl, 2 MgCl2, 10 HEPES, 0.5 EGTA, 2 ATPNa2, 0.4 GTPNa, and 5 phosphocreatine (pH 7.3). Leptin was dissolved in the extracellular solution and applied to the cells. To avoid potential desensitization induced by repeated applications of the agonist, one slice was limited to only one application of leptin. Data were filtered at 1 kHz, digitized at 10 kHz, acquired online and subsequently analyzed using pCLAMP 10.7 software (Molecular Devices, Sunnyvale, CA). Action potentials (APs) were evoked by injection of a series of positive currents from 50 pA to 700 pA at an increment of 50 pA and a duration of 600 ms every 7 s. This protocol was applied to the same cells before and after the application of leptin when the maximal depolarizing effect of leptin was observed. For the recordings of resting membrane potentials (RMPs), holding currents (HCs, at −60 mV) and current-voltage (I-V) relationship, the extracellular solution was supplemented with tetrodotoxin (TTX, 0.5 μM) to block AP firing.

2.3. Recordings of the inwardly rectifying K+ (Kir) currents from BLA neurons

The above-mentioned K+-gluconate-containing intracellular solution was used to record Kir currents from BLA neurons. TTX (0.5 μM) was supplemented in the extracellular solution to block Na+ channel currents. Cells were held at −60 mV and stepped from −140 mV to −30 mV for 400 ms at a voltage interval of 10 mV every 10 s. Steady-state currents were measured within 5 ms prior to the end of the step voltage protocols.

2.4. Recordings of spontaneous synaptic events

Spontaneous synaptic events were recorded from the BLA principal neurons with intracellular solution containing (in mM) 120 Cs+-gluconate, 10 CsCl, 5 NaCl, 2 MgCl2, 10 HEPES, 0.5 EGTA, 2 ATPNa2, 0.4 GTPNa, and 5 phosphocreatine (pH 7.3). sIPSCs were recorded at +30 mV in the normal extracellular solution supplemented with dL-2-amino-5-phosphonovaleric acid (dl-APV) (100 μM) and 6,7-dinitroquinoxaline-2,3(1H, 4H)-dione (DNQX) (10 μM) to block NMDA and AMPA receptor-mediated responses, respectively, as described previously (Deng, Porter, Shin, & Lei, 2006). sEPSCs were recorded at −65 mV in the normal extracellular solution supplemented with bicuculline (10 μM) to block GABAergic transmission. Synaptic events were recorded in Clampex 10.7 and analyzed in Clampfit 10.7 with ‘Event Detection’ and ‘Template Search’. The numbers of sIPSCs and sEPSCs were binned per min in Excel with a custom-made experience formula. The cumulative probability histograms of frequency and amplitude were constructed in GraphPad Prism 9.

2.5. Measurement of food intake

Surgical procedures and cannulation were performed as described previously (Boyle, Hu, Quaintance, Mastrud, & Lei, 2022). Male (349.9 ± 70.4 g, n = 45) and female (234.1 ± 35.9 g, n = 45) Sprague-Dawley (SD) rats at the age of 8–11 weeks were anaesthetized with 5% vaporized isoflurane until the loss of the righting response and placed in a stereotaxic frame (Stoelting Co. Wood Dale, IL, USA). Guide cannula (23 GA, 8.5 mm length; P1 Technologies Inc., Roanoke, VA, USA) were bilaterally implanted to target the BLA with coordinates from the rat brain atlas (from bregma, anteroposterior: −2.4 ~ −2.6 mm, mediolateral: ± 4.9 ~ 5 mm, dorsoventral: −7.3 mm) (Boyle et al., 2022). The tip of the guide was positioned within or ~0.5 mm above the BLA. Three stainless steel screws (4.8 mm, P1 Technologies Inc.) placed in the skull served as anchor points to secure the cannula in place with dental acrylic. Stainless steel stylets were screwed into the guide cannula to prevent occlusion during the 7–10 day recovery period. During the recovery period, animals were handled daily for habituation to the microinjection and daily assessment procedures. Saline or leptin (1 μL/side) was bilaterally injected into the BLA through an internal cannula (30 GA, 8.5 mm; P1 Technologies Inc.). Compounds were administered at a rate of 0.2 μL/min using an automated pump (Harvard Apparatus, Holliston, MA, USA) connected via polyethylene tubing to two Hamilton syringes. Following the completion of the injection, the internal cannula was left in the guide cannula for an additional 2 minutes to allow for adequate diffusion into the BLA. Following experiments, correct cannula placement was confirmed. Briefly, animals were anaesthetized and bilaterally injected with 3 % Chicago Sky Blue 6B (Sigma-Aldrich, St. Louis, MO, USA). Sections in the coronal plane (80 μm) were collected on a vibratome (VT1200, Leica Biosystems) to verify correct tip placement. Animals with incorrect cannula placement were not included in our analysis.

A single SD rat was housed in Nalgene metabolic cages for the duration of the experiment. Animals were allowed a 3-day habituation period in the metabolic cage prior to microinjection and a chew toy was present for environmental enrichment. Rats were allowed ad libitum access to a powdered-food hopper and water during habituation. Animals received daily handling and cages were meticulously cleaned each day. On testing days, animals were food restricted 10 h prior to microinjection of either saline or leptin. Intake of powdered chow were measured by subtracting the amount remaining from the initially premeasured amount at 1 h later.

2.6. Data analysis

Data are presented as the means ± SD. Wilcoxon matched-pair signed rank test (abbreviated as Wilcoxon test in the text), the Mann-Whitney test, one-way or two-way ANOVA were used for statistical analysis as appropriate. For the ex vivo experiments, “n” number is the number of cells recorded. Because we recorded only one cell from each slice, the “n” number is also the number of slices used. To minimize potential influences of variations from individual animals, each experiment was performed from slices attained from at least four animals and one-way ANOVA was performed to ensure there was no significant difference for the data obtained from individual animals under the same treatment. One-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison test was used for statistical analysis when the pooled control data were used for comparison. Two-way repeated measures ANOVA followed by Sidak’s multiple comparison test was used for statistical analysis for the AP firing frequency elicited by injections of positive currents and for the data used to construct the voltage-current relationship. P values were reported throughout the text and significance was set as P < 0.05.

