Kisspeptin knockout (Kiss−/−)22, Kisspeptin-IRES-Cre (KissIC)26, GnRH::Cre12; DicerloxP/loxP59, R26-BIZ31, and nNOS knockout (nNOS−/−)54,60 mouse strains have been previously described and validated, and are on different genetic backgrounds (see Supplementary Table 1). All experiments were performed on adult (>8 weeks of age) female mice unless otherwise stated.
Animal care and experimental procedures were performed in accordance with the guidelines established by the institutional animal care and use committee of the Royal Netherlands Academy of Arts and Science and by the National Institutes of Health “Guide for the Care and Use of Research Animals, Eight Edition,” and were approved by the Ethical Committee for Animal Use of the Universities of Liege (Belgium), Saarland (Germany) and of Otago (New Zealand). Female mice were placed into individual cages under a reversed light/dark cycle (12 h:12 h light/dark; 21.00 h lights on and 9.00 lights off) with food and water ad libitum.
Ovariectomy and hormone supplementation
Unless otherwise stated, females were ovariectomized in adulthood (>8 weeks of age) under general anesthesia after either subcutaneous (sc) injections of ketamine (80 mg kg−1 per mouse) and medetomidine (Domitor, Pfizer, 1 mg kg−1 per mouse) or under 5% isoflurane, in order to control for endogenous hormone concentrations and to prevent pregnancies upon repeated testing. At the same time, all females received a 5-mm-long silastic capsule (inner diameter: 1.57 mm; outer diameter: 2.41 mm) containing crystalline 17β-estradiol (diluted 1:1 with cholesterol) subcutaneously in the neck. The dose of E2 (E8875, Sigma) was based on a previous study61 showing that this treatment leads to estradiol levels similar to mice in estrus. At the end of surgery, females under ketamine/medetomidine anesthesia received an sc injection of atipamezole (Antisedan, Pfizer, 4 mg kg−1 per mouse) to antagonize medetomidine-induced effects and accelerate recovery. In order to induce sexual receptivity at the day of testing, all females received a subcutaneous injection with progesterone (500 µg, P0130, Sigma) 3 h before the onset of the behavioral test, unless stated otherwise (for overview of all the different hormone treatments, see Supplementary Table 1).
Viruses and stereotaxic injections for behavioral testing
The AAV5-flex-taCasp3-TEVp virus (abbreviated to “AAV-Casp3” (Vector Core, University of North Carolina)) was stereotaxically injected bilateral into the RP3V in KissIC mice (total: Cre−: n = 14; Cre+: n = 14). AAV-Casp3 uses the T2A peptide encoding sequence to ensure bicistronic expression of pro-taCasp3 and TEVp after Cre-mediated recombination. taCasp3 triggers cell autonomous apoptosis, thereby minimizing toxicity to adjacent Cre− cells23.
AAV5-EF1a-DIO-hChR2(H134R)-mCherry-WPRE-pA virus (abbreviated to “AAV-ChR2” (Vector Core, University of North Carolina)) was stereotaxically (see below) injected bilaterally into the RP3V in KissICmice (Cre−: n = 8; Cre+: n = 8) to selectively express the light-activated cation channel channelrhodopsin-2 and mCherry62 in kisspeptin neurons.
Mice were placed in a motorized stereotaxic frame (Neurostar, Germany) under 5% isoflurane anesthesia. The skull was exposed by a midline scalp incision, and the stereotaxic frame was aligned at Bregma using visual landmarks. After alignment of the head of the mice, a drill was placed above the skull at coordinates (according to the Paxinos Brain Atlas63) corresponding to the rostral periventricular area of the third ventricle (RP3V; rostrocaudal, 0.2 mm; mediolateral, ±0.1 mm) and a hole drilled through the skull bone to expose the brain. A 33-gauge steel needle loaded with virus (AAV-Casp3 or AAV-ChR2) was slowly inserted through the hole until it penetrated to a depth of 5.8 mm. Virus (1 μl per brain site injected) was delivered at 100 nl min−1 through a Hamilton syringe using a syringe pump (Harvard Apparatus). The needle was left in place for an additional 10 min to allow diffusion of the virus before being slowly removed. Following AAV-Casp3 injection, the hole was filled with dental cement and the skin was sutured. Following AAV-ChR2 injection, a bilateral cannula (200 µm core diameter; Doric Lenses) holding optical fibers with 45°-oriented mirror tips was inserted into the RP3V at a distance of 0.25 mm (mediolateral) from the center of injection and further fixed to the skull with dental cement. Mice were allowed to recover on a heating pad and returned to their home cage after waking up. All mice received an sc injection with Caprofen (5 mg kg−1) for post-operative analgesia.
