High-frequency stimulation-induced peptide release synchronizes arcuate kisspeptin neurons and excites GnRH neurons
Pulsatile secretion of gonadotropin-releasing hormone 1 ( GnRH ) , and consequently pulsatile release of pituitary luteinizing hormone ( LH ) , is required for normal reproductive function , and there is increasing evidence that kisspeptin ( Kiss1 ) neurons in the hypothalamic arcuate nucleus ( ARH ) are a component of the “GnRH pulse generator” ( Navarro et al . , 2009; Navarro et al . , 2011a; Lehman et al . , 2010a; Okamura et al . , 2013 ) . The “GnRH pulse generator” consists of bursts of multiunit activity ( MUA ) in the arcuate nucleus that are timed with pulses of LH secretion , and this phenomenon has been demonstrated in the monkey ( Knobil , 1989 ) , goat ( Wakabayashi et al . , 2010; Okamura et al . , 2013 ) , and rat ( Kimura et al . , 1991; Kinsey-Jones et al . , 2008 ) . In mammals , two major populations of kisspeptin-synthesizing neurons exist - one in the anterior preoptic area and the other in the ARH ( Lehman et al . , 2010b ) . In rodents , the anterior preoptic cell group is a periventricular continuum within the anteroventral periventricular nucleus ( AVPV ) and the periventricular preoptic nucleus ( PeN ) and is responsible for the GnRH/LH surge ( Clarkson and Herbison , 2006 ) . Both the AVPV/PeN and ARH Kiss1 neurons ( Kiss1AVPV/PeN; Kiss1ARH ) co-express other peptide and amino acid neurotransmitters , and the Kiss1ARH neurons co-synthesize and potentially co-release neurokinin B ( NKB ) and dynorphin ( Dyn ) in most mammals ( Goodman et al . , 2007; Navarro et al . , 2011b; Bartzen-Sprauer et al . , 2014 ) , forming the basis for the “KNDy neuron” terminology ( Lehman et al . , 2010a ) . Importantly , Kiss1 , NKB and their respective receptors are absolutely essential for pubertal development and the control of reproduction ( Seminara et al . , 2003; d'Anglemont de Tassigny et al . , 2007; Topaloglu et al . , 2009 ) . Morphological studies have provided evidence that Kiss1ARH neurons can communicate with each other ( Lehman et al . , 2010a; Rance et al . , 2010; Navarro et al . , 2011a; Navarro et al . , 2011b ) , and NKB/tachykinin receptor 3 ( Tacr3 ) and Dyn/κ-opioid receptor ( KOR ) signaling have been proposed to play a critical role in the generation of episodic GnRH/LH pulses via direct and synchronized action on GnRH nerve terminals in the median eminence ( Navarro et al . , 2009; Ohkura et al . , 2009; Wakabayashi et al . , 2010 ) . Anatomical tract-tracing studies have shown that Kiss1ARH neurons are bilaterally interconnected within the ARH and project to GnRH terminals in the median eminence and to Kiss1AVPV/PeN neurons ( Rance et al . , 2010; Yip et al . , 2015 ) . Therefore , we hypothesized that Kiss1ARH neurons could coordinate ( synchronize ) their activity to drive GnRH secretion via these contralateral projections . Recent findings also indicate that Kiss1ARH neurons co-express the vesicular glutamate transporter 2 ( vGluT2 ) and release the neurotransmitter glutamate ( Cravo et al . , 2011; Nestor et al . , 2016 ) , which could also provide an excitatory drive to hypothalamic neurons ( Van den Pol et al . , 1990 ) . Currently , we explored the synaptic mechanisms by which Kiss1ARH neurons synchronize their burst firing activity and how their unified activity was conveyed to the rostral Kiss 1AVPV/PeN neurons and hence excitation of GnRH neurons . We used a combination of whole-cell patch recording , optogenetics , molecular pharmacology and single cell RT-PCR to elucidate the underlying synaptic mechanisms at multiple sites . We discovered that high-frequency photoactivation of Kiss1ARH neurons induced a NKB-mediated slow excitatory postsynaptic potential ( EPSP ) , which was modulated by co-released dynorphin , and caused synchronization of Kiss1ARH neurons . Interestingly , activation of Kiss1ARH neurons co-released glutamate that excited the rostral Kiss1AVPV/PeN neurons . The combination of synchronization and unified excitation of Kiss1AVPV/PeN neurons resulted in robust excitation of GnRH neurons .
To investigate the firing properties of Kiss1 neurons necessary for peptide release , we did whole-cell recordings of photostimulated ARH and AVPV/PeN Kiss1 neurons expressing ChR2:YFP in slices obtained from OVX females ( Figures 1A–C , 2A–D ) . These neurons were confirmed to express Kiss1 mRNA based on scRT-PCR ( Figure 2D ) . High fidelity responses to light ( 470 nm ) were measured as evoked inward currents in voltage clamp ( Vhold = -60 mV ) or depolarizations in current clamp ( Figures 1 and 2 ) . A 10-s photostimulation at 1 or 5 Hz had no lasting effect on Kiss1ARH neuron activity; however , with 10 Hz stimulation there was a prominent depolarization of ~10 mV that peaked about a minute later , and it was accompanied by a train of action potentials during that time ( Figure 2E ) . We also varied the duration of photostimulation at 20 Hz , which revealed that 1 s was ineffective but with 5 or 10 s of stimulation there was again a prominent depolarization accompanied by a barrage of action potentials that continued for about a minute after stimulation ( Figure 2F ) . The results from varying frequency and/or duration of photostimulation revealed that a maximum depolarization of 22 ± 1 . 4 mV was obtained with a 10 s stimulation at 20 Hz ( Figure 2G , H ) . We refer to this depolarization as a slow excitatory postsynaptic potential ( EPSP ) . To determine whether the slow EPSP is a unique property of Kiss1ARH neurons , we also tested the AVPV/PeN population of Kiss1 neurons with 10-s stimulation at 20 Hz ( Figure 2I ) ; these neurons had a transient hyperpolarization after stimulation and a return to continuous firing ~30 s after the stimulation . The depolarization of Kiss1ARH neurons was also distinct from POMC and AgRP/NPY neurons in the ARH , even though they are probably all derived from a common precursor ( Sanz et al . , 2015 ) . The relatively quiescent POMC neurons fired action potentials during the 10-s , 20-Hz stimulation and exhibited a prolonged hyperpolarization afterwards ( Figure 2J ) ; whereas the spontaneously active AgRP neurons exhibited a brief pause in firing in conjunction with a slight hyperpolarization ( Figure 2K ) . Thus , Kiss1ARH neurons are unique among the neurons tested in the generation of the slow EPSP . Given that the expression of Kiss1 , dynorphin , NKB and Tacr3 are all down-regulated in kisspeptin neurons by E2-treatment ( Smith et al . , 2005; Gottsch et al . , 2009; Navarro et al . , 2011a; Ruiz-Pino et al . , 2012 ) , we asked the question whether the slow EPSP would also be reduced by E2 or even vary during the ovulatory cycle . The slow EPSP was induced in slices obtained from diestrous or proestrous animals or OVX females treated with either oil vehicle or E2 . The slow EPSP was the most robust in OVX females ( 13 . 8 ± 1 . 6 mV , n= 17 ) , which was to be expected if this current was peptidergic in nature—i . e . , correlates with the up-regulation of NKB ( Figure 2L ) . Moreover , in vivo E2 treatment resulted in a significant reduction of the slow EPSP amplitude ( 4 . 8 ± 1 . 1 mV , n = 9 , p<0 . 001 ) . Importantly , the slow EPSP was also of greater magnitude in Kiss1ARHneurons from diestrous animals ( 9 . 2 ± 1 . 1 mV , n=14 ) and significantly reduced in Kiss1ARH neurons from proestrous animals ( 3 . 3 ± 0 . 8 mV , n=15 , p<0 . 01 ) ( Figure 2L ) . 10 . 7554/eLife . 16246 . 003Figure 1 . High fidelity response to light ( 470 nm ) stimulation of ARH Kiss1 ChR2-expressing neurons . ( A–B ) Examples of evoked inward currents in voltage-clamp ( A ) and depolarization in current-clamp ( B ) with varied light intensity ( 33–330 µW ) . ( C ) Trains of light pulses ( 0 . 9 mW , 10 ms pulse-width ) at various frequencies and the resulting action potentials in the same neuron from A and B . DOI: http://dx . doi . org/10 . 7554/eLife . 16246 . 00310 . 7554/eLife . 16246 . 