Estrogenic-dependent glutamatergic neurotransmission from kisspeptin neurons governs feeding circuits in females - PubMed (original) (raw)

Estrogenic-dependent glutamatergic neurotransmission from kisspeptin neurons governs feeding circuits in females

Jian Qiu et al. Elife. 2018.

Abstract

The neuropeptides tachykinin2 (Tac2) and kisspeptin (Kiss1) in hypothalamic arcuate nucleus Kiss1 (Kiss1ARH) neurons are essential for pulsatile release of GnRH and reproduction. Since 17β-estradiol (E2) decreases Kiss1 and Tac2 mRNA expression in Kiss1ARH neurons, the role of Kiss1ARH neurons during E2-driven anorexigenic states and their coordination of POMC and NPY/AgRP feeding circuits have been largely ignored. Presently, we show that E2 augmented the excitability of Kiss1ARH neurons by amplifying Cacna1g, Hcn1 and Hcn2 mRNA expression and T-type calcium and h-currents. E2 increased Slc17a6 mRNA expression and glutamatergic synaptic input to arcuate neurons, which excited POMC and inhibited NPY/AgRP neurons via metabotropic receptors. Deleting Slc17a6 in Kiss1 neurons eliminated glutamate release and led to conditioned place preference for sucrose in E2-treated KO female mice. Therefore, the E2-driven increase in Kiss1 neuronal excitability and glutamate neurotransmission may play a key role in governing the motivational drive for palatable food in females.

Keywords: 17-beta-estradiol; T-type calcium current; h-current; mouse; neuroscience; optogenetics; vGluT2.

© 2018, Qiu et al.

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Conflict of interest statement

JQ, HR, MB, SP, TS, MK, OR No competing interests declared, RP Reviewing editor, eLife

Figures

Figure 1.

Figure 1.. qPCR amplification assay illustrating the cycle threshold (CT) for the different neuropeptides and vGluT2 in Kiss1ARH neurons.

Cycle number is plotted against the normalized fluorescence intensity (∆RN) to visualize the PCR amplification of Tac2, Pdyn, Kiss1, Tacr3, Slc17a6 and the reference gene Gapdh in 5 cell Kiss1ARH pools obtained from oil- and E2-treated, OVX animals. The amplification efficiency for each primer pair is listed in Table 1. These efficiencies allowed us to use the comparative ∆∆CT methods for quantification. The cycle threshold (CT; horizontal dashed line) is the point in the amplification from which sample values were calculated using the 2-∆∆CT equation as described in the Methods.

Figure 2.

Figure 2.. Estradiol regulation of ion channel mRNA expression and excitability of Kiss1ARH Neurons.

(A) Quantitative real-time PCR measurements of Cacna1g (Cav3.1), HCN1, HCN2, Kiss1 and Pdyn mRNAs in Kiss1ARH neuronal pools (3 pools of 5 cells each per animal) from OVX oil- and E2-treated mice (n = 5–6 animals per group). Note that E2 increased the mRNA expression of Cacna1g, Hcn1, Hcn2, but as expected decreased the mRNA expression of Kiss1 and Pdyn in the same Kiss1 neuronal pools (for, Cacna1g, Unpaired t-test t(10) = 6.037, p<0.001; Hcn1, Unpaired t-test, t(8) = 10.13, p<0.0001; Hcn2, Unpaired t-test, t(8) = 3.420, p<0.01; Kiss1, Unpaired t-test, t(8) = 6.348, p<0.001; Pdyn, Unpaired t-test, t(8) = 6.118, p<0.001). (B) T-type calcium current and h-current density (pA/pF) in Kiss1ARH neurons from OVX oil- and E2-treated mice (for T-current, t(19) = 6.956, p<0.0001; for h-current, t(19) = 6.964, p<0.0001; n = 9–12 neurons from 8 animals). Current densities were measured as previously described (Zhang et al., 2013). (C) Example of rebound burst firing in Kiss1ARH neurons (left), which increased fast Na+ spiking with E2, and summary data (right) from oil- versus E2-treated females (n = 10 and 25 neurons, respectively). Rebound firing was measured as previously described (Zhang et al., 2013). Bar graphs represent the mean ±SEM, (Unpaired t-test, t(33) = 4.455, p<0.0001). **p<0.01, ***p<0.001.