2.7. Chemicals

Leptin and leptin antagonist triple mutant (leptin tA) were purchased from Prospec (Rehovot, Israel). AG490, S3I-201 and glibenclamide were provided by MedChemExpress. The following chemicals were products of R&D Systems: ML 133, SCH23390, PD98059, wortmannin and LY294002. Drugs were initially prepared in stock solution, aliquoted and stored at −20°C. For those chemicals which are only soluble in dimethyl sulfoxide (DMSO), the concentration of DMSO was less than 0.1%.

3. Results

3.1. Leptin excites BLA neurons:

Because the BLA expresses leptin receptors (Han et al., 2003; Udagawa et al., 2000; W. Wang et al., 2015) and leptin is a neuromodulator, we probed the effects of leptin on neuronal activity in the BLA. The extracellular solution contained kynurenic acid (1 mM) to block glutamatergic transmission and picrotoxin (100 μM) to block GABAergic transmission and K+-gluconate intracellular solution was used. We recorded RMPs from the principal BLA neurons in current-clamp. Under these circumstances, bath application of leptin at 100 nM, a saturating concentration, induced subthreshold depolarization of BLA neurons (Control: −68.1 ± 3.2 mV, Leptin: −64.7 ± 5.8 mV, net depolarization: 3.4 ± 4.1 mV, n = 18, P < 0.0001, Wilcoxon test, Fig. 1a, b). We further tested the roles of leptin on the excitability of BLA principal neurons by injecting a series of positive currents from 50 pA to 700 pA at an increment of 50 pA. This current injection protocol was applied to the same neurons prior to and after the application of leptin when the maximal depolarizing effect of leptin was observed. Bath application of leptin significantly increased the number of APs elicited by the positive current injection protocol (n = 12, F(1,11) = 30.48, P = 0.0002, two-way repeated measures ANOVA followed by Sidak multiple comparison tests, Fig. 1c, d). We then persistently injected a depolarizing current to elevate the membrane potential to just above the firing threshold (−50.0 ± 3.2 mV, n = 13) to elicit sparse AP firing and further tested the effect of leptin on the excitability of BLA neurons. Bath application of the same concentration of leptin for 5 min significantly augmented the AP firing frequency (Control: 0.58 ± 0.42 Hz, Leptin: 2.00 ± 1.35 Hz, n = 13, P = 0.002, Wilcoxon’s test, Fig. 1e, f) and the persistent increases of AP firing were still observed after wash in leptin-free extracellular solution for 30 min (3.18 ± 3.61 Hz, n = 13, P = 0.013 vs. Control, Wilcoxon’s test, Fig. 1e, f). These results together indicate that bath application of leptin significantly enhances the excitability of BLA neurons.

Figure 1. Leptin excites BLA principal neurons.

Figure 1.

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(a-b), Bath application of leptin (100 nM) elicited subthreshold depolarization of BLA principal neurons. (a), RMP recorded from a BLA principal neuron prior to, during and after application of leptin. (b), Summary data for leptin-induced subthreshold depolarization. Empty symbols were data from individual cells and solid symbols were their averages (n = 18) for control (red) and leptin (green). (c-d), Bath application of leptin (100 nM) augmented the number of AP firing elicited by injection of a series of positive currents from 50 pA to 700 pA at an increment of 50 pA and duration of 600 ms every 7 seconds. (c), APs recorded from a BLA neuron evoked by the positive current injection protocol prior to (left, red) and after (right, green) the application of leptin. (d), Relationship between the injected currents and the elicited AP numbers from 12 BLA neurons. * P < 0.05, ** P < 0.01, two-way repeated measures ANOVA followed by Sidak multiple comparison tests. (e), APs recorded from a BLA neuron prior to, during and after application of leptin when a positive current was injected persistently to induce basal sparse AP firing. Lower panels showed the expanded traces recorded at the time points indicated in the upper graph. (f), Summary data showing leptin-induced excitation of BLA neurons (n = 13).

We then recorded the HCs at −60 mV in voltage clamp. Bath application of leptin (100 nM) induced an inward current (−30.4 ± 25.4 pA, n = 20, P < 0.0001 vs. baseline, Wilcoxon test, Fig. 2a1, a2), further supporting that leptin excites BLA neurons via membrane depolarization. The EC50 of leptin measured by using the HCs as an indicator was 3.2 nM (Fig. 2a3).

Figure 2. Leptin induces an inward current, increases the input resistance, and increases membrane time constants.

Figure 2.

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(a1), an inward current recorded from a BLA neuron in response to bath application of leptin (100 nM). (a2), summary graph showing leptin-elicited inward currents (n = 20). Left: The holding current before and after application of leptin recorded from the same cells are shown in empty circles and the solid circles are their averages. Right: Leptin-induced net current was obtained by subtraction. **** P < 0.0001. (a3) Concentration-response curve of leptin. The numbers in the parentheses were the numbers of neurons recorded at each concentration. (b1-b3), Leptin increased the input resistance (Rin) of BLA neurons. (b1), voltage responses evoked by injection of negative currents from −150 to −50 pA at an interval of 50 pA prior to (left, red) and after (right, green) the application of leptin from the same cell. (b2), current-voltage relationship averaged from 10 cells. Rin was obtained by linear fitting of the current-voltage relationship. (b3), summary graph for Rin prior to and after the application of leptin (n = 10). The open circles represent the values from individual cells and the filled symbols are their averages for control (red) and leptin (green). (c1-c3), Leptin increased the time constants of BLA neurons. (c1), voltage response evoked by −150 pA prior to and after application of leptin. (c2), expansion of the voltage transient shown in the box in (b1) to show leptin-elicited augmentation of membrane time constants. (c3), summary graph for membrane time constants before and after the application of leptin (n = 8).