004Figure 2 . Slow excitatory postsynaptic potential ( slow EPSP ) is frequency and duration dependent . ( A ) Illustration of experimental approach . ( B–C ) , Overlay of epifluorescence ( YFP ) and differential interference contrast ( DIC ) images during whole-cell recording ( pipette tip can be seen ) from Kiss1 neurons in ARH ( B ) and AVPV ( C ) brain slices from a Kiss1-Cre::Ai32 mouse ( scale bar = 200 µm ) . ( B , C ) Insets show high power magnification of labeled cells in each area ( scale bar = 10 µm ) . ARH , hypothalamic arcuate nucleus; AVPV , anteroventral periventricular nucleus . ( D ) Confirmation of Kiss1 expression in YFP-expressing neurons from Kiss1-Cre::Ai32 . Representative gel illustrating single-cell RT-PCR identification of Kiss1 mRNA following whole-cell recording in ChR2-YFP neurons . RNA extracted from the medial basal hypothalamic tissue control ( TC ) was included as positive ( + , with RT ) and negative ( - , without RT ) controls . MM , molecular marker . ( E ) Slow EPSP induced by a 10-s photostimulation ( light intensity 0 . 9 mW and pulse duration , 10 ms ) with varied frequencies in the same neuron . The insets show the measurement of slow EPSP after low-pass filtering . 1–5 Hz photostimulation did not induce any post-stimulus depolarization ( arrow , middle trace ) ; but ≥10 Hz stimulation generated a significant post-stimulus depolarization ( double arrow , lower trace ) . ( F ) , Examples of synaptic responses induced by a train of stimuli ( 0 . 9 mW , 10 ms pulse-width ) delivered at 20 Hz with varied duration in the same Kiss1ARH neuron . ( G–H ) , Bar graphs summarizing slow EPSP responses induced by a train of stimulation at 10 , 20 , 30 and 50 Hz with duration of 10 s ( G ) and by a 20 Hz-stimulation delivered at 5 , 10 and 15 s ( H ) , current-clamped to -70 mV . The slow EPSP was larger when induced by 10-s and 20 Hz photostimulation ( one-way ANOVA , effect of treatment , F ( 3 , 53 ) = 9 . 912 , p<0 . 0001 ( G ) ; F ( 2 , 44 ) = 12 . 69 , p<0 . 0001 ( H ) ; Newman-Keuls’s Multiple-comparison test , *** , ** , * indicates p<0 . 005 , 0 . 01 and 0 . 05 , respectively . ( I–K ) , Slow EPSP is unique to arcuate Kiss1 neurons . ( I ) Photostimulation induced auto-inhibition in AVPV Kiss1 neuron , ( J ) ARH POMC neuron and ( K ) AgRP neuron from Kiss1-Cre::Ai32 , POMC-Cre::Ai32 and AgRP-Cre::Ai32 mice , respectively . ( L ) Light evoked slow EPSP in Kiss1ARH neurons was reduced by E2 treatment and varied during the ovulatory cycle . Bar graphs summarizing slow EPSP responses induced by 20 Hz , 10 s photostimulation in slices obtained from diestrous or proestrous females , or OVX females treated with either oil vehicle or E2 that had received injection of AAV-DIO-ChR2:YFP into ARH . Slow EPSP was larger in low estrogen states ( one-way ANOVA , effect of treatment , F ( 3 , 51 ) = 15 . 43 , p<0 . 0001; Newman-Keuls’s Multiple-comparison test . **** , ** indicates p<0 . 001 and 0 . 01 , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 16246 . 004 To investigate the properties of the slow EPSP , we used a ratio method in which the same neuron was stimulated ( 10 s , 20 Hz ) and then tested again 10 min later after experimental manipulation ( Figure 3A ) . Under control conditions the R2/R1 ratio was 0 . 65 ± 0 . 04 ( n = 12 ) ( Figure 3A , F ) . The slow EPSP was abolished by perfusing CSF containing low Ca2+ ( 0 . 5 mM ) and high Mg2+ ( 10 mM ) or tetrodotoxin ( TTX , 0 . 5 µM ) ( Figure 3B , D , F ) . We could rescue TTX blockade of the light-induced response with the addition of K+ channel blockers 4-aminopyridine ( 4-AP , 0 . 5 mM ) and tetraethylammonium ( TEA , 7 . 5 mM ) to facilitate ChR2- mediated depolarization of nerve terminals and neurotransmitter release ( Cousin and Robinson , 2000; Petreanu et al . , 2009 ) , arguing that the response is dependent on direct pre-synaptic input from neighboring Kiss1 neurons ( Figure 3C , F ) . Although the majority of Kiss1ARH neurons are glutamatergic ( Cravo et al . , 2011 ) , release glutamate locally in ARH ( Nestor et al . , 2016 ) and the fast EPSP was blocked by CNQX ( 10 µM ) and AP5 ( 50 µM ) , the slow EPSP was unaffected by these ionotropic glutamate receptor antagonists ( Figure 3E , F ) . Action potentials were abrogated by dialyzing Kiss1ARH neurons with the Na+ channel blocker QX314 ( 0 . 5 mM in the internal solution ) ( Isaac and Wheal , 1993 ) . The expression of the slow EPSP was reduced but not blocked by QX314 ( Figure 4A , E ) . The reduction in the amplitude of the slow EPSP was probably due to QX314 suppression of calcium currents in the Kiss1 neurons ( Hu et al . , 2002 ) . Moreover , postsynaptic electrical stimulation ( 20 Hz , 10 s ) of individual neurons , even in the presence of 4-AP and TEA , did not mimic the light-induced EPSP ( Figure 4B , C , E ) , suggesting that the response was not dependent on the generation of action potentials in the cell soma . 10 . 7554/eLife . 16246 . 005Figure 3 . Slow EPSP is dependent on direct synaptic input from neighboring Kiss1 neurons . ( A ) To reduce the variability between Kiss1 neurons from different animals , a response ratio ( R2/R1 ) protocol was used in which the magnitude of second light response ( A2 ) was compared to first response ( A1 ) . Representative traces showing that the R2/R1 ratio of two photostimuli is 0 . 65 in Kiss1ARH neurons . ( B–E ) , Representative traces showing that the slow EPSP was abolished by perfusing TTX ( B ) , and the TTX blockade was rescued by the addition of the K+ channel blockers 4-AP and TEA , suggesting a direct synaptic input from other Kiss1 neurons ( C ) ; the slow EPSP was abolished by perfusing low Ca2+/high Mg2+ ( D ) ; and the slow EPSP was unaffected by the ionotropic glutamate receptor antagonists CNQX and AP5 ( E ) . ( F ) Bar graphs summarizing the effects of drugs on the direct synaptic input ( EPSP ) to Kiss1 neurons . Comparisons between different treatments were performed using a one-way ANOVA analysis ( F ( 4 , 30 ) = 12 . 18 , p<0 . 0001 ) with the Newman-Keuls’s post hoc test . ** indicates p<0 . 01 vs . control . DOI: http://dx . doi . org/10 . 7554/eLife . 16246 . 00510 . 7554/eLife . 16246 . 006Figure 4 . The slow EPSP is dependent upon G-protein activation . ( A ) Intracellular dialysis with low concentrations of QX-314 ( 0 . 5 mM ) in the internal solution ( IS ) blocked Na+ channels , but the slow EPSP induced by light stimulation was not blocked . ( B ) Slow EPSP induced by photostimulation ( upper ) was not mimicked by electrical stimulation ( lower ) at 20 Hz for 10 s in the same cell . ( C ) Electrical stimulation in the presence of 4-AP and TEA did not mimic the effects of photostimulation to induce a slow EPSP in Kiss1ARH neurons; a hyperpolarizing bias ( current injection ) was used to repolarize membrane potential to resting membrane potential when the 4-AP/TEA response reached the maximal depolarization . ( D ) Intracellular dialysis with GDPβS ( 2 mM ) blocked the slow EPSP , demonstrating that the slow EPSP was mediated by G protein-coupled receptors . ( E ) Bar graphs summarizing the effects of Na+ channel and G-protein blockers and electrical stimulation on the slow EPSP in Kiss1 neurons . Comparisons between different treatments were performed using a one-way ANOVA analysis ( F ( 3 , 44 ) = 22 . 39 , p<0 . 0001 ) with the Newman-Keuls’s post hoc test . *** and * indicates p<0 . 005 and p<0 . 05 vs . control , respectively; ## indicates p<0 . 01 , QX314 group vs electrical stimulation and GDPβS groups . DOI: http://dx . doi . org/10 . 7554/eLife . 16246 . 