Figure 3.

Figure 3.. Estradiol regulation of Slc17a6 mRNA expression in Kiss1ARH neurons.

Quantitative real-time PCR measurements of Slc17a6, Tac2 and Tacr3 mRNAs in Kiss1ARH neuronal pools (3–6 pools of 5 cells each per animal) from OVX oil- and E2-treated mice (n = 4–7 animals per group). Note that E2 increased the mRNA expression of Slc17a6, but as expected decreased the mRNA expression of Tac2 and Tacr3 in the same Kiss1 neuronal pools. Bar graphs represent the mean ±SEM (for Slc17a6, Unpaired t-test, t(8) = 4.522, p<0.001; Tac2, Unpaired t-test, t(8) = 6.350, p<0.001; Tacr3, Unpaired t-test, t(6) = 7.161, p<0.001). ***p<0.001.

Figure 4.

Figure 4.. Optogenetic activation of Kiss1-ARH neurons directly excites POMC and NPY/AgRP neurons via glutamate release.

(A) AAV1-DIO-ChR2:mCherry was bilaterally injected into ARH of Kiss1Cre: : _Npy_GFP mice or Kiss1Cre: : PomcEGFP (not shown). (B), schematic of experimental design; whole-cell, voltage-clamp (Vhold = −60 mV) recordings were made in POMCEGFP or NPYGFP neurons and a single pulse (intensity, 660 μW; 10 ms duration) of blue light (470 nm) was delivered to the ARH. (C), a fast inward current was recorded in both POMC and NPY neurons (yellow trace is average) that was antagonized by CNQX (10 µM) and AP5 (50 µM) (red trace). (D,E), the optogenetic (glutamate) response (green trace) was abrogated in the presence of TTX (1 µM, black trace) but rescued with the addition of the K+ channel blocker 4-AP (100 µM, magenta trace) in both POMC (D) and NPY (E) neurons, n = 3 and 2, respectively. Insets show the scRT-PCR post hoc identification of representative recorded Pomc and Npy neurons. RC, recorded cell; +, positive control (with reverse transcriptase); -, negative control (without reverse transcriptase); MM, molecular marker.

Figure 5.

Figure 5.. E2-treatment increases glutamate release from Kiss1ARH neurons onto POMC and NPY neurons.

(A) schematic of photostimulation of cells/terminals of Kiss1ARH neurons and recording from POMC or NPY/AgRP neurons. (B), illustration of a paired-pulse regime (two blue light pulses of 5 ms duration separated by 50 ms); fast glutamatergic inward currents (P1 and P2) were recorded to measure the probability of neurotransmitter release in postsynaptic neurons. (C), AAV1-DIO-ChR2:mCherry was bilaterally injected into ARH of Kiss1Cre::_Npy_GFP mice or Kiss1Cre::PomcEGFP mice. Using a paired-pulse regime, fast glutamatergic inward currents were recorded in POMCEGFP neurons (Vhold = −60 mV) from both oil-treated, OVX (upper trace, black) and E2-treated, OVX (lower trace, red) females. The averaged responses (50 sweeps) are shown. (D) E2-treatment significantly decreased the paired-pulse ratio (P2/P1; indicating that there was a higher probability of glutamate release from Kiss1Cre:GFP-ChR2 neurons (Unpaired t-test, t(20) = 4.184, p<0.001). (E) similarly using a paired-pulse regime, fast glutamatergic inward currents were recorded in NPYGFP neurons (Vhold = −60 mV) from both oil-treated, OVX (upper trace, black) and E2-treated, OVX (lower trace, red) females. The averaged responses (50 sweeps) are shown. (F) E2-treatment significantly decreased the paired-pulse ratio (P2/P1) indicating that there was a higher probability of glutamate release from Kiss1Cre:GFP-ChR2 neurons (Unpaired t-test, t(14) = 3.255, p<0.01). **p<0.01, ***p<0.001.

Figure 6.

Figure 6.. E2 treatment increases the probability of glutamate release from Kiss1ARH neurons onto Kiss1AVPV/PeN neurons.