Viruses and stereotaxic injections for electrophysiology
Adult female (>2 months old) heterozygous KissIC mice were group-housed under conditions of controlled temperature (22 ± 2 °C) and lighting (12-h light, 12-h dark cycles) with ad libitum access to food and water. Mice were anesthetized, placed in a stereotaxic apparatus, and given simultaneous bilateral 0.5 µl injections of AAV9-EF1-DIO-hChR2-(H134R)-mCherry-WPRE-hGH (2.2 × 1013 GC ml−1; Penn Vector Core) into the RP3V (coordinates according to the Paxinos Brain Atlas63), 0.2 mm anterior to Bregma and 5.8 mm in depth) at a rate of 100 nl min−1. The syringes were left in situ for 3 min before and 10 min after the injections. Following a recovery period, mice were bilaterally ovariectomized under anesthesia and, after >2 weeks, received subcutaneous silastic implants containing 17-β-estradiol (1 µg per 20 g body weight) according to Bronson64. Implants were made of 17-β-estradiol dissolved in ethanol and mixed with medical grade adhesive (0.1 mg ml−1 adhesive), which is then injected into 1 mm internal diameter silastic tubing. Six days later, mice received a subcutaneous injection of estradiol benzoate (1 µg per 20 g body weight) in the morning and were used for electrophysiology the following day
Cannula implantation for ICV kisspeptin administration
Mice were placed in a motorized stereotaxic frame (Neurostar, Germany) under 5% isoflurane anesthesia. The skull was exposed by a midline scalp incision, and the stereotaxic frame was aligned at Bregma using visual landmarks. After alignment of the head of the mice, a drill was placed above the skull at coordinates corresponding to the lateral ventricle (lateral+1, anterior–posterior: −0.34; dorsoventral: −2.5) and a hole drilled through the skull bone to expose the brain. Then, a 26-gauge cannula cut at 2 mm from pedestal was implanted and fixed to the skull with dental cement. A dummy was inserted to close the cannula until the behavioral experiment. Mice were allowed to recover on a heating pad and returned to their home cage after waking up. All mice received an sc injection with Temsegic (0.05 mg kg−1) for post-operative analgesia.
Removal of the VNO
One week after ovariectomy, subjects underwent either bilateral removal of the VNO or sham surgery (VNOx or VNOi groups)65. Briefly, animals were placed on their back and the lower jaw was gently opened after general anesthesia. A midline incision was made in the soft palate extending rostrally from behind the first palatal ridge to the incisors, and the underlying bone was exposed by blunt dissection. In VNOi animals, the incision was then closed with reabsorbable sutures. For VNOx animals, the rostral end of the VNO was exposed by drilling, the caudal end of the vomer bone was cut, and the VNO was removed bilaterally with a gentle twisting motion. Bleeding was controlled using a blunted 18-gauge needle attached to a vacuum. Animals were carefully monitored after surgery for bleeding and or breathing difficulties.
Ablation of the MOE
Two weeks after the removal of the VNO (VNOx females) or sham surgery (VNOi females), mice received an intranasal application of 10% ZnSO4 to lesion the main olfactory epithelium (MOEx) or saline solution (MOEi) under general anesthesia66.