3.2. Leptin increases the input resistance and membrane time constants of BLA neurons

We then assessed the effects of leptin on the input resistance (Rin) of BLA neurons by injecting negative currents from 0 to −150 pA with 50 pA steps for a duration of 600 ms before and after the application of leptin at the maximal effect of leptin. We fit the current-voltage (I-V) relationship with a linear function for each cell to obtain Rin, which equals the slope of the linear fitting (Fig. 2b1–b3). Application of leptin significantly increased Rin (Control: 75 ± 17 MΩ, Leptin: 91 ± 28 MΩ, n = 10, P = 0.004, Wilcoxon test, Fig. 2b3), indicating that leptin increases Rin. The membrane time constant obtained by fitting a single exponential function to the voltage transient (100 ms from the baseline) induced by −150 pA current step was significantly increased (Control: 13.2 ± 3.5 ms, Leptin: 22.1 ± 4.7 ms, n = 8, P = 0.008, Wilcoxon test, Fig. 2c1–c3). These results suggest that leptin excites BLA via inhibiting a membrane conductance.

3.3. LepRb, JAK2 and PI3K are involved in leptin-induced inward currents

The effects of leptin are mediated by LepRb. We therefore tested the roles of LepRb in leptin-elicited inward currents by applying the selective LepRb antagonist, leptin antagonist triple mutant (Leptin tA) (F. Huang, Xiong, Wang, You, & Zeng, 2010; Shpilman et al., 2011). Prior to and concomitant application of Leptin tA (100 nM) in the bath blocked leptin-induced inward currents (−3.2 ± 7.9 pA, n = 14, P = 0.217 vs. baseline, Wilcoxon test; P < 0.0001 vs. leptin alone, one-way ANOVA followed by Dunnett’s test, Fig. 3a, 3g), indicating the requirement of LepRb.

Figure 3. LepRb, JAK2 and PI3K are involved in leptin-induced inward currents.

Figure 3.

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(a), Current trace recorded from a BLA neuron in response to bath application of leptin (100 nM) in the continuous presence of leptin tA (100 nM). (b), Leptin-induced inward current recorded from a BLA neuron treated with AG490 (10 μM). (c), Current trace elicited by leptin in a BLA neuron treated with wortmannin (200 nM). (d), Leptin-evoked current trace in a BLA neuron treated with LY294002 (10 μM). (e), Current trace elicited by leptin in a BLA neuron in the presence of PD98059 (50 μM). (f), Inward current evoked by leptin in a BLA neuron treated with S3I-201 (300 μM). (g), summary graph. **** P < 0.0001 vs. leptin alone, one-way ANOVA followed by Dunnett’s multiple comparison test.

Leptin binding to LepRb results in dimerization and phosphorylates and activates JAK2, which phosphorylates itself and Tyr985, Tyr1077, and Tyr1138 in the cytoplasmic domain of the LepRb (Allison & Myers, 2014; Bjorbaek & Kahn, 2004; Park & Ahima, 2014). We therefore probed the roles of JAK2 by utilizing AG490, the tyrosine kinase inhibitor that selectively blocks JAK2 phosphorylation (Arbel et al., 2003). Pretreatment of slices with and continuous bath application of AG490 (10 μM) annulled leptin-elicited inward currents recorded from BLA neurons (−4.7 ± 8.5 pA, n = 14, P = 0.153 vs. baseline, Wilcoxon test; P < 0.0001 vs. leptin alone, one-way ANOVA followed by Dunnett’s test, Fig. 3b, 3g).These results indicate that the function of JAK2 is required for leptin-induced inward currents in the BLA.

JAK2 activation by leptin binding to LepRb phosphorylates insulin receptor substrate, which further activates PI3K (Allison & Myers, 2014; Bjorbaek & Kahn, 2004; Park & Ahima, 2014). We then tested the roles of PI3K in leptin-mediated inward currents. Pretreatment of slices with and continuous bath application of the PI3K inhibitor, wortmannin (200 nM) blocked leptin-induced inward currents (−2.4 ± 5.5 pA, n = 15, P = 0.135 vs. baseline, Wilcoxon test; P < 0.0001 vs. leptin alone, one-way ANOVA followed by Dunnett’s test, Fig. 3c, 3g). Likewise, prior to and continuous bath application of LY294002 (10 μM), another PI3K inhibitor (Adi, Wu, & Rosenthal, 2001), blocked leptin-elicited inward currents (−4.0 ± 9.8 pA, n = 14, P = 0.194 vs. baseline, Wilcoxon test; P < 0.0001 vs. leptin alone, one-way ANOVA followed by Dunnett’s test, Fig. 3d, 3g). These data together suggest that PI3K is involved in leptin-induced inward currents.

3.4. MAPK signaling cascade is unnecessary for leptin-induced inward currents

Activated JAK2 also phosphorylates Tyr985 to recruit SHP2, which activates the MAPK signaling cascade (Allison & Myers, 2014; Bjorbaek & Kahn, 2004; Dance, Montagner, Salles, Yart, & Raynal, 2008; Park & Ahima, 2014). We tested the roles of MAPK signaling cascade in leptin-induced inward currents. Pretreatment of slices with and continuous bath application of the selective MAPK inhibitor, PD98059 (50 μM), did not alter significantly leptin-evoked inward currents (−38.0 ± 16.8 pA, n = 13, P = 0.0002 vs. baseline, Wilcoxon test; P = 0.595 vs. leptin alone, one-way ANOVA followed by Dunnett’s test, Fig. 3e, 3g), suggestive of unnecessity of the MAPK signaling cascade.