006 We have measured Tacr1 and Tacr3 , but not Tacr2 , mRNA expression in Kiss1ARH neurons using single cell RT-PCR ( Navarro et al . , 2015 ) , and the Tacr3 agonist senktide depolarizes Kiss1ARH neurons ( Navarro et al . , 2011b ) . Firstly , in order to inhibit G protein activation we dialyzed Kiss1 neurons with the GDP analog GDP-β-S ( Guanosine 5′-[β-thio]diphosphate trilithium , 2 mM in the internal solution ) and completely abrogated the response ( Figure 4D and E ) , demonstrating that the slow EPSP was mediated by a G protein-coupled receptor like Tacr3 . Secondly , we blocked the slow EPSP with a Tacr3 antagonist SB222 , 200 ( 3 µM ) or with a cocktail of antagonists against all 3 tachykinin receptors ( Figure 5A , B , D ) . The slow EPSP was also occluded by pretreatment with Tacr3 agonist senktide that depolarized Kiss1ARH neurons and pharmacologically prevented any effects of the photostimulated release of NKB ( Figure 5C–D ) . Finally , To see if dual photostimulation induced desensitization , we perfused senktide repeatedly ( 2x ) , and the averaged ratio of the responses ( 0 . 65 ± 0 . 01 , n = 4 ) was similar to that obtained by photostimulation , suggesting that desensitization was caused by internalization and downregulation of Tacr3 following binding of NKB ( Steinhoff et al . , 2014 ) ( Figure 5E , F ) . 10 . 7554/eLife . 16246 . 007Figure 5 . Tacr3 agonist mediates the slow EPSP . ( A–C ) The slow EPSP was abrogated by an NKB receptor ( Tacr3 ) antagonist SB222 , 200 ( A ) and the NKB receptor antagonist cocktail SDZ-NKT 343 ( 1 µM ) , GR94 , 800 ( 1 µM ) and SB222 , 200 ( 3 µM ) ( Tacr1 , 2 and 3 antagonists , respectively ) ( B ) . Furthermore , Tacr3 agonist senktide pretreatment completely occluded the slow EPSP ( C ) , demonstrating that the slow EPSP was mediated by the Tacr3 . A hyperpolarizing bias was used to repolarize the membrane potential to −70 mV when the senktide response reached the maximal depolarization ( C2 ) . ( D ) Bar graphs summarizing the effects of Tacr3 agonist and antagonists on the slow EPSP . Comparisons between different treatments were performed using a one-way ANOVA analysis ( F ( 3 , 20 ) = 31 . 6 , p<0 . 0001 ) with the Newman-Keuls’s post hoc test . *** indicates p<0 . 005 vs . control . ( E–F ) Depolarization induced by senktide ( 250 nM ) perfused on two separate occasions in a Kiss1ARH neuron . The R2/R1 ratio was 0 . 65 ( E , upper trace ) , which was not different from the ratio from photostimulation ( E , lower trace ) . ( F ) Bar graphs summarizing R2/R1 ratios ( Un-paired t-test , t ( 14 ) = 0 . 0408 , p = 0 . 9680 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 16246 . 007 Since dynorphin is co-localized to over 90% of Kiss1ARH neurons ( Goodman et al . , 2007; Navarro et al . , 2009 ) , and κ-opioid receptor mRNA is expressed in a subset of Kiss1ARH neurons ( Navarro et al . , 2009 ) , we hypothesized that dynorphin would be co-released with high-frequency photostimulation and modulate NKB release from Kiss1 terminals . Indeed , blockade of κ-opioid receptors by the selective antagonist nor-BNI ( 1 µM ) potentiated the slow EPSP ( Figure 6A , C ) . Moreover , the κ-opioid receptor agonist U69 , 593 ( 1 µM ) abrogated the slow EPSP through its presynaptic actions , but Kiss1ARH neurons ( n = 4 ) still responded directly to senktide ( Figure 6B ) . Finally , U69 , 593 ( 1 μM ) reduced the frequency but not the amplitude of miniature EPSCs ( mEPSCs ) ; U69 , 593 did not alter the holding current ( or input resistance ) of Kiss1ARH neurons ( Figure 6D–F ) . Therefore , co-released Dyn is acting presynaptically to inhibit the peptidergic ( NKB ) and glutamatergic excitatory input to Kiss1ARH neurons . 10 . 7554/eLife . 16246 . 008Figure 6 . The κ-opioid receptor agonist blocks the slow EPSP . ( A–B ) The κ-opioid receptor antagonist nor-BNI ( 1 μM ) potentiated the slow EPSP ( A ) and the agonist U69 , 593 ( 1 µM ) attenuated the slow EPSP ( B2 versus B1 ) . However , the Tacr3 agonist senktide is able to excite Kiss1ARH neurons in the presence of U69 , 593 , indicative that the kappa-opioid effects are pre-synaptic to inhibit NKB release ( B ) . ( C ) Bar graphs summarizing the effects of κ-opioid receptor agonist and antagonist on the slow EPSP . Comparisons between different treatments were performed using a one-way ANOVA analysis ( F ( 2 , 22 ) = 9 . 784 , p = 0 . 0009 ) with the Newman-Keuls’s post hoc test . ** and * indicates p<0 . 01 and p<0 . 05 vs . control , respectively . ( D–F ) Effects of U69 , 593 on mEPSCs in Kiss1ARH neurons . Representative traces showing mEPSCs recorded at a holding potential of −60 mV under control conditions ( TTX ) , and after TTX plus 1 µM U69 , 593 . Summary data showing the effects of U69 , 593 on the frequency ( E ) and the amplitude ( F ) of mEPSCs in Kiss1ARH neurons ( n = 6 ) . Paired t-test , t ( 5 ) = 8 . 104 , ***p<0 . 001 ( E ) and Paired t-test , t ( 5 ) =1 . 580 , p = 0 . 1750 ( F ) compared to the control . DOI: http://dx . doi . org/10 . 7554/eLife . 16246 . 008 To study the ChR2-expressing neuronal input to non-ChR2-expressing Kiss1 neurons , an AAV-DIO-ChR2:mCherry virus was unilaterally injected into one side of the ARH ( Figure 7A ) . As expected , these injections labeled mCherry-positive ( Kiss1 ) cells and fibers ipsilaterally , but only mCherry-positive fibers in the contralateral ARH ( Figure 7A ) . In addition to the ChR2:mCherry labeling ipsilaterally , control AAV-DIO-YFP was injected into the contralateral ARH in order to identify Kiss1 neurons for whole cell recordings ( Figure 7B–D ) . With low-frequency photostimulation of the ChR2 fibers in the contralateral ARH ( Figure 7E–G ) , we detected a glutamatergic fast EPSC in voltage clamp ( 22 . 9 ± 8 . 3 pA , n=6 ) ( Figure 7E , upper trace ) and with high-frequency stimulation a peptidergic slow EPSP in current clamp ( 19 . 2 ± 4 . 1 mV , n = 6 ) ( Figure 7E , lower trace ) . There was no ChR2-mediated inward current and/or depolarization with photostimulation . The slow EPSP was blocked by TTX and rescued by TTX plus 4-AP and TEA , demonstrating direct communication between Kiss1ARH neurons on both sides ( Figure 7F ) . Similar to the recordings in ipsilateral ChR2-expressing Kiss1ARH neurons , the Tacr3 antagonist SB222 , 200 ( 3 µM ) blocked the optogenetic response ( reduced R2/R1 by 75% from 0 . 64 ± 0 . 06 , n = 4 to 0 . 16 ± 0 . 04 , n = 5; Un-paired t-test , t ( 7 ) = 7 . 193 , ****p<0 . 001 ) ( Figure 7G ) . Moreover , with dual patch recording we were able to simultaneously depolarize and excite the contralateral Kiss1ARH neurons ( n = 2 pair of neurons ) following photostimulation ( Figure 8A–C ) . To ensure that the response was due to activation of the axonal terminal field of ChR2-projecting Kiss1ARH neurons , we acutely cut the median eminence to isolate the contralateral Kiss1ARH neurons , which did not express ChR2 , and found that high-frequency photostimulation of the ChR2-expressing terminal field simultaneously depolarized and increased the firing of Kiss1ARH neurons ( n = 3 pair of neurons ) ( Figure 8D–F ) . Thus , high-frequency autoexcitation of Kiss1ARH neurons ipsilaterally is able to recruit Kiss1ARH neurons bilaterally to induce synchronization of this critical neural network . 10 . 7554/eLife . 16246 . 