Kisspeptin-10 (Kp-10) was synthesized in Strasbourg, France (Sequence: Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Tyr-NH2, NeoMPS; weight = 25.7 mg). To determine whether kisspeptin-stimulated female sexual behavior in wild-type C57BL/6J mice, females received an sc injection of Kp-10 at the dose of 0.52 µg kg−1 (injection volume 100 µl) 2 h before the lordosis test. Each animal was used as its own control, i.e., the animal was injected 1 day with Kp-10 and the other day with saline. Females were not injected with progesterone in this particular experiment. Injections were separated by at least 3 days.
When injected intracerebroventricularly, females were injected with 10.4 ng kg−1 of Kp-10 (injection volume 2 µl), 1 h before the lordosis test through a cannula inserted into the lateral ventricle.
Two hours before mate preference test, female mice received a single sc injection of GnRH (0.025 mg kg−1, Polypeptide Laboratories France SAS, SC087).
S-nitroso-N-acetyl-DL-Penicillamine (SNAP) (N398-Sigma) is a NO donor. In order to ascertain the most efficient activity of SNAP (8 mg kg−1) was combined with the guanylate cyclase agonist BAY-41-2272 (10 mg kg−1; B8810—Sigma) 1 h before injection. One hour before behavioral tests, female mice received a single subcutaneous injection (100 µl) of the cocktail SNAP+BAY 41-2272.
Brain slice preparation for electrophysiology
Mice were killed by cervical dislocation, decapitated and brains quickly removed. Coronal brain slices (200–250 μm) containing the rostral periventricular area of the third ventricle (RP3V) were cut with a vibratome (VT1000S; Leica) in an ice-cold solution containing (in mM): NaCl 87, KCl 2.5, NaHCO3 25, NaH2PO4 1.25, CaCl2 0.5, MgCl2 6, glucose 25, and sucrose 75. Slices were then incubated at 30 °C for at least 1 h in artificial cerebrospinal fluid (aCSF; in mM): NaCl 120, KCl 3, NaHCO3 26, NaH2PO4 1, CaCl2 2.5, MgCl2 1.2, and glucose 10. All solutions were equilibrated with 95%O2/5%CO2.
Cell-attached recordings and light stimulation
Slices were placed under an upright microscope fitted for epifluorescence (Olympus, Tokyo, Japan) and constantly perfused (1.5 ml min−1) with warm (~30 °C) aCSF. mCherry-expressing RP3V neurons were first visualized by brief fluorescence illumination and subsequently approached using infrared differential interference contrast optics. Action potential firing was recorded in voltage clamp mode in the cell-attached loose patch configuration. Recording electrodes (3–5 MΩ) pulled from borosilicate capillaries (Warner Instruments, Hamden, CT) with a horizontal puller (Sutter Instruments, Navato, CA) were filled with aCSF including 10 mM HEPES. Low resistance seals (10–30 MΩ) were achieved by applying either no suction or the lowest amount of suction required to detect spikes. For ChR2 activation, blue light was delivered to the slice through a ×40 immersion objective (0.8 NA, Olympus) via a 470 nm light-emitting diode (LED, CoolLED) connected to the vertical illumination port of the microscope. Stimulation consisted of 1–15 s trains of blue light pulses (2 ms duration; ~0.25 mW) delivered at 10 Hz, repeated ten times every 60 s in each cell. Electrophysiological signals were recorded using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA) connected to a Digidata 1440A digitizer (Molecular Devices). Signals were low-pass filtered at 3 kHz before being digitized at a rate of 10 kHz and stored on a personal computer. Signal acquisition and analysis was carried out with pClamp 10 (Molecular Devices). Spikes were detected using the threshold crossing method. In each cell, spike fidelity was calculated by dividing the number of light-evoked spikes by the number of blue light stimuli and expressed as a percentage.
All experimental females were brought into behavioral estrus by ovariectomy (OVX) in adulthood and combined treatment with estradiol (E) through a silastic capsule and an acute injection (3 h before testing) with progesterone (P), unless stated otherwise (for all details on hormone treatments, see Supplementary Table 1). Females were always tested during the dark phase of the light/dark cycle. Finally, levels of female sexual behavior displayed by the control (wild type) females vary as function of the background strain with 129SvJ females (Kiss+/+) showing relatively low levels compared to C57BL6/j females.