3.5. STAT3 is not involved in leptin-evoked inward currents

JAK2-mediated phosphorylation of Tyr1138 in the cytoplasmic domain of the LepRb results in phosphorylation and activation of STAT3 (Allison & Myers, 2014; Bjorbaek & Kahn, 2004; Park & Ahima, 2014). We also tested whether this signaling pathway is involved in leptin-evoked inward currents in BLA neurons. Application of S3I-201, a STAT3 inhibitor, at a concentration range of 50–300 μM, has been shown to inhibit STAT3 (Choi, Kwon, & Joe, 2018; Gurbuz et al., 2014). Pretreatment of slices with and continuous bath application of S3I-201 (300 μM) did not significantly alter leptin-mediated inward currents (−28.1 ± 18.3 pA, n = 14, P = 0.0001 vs. baseline, Wilcoxon test; P = 0.997 vs. leptin alone, one-way ANOVA followed by Dunnett’s test, Fig. 3f, 3g), suggesting that STAT3 is not necessary for leptin-elicited inward currents.

3.6. Leptin-evoked inward currents are mediated by depression of an inwardly rectifying K+ channel

We then determined the ionic mechanisms whereby activation of LepRb elicited the inward currents by constructing the I-V relationship. BLA neurons were held at −60 mV and stepped from −140 mV to −30 mV for 400 ms at a voltage step of 10 mV every 10 s. Steady-state currents were measured within 5 ms prior to the end of the step voltage protocol. Under these circumstances, the leptin-elicited currents showed inward rectification with a reversal potential at −90.7 ± 13.7 mV (n = 14, Fig. 4a–c), close to the calculated K+ reversal potential in our recording condition (−94.4 mV), suggesting that activation of LepRb induced an inward current in the BLA neurons by suppressing an inwardly rectifying K+ (Kir) channel. We further tested the roles of Kir channels by using Ba2+. Bath application of Ba2+ (500 μM) by itself induced an inward current (−40.5 ± 18.0 pA, n = 10, P = 0.002, Wilcoxon test, Fig. 5a, 5e) and blocked leptin-elicited inward currents (−3.0 ± 5.6 pA, n = 10, P = 0.193 vs. Ba2+ alone, Wilcoxon test, P = 0.0001 vs. leptin alone, one-way ANOVA followed by Dunnett’s test, Fig. 5a, 5f), further confirming the involvement of Kir channels.

Figure 4. Leptin suppresses an inwardly rectifying K+ channel in BLA principal neurons revealed by a voltage-step protocol.

Figure 4.

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(a), Currents elicited by the voltage-step protocol before (left, red) and after (middle, green) bath application of leptin and the net current obtained by subtraction (right, blue) from a BLA neuron. Cells were held at −60 mV and stepped from −140 to −30 mV for 400 ms at a voltage interval of 10 mV in every 10 s. (b), I-V curve averaged from 14 cells before (red) and after (green) application of leptin. (c), I-V curve of the net current obtained by subtracting the currents in control condition from those after application of leptin (n = 14).

Figure 5. GIRK channels are involved in leptin-mediated excitation of BLA neurons.

Figure 5.

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(a), Current trace recorded from a BLA neuron in response to bath application of Ba2+ (500 μM) alone and concomitant application of Ba2+ with leptin. (b), Current trace in response to bath application of ML 133 (30 μM) alone and concurrent application of leptin. (c), Current trace in response to bath application of glibenclamide (100 μM) and co-application of leptin. (d), Current trace recorded from a BLA neuron in response to bath application of SCH23390 (40 μM) alone and co-application of leptin. (e), Effects of Kir channel blockers on holding currents at −60 mV recorded from BLA neurons. ** P < 0.01 vs. baseline, Wilcoxon test. (f), Effects of Kir channel blockers on leptin-induced inward currents. ** P < 0.01, *** P < 0.001 vs. leptin alone, one-way ANOVA followed by Dunnett’s multiple comparison test. Leptin was applied for a 5-minute period as indicated by the bar.

Kir channels include Kir2 subfamily (Kir2.1, Kir2.2, Kir2.3, Kir2.4), Kir3 subfamily (GIRK channels, Kir3.1, Kir3.2, Kir3.3, Kir3.4,), Kir6 subfamily (ATP-sensitive K+ channels, KATP channels, Kir6.1, Kir6.2) and K+ transport channels (Kir1.1, Kir4.1, Kir4.2, Kir7.1) (Hibino et al., 2010). We used ML 133, a selective blocker for Kir2 subfamily (Ford & Baccei, 2016; X. Huang, Lee, Lu, Sanders, & Koh, 2018; Kim et al., 2015; Sonkusare, Dalsgaard, Bonev, & Nelson, 2016; H. R. Wang et al., 2011) and tested the roles of the Kir2 subfamily in leptin-elicited inward currents. Bath application of ML 133 (30 μM) by itself did not significantly change the holding currents (−0.03 ± 7.06 pA, n = 10, P = 0.770 vs. baseline, Wilcoxon test, Fig. 5b, 5e) and exerted no significant effect on leptin-induced inward currents (−31.1 ± 17.9 pA, n = 10, P = 0.002 vs. ML 133 alone, Wilcoxon test; P = 0.999 vs. leptin alone, one-way ANOVA followed by Dunnett’s test, Fig. 5b, 5f), suggesting that Kir2 subfamily is not involved in leptin-elicited excitation of BLA neurons. We then used the KATP channel blocker, glibenclamide (100 μM) and tested whether KATP channels are involved in leptin-induced inward currents. Bath application of glibenclamide failed to alter significantly the holding currents (−5.0 ± 7.7 pA, n = 10, P = 0.106 vs. baseline, Wilcoxon test, Fig. 5c, 5e) and subsequent application of leptin still elicited a comparable inward current (−27.2 ± 6.8 pA, n = 10, P = 0.002 vs. glibenclamide alone, Wilcoxon test; P = 0.976 vs. leptin alone, one-way ANOVA followed by Dunnett’s test, Fig. 5c, 5f), suggesting that KATP channels are not related to leptin-evoked inward currents. We next tested the roles of GIRK channels in leptin-induced inward currents. Bath application of the GIRK channel blocker, SCH23390 (40 μM) (Kuzhikandathil & Oxford, 2002), induced an inward current (−18.0 ± 10.3 pA, n = 11, P = 0.001 vs. baseline, Wilcoxon test, Fig. 5d, 5e) and following application of leptin significantly reduced leptin-induced inward currents (−5.6 ± 7.1 pA, n = 11, P = 0.049 vs. SCH23390 alone, Wilcoxon test; P = 0.002 vs. leptin alone, one-way ANOVA followed by Dunnett’s test, Fig. 5d, 5f), indicating that GIRK channels are involved in leptin-elicited excitation of BLA neurons.