009Figure 7 . Kiss1ARH neurons send contralateral projections to Kiss1ARH neurons inducing circuit excitation . ( A ) Confocal image of a coronal section through ARH from a Kiss1-Cre mouse following a unilateral ARH injection of AAV-DIO-ChR2:mCherry . Kiss1 neurons expressing ChR2-mCherry were observed on the injected left side , whereas only fibers were visible in the internal zone of the median eminence ( ME ) and in the contralateral ARH . ( Scale bar = 100 μm ) . Insets on the ipsilateral and contralateral sides illustrate high power images of dual labeling of Kiss1-GFP and mCherry . On the ipsilateral side , a representative Kiss1-GFP neuron show co-expression of GFP and mCherry . ( Scale bar = 10 μm ) . On the contralateral side , mCherry-positive fibers formed close contacts with a representative Kiss1-GFP neuron . ( Scale bar = 10 μm ) . ( B ) , Schematic of coronal sections through the ARH illustrating ChR2-mCherry and control YFP injections , in which one side of the ARH was injected with AAV-DIO-ChR2:mCherry and the contralateral side with AAV-DIO-YFP . ( C ) Illustration of the experimental approach . The whole-cell recorded neurons were YFP- expressing neurons . ( D ) Higher power images of a coronal section through the ARH from a Kiss1-Cre female showing the Kiss1 neurons expressing ChR2-mCherry ( left side , red ) ( D1 ) , versus AAV-DIO-YFP ( right side , yellow ) and overlay of epifluorescence ( YFP and mCherry ) images in YFP positive neuron area showing mCherry-expressing terminals ( D2 ) . Only mCherry expressing fibers were observed in the contralateral ARH ( D2 ) . Scale bars = 20 µm . ARH , hypothalamic arcuate nucleus; 3V , third ventricle . ( E ) Representative trace showing that photostimulation induced not only a fast glutamate response ( upper traces ) with a single 10 ms pulse , but also a slow EPSP ( lower trace ) with 20 Hz for 10 s photostimulation in the same cell . Red trace indicates averaged sweeps . Blue trace illustrates that the AMPA- and NMDA-mediated currents ( EPSCs ) were blocked with CNQX ( 10 μM ) and D-AP5 ( 50 μM ) . ( F ) Representative traces showing that the slow EPSP was abolished by perfusing TTX , and the TTX blockade was rescued by the addition of non-selective K+ channel blockers 4-AP and TEA suggesting a direct input from other Kiss1 ChR2-expressing neurons . All Kiss1 neurons ( n = 7 ) were rescued by the combination of K+ channel blockers . ( G ) Blue light stimulation induced a slow EPSP in a contralateral Kiss1ARH neuron ( upper trace ) , which was antagonized by SB222 , 200 ( 3 µM ) ( lower trace ) . DOI: http://dx . doi . org/10 . 7554/eLife . 16246 . 00910 . 7554/eLife . 16246 . 010Figure 8 . Dual patch recording reveal that high-frequency auto-excitation of ipsilateral Kiss1ARH neurons recruit contralateral Kiss1ARH neurons . ( A ) Image of a coronal section through the ARH from Kiss1-Cre mouse that received dual injection of AAV-DIO-ChR2:mCherry ( left side , red ) and AAV-DIO-YFP ( right side , yellow ) . Scale bar = 200 µm . ( B ) DIC and fluorescent image ( YFP ) of dual patch recording from Kiss1ARH neurons in the contralateral ARH; whole cell recordings were made with electrode 1 ( E1 ) and electrode 2 ( E2 ) in the current-clamp mode . Scale bar = 20 µm . ( C ) High-frequency photostimulation induced a simultaneous increase in firing and a slow EPSP in a pair of Kiss1ARH neurons ( Cell 1 and Cell 2 ) recorded in the contralateral ARH . ( D ) , Image of a coronal section through the ARH from Kiss1-Cre mouse that received dual injection of AAV-DIO-ChR2:mCherry ( left side , red ) and AAV-DIO-YFP ( right side , yellow ) . In this slice , the ME was cut to disconnect the two sides . Scale bar = 200 µm . ( E and F ) Examples of synchronized activity obtained using dual patch recording from two pairs of Kiss1ARH neurons in the contralateral ARH ( D ) . ARH , hypothalamic arcuate nucleus; ME , median eminence; 3V , third ventricle . DOI: http://dx . doi . org/10 . 7554/eLife . 16246 . 010 In a number of animal models including mouse , GnRH neurons are primarily located in the POA , and the mechanism by which Kiss1ARH neurons regulate GnRH neurons is not well understood ( Navarro et al . , 2015 ) . Currently , we tested whether pharmacological activation of Tacr3 within Kiss1-GnRH circuits would directly and/or indirectly excite GnRH neurons ( Figure 9A , C ) . The Tacr3 agonist senktide robustly excited GnRH neurons in the horizontal slice which included both ARH kisspeptin and the preoptic GnRH neurons ( Figure 9A , B ) , but not in the coronal slice in which the ARH kisspeptin neurons were separated from the preoptic ( rostral ) GnRH neurons ( Figure 9C , D ) . As expected , kisspeptin excited GnRH neurons irrespective of slice orientation ( Figure 9B , D ) . Therefore , NKB/Tacr3 signaling is upstream from kisspeptin/GPR54 signaling , and activation of kisspeptin/GPR54 signaling via Tacr3 stimulation results in excitation of GnRH neurons . 10 . 7554/eLife . 16246 . 011Figure 9 . Tacr3 agonist senktide excites GnRH neurons indirectly through ARH Kiss1 neurons , and a slow EPSP in GnRH neurons is evoked with photoactivation of ChR2 Kiss1ARH neurons . ( A ) Schematic drawing of the Kiss1ARH , Kiss1AVPV/PeN and GnRH neuronal circuit ( A1 ) and a high power image ( A2 ) of a parahorizontal slice from a GnRH-EGFP mouse illustrating whole-cell recording of GnRH neuron . Scale bar = 20 µM . ( B ) Representative traces showing that perfusing the slice with senktide ( 1 µM ) induced depolarization and increased firing in a GnRH neuron ( left ) , which was mimicked by perfusion with kisspeptin ( 10 nM , right ) . ( C ) Schematic drawing ( C1 ) and highpower image ( C2 ) of a coronal slice through the POA from a GnRH-EGFP mouse illustrating whole-cell recording of GnRH neuron . ( D ) Representative trace showing that perfusing the slice with senktide ( 1 µM ) did not depolarize or increase firing of a GnRH neuron , but kisspeptin could excite the same POA GnRH neuron . E , Schematic drawing of the Kiss1ARH , Kiss1AVPV/PeN and GnRH neuronal circuit ( E1 ) and a high power image ( E2 ) of a parahorizontal slice from a bilateral arcuate AAV-DIO-ChR2:mCherry injected Kiss1-Cre::GnRH-EGFP mouse illustrating whole-cell recording of GnRH neuron . Scale bar , 20 µM ( E2 ) . ( F–G ) High-frequency photoactivation of ChR2-labeled ARH neurons with low ( 0 . 45 mW , left panel ) and higher ( 0 . 9 mW , right panel ) intensities evoked slow EPSPs and increased firing in the same GnRH neuron ( F ) which was mimicked by perfusion with kisspeptin ( 10 nM ) ( G ) but low-frequency photostimulation ( 0 . 5 Hz ) did not evoke a fast EPSC in this GnRH neuron ( H ) . ( I–J ) High-frequency photoactivation of ChR2 evoked a slow EPSP ( I ) in another GnRH neuron , and the I-V relationship before and during the peak response from the same cell indicated that the reversal potential of the nonselective cation current was ~ -40 mV ( J ) . AC , anterior commissure; ARH , hypothalamic arcuate nucleus; AVPV , anteroventral periventricular nucleus; mPOA , medial preoptic area . DOI: http://dx . doi . org/10 . 7554/eLife . 16246 . 011 As proof of principle in a horizontal slice that preserved the connections between the Kiss1ARH neurons , Kiss1AVPV/PeN neurons and the preoptic area containing GnRH neurons , we found that highfrequency ( 20 Hz ) photostimulation of Kiss1ARH neurons depolarized and excited GnRH neurons ( 5 . 5 ± 1 . 4 mV , n = 5 ) via activation of a non-selective cation channel with double rectifying properties typical of TRPC channels ( Zhang et al . , 2008 ) ( Figure 9E-F , I-J ) , and this excitation was mimicked by bath application of 10 nM kisspeptin ( Figure 9G ) . Importantly , there were no fast EPSCs detected in GnRH neurons with photostimulation of Kiss1ARH ChR2- expressing neurons ( Figure 9H ) . Given that Tacr3 is essentially not expressed in GnRH or in Kiss1AVPV/PeN neurons , and Kiss1AVPV/PeN neurons do not respond to kisspeptin ( Navarro et al . , 2015; Ducret et al . , 2010 ) , the question became how does activation of Kiss1ARH neurons excite GnRH neurons ? A recent study has shown that mouse Kiss1ARH neurons project to Kiss1AVPV/PeN neurons ( Yip et al . , 2015 ) . Indeed , we recorded light-evoked EPSCs in Kiss1AVPV/PeN neurons ( 33 . 2 ± 9 . 7 pA , n = 5 ) following photostimulation of fibers from ChR2-mCherry or from ChR2-YFP expressing Kiss1ARH neurons ( Figure 10A–D ) . The fast EPSC was blocked with CNQX ( 10 μM ) and D-AP5 ( 50 μM ) , documenting a glutamatergic input from Kiss1ARH neurons to Kiss1AVPV/PeN neurons ( Figure 10D ) . Furthermore , we bilaterally injected ChR2:mCherry-expressing viral vector into the AVPV/PeN of Kiss1-Cre::GnRH-EGFP female mice . The anterograde transport revealed close contacts with GnRH cell bodies in the mPOA ( Figure 11A , B1–3 ) . Viral-labeled Kiss1AVPV/PeN neurons also revealed projections caudally to the ARH ( Figure 11B4 ) . High-frequency photostimulation of the Kiss1AVPV/PeN neurons evoked a slow EPSP in GnRH neurons in both parahorizontal ( 8 . 0 ± 2 . 9 mV , n = 4 ) ( Figure 11C–E ) and coronal slices ( 15 . 