(A1-A2) Photomicrographs showing the pronounced projections of ChR2:mCherry fibers to the preoptic area including the PeN following bilateral injections of AAV1-DIO-ChR2:mCherry in the ARH of Kiss1CreGFP V2 mice (note that the GFP was not visible in the V2 animals). Therefore, some POA sections from the same animals were stained for kisspeptin using the Caraty 564 antibody and revealed immunoreactive Kiss1 neurons in the PeN (green cells) (A3). Essentially none of the POA somas including the Kiss1 cells expressed ChR2-mCherry. Scale bars = 100 µM (A1,A2); 50 μm (A3). (B) schematic of photostimulation of the terminals of Kiss1ARH neurons and recording of Kiss1AVPV/PeN neurons. (C) following AAV1-DIO-ChR2:YFP (or mCherry) injection into the ARH, a fast inward current was recorded in Kiss1AVPV/PeN neurons following blue light stimulation (green trace). The response was antagonized by CNQX (10 µM) and AP5 (50 µM) (not shown) and was abrogated in the presence of TTX (1 µM, black trace) but rescued with the addition of the K+ channel blocker 4-AP (100 µM, magenta trace; n = 4 neurons). (D) using a paired-pulse regime (two blue light pulses of 5 ms duration separated by 50 ms), fast glutamatergic inward currents were recorded in Kiss1AVPV/PeN neurons (Vhold = −60 mV) from both oil-treated, OVX (upper trace, black) and E2-treated, OVX (lower trace, red) females. The averaged responses (50 sweeps) are shown. (E) E2-treatment significantly decreased the paired-pulse ratio (P2/P1) indicating that there was a higher probability of glutamate release from arcuate Kiss1Cre:ChR2 neurons (Unpaired t-test, t(14) = 4.748, p<0.001). ***p<0.001. Inset shows scRT-PCR post hoc identification of representative recorded Kiss1AVPV/PeN neurons. RC, recorded cell; +, positive control (with reverse transcriptase); -, negative control (without reverse transcriptase); MM, molecular marker.

Figure 7.

Figure 7.. High frequency stimulation of Kiss1Cre:GFP neurons inhibits NPY neurons but excites POMC neurons.

(A) Schematic of photostimulation of the terminals of Kiss1ARH neurons and recording of POMC or NPY/AgRP neurons. (B) high-frequency optogenetic stimulation (20 Hz, 10 s) of Kiss1Cre:GFP neurons/fibers, which were labeled with AAV-DIO-ChR2-mCherry, generated a slow EPSC in POMCEGFP neurons (in the presence of CNQX, 10 μM and AP5, 50 μM). (C) in current clamp the same stimulus depolarized and increased the firing frequency of POMC neurons. (D) in voltage clamp (Vhold = −60 mV) high-frequency stimulation of Kiss1Cre:GFP-ChR2 neurons/fibers generated a slow IPSC in NPYGFP neurons (CNQX, 10 μM; AP5, 50 μM). (E) in current clamp the same stimulus hyperpolarized and inhibited the firing frequency of NPY neurons. (F) summary of the effects of high-frequency stimulation of Kiss1Cre:GFP-ChR2 neurons on POMC neurons (depolarized 9.5 ± 1.5 mV, n = 7) and on NPY neurons (hyperpolarized 6.4 ± 1.4 mV, n = 6). The responses (change in membrane potential, Delta Vm) were significantly different in POMC versus NPY/AgRP neurons (Unpaired t-test, t (11)=7.685, p<0.0001). ***p<0.001.

Figure 8.

Figure 8.. Metabotropic glutamate receptor expression in POMC and NPY neurons.

(A) representative gels illustrating mRNA expression of Pomc, Grm1 (encoding mGluR1) and Grm5 (encoding mGluR5) in individual POMCEGFP neurons. The expected base pair (bp) sizes for Pomc, Grm1 and Grm5 are 200 bp, 186 bp, 247 bp, respectively. (B) bar graphs summarizing the percentage (mean ±SEM) of POMCEGFP cells (24 cells each from 5 animals) that expressed Pomc, Grm1, and Grm5 mRNAs. (C) representative gels illustrating mRNA expression of NPY, Grm2 (encoding mGlur2) and Grm7 (encoding mGlur7) in individual NPYGFP neurons. The expected base pair (bp) sizes for Npy, Grm2 and Grm7 are 182 bp, 145 bp, 109 bp, respectively. (A,C) exclusion of reverse transcriptase (-RT) in a reacted cell was used as negative control. RNA extracted from medial basal hypothalamic tissue was also included as positive (+, with RT) and negative (-, without RT) tissue controls. (D) bar graphs summarizing the percentage (mean ± SEM) of NPYGFP cells (24 cells each from 5 animals) that expressed Npy, Grm2, and Grm7 mRNAs.