3.7. LepRb, JAK2 and PI3K are required for leptin-induced depression of Kir channels

If leptin-elicited inward currents are mediated by inhibition of Kir channels, LepRb, JAK2 and PI3K should also be involved in leptin-elicited depression of Kir currents. We further tested the roles of LepRb, JAK2 and PI3K in leptin-induced inhibition of Kir currents. Pretreatment of slices with and continuous bath application of the selective LepRb antagonist, leptin tA (100 nM), blocked leptin-elicited suppression of Kir currents at all the voltages except at −140 mV (two-way repeated measures ANOVA followed by Sidak’s multiple comparison test, Drug: F(1,13) = 0.098, P = 0.759; Voltage: F(11,143) = 85.67, P < 0.0001; Drug x Voltage: F(11,143) = 2.749, P = 0.003, Fig. 6a–c), suggesting that LepRb is required for leptin-induced depression of Kir channels.

Figure 6. LepRb and JAK2 are required for leptin-elicited depression of Kir channels.

Figure 6.

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(a), Currents elicited by a voltage-step protocol prior to (left, red) and after (middle, green) bath application of leptin and the net current obtained by subtraction (right, blue) from a BLA neuron in a slice pretreated with leptin tA (100 nM). The bath was continuously perfused with the same concentration of leptin tA. Note the differences of the scale bars. The dashed line was the zero current level. (b), I-V curve averaged from 14 cells before (red) and after (green) application of leptin in the presence of leptin tA (two-way repeated measures ANOVA followed by Sidak’s multiple comparison test; Drug: F(1,13) = 0.098, P = 0.759; Voltage: F(11,143) = 85.67, P < 0.0001; Drug x Voltage: F(11,143) = 2.749, P = 0.003). There was no significant difference except at −140 mV (* P = 0.026). (c), I-V curve of the net current obtained by subtracting the currents in control condition from those after application of leptin in the presence of leptin tA. (d), Currents elicited by the voltage-step protocol prior to (left, red) and after (middle, red) bath application of leptin and the net current obtained by subtraction (right, blue) from a BLA neuron in a slice pretreated with AG490 (10 μM). The bath was continuously perfused with the same concentration of AG490. Note the differences of the scale bars. The dashed line was the zero current level. (e), I-V curve averaged from 12 cells before (red) and after (green) application of leptin in the presence of AG490 (two-way repeated measures ANOVA followed by Sidak’s multiple comparison test; Drug: F(1,11) = 0.934, P = 0.355; Voltage: F(11,121) = 92.79, P < 0.0001; Drug x Voltage: F(11,121) = 1.901, P = 0.046). (f), I-V curve of the net current obtained by subtracting the currents in control condition from those after application of leptin in the presence of AG490.

We further tested the roles of JAK2 in leptin-elicited depression of Kir channels. Pretreatment of slices with and continuous bath application of the JAK2 inhibitor, AG490 (10 μM) annulled leptin-induced depression of Kir channels (two-way repeated measures ANOVA followed by Sidak’s multiple comparison test, Drug: F(1,11) = 0.934, P = 0.355; Voltage: F(11,121) = 92.79, P < 0.0001; Drug x Voltage: F(11,121) = 1.901, P = 0.046, Fig. 6d–f), suggesting that JAK2 is necessary for leptin-induced depression of Kir channels.

We further probed the roles of PI3K in leptin-mediated depression of Kir channels. Pretreatment of slices with and continuous bath application of wortmannin (200 nM) also blocked leptin-mediated suppression of Kir channel currents (two-way repeated measures ANOVA followed by Sidak’s multiple comparison test; Drug: F(1,11) = 0.017, P = 0.900; Voltage: F(11,121) = 294.3, P < 0.0001; Drug x Voltage: F(11,121) = 2.381, P = 0.011, Fig. 7a–c). Likewise, application of LY294002 (10 μM), another PI3K inhibitor, blocked leptin-induced depression of Kir channel currents (two-way repeated measures ANOVA followed by Sidak’s multiple comparison test; Drug: F(1,12) = 0.411, P = 0.534; Voltage: F(11,132) = 305.7, P < 0.0001; Drug x Voltage: F(11,132) = 0.771, P = 0.668, Fig. 7d–f). These results together suggest that PI3K is involved in leptin-elicited depression of Kir channels.

Figure 7. PI3K is necessary for leptin-induced inhibition of Kir channels.

Figure 7.

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(a), Currents elicited by the voltage-step protocol prior to (left, red) and after (middle, green) bath application of leptin and the net current obtained by subtraction (right, blue) from a BLA neuron in a slice pretreated with wortmannin (200 nM). The bath was continuously perfused with the same concentration of wortmannin. The dashed line was the zero current level. (b), I-V curve averaged from 12 cells before (red) and after (green) application of leptin with concomitant application of wortmannin (two-way repeated measures ANOVA followed by Sidak’s multiple comparison test; Drug: F(1,11) = 0.017, P = 0.900; Voltage: F(11,121) = 294.3, P < 0.0001; Drug x Voltage: F(11,121) = 2.381, P = 0.011). (c), I-V curve of the net current obtained by subtracting the currents in control condition from those after application of leptin in the presence of wortmannin. (d), Currents elicited by the voltage-step protocol prior to (left, red) and after (middle, green) bath application of leptin and the net current obtained by subtraction (right, blue) from a BLA neuron in a slice pretreated with LY294002 (10 μM). The bath was continuously perfused with the same concentration of LY294002. The dashed line was the zero current level. (e), I-V curve averaged from 13 cells before and after application of leptin in the presence of LY294002 (two-way repeated measures ANOVA followed by Sidak’s multiple comparison test; Drug: F(1,12) = 0.411, P = 0.534; Voltage: F(11,132) = 305.7, P < 0.0001; Drug x Voltage: F(11,132) = 0.771, P = 0.668). (f), I-V curve of the net current obtained by subtracting the currents in control condition from those after application of leptin in the presence of LY294002.