6 ± 4 . 8 mV , n = 4 ) ( Figure 11F–H ) . The slow EPSP was mediated by the kisspeptin release from Kiss1AVPV/PeNneurons based on the double rectifying , dual reversal potential I/V ( Figure 11E ) ( Zhang et al . , 2008 ) and occlusion of the response by the partial agonist peptide 234 ( Liu et al . , 2011 ) , which blocked the evoked kisspeptin response by 85% ( from 15 . 6 ± 4 . 8 mV to 2 . 4 ± 0 . 9 mV , n = 4 ) in coronal slices ( Figure 11H ) . Finally , we harvested Kiss1 AVPV/PeN neurons and measured Slc17a6 ( vGluT2 ) mRNA expression using scRT-PCR . Based on the analysis of 71 Kiss1 neurons from 3 females ( 23–24 cells/ animal ) , only 2 cells ( 2 . 9% ) were found to express Slc17a6 mRNA , an indication that glutamate is not packaged into vesicles and released by Kiss1AVPV/PeN neurons . 10 . 7554/eLife . 16246 . 012Figure 10 . Photoactivation of Kiss1ARH fibers excites Kiss1AVPV/PeN neurons via glutamate release . ( A ) Schematic drawing of a coronal section showing the bilateral viral injections in the ARH with AAV-DIO-ChR2:mCherry . ( B ) Sagittal brain slice ( B1 ) and confocal images from the slice ( B2 , 3 ) of mouse bilaterally injected with a ChR2-mCherry-expressing viral vector into the ARH of Kiss1-Cre::GnRH-EGFP female mouse . Projections from Kiss1ARH neurons ( B3 ) to the AVPV/PeN are illustrated ( B2 ) . Green neurons in the POA illustrate the location of GnRH neurons in the sagittal brain slice . Scale bars , 100 µm ( B2 , B3 ) . ( C ) Overlay of epifluorescence ( YFP ) and differential interference contrast ( DIC ) images of a coronal arcuate slice showing injection site ( C1 ) in a Kiss1-Cre:ChR2:YFP mouse ( arrow points to high power image of labeled neurons in the ARH ) and fiber-projections to the AVPV ( C2; arrow points to labeled fibers in the AVPV area that are being photostimulated ) . Scale bars = 10 µm . ( D ) Example of light-evoked fast EPSCs in a Kiss1AVPV/PeN neuron ( mean response: 33 . 2 ± 9 . 7 pA , n = 5 ) . Red trace indicates averaged sweeps . Blue trace illustrates that the AMPA- and NMDA-mediated currents ( EPSCs ) were blocked with CNQX ( 10 μM ) and D-AP5 ( 50 μM ) . Following recording , cells were harvested for scRT-PCR determination of Kiss1 mRNA expression . All recorded AVPV/PeN cells ( n = 5 ) expressed Kiss1 mRNA ( data not shown ) . ARH , hypothalamic arcuate nucleus; AVPV/PeN , anteroventral periventricular and periventricular preoptic nuclei; POA , preoptic area . DOI: http://dx . doi . org/10 . 7554/eLife . 16246 . 01210 . 7554/eLife . 16246 . 013Figure 11 . High-frequency photoactivation of Kiss1AVPV/PeN neurons excites GnRH neurons via kisspeptin release . ( A ) Schematic drawing of coronal section showing bilateral viral injections in the AVPV with AAV-DIO-ChR2:mCherry . ( B ) Parahorizontal brain slice of mouse bilaterally injected with a ChR2-mCherry-expressing viral vector into the AVPV/PeN of Kiss1-Cre::GnRH-EGFP female mouse . Injection sites into AVPV/PeN are illustrated in low power ( red , B1 ) and high power ( B2 ) . Kiss1AVPV/PeN neurons send projections rostrally to GnRH neurons ( B3 ) and also caudally to the ARH ( B4 ) . Note that following AVPV/PeN injections , only mCherry fibers but no labeled cells were detected in the ARH . Scale bars , 200 µM ( B1 ) , 20 µm ( B2 ) , 10 µm ( B3 ) and 50 µm ( B4 ) . ( C ) , Low power ( C1 ) and high power ( C2 ) images of whole-cell recordings in an angled , parahorizontal slice from bilateral AVPV AAV-injected Kiss1-Cre::GnRH-GFP mouse . ( D–E ) High-frequency photoactivation of AVPV/PeN with durations of 10 s ( upper ) and 30 s ( lower ) evoked a slow EPSP ( mean slow EPSP response following 30 s duration stimulation: 8 . 0 ± 2 . 9 mV , n = 4 ) and increased firing ( D ) ; typical I-V relationship for a kisspeptin-evoked response in GnRH neurons in the same cell ( E ) . Scale bars , 200 µm ( C1 ) and 20 µm ( C2 ) . ( F ) , Coronal slice showing ChR2-mCherry expression in Kiss1AVPV/PeN neurons following bilateral viral injections in the AVPV/PeN area in a Kiss1-Cre::GnRH-EGFP mouse ( F1 ) . Scattered GnRH-EGFP neurons can be seen dorsal and lateral to the injection site , one of which is being recorded from ( F1 , low power; F2 , high power ) . Scale bars , 200 µm ( F1 ) and 20 µm ( F2 ) . ( G ) High-frequency photostimulation of Kiss1AVPV/PeN neurons causes a slow EPSP in a GnRH neuron that induces a high-frequency discharge . ( H ) , High-frequency-induced depolarization/excitation of GnRH neurons is blocked ( occluded ) by the partial agonist peptide 234 ( 100 nM ) . ARH , hypothalamic arcuate nucleus; AVPV/PeN , anteroventral periventricular and periventricular preoptic nuclei; ME , median eminence; POA , preoptic area . DOI: http://dx . doi . org/10 . 7554/eLife . 16246 . 013
We have shown that high-frequency photostimulation of Kiss1ARH neurons expressing ChR2 evokes a slow excitatory postsynaptic potential ( autoexcitation ) in these Kiss1 neurons . The slow EPSP was abolished with Na+ channel blocker TTX treatment but rescued by the addition of K+ channel blockers , which is physiological evidence for Kiss1ARH synaptic contacts on Kiss1ARH neurons . Furthermore , the slow EPSP was attenuated by the Tacr3 antagonist SB222 , 200 and occluded by the Tacr3 agonist senktide pretreatment , demonstrating that the slow EPSP was mediated by the Gq-coupled Tacr3 . In addition , the κ-opioid receptor agonist U69 , 593 markedly attenuated the slow EPSP , whereas the selective κ-antagonist nor-BNI enhanced the ChR2-evoked slow EPSP , indicative of κ-opioid receptor-mediated presynaptic inhibition of NKB release . As demonstrated with dual whole-cell recordings , there was a simultaneous activation and recruitment of Kiss1ARH neurons bilaterally to provide a synchronous drive to GnRH neurons . Finally , in horizontal but not the coronal hypothalamic slices , the Tacr3 agonist senktide indirectly excited GnRH neurons , and high-frequency photoactivation of Kiss1ARH neurons depolarized/excited GnRH neurons via a multisynaptic pathway that involved glutamatergic excitation of Kiss1AVPV/PeN neurons , which excited GnRH neurons via kisspeptin release . High-frequency stimulation is required to evoke peptide release as originally demonstrated in the frog ganglion by Jan et al . ( 1979 ) ( see Arrigoni and Saper ( 2014 ) for review ) . Indeed , high-frequency photostimulation of ChR2-expressing Kiss1ARH neurons elicited postsynaptic responses in neighboring Kiss1 neurons that entrained to stimulation frequencies up to 20 Hz . We observed a slowly developing depolarization over tens of seconds that was unaffected by ionotropic glutamate receptor blockade , but was antagonized by a Tacr3 antagonist . Schöne et al . ( 2014 ) evoked a slow EPSC in tuberomammillary histamine neurons following high-frequency ( ≥20 Hz ) photostimulation of ChR2-expressing orexin neurons that was abrogated by a selective orexin receptor two antagonist . Therefore , in both kisspeptin and histamine neurons there is glutamate-dependent ( low-frequency ) and peptide-dependent ( high-frequency ) release that transmit distinct postsynaptic excitatory effects; but in the case of Kiss1ARH neurons , it is a recurrent auto-excitation that allows recruitment of neighboring Kiss1ARH neurons for synchronizing firing . Also unique to Kiss1ARH neurons is that high-frequency stimulation co-released dynorphin , which modulated the NKB-Tacr3 response . Dynorphin is co-localized in the vast majority of Kiss1ARH neurons ( Navarro et al . , 2009; Lehman et al . , 2010a ) , and loose-cell attached recordings have shown that dynorphin and κ-opioid receptor agonist U69 , 593 inhibit the activity of spontaneously firing Kiss1ARH neurons , although it has not been established whether this was a postsynaptic or presynaptic action on excitatory input to Kiss1ARH neurons ( de Croft et al . , 2013; Ruka et al . , 2013 ) . In our studies , dynorphin appeared to be co-released with high-frequency stimulation based on the fact that the slow EPSP