Figure 9.

Figure 9.. Metabotropic glutamate response is excitatory in POMC neurons.

(A) metabotropic glutamate receptor 1/5 agonist DHPG (50 µM) depolarized and induced firing of a POMC neuron. (B) Rapid bath application of DHPG induced an inward current in the presence of fast sodium channel and ionotropic glutamatergic blockade, Vhold = −60 mV. (C) voltage ramps from 0 to −100 mV were applied (over 2 s) before and during the treatment with DHPG, showed a reversal at −30 mV. (D) summary of the depolarizing effects of DHPG in POMC neurons in oil-treated and E2-treated, OVX females. There was not a significant difference in the response (Unpaired t-test, t(13) = 0.4168, p=0.6831).

Figure 10.

Figure 10.. Metabotropic glutamate response is inhibitory in NPY neurons and augmented by E2.

(A) the group II mGluR agonist DCG-IV (10 µM) hyperpolarized a NPYGFP neuron. (B) DCG-IV activated an outward current in a NPYGFP neuron in the presence of fast sodium channel (TTX, 0.5 μM), ionotropic glutamatergic (CNQX, 10 μM and AP5, 50 μM) and GABAergic (picrotoxin, 100 μM) blockers (V hold = −60 mV). (C) I-V relationship for DCG-IV- induced current showed a reversal at EK+ (−95 mV). (G) DCG-IV was more efficacious to hyperpolarize NPY neurons in E2-treated versus oil-treated, OVX females (Unpaired _t_-test, t(32) = 2.261, p=0.031). *p<0.05. (D) the mGluR7 allosteric agonist AMN082 (10 µM) hyperpolarized and inhibited firing of an NPYGFP neuron. (E) AMN082 generated a 25 pA outward current in a NPYGFP neuron in the presence of fast sodium channel and ionotropic glutamatergic blockade (V hold = −60 mV). (F) I-V relationship for AMN082-induced current showed a reversal close to EK+. (H) AMN082 was more efficacious than DCG-IV to hyperpolarize NPY neurons in E2-treated versus oil-treated, OVX females (Unpaired _t_-test, t(17) = 3.747, p=0.002). (I) Quantitative real-time PCR measurements of Grm7 mRNA in NPYGFP neuronal pools (4 pools of 5 cells each per animal) from oil- and E2-treated, OVX mice (n = 4–5 animals per group). Bar graphs represent the mean ± SEM (Unpaired _t_-test, t(7)=6.020, p=0.0005). ***p<0.001.

Figure 11.

Figure 11.. Deletion of Slc17a6 in Kiss1ARH neurons attenuates the slow EPSP in Kiss1ARH neurons from E2-treated mice.

(A) representative gels illustrating mRNA expression of Slc17a6, Kiss1 and Tac2 in Slc17a6 KO Kiss1ARH neurons and in control Kiss1ARH cells. The expected base pair (bp) sizes for Slc17a6, Kiss1 and Tac2 are 194, 120 and 147 bp, respectively. RNA extracted from the medial basal hypothalamic tissue was used as positive (+, with RT) and negative (-, without RT) tissue controls. MM, molecular marker. (B) Experimental protocol: AAV1-DIO-ChR2:mCherry was bilaterally injected into ARH of Kiss1Cre:GFP control and Slc17a6 KO mice, followed by high-frequency photostimulation of Kiss1ARH neurons/terminals and recording of Kiss1ARH neurons. (C) high-frequency optogenetic stimulation (20 Hz, 10 s) generated a slow EPSP in an arcuate Kiss1Cre:GFP neuron from OVX, control Kiss1 mice. (D), high-frequency response (slow EPSP) in arcuate Kiss1Cre:GFP neurons from OVX, Kiss1Cre:GFP::Slc17a6lox/∆ (KO) mice.(E) high-frequency response (slow EPSP) in arcuate Kiss1Cre:GFP neuron from E2-treated, OVX control Kiss1 mice. Inset shows full amplification of sEPSP. (F) high-frequency response in arcuate Kiss1Cre:GFP neuron from E2-treated, OVX Kiss1Cre:GFP::Slc17a6lox/∆ mice. Inset shows full amplification of sEPSP. (G) summary of the effects of vGluT2 deletion on slow EPSP amplitude: (one-way ANOVA, effect of treatment, F(3, 50)=14.13, p<0.0001; Newman-Keuls’ Multiple-comparison test, *** or ###, indicates p<0.005). Although knockout of vGluT2 did not significantly diminish the slow EPSP amplitude in OVX females, it did attenuate the response in E2-treated, OVX females (Unpaired t-test, t (25)=2.735, p=0.0113). a-a, p<0.05.