3.8. Leptin does not modulate the synaptic transmission onto the BLA principal neurons

The above experiments were performed by including kynurenic acid to block glutamatergic transmission and picrotoxin (100 μM) to block GABAergic transmission. The BLA principal neurons also receive glutamatergic transmission from the LA neurons and GABAergic transmission from local GABAergic interneurons. We then studied whether leptin modulates synaptic transmission by recording sIPSCs and sEPSCs to assess the effects of leptin on GABAergic and glutamatergic transmission, respectively. Bath application of leptin (100 nM) exerted no significant effects on the frequency (Control: 3.9 ± 2.9 Hz, Leptin: 3.8 ± 2.8 Hz, n = 10, P = 0.432, Wilcoxon test, Fig. 8a1–a5) and amplitude (Control: 29.8 ± 12.8 pA, Leptin: 28.5 ± 13.2 pA, n = 10, P = 0.375, Wilcoxon test, Fig. 8a1–a5) of sIPSCs. Likewise, application of 100 nM leptin failed to alter significantly the frequency (Control: 3.6 ± 2.4 Hz, Leptin: 3.1 ± 2.0 Hz, n = 9, P = 0.203, Wilcoxon test, Fig. 8b1–b5) and amplitude of sEPSCs (Control: 11.8 ± 4.1 pA, Leptin: 11.6 ± 4.4 pA, n = 9, P = 0.734, Wilcoxon test, Fig. 8b1–b5).

Figure 8. Leptin does not modulate sIPSCs and sEPSCs recorded from BLA neurons.

Figure 8.

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(a1–a5), Leptin exerted no effects on sIPSC frequency and amplitude. (a1), sIPSC traces recorded from a BLA principal neuron prior to (red) and after (green) the application of leptin. (a2), Time course of sIPSC frequency before, during and after bath application of leptin (n = 10). (a3), Cumulative frequency distribution from 10 BLA neurons before (red) and after (green) the application of leptin. (a4), Cumulative amplitude distribution from 10 BLA neurons before (red) and after (green) the application of leptin. (a5), Summary graph showing that leptin exerted no effects on sIPSC frequency and amplitude. (b1–b5), Leptin exerted no effects on sEPSC frequency and amplitude. (b1), sEPSC traces recorded from a BLA principal neuron prior to (red) and after (green) the application of leptin. (b2), Time course of sEPSC frequency before, during and after bath application of leptin (n = 9). (b3), Cumulative frequency distribution from 9 BLA neurons before (red) and after (green) the application of leptin. (b4), Cumulative amplitude distribution from 9 BLA neurons before (red) and after (green) the application of leptin. (b5), Summary graph showing that leptin exerted no effects on sEPSC frequency and amplitude.

3.9. Microinjection of leptin into the BLA decreases food intake

Both leptin (Park & Ahima, 2014, 2015; Tartaglia, 1997) and the amygdala (Petrovich, 2011, 2013; Smith & Lawrence, 2018; Zanchi et al., 2017) modulate appetite. The BLA is interconnected between brain regions that influence eating behavior, and the BLA projects directly to hypothalamic feeding circuits and motor output pathways that are involved in triggering feeding behaviors (McDonald, 2020; Ono, Luiten, Nishijo, Fukuda, & Nishino, 1985). BLA has been involved in food intake (Sun et al., 2015) and animals with lesions of the BLA showed altered feeding behaviors (Holland & Petrovich, 2005; Petrovich & Gallagher, 2003; Y. Wang et al., 2006). We therefore studied the effects of microinjection of leptin into the BLA on food intake (Fig. 9a). We measured the food intake within 1 h after microinjection of rats with saline or leptin. Microinjection of leptin at 0.1 nmol, an effective dose in the brain (Lu, Kim, Frazer, & Zhang, 2006; Morrison et al., 2007), into the BLA significantly reduced food intake (1.6 ± 1.0 g, n = 10 rats), compared with the food intake of rats microinjected with saline (4.6 ± 1.8 g, n = 14 rats, P =0.003, One-way ANOVA followed by Turkey test, Fig. 9b). The effects of leptin were not sex-dependent as comparison of the food intake from male and female rats showed no significant difference (P = 0.81, Mann-Whitney test). Because our electrophysiological data indicate that LepRb, JAK2 and PI3K are necessary for leptin-mediated excitation of BLA neurons, we further tested the roles of these intracellular signaling molecules in leptin-mediated reduction of food intake. Microinjection of the LepRb antagonist, Leptin tA at 80 pmol, an effective microinjection dose (Arnold & Diz, 2014), by itself did not significantly alter food intake (4.7 ± 2.1 g, n = 11 rats, P > 0.999 vs. saline group, One-way ANOVA followed by Turkey test, Fig. 9b), but blocked leptin-induced reduction of food intake (Leptin tA + Leptin: 5.1 ± 1.9 g, n = 11 rats, P = 0.999 vs. Leptin tA alone, One-way ANOVA followed by Turkey test, Fig. 9b), indicating that the function of LepRb is required for leptin-elicited decreases of food intake. Furthermore, microinjection of the JAK2 inhibitor, AG490 at 1 nmol, an effective microinjection dose (Morrison et al., 2007), failed to change food intake significantly (3.8 ± 1.5 g, n = 11 rats, P = 0.962 vs. Saline, One-way ANOVA followed by Turkey test, Fig. 9b), but blocked leptin-induced decreases in food intake (AG490 + Leptin: 4.1 ± 1.7 g, n = 9 rats, P > 0.999 vs. AG490 alone, One-way ANOVA followed by Turkey test, Fig. 9b), suggesting that JAK2 activity is required for leptin-mediated reduction of food intake. Finally, microinjection of PI3K inhibitor, LY294002 (10 nmol) (Cui et al., 2010) did not change food intake significantly (5.6 ± 1.9 g, n = 12 rats, P = 0.824 vs. saline, One-way ANOVA followed by Turkey test, Fig. 9b), but annulled leptin-mediated reductions of food intake (LY294002 + Leptin: 4.7 ± 1.9 g, n =12 rats, P = 0.904 vs. LY294002 alone, One-way ANOVA followed by Turkey test, Fig. 9b). These results together indicate that leptin-elicited excitation of BLA principal neurons is responsible for leptin-mediated reductions of food intake.