evoked by high-frequency photostimulation was augmented with κ-opioid receptor antagonism ( nor-BNI treatment ) , and abrogated by the κ-agonist U69 , 593 . Moreover , although the κ-opioid receptor agonist U69 , 593 directly inhibits Kiss1AVPV/PeN neurons via activating G protein-coupled inwardly rectifying K+ channels ( Zhang et al . , 2013 ) , we did not see a similar direct response in Kiss1ARH neurons . However , the κ-agonist U69 , 593 did reduce the frequency of miniature EPSCs in Kiss1ARH neurons . Therefore , co-released dynorphin most likely acts presynaptically to modulate NKB release . This is analogous to the co-release of dynorphin from vasopressin neurons ( Iremonger and Bains , 2009; Iremonger et al . , 2011 ) in which it is co-packaged in the same dense core vesicles ( Shuster et al . , 2000 ) . However , kisspeptin , NKB and dynorphin are packaged in separate vesicles as documented by electron microscopy ( Murakawa et al . , 2015 ) so there must be a coordinated co-release of the segregated peptide neurotransmitters ( Vaaga et al . , 2014 ) from Kiss1ARH neurons . Once released , dynorphin can inhibit synaptic vesicle mobilization/release via inhibiting presynaptic calcium channels ( Rusin et al . , 1997 ) , activating K+ channels ( Simmons and Chavkin , 1996; Vaughan and Christie , 1997 ) or directly inhibiting the presynaptic release machinery ( Iremonger and Bains , 2009 ) . Presently , we do not know which one of the three mechanisms mediates the κ-opioid receptor-mediated actions on the presynaptic Kiss1ARH terminals , but it is clear that co-released dynorphin feeds back to modulate NKB release . It is known that nerve fibers expressing kisspeptin or co-expressing kisspeptin and NKB are localized in the internal zone of the median eminence in mice and rats ( Clarkson et al . , 2009; True et al . , 2010 ) , and a recent report has shown that simultaneous activation of Kiss1ARH neurons with optogenetic stimulation generates LH release in vivo , which is thought to be via kisspeptin-mediated depolarization of GnRH nerve terminals ( Han et al . , 2015 ) . Moreover , morphological tract-tracing studies have shown that ARH NKB neurons , some of which are presumed to be Kiss1 neurons , are bilaterally interconnected , and NKB axons cross in the internal zone of the median eminence and project to the contralateral ARH ( Krajewski et al . , 2010 ) . However , a direct synaptic connection between Kiss1ARH neurons has not been demonstrated . Herein , we show that ChR2:mCherry-labeled fibers cross over to the opposite side , and both a fast EPSC and a slow synaptic response ( slow EPSP ) were evoked with low- and high-frequency photostimulation , respectively , in the contralateral Kiss1ARH neurons . In fact , dual patch recording revealed simultaneous activation of Kiss1ARH neurons . Importantly , the contralateral Kiss1ARH neurons did not exhibit a direct ChR2 response thus confirming the confocal analysis that only ChR2 expressing fibers ( axons ) and control YFP expressing cell bodies were detected on the contralateral side of the ARH . Moreover , the slow EPSP was blocked by Na+ channel blocker TTX and rescued with the addition of K+ blockers , which allowed robust depolarization of nerve terminals with photoactivation ( Cousin and Robinson , 2000; Petreanu et al . , 2009 ) , and in our studies release of NKB . Therefore , it appears that Kiss1ARH neurons are contacted by other Kiss1ARH nerve terminals , and the activation of the Kiss1ARH network is dependent on action potential-driven synaptic release of NKB . Moreover , Kiss1ARH fibers crossing within the median eminence allow for synaptic communication between the arcuate nuclei for recruiting bilaterally additional Kiss1 neurons to generate a robust excitatory drive to GnRH neurons . We used a horizontal slice preparation from Kiss1-Cre-ChR2::GnRH-EGFP mice , which contains both the ARH and AVPV/PeN Kiss1 neurons and preoptic GnRH neurons , to determine if an NKB agonist could excite GnRH neurons via a multi-synaptic pathway . Given that essentially all Kiss1ARH neurons , but not Kiss1AVPV/PeN or GnRH neurons , express Tacr3 mRNA ( Navarro et al . , 2011b; Navarro et al . , 2015 ) and Kiss1AVPV/PeN neurons do not respond directly to kisspeptin ( Ducret et al . , 2010 ) , we applied senktide to activate Kiss1ARH neurons and record changes in GnRH cell activity . Senktide induced robust firing in GnRH neurons in horizontal slices that included the Kiss1ARH neurons , but not in coronal slices excluding these neurons . These physiological findings support previous neuroanatomical tract tracing/immunocytochemical studies in mice showing that Kiss1ARH neurons project to AVPV/PeN ( Kiss1 ) neurons but not to GnRH somata in the preoptic area ( Yip et al . , 2015 ) . In addition , photoactivation of Kiss1ARH neurons excited Kiss1AVPV/PeN neurons via glutamate , which we have shown generates burst firing in these neurons ( Zhang et al . , 2013 ) , and transsynaptically depolarized and excited GnRH neurons via kisspeptin activation of GPR54 and the opening of TRPC channels in a horizontal slice preparation that preserved all of the interconnections . These results are summarized in Figure 12 . Therefore , our findings provide the first evidence that Kiss1ARH neurons can synchronize via NKB/Dyn auto-excitation/inhibition to provide an excitatory drive ( via Kiss1AVPV/PeN neurons ) to GnRH neurons for stimulating GnRH neuronal activity and subsequent GnRH release . This does not preclude the fact that Kiss1ARH neurons also project to the median eminence ( True et al . , 2010 ) and may stimulate pulsatile release of GnRH in the ovariectomized state ( Yip et al . , 2015 ) . Hence , Kiss1ARH neurons probably play a dual role of driving pulsatile ( episodic ) secretion of GnRH through kisspeptin release into the external zone of the median eminence , but also excite Kiss1AVPV/PeN neurons via glutamate release to help generate the preovulatory surge of GnRH through a robust di-synaptic pathway ( Figure 12 ) . What is intriguing is the possibility that the differential release of amino acid and peptide neurotransmitters ( segregated co-transmission ) ( Vaaga et al . , 2014 ) occurs at these different release sites ( ARH , median eminence , and AVPV/PeN ) to coordinate reproductive function . 10 . 7554/eLife . 16246 . 014Figure 12 . Proposed model by which activation of Kiss1 neurons governs GnRH neuronal excitability . High-frequency photostimulation of Kiss1 neurons in the ARH releases NKB that depolarizes and recruits other Kiss1ARH neurons . Dynorphin is co-released and acts presynaptically to modulate ( inhibit ) the release of NKB . Together the two peptides govern the synchronous activity of Kiss1ARH neurons and promote kisspeptin release that stimulates GnRH release in the median eminence ( ME ) . Kiss1ARH neurons also communicate with the Kiss1AVPV/PeN neurons via the fast neurotransmitter glutamate , which stimulates burst-firing of Kiss1AVPV/PeN neurons . Activation of these rostral Kiss1 neurons releases kisspeptin to robustly excite GnRH neurons via activation of the GPR54 signaling cascade , thereby stimulating the release of GnRH at the time of the preovulatory surge . Kisspeptin , GPR54 , NKB , Tacr3 and GnRH are all required for normal fertility . DOI: http://dx . doi . org/10 . 7554/eLife . 16246 . 014