Figure 12.

Figure 12.. Deletion of Slc17a6 in Kiss1ARH neurons abrogates fast glutamatergic responses in Kiss1AVPV/PeN, POMC and NPY/AgRP neurons.

(A) Experimental protocol: AAV1-DIO-ChR2:mCherry (or YFP) was bilaterally injected into ARH of Kiss1Cre:GFP mice. Thereafter, low-frequency photostimulation of the terminals of Kiss1ARH neurons were done, and postsynaptic responses in Kiss1AVPV/PeN, POMC or NPY/AgRP neurons were recorded. (B,C) whole-cell, voltage clamp (Vhold = −60 mV) recordings in Kiss1AVPV/PeN neurons show that low-frequency optogenetic stimulation (0.5 Hz) evoked fast glutamatergic postsynaptic responses in control female Kiss1Cre mice (B, red trace), but failed in Kiss1AVPV/PeN cells (n = 10) from Kiss1Cre:GFP::Slc17a6lox/∆ mice (C, black trace). (D,E) and F,G) similarly, the response could be induced in POMC neurons (D, yellow trace) or NPY/AgRP neurons (F, green trace) from control Kiss1Cre:GFP mice, but abrogated in POMC neurons (E, black trace) (n = 28) or NPY/AgRP neurons (G, black trace) (n = 30) from Kiss1Cre:GFP::Slc17a6lox/∆ mice. (H) Experimental protocol: high-frequency photostimulation of the terminals of Kiss1ARH neurons and recording of POMC or NPY/AgRP neurons. (I) high-frequency stimulation (20 Hz, 10 s) of arcuate Kiss1 neurons from Kiss1Cre:GFP::Slc17a6lox/∆ mice evoked a small inward current (2.8 ± 0.5 pA, n = 7) in POMC neurons (identified post hoc by scRT-PCR, gel inset). (J) likewise, high-frequency stimulation evoked a small outward current (4.0 ± 1.6 pA, n = 4) in NPY/AgRP neurons (identified post hoc by scRT-PCR, gel inset). Insets show scRT-PCR post hoc identification of representative recorded POMC and NPY neurons. RC, recorded cells; +, positive tissue control reacted with RT; -, negative tissue control reacted without RT; MM, molecular marker.

Figure 13.

Figure 13.. Kisspeptin and RFRP-3 inhibit NPY neurons.

(A,B) kisspeptin (200 nM) inhibited the firing and hyperpolarized NPY neurons even in the presence of GABAA blocker bicuculline (BIC) (10 μM). (C) Similar to GABAB receptor agonist baclofen (10 μM), kisspeptin induced an outward, albeit smaller, current with a reversal potential at EK+ (−90 mV). (D) scRT-PCR expression of Npffr1 in NPY/AgRP neurons. (E) RFRP-3 (10 μM), selective agonist for NPFFR1/NPFFR2, hyperpolarized and inhibited firing in NPY neurons. (F) the I/V plots of the RFRP-3 current showed a reversal potential close to EK+ (−85 mV).

Figure 14.

Figure 14.. Kisspeptin excites POMC neurons by activating a non-selective cation conductance.

(A) representative gel illustrating the scRT-PCR expression of Kiss1r (GPR54) transcript in POMC neurons. (B) kisspeptin (200 nM) depolarized and increased firing of POMC neurons (n = 8). (C) I/V (digital subtraction of control I/V from I/V with kisspeptin using a Cs+-based internal solution; see Materials and Methods) showed that kisspeptin activated a non-selective cationic channel that reversed at −10 mV.