Figure 9. Microinjection of leptin into the BLA suppresses food intake.

Figure 9.

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(a), Cannula tip placement in the BLA displayed onto an atlas figure modified from Paxinos & Watson (2007) (left) and accurate cannula placement is shown with the arrow (right).Asterisks indicate the cannula track. Note, all animal cannula placements were verified, dots represent a subset of the rats used for the experiments. (b), microinjection of leptin into the BLA significantly reduced food intake in 1 h and microinjection of leptin tA, AG490 and LY294002 blocked leptin-induced reduction of food intake. ** P < 0.01, one-way ANOVA followed by Dunnett’s multiple comparisons test. N.S. indicates a non-significant difference compared to saline-treated rats.

4. Discussion

Whereas the BLA expresses LepRbs (Han et al., 2003; Udagawa et al., 2000; W. Wang et al., 2015), the functions of leptin in the BLA have not been determined. Because leptin is a neuromodulator which regulates hunger and energy balance, we tested the hypothesis that activation of LepRbs modulates the excitability of BLA neurons, which contributes to food intake. Our results indicate that application of leptin facilitates the excitability of BLA principal neurons by depressing the GIRK type of the Kir channels. Leptin-mediated excitation of BLA neurons requires the functions of LepRb, JAK2 and PI3K. Furthermore, microinjection of leptin into the BLA reduces food intake via LepRb, JAK2 and PI3K signaling molecules. Our results may provide a signaling and ionic mechanism to explain the physiological functions of leptin in the BLA.

Leptin modulates the functions of a variety of neurons via distinct ionic mechanisms. For instance, leptin inhibits the activity of neurons expressing neuropeptide Y or agouti-related peptide by enhancing Kv2.1 channels (Baver et al., 2014), whereas stimulation of ATP-sensitive K+ channels is the ionic mechanism involved in leptin-induced depression of hypothalamic neurons (Spanswick, Smith, Groppi, Logan, & Ashford, 1997) and a subset of the hypothalamic ventral premammillary nucleus neurons (Williams et al., 2011). Activation of nonspecific cation channels is responsible for leptin-elicited excitation of the anorexigenic proopiomelanocortin neurons (Cowley et al., 2001; Hill et al., 2008) and the paraventricular nucleus neurons (Powis, Bains, & Ferguson, 1998) in the hypothalamus. It seems that TRPC channels are the type of the cation channels involved in leptin-induced excitation of proopiomelanocortin neurons (Qiu, Fang, Ronnekleiv, & Kelly, 2010) and the majority of ventral premammillary nucleus neurons (Williams et al., 2011). Moreover, leptin depolarizes rat nodose ganglia via inhibition of a K+ channel (Heldsinger, Grabauskas, Song, & Owyang, 2011). Added to this spectrum of ion channels modulated by leptin, our results indicate that activation of LepRb excites BLA neurons by depressing Kir channels. Kir channels include Kir2, Kir3 (GIRK channels), Kir6 (KATP channels), and K+ transport channels (Hibino et al., 2010). Our results suggest that Kir2 channels are unlikely to be involved because application of ML133, the selective inhibitor for Kir2 subfamily (Ford & Baccei, 2016; X. Huang et al., 2018; Kim et al., 2015; Sonkusare et al., 2016; H. R. Wang et al., 2011), by itself failed to alter the basal holding currents and exerted no significant effects on leptin-elicited inward currents. Our results further suggest that neither are KATP channels involved in leptin-elicited excitation of BLA neurons as bath application of the selective ATP channel blocker, glibenclamide, by itself did not change the holding currents in BLA neurons and failed to alter significantly leptin-mediated inward currents. These results suggest that there was no or sparse basal opening of KATP channels and leptin-mediated excitation of BLA neurons was not mediated by depressing KATP channels. Our results indicate that the GIRK type of Kir channels are required for leptin-elicited excitation of BLA neurons. GIRK channels comprise four isoforms, namely GIRK1, GIRK2, GIRK3 and GIRK4. Consistent with our results, amygdala abundantly express GIRK1, GIRK2 and GIRK3 without expression of GIRK4 (Karschin, Dissmann, Stuhmer, & Karschin, 1996; Sosulina, Schwesig, Seifert, & Pape, 2008). GIRK channels exist as predominantly heterotetramers of GIRK1, GIRK2 and/or GIRK3, or as homotetramers of the GIRK2 subunit (Hibino et al., 2010; Luscher & Slesinger, 2010), although there is general agreement that GIRK1/GIRK2 heterotetramers are the prototypical neural GIRK channel (Fernandez-Alacid, Watanabe, Molnar, Wickman, & Lujan, 2011; Luscher, Jan, Stoffel, Malenka, & Nicoll, 1997). LepRb-elicited excitation of BLA neurons might be mediated by suppression of the heterotetramers formed by GIRK1, GIRK2 and/or GIRK3, or the homotetramers of the GIRK2 subunit. Future experiments are required to identify the isoform(s) of GIRK channels involved in LepRb-elicited excitation of BLA neurons.