Primarily female mice were used in this study . In a few instances male mice were also used for pharmacological analysis after it was determined that male and female neurons responded similarly . All animal procedures were conducted at Oregon Health and Science University ( OHSU ) according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with approval from the OHSU Animal Care and Use Committee . Kiss1-CreGFP mice ( RRID:IMSR_JAX:017701 ) ( Gottsch et al . , 2011 ) were housed under constant temperature ( 21–23°C ) and 12-h light , 12-h dark cycle schedule ( lights on at 0600 and lights off at 1800 h ) , with free access to food ( Lab Diets 5L0D ) and water . Kiss1-CreGFP mice were used for viral injection to express ChR2 in ARH or AVPV/PeN Kiss1 neurons or they were crossed with heterozygous Ai32 mice ( RRID:IMSR_JAX:024109 , C57BL/6 background ) purchased from The Jackson Laboratory . These Ai32 mice carry the ChR2 ( H134R ) –EYFP gene in their Gt ( ROSA ) 26Sor locus ( Madisen et al . , 2012 ) . The gene is separated from its CAG promoter by a loxP-flanked transcriptional STOP cassette , allowing its expression in a Cre-dependent manner . In the Kiss1-CreGFP::Ai32 ( R26-flox-stop-ChR2 ( H134R ) -EYFP ) cross , sometimes the Kiss1-CreGFP gene was expressed early in development resulting in expression of ChR2 ( H134R ) -EYFP gene ectopically in many cells ( Gottsch et al . , 2011 ) . Those mice were not used for experiments . All other Kiss1-Ai32 crossed mice exhibited EYFP expression only in the expected locations ( ARH and AVPV/PeN ) as illustrated in Figure 2B , C and were used for the experiments . In some studies , Kiss1-Cre:: GnRH-EGFP , AgRP-Cre::Ai32 and POMC-Cre::Ai32 female mice were used . These mice were produced by breeding Kiss1-CreGFP mice with GnRH-EGFP transgenic mice ( Suter et al . , 2000 ) , and AgRP-Cre ( RRID:IMSR_JAX:012899 ) or POMC-Cre mice ( RRID:IMSR_JAX:010714 ) were bred with Ai32 mice , respectively . Fourteen to twenty-one days prior to each experiment , Kiss1-CreGFP mice or Kiss1-Cre::GnRH-EGFP mice ( >60 days old ) received bilateral or unilateral ARH or AVPV injections of a Cre-dependent adeno-associated viral ( AAV; serotype 1 ) vector encoding ChR2-mCherry ( AAV1-Ef1α-DIO-ChR2:mCherry ) , ChR2-YFP ( AAV1-Ef1α-DIO-ChR2:YFP ) or YFP alone ( AAV1-Ef1α-DIO-YFP ) . Using aseptic techniques , anesthetized female mice ( 1 . 5% isoflurane/O2 ) received a medial skin incision to expose the surface of the skull . The glass pipette ( Drummond Scientific #3-000-203-G/X; Broomall , PA ) with a beveled tip ( diameter = 45 µm ) was filled with mineral oil , loaded with an aliquot of AAV using a Nanoject II ( Drummond Scientific ) . ARH injection coordinates were anteroposterior ( AP ) : −1 . 20 mm , mediolateral ( ML ) : ± 0 . 30 mm , dorsoventral ( DL ) : −5 . 80 mm ( surface of brain z = 0 . 0 mm ) ; AVPV injection coordinates were AP: 0 . 53 mm , ML: ± 0 . 30 mm , DL: −4 . 70 and −5 . 10 mm ( surface of brain z = 0 . 0 mm ) ; 500 nl of the AAV ( 2 . 0 x 1012 particles/ml ) was injected ( 100 nl/min ) into each position , left in place for 10 min post-injection , then the pipette was slowly removed from the brain . The skin incision was closed using skin adhesive , and each mouse received analgesia ( Rimadyl; 4 mg/kg ) for two days post-operation . It should be noted that the fluorescence intensity of YFP in Kiss1ARH neurons following viral injections or as a result of Kiss1-CreGFP::Ai32 cross masked the weaker GFP signal in Kiss1-CreGFP neurons , i . e . no weak GFP cells were observed but only intensely labeled YFP cells . Therefore , in initial experiments these ARH YFP cells were dispersed and harvested , or harvested in the slice following whole cell recording and subsequently subjected to single cell ( sc ) RT-PCR ( see below ) . In all instances , the ARH YFP cells were found to express Kiss1 mRNA ( see Figure 2 ) . Females were used with gonads intact or were subjected to ovariectomy ( OVX ) . It is well known that OVX animals exhibit increased GnRH and LH pulsatile secretion that coincide with increased expression of neuropeptides ( including kisspeptin , neurokinin B and dynorphin ) in ARH Kiss1 neurons ( Caraty et al . , 1989; Smith et al . , 2005; Dellovade and Merchenthaler , 2004; Gottsch et al . , 2009 ) . Since we were interested in determining the cellular mechanism by which ARH Kiss1 neurons synchronize to induce pulsatile GnRH and LH secretion , as well as determining the stimulus needed for peptide release , we primarily used OVX females for these experiments . However , we also used intact females at different stages of the estrous cycle in order to compare the magnitude of the high-frequency response from these animals to the response in OVX females ( see Figure 2 ) . For studies involving AVPV/PeN Kiss1 neurons and GnRH neurons , we used OVX females treated with E2 as a model for the proestrous stage of the cycle ( Zhang et al . , 2013 , 2015; Rønnekleiv et al . , 2015 ) . Seven days prior to each experiment , ovariectomies were performed on female mice under inhalant isofluorane anesthesia . Carprofen ( Rimadyl; Pfizer ) was given immediately after a surgery at a dose of 4 mg/kg for relief of postoperative pain . Two to three days prior to electrophysiological experiments , OVX females were given oil-vehicle injections or low dose followed by a high dose one day later of 17β-estradiol ( s . c . ) , which produces an LH surge on the experimental day ( Bosch et al . , 2013 ) . Female mice were prepared for immunocytochemistry as described previously ( Roepke et al . , 2011 ) . Briefly , two mm coronal hypothalamic blocks were fixed by immersion in 4% paraformaldehyde , cryoprotected in 20% sucrose solution , frozen at −55ºC , sectioned coronally on a cryostat at 20 μm , and thaw-mounted on Superfrost Plus slides ( Thermo Fisher Scientific ) . Sections were rinsed in PB ( 0 . 1 M phosphate buffer , pH 7 . 4; all rinses were in PB for at least 30 min ) , and then incubated for 24 hr at 4°C in rabbit polyclonal antiserum against mCherry ( 1:5000–1:10 , 000; ab167453 Abcam Inc , Cambridge MA ) . Some sections were also reacted with goat polyclonal antiserum against GFP conjugated to FITC ( RRID:AB_305635 ) ( 1:1000; ab6662 Abcam ) . After rinsing , sections stained for mCherry were incubated in donkey-antirabbit IgG antibody conjugated to Cy3 ( 1:300; Jackson Immunoresearch ) . Following a final rinse , slides were coverslipped using vectashield ( Vector Laboratories ) . Immunocytochemical-stained sections and slices used for recording were acquired and analyzed using confocal microscopy . Photomicrographs were acquired by an Olympus BX51W1 upright microscope equipped with a Rolera XR Fast 1394 camera . Confocal photomicrographs were acquired using a Zeiss LSM 510 and a Zeiss LSM 780 confocal microscopes with Zen software ( RRID:SCR_013672 ) . Coronal brain slices ( 250 µm ) containing the ARH or AVPV/PeN from gonadectomized or intact mice were prepared as previously described ( Qiu et al . , 2003 ) . Also in some experiments , angled parahorizontal brain slices were cut as previously described ( Liu et al . , 2011 ) , and the vibratome blade was positioned to just touch the optic chiasm and a single 400 µm-thick horizontal slice prepared . Whole-cell , patch recordings were performed in voltage clamp and current clamp using an Olympus BX51W1 upright microscope equipped with video-enhanced , infrared-differential interference contrast ( IR-DIC ) and an Exfo X-Cite 120 Series fluorescence light source . Electrodes were fabricated from borosilicate glass ( 1 . 5 mm outer diameter; World Precision Instruments , Sarasota , FL ) and filled with a normal internal solution ( in mM ) : 128 potassium gluconate , 10 NaCl , 1 MgCl2 , 11 EGTA , 10 HEPES , 2 ATP , and 0 . 25 GTP ( pH was adjusted to 7 . 3–7 . 4 with 1N KOH , 290–300 mOsm ) . Pipette resistances ranged from 3–5 MΩ . In whole cell configuration , access resistance was less than 20 MΩ; access resistance was 80% compensated . For optogenetic stimulation , a light-induced response was evoked using a light-emitting diode ( LED ) 470 nm blue light source controlled by a variable 2A driver ( ThorLabs , Newton , NJ ) with the light path delivered directly through an Olympus 40 × water-immersion lens . High fidelity response to light ( 470 nm ) stimulation of Kiss1ARH ChR2-expressing neurons was observed , and both evoked inward currents ( in voltage clamp , Vhold = −60 mV ) or depolarization ( in current clamp ) were measured ( Figures 1 and 2 ) . Electrophysiological signals were amplified with an Axopatch 200A and digitized with Digidata 1322A ( Molecular Devices , Foster City , CA ) , and the data were analyzed using p-Clamp software ( RRID:SCR_011323 , version 9 . 