Figure 15.

Figure 15.. Protocol for inducing CPP with sucrose.

The protocol for conditioning and preference testing consisted of four phases over the course of 11 days (sucrose habituation, a baseline place preference (BPP) test, sucrose conditioning, and a conditioned place preference (CPP) test). Food-motivated behavior was assessed during the dark cycle using an unbiased procedure. On the day before BPP, Day −1, sucrose habituation occurred where mice received sucrose pellets overnight (O/N) in their home cage to prevent neophobia. The initial BPP (black vs. white chamber) was assessed on Day 1 in a three-chamber place preference apparatus and the chamber pairing was assigned in an unbiased manner. During sucrose conditioning, mice were given access to sucrose-filled (CS+, Days 2, 4, 6, 8) or empty (CS-, Days 3, 5, 7, 9) lids on alternating days. Mice were given access to sucrose-filled lids in one chamber (e.g. white), then on alternating days they were presented with empty lids in the other chamber (e.g. black). Mice were tested for acquisition of a CPP to sucrose on Day 10, which was indicated by increased time spent in the sucrose-conditioned chamber. Animals were fed ad lib standard low-fat chow in their home cage throughout the study. For cyclical estradiol treatment, animals were given a priming (0.25 μg) and a surge (1.5 μg) dose of 17β-estradiol Benzoate (E2) at 9 AM prior to the BPP and prior to the CPP as indicated. During the sucrose-conditioning (phase 3), the animals were treated twice with a 1 μg maintenance dose of E2.

Figure 16.

Figure 16.. Female mice lacking Slc17a6 in Kiss1ARH neurons develop a conditioned place preference for sucrose.

(A) Time spent in sucrose-paired chamber by control Kiss1 female mice (n = 8), Slc17a6 Het (n = 4) and Slc17a6 KO Kiss1 females (n = 8) was measured during the Pretest (Day 1, Baseline Place Preference) and the Posttest (Day 10, Conditioned Place Preference). All animals were OVX and E2-treated, and had free access to standard mouse chow in their home cage throughout the study. After sucrose conditioning, Slc17a6 KO mice developed a preference for the sucrose-paired chamber (Bonferroni post hoc test, p=0.001). Slc17a6 Het mice displayed a trend to develop a preference for the sucrose-paired chamber (Bonferroni post hoc test, p=0.086). Control Kiss1Cre female mice, however, failed to develop a preference (Bonferroni post hoc test, p=0.619). [Also, see Figure 16—figure supplement 1A for comparison between E2-treated, OVX Kiss1 female and intact Kiss1 male mice]. Two-way ANOVA: main effect of experimental group (F(2,17) = 0.298, p=0.746), main effect of protocol day (F(1,17) = 20.34, p=0.0003), and interaction (F(2,17) = 2.33, p=0.128); **p<0.01.(B) Sucrose consumption during the CPP. Sucrose intake (mg) was measured during the four sucrose-paired days (Days 2, 4, 6, and 8). Slc17a6 KO mice slightly increased their sucrose intake on Day 6 and this was significantly increased by Day 8 (Bonferroni post hoc test, p<0.0001). Slc17a6 Het mice displayed a smaller, but significant increase in sucrose intake on Day 6 (Bonferroni post hoc test, p=0.0464). [Also, see Figure 16—figure supplement 1B for comparison between E2-treated, OVX Kiss1 females and intact Kiss1 males]. Two-way ANOVA: main effect of experimental group (F(2,17) = 3.788, p=0.0436), main effect of protocol day (F(3,51) = 12.75, p<0.0001), and interaction (F(6,51) = 5.763, p<0.0001). *p<0.05, Het mice versus Kiss1 control; ***p<0.001, Slc17a6 KO mice versus Kiss1 control. (C) Body weight-gain during the ten-day CPP period. Despite that both the Slc17a6 KO and Het mice gained weight in comparison to control Kiss1 mice, only Slc17a6 Het mice were significantly different (Bonferroni post hoc test, p=0.0312, Slc17a6 Het vs Kiss1 control; p=0.066, Slc17a6 KO vs Kiss1 control;). One-way ANOVA: main effect of experimental group (F(2,17) = 5.232, p=0.017). *p<0.05, Het mice versus Kiss1 control. (D) Quantitative real time PCR measurement of Slc17a6 in Kiss1ARH neuronal pools from control Kiss1Cre:GFP mice (5 Kiss1 neurons in each pool and 5 pools from each of 5 animals) and Slc17a6 Het Kiss1 mice (5 Kiss1 neurons in each pool and 5 pools from each of 4 animals). Slc17a6 KO Kiss1 mice did not express Slc17a6 in Kiss1ARH neurons. (Unpaired t-test, t(7) = 5.791, p=0.0007). ***p<0.001, Het mice versus Kiss1 control.