Whereas there are at least five splice variants of leptin receptors, the long form leptin receptor (LepRb) is the major form capable of signal transduction (Allison & Myers, 2014; Park & Ahima, 2014; Tartaglia, 1997). Our result showed that pretreatment of slices with and concomitant bath application of the selective LepRb antagonist, leptin tA, blocked leptin-elicited inward currents, indicating that leptin-induced excitation of BLA neurons is mediated by activation of LepRb, consistent with leptin-mediated enhancement of synaptic transmission (Murayama et al., 2019). Stimulation of LepRb activates various intracellular signaling pathways, including JAK2/signal transducer and activator of transcription 3 (STAT3), IRS/PI3K, SHP2/MAPK, and AMPK/ACC, in the central nervous system and peripheral tissues (Allison & Myers, 2014; Bjorbaek & Kahn, 2004; Chua, 2009; Park & Ahima, 2014). Among these signaling pathways, PI3K is required for leptin-mediated modulation of neural activity in a variety of tissues. For instance, PI3K is required for leptin-elicited excitation of the anorexigenic proopiomelanocortin neurons (Hill et al., 2008) and the majority of ventral premammillary nucleus neurons (Williams et al., 2011), whereas leptin-elicited inhibition of the nucleus tractus solitarus neurons (Williams & Smith, 2006) and a subset of ventral premammillary nucleus neurons (Williams et al., 2011) are also mediated by PI3K. PI3K is also responsible for leptin-mediated activation of KATP channels in neurons of dorsal motor nucleus of the vagus (Williams, Zsombok, & Smith, 2007). Leptin-induced inhibition of K+ channels in rat nodose ganglia requires PI3K and STAT3 signaling pathway (Heldsinger et al., 2011), whereas Src kinase is necessary for leptin-mediated enhancement of Kv2.1 channels (Baver et al., 2014). JAK2 and PI3K-PKB/Akt pathways are involved in leptin-induced unitary IPSC enhancement (Murayama et al., 2019). LepRb-JAK2-PI3K-PLCγ1 signaling pathway is responsible for leptin-mediated activation of TRPC channels in proopiomelanocortin neurons (Qiu et al., 2010). Our results indicate that LepRb-JAK2-PI3K signaling pathway is responsible for leptin-elicited excitation of BLA neurons and depression of the GIRK type of Kir channels.

How does PI3K modulate GIRK channels in the BLA? PI3K catalyzes the phosphorylation of PI(4,5)P2 (PIP2) to PI(3,4,5)P3 (PIP3), leading to a decreased level of PIP2. It is well-established that decreased level of PIP2 is responsible for Gq/11 receptor-elicited depression of GIRK channels (Cho, Lee, Lee, & Ho, 2005; Cho, Nam, Lee, Earm, & Ho, 2001; Keselman, Fribourg, Felsenfeld, & Logothetis, 2007; Lei, Jones, Talley, Garrison, & Bayliss, 2003; Mark & Herlitze, 2000; Meyer et al., 2001; Whorton & MacKinnon, 2011). Reduced levels of PIP2 mediated by PI3K in response to leptin could result in depression of GIRK channels, leading to excitation of BLA neurons.

What are the physiological significances underlying leptin-mediated excitation of BLA neurons ? Because appetite is related to the functions of both leptin (Park & Ahima, 2014, 2015; Tartaglia, 1997) and BLA (Holland & Petrovich, 2005; Petrovich & Gallagher, 2003; Sun et al., 2015; Y. Wang et al., 2006), we therefore studied the effect of microinjection of leptin in the BLA on food intake. Our results indicate that leptin-mediated activation of LepRbs in the BLA reduces food intake via activation of JAK2 and PI3K. Our results are consistent with a study showing that activation of calcium-sensing receptors in the BLA inhibits food intake (Guo, Jiang, Jin, Huang, & Sun, 2023), suggesting that elevation of intracellular Ca2+ level in the BLA neurons suppresses food intake. Our results indicate that leptin-mediated activation of LepRb suppresses GIRK channels to depolarize BLA neurons, which subsequently facilitates Ca2+ channels to increase intracellular Ca2+ levels resulting in reduction of food intake. This scenario is similar to the working mode of another type of Kir channels, KATP channels in β cells of the pancreas where depression of KATP channels results in membrane depolarization to augment Ca2+ channel opening to elevate intracellular Ca2+ leading to insulin release. Increases in insulin release would keep maintaining body weight, as leptin does. In addition to appetite, both leptin and BLA are involved in modulation of fear responses. Infusion of leptin into the LA has been shown to facilitate extinction of conditioned fear responses (W. Wang et al., 2015) and leptin is involved in neuronal communication for conditioned taste aversion (CTA) (Han et al., 2003) and alcohol use (Bach, Koopmann, & Kiefer, 2021). Other functions of leptin in the BLA could be modulate fear responses and alcohol intake, which awaits further investigation.

In the amygdala, LepRb is expressed in the BLA (Han et al., 2003; Udagawa et al., 2000; W. Wang et al., 2015) and LA (Udagawa et al., 2000; W. Wang et al., 2015), without expression in the CeA (Leshan et al., 2010; Udagawa et al., 2000). However, the CeA is innervated by the LepRb-expressing projections from the neurons in the ventral tegmental area (VTA) in mice (Leshan et al., 2010). An alternative possibility is that leptin microinjected into the BLA diffused to the CeA region and activated the presynaptic LepRb located on the VTA projections to reduce food intake instead. While we excluded the data with incorrect injection sites from analysis and used the acceptable microinjection volume (1 μL) and rate (0.2 μL/min) to limit the diffusion of leptin (Liu, Perez, Zhang, Lodge, & Lu, 2011; Myers, 1966) in the current study, we cannot eliminate the possibility that leptin-induced depression of food intake could be related to the CeA and VTA, given that both CeA (Izadi & Radahmadi, 2022) and VTA (Meye & Adan, 2014) are closely associated with food intake. Further studies are welcome to elucidate the roles and mechanisms of LepRb in the amygdala.

Acknowledgements

We thank Dr. John Watt for providing the Nalgene metabolic cages for the experiments regarding the food intake.

Funding

This work was supported by the National Institute Of General Medical Sciences (NIGMS) and National Institute Of Mental Health (NIMH) grant R01MH118258 to S.L.

Footnotes

Competing interests

None.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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