2 , Molecular Devices ) . A MultiClamp 700B amplifier ( Molecular Devices , Foster City , CA ) was used for the dual patch recording , and the signals were similarly digitized and analyzed . The amplitude of the slow EPSP was measured after low pass filtering in order to eliminate the barrage of action potentials riding on the depolarization ( Figure 2E ) . The liquid junction potential was corrected for all data analysis . After electrophysiological recording , the cytosol of recorded arcuate YFP-expressing cells from Kiss1-Cre::Ai32 mice was harvested and used for post-hoc identification of Kiss1 mRNA . Single-cell harvesting and RT-PCR was conducted as described ( Bosch et al . , 2013; Zhang et al . , 2013 ) . Briefly , the recorded cells were harvested with gentle suction to the recording pipette and expelled into a siliconized 0 . 65 ml microcentrifuge tube containing a solution of 1X Invitrogen Superscript III Buffer , 15U of RNasin ( Promega ) , 10 mM of dithiothreitol ( DTT ) and diethylpyrocarbonate ( DEPC ) -treated water in a total of 5 μl for a single cell . cDNA synthesis was performed on single cells and stored at −20°C . Controls included harvested cells processed without reverse transcriptase ( −RT ) and hypothalamic tissue RNA reacted with and without reverse transcriptase . Primers were designed using Clone Manager software ( Sci Ed Software ) to cross at least one intron-exon boundary and optimized as previously described ( Bosch et al . , 2013 ) . The primers for Kiss1 were as described previously ( Zhang et al . , 2013 ) . In addition , Kiss1 neurons in the preoptic area ( POA ) were dispersed and harvested as described ( Zhang et al . , 2013 ) and the mRNA expression of the vesicular glutamate transporter 2 ( vGluT2 ) was analyzed . The Slc17a6 ( vGluT2 ) primers were as follows: 136 bp product , accession number NM_080853 , forward primer 1677–1694 nt , reverse primer 1795–1812 nt . Single-cell RT-PCR was performed on 3 µl of cDNA in a 30 µl reaction volume and amplified 40–50 cycles using a C1000 Thermal Cycler ( Bio-Rad ) . The PCR products were visualized with ethidium bromide on a 2% agarose gel . The scRT-PCR products for Kiss1 and Slc17a6 ( vGluT2 ) have been confirmed by sequencing . All drugs were purchased from Tocris Bioscience ( Minneapolis , MN ) unless otherwise specified . DL-2-amino-5-phosphonopentanoic acid sodium salt ( AP5 ) ( 50 mM ) , 6-cyano-7-nitroquinoxaline-2 , 3-dione disodium ( CNQX ) ( 10 mM ) , 4-Aminopyridine ( 4-AP ) ( 500 mM ) and Nor-Binaltorphimine dihydrochloride ( 1 mM ) and were dissolved in H2O . Tetrodotoxin ( TTX ) was purchased from Alomone Labs ( Jerusalem , Israel ) ( 1 mM ) and dissolved in H2O . Tetraethylammonium chloride ( TEA , 7 . 5 mM ) and U69 , 593 ( 20 mM ) were purchased from Sigma-Aldrich ( St . Louis , MO ) and dissolved in aCSF or ethanol , respectively . Tacr1 antagonist SDZ-NKT-343 ( 20 mM ) , Tacr2 antagonist GR94 , 800 ( 2 mM ) , Tacr3 antagonist SB222 , 200 ( 50 mM ) and Tacr3 agonist senktide ( 1 mM ) and GABAA receptor antagonist Bicuculline methiodide ( 50 mM ) were prepared in DMSO . Guanosine 5′-[β-thio] diphosphate trilithium salt ( GDP-β-S , 2 mM ) from Calbiochem ( La Jolla , CA ) and QX 314 chloride ( 0 . 5 mM ) were dissolved in pipette solution . Kisspeptin-10 [Mouse Kiss-1 ( 110–119 ) -NH2; Kp-10 , from Phoenix Pharmaceuticals ( Belmont , CA ) ] ( 100 μM ) and peptide 234 ( 2 mM ) ( gift from Dr . Robert Millar , U . Pretoria , South Africa ) were dissolved in H2O . Aliquots of the stock solutions were stored as appropriate until needed . Comparisons between different treatments were performed using a one-way ANOVA analysis with the Newman-Keuls’s post hoc test . Differences were considered statistically significant if p<0 . 05 . All data are expressed as mean ± SEM .
Kisspeptin ( Kiss1 ) and neurokinin B ( NKB ) neurocircuits are essential for pubertal development and fertility . Kisspeptin neurons in the hypothalamic arcuate nucleus ( Kiss1ARH ) co-express Kiss1 , NKB , dynorphin and glutamate and are postulated to provide an episodic , excitatory drive to gonadotropin-releasing hormone 1 ( GnRH ) neurons , the synaptic mechanisms of which are unknown . We characterized the cellular basis for synchronized Kiss1ARH neuronal activity using optogenetics , whole-cell electrophysiology , molecular pharmacology and single cell RT-PCR in mice . High-frequency photostimulation of Kiss1ARH neurons evoked local release of excitatory ( NKB ) and inhibitory ( dynorphin ) neuropeptides , which were found to synchronize the Kiss1ARH neuronal firing . The light-evoked synchronous activity caused robust excitation of GnRH neurons by a synaptic mechanism that also involved glutamatergic input to preoptic Kiss1 neurons from Kiss1ARH neurons . We propose that Kiss1ARH neurons play a dual role of driving episodic secretion of GnRH through the differential release of peptide and amino acid neurotransmitters to coordinate reproductive function .
Puberty and fertility are necessary for survival of the species . An evolutionarily ancient region of the brain called the hypothalamus regulates these processes . The hypothalamus releases a chemical messenger called gonadotropin-releasing hormone ( or GnRH for short ) , which is then transported from the brain to the pituitary gland . GnRH activates the pituitary gland , which in turn releases reproductive hormones that control ovulation in females and sperm production in males . For this process to work correctly in females , the hypothalamus must release GnRH in appropriately timed pulses and produce one massive release , or “surge” , of GnRH to trigger ovulation . Two populations of neurons within the hypothalamus produce a peptide molecule called Kisspeptin and drive the activity and subsequent release of GnRH . One population resides in an area called the arcuate nucleus and the other in the preoptic nucleus . Recent findings suggest that the arcuate nucleus is the “pulse generator” responsible for triggering the rhythmic release of GnRH by the hypothalamus , whereas the preoptic nucleus induces the surge of GnRH . However , how these brain regions do this remains unclear . Using a technique called optogenetics , Qiu , Nestor et al . explored whether kisspeptin-producing neurons in the arcuate nucleus are able to communicate with each other to drive pulses of GnRH release . The idea was to selectively activate a subset of kisspeptin neurons in mice and determine whether this would activate the remaining neurons at the same time . Qiu , Nestor et al . introduced a light-sensitive protein into the kisspeptin-producing neurons on one side of the arcuate nucleus , and then used light to activate those neurons . As predicted , this caused kisspeptin neurons throughout the arcuate nucleus to coordinate their activity . In addition to their namesake peptide , kisspeptin-producing neurons also make the neurotransmitter glutamate , the excitatory peptide neurokinin B , and the inhibitory peptide dynorphin . Light-induced stimulation of the arcuate nucleus caused its kisspeptin neurons to also release neurokinin B and dynorphin , which synchronized the firing of the kisspeptin neurons . The hypothalamus then translates this coordinated activity into pulses of GnRH release . The light-induced stimulation also triggered the release of glutamate , which caused kisspeptin neurons within the preoptic nucleus to fire in bursts . This in turn robustly excited the GnRH neurons , giving rise to the surge of GnRH . These findings show that peptide and classical neurotransmitters collaborate to control GnRH neuron activity and , consequently , fertility . The results obtained by Qiu , Nestor et al . can be used to further explore kisspeptin-GnRH neuronal circuits , and to obtain insights into the role of neuronal peptide signaling in healthy as well as diseased states .