Figure 16—figure supplement 1.

Figure 16—figure supplement 1.. Studies documenting that intact male mice develop a conditioned place preference for sucrose.

(A) Time spent in sucrose-paired chamber by intact Kiss1Cre male mice (n = 15) and E2-treated, OVX Kiss1Cre female mice (n = 8) was measured during the Pretest (Day 1, Baseline Place Preference; BPP) and the Posttest (Day 10, Conditioned Place Preference; CPP). Male mice did not receive any treatment, whereas female mice were OVX and E2-treated. All animals were handled daily prior to the BPP/CPP tests. After sucrose conditioning intact Kiss1 males, which expressed lower levels of vGluT2 in Kiss1ARH neurons in comparison to E2-treated, OVX females, developed a preference for the sucrose-paired chamber (Bonferroni post hoc test, p<0.01). E2-treated, OVX Kiss1 female mice failed to develop a preference for the sucrose-paired chamber (Bonferroni post hoc test, p=0.124). Two-way ANOVA: main effect of experimental group (F(1, 21)=0.823, p=0.375), main effect of protocol day (F(1, 21)=14.89, p<0.001), and interaction (F(1, 21)=0.457, p=0.506); **p<0.01. (B) Sucrose consumption (mg) during the CPP was measured during the four sucrose-paired days (Days 2, 4, 6, and 8). The intact males consumed significant more sucrose on Day 2 of the test as compared to E2-treated, OVX females. Two-way ANOVA: main effect of experimental group (F(1, 21)=8.373, p<0.01), main effect of protocol day (F(3,63) = 2.49, p=0.068), and interaction (F(3,63) = 3.062, p<0.05); ***p<0.001. During this time, the animals had free access to standard low-fat mouse chow in their home cage.

Figure 17.

Figure 17.. Working Model. KNDy (Kisspeptin, NKB, Dynorphin) neurons in the ARH express CaV3 (IT) and HCN (Ih) channels (currents) that are upregulated by E2 and contribute to increased excitability of Kiss1ARH neurons.

Kiss1AVPV/PeN neurons also express CaV3 (IT), HCN (Ih) and Nav (INaP) channels that are highly up-regulated by E2 along with Kiss1 mRNA expression. Notably, E2 induces spontaneous, repetitive burst firing activity in Kiss1AVPV/PeN neurons necessary for the release of GnRH (Wang et al., 2016; Zhang et al., 2015). E2 also directly excites POMC neurons via inhibition of GIRK current, but inhibit NPY/AgRP neurons via activation of GIRK current (Kelly and Rønnekleiv, 2015). These congruent actions of E2 on POMC and NPY/AgRP neurons contribute to the control of homeostatic feeding. High frequency photo-stimulation (focal light stimulation of channel rhodopsin, ChR2) in Kiss1ARH neurons releases glutamate to further excite POMC neurons via mGluRs group I and inhibit NPY/AgRP neurons via mGluRs group II/III; and excite Kiss1AVPV/PeN neurons via NMDA/AMPA receptors. Ablating Slc17a6 from Kiss1ARH neurons, results in the abrogation of glutamate release onto POA and ARH neurons. The lack of glutamate release from Kiss1ARH neurons appears to have little or no effect on estrous cyclicity, an indication that the direct effects of E2 to increase the excitability of Kiss1AVPV/PeN neurons is sufficient to drive the reproductive cycle. However, E2-treated Slc17a6 KO Kiss1 mice develop a condition place preference for sucrose indicative of positive motivational effect of sucrose in these females.

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