Assessment of the MOE lesion
Anosmia was assessed by submitting females to the hidden cookie test66. Briefly, female mice were food-deprived overnight. A small piece of a chocolate chip cookie was buried (~1 cm deep) at a random location in a clean Plexiglas aquarium (35 cm long × 25 cm high × 19 cm wide) containing fresh sawdust. The time it took each mouse to find the cookie was recorded. The test lasted until the mouse had located the cookie or 10 min if the cookie was not found. All mice treated with ZnSO4 failed to find the hidden cookie and were thus considered to be anosmic.
Exposure to odors in bedding
Four groups of gonadally intact males (n = 5 each) were placed in clean cages containing fresh sawdust. Bedding was collected 12 h later and directly used as olfactory stimulus for the experimental females. Thirty-six hours before bedding exposure, all experimental females (singly housed) were placed on clean sawdust in two separate housing units to separate females, which were going to be exposed to male bedding or to clean bedding as control (and thus to prevent the controls from being exposed to male odors). On the day of testing, females were injected with P (500 µg) to induce behavioral estrus. This hormonal treatment made the experimental females behaviorally receptive at the time of odor exposure. Three hours after P injection, 15 g of fresh male-soiled or clean bedding was placed into the subject’s own cage. Ninety minutes after bedding exposure, females were perfused with paraformaldehyde and brains were collected.
Mate preference tests
To assess mate preferences shown in response to auditory and olfactory stimuli, we used a box (60 cm long × 30 cm high × 13 cm wide) that was divided into three compartments using perforated opaque partitions. The partitions contained perforated holes at a height of 8 cm to facilitate the diffusion of odors from the two-side compartments to the middle compartment. Tests were performed during the dark phase of the light cycle (5 h after lights out). Animals were habituated to the three compartment box only once on the day before the behavioral experiments by placing them in the middle compartment for 10 min (with no stimulus animals placed in the two-side compartments). On the day of testing, an intact male stimulus and an estrous female stimulus were placed in the lateral compartments with their own bedding to make the stimuli as odorous as possible. Three hours after receiving a progesterone injection (500 µg), the female subject was introduced into the middle compartment, and was observed for 10 min. The time the subject spent poking her nose through the holes of the partition or actively sniffing the bottom of the partition in front of the female vs. male stimulus animal was recorded. A preference score was calculated by dividing the time spent investigating the male compartment minus the time spent investigating the female compartment by the total time spent investigating both compartments. A positive value of the preference score indicates a mate preference directed toward the stimulus male, whereas a negative value indicates a mate preference directed toward the stimulus female (for details, see ref. 67).
Lordosis tests without photostimulation
Females were subjected to weekly lordosis tests in a Plexiglas aquarium (37 cm long × 17 cm high × 21 cm wide). A sexually experienced male was placed alone in the aquarium and allowed to adapt for 15 min. Subsequently, 3 h after receiving a subcutaneous progesterone injection (500 µg, P0130, Sigma) to induce behavioral estrus, the lordosis responses of the female to the mounts of the stimulus male were recorded. The test lasted until the female received 10 mounts or 10 min had elapsed. For the first experiment (mating-induced Fos activation), ovary intact females were paired with males during 30 min. A lordosis quotient (LQ) was calculated by dividing the number of lordosis responses displayed by the female subjects by the number of mounts received (x100). Before each experimental condition (drug injection, cell ablation, and optogenetic stimulation), all females were subjected to at least three lordosis tests (with progesterone) in order to acquire sufficient sexual experience and thus a significant LQ. Tests were performed during the dark phase of the light cycle (5 h after lights out; for details, see ref. 67). For all details on the different hormone treatments (estradiol vs. estradiol+progesterone), see Supplementary Table 1.
Lordosis tests with photostimulation
Prior to the lordosis test, the cannula was connected to an optical fiber (Doric Lenses), which in turn was connected to a blue laser (wavelength = 473 nm) via an optical rotatory join allowing free movements of the animal. The optic fiber was flexible and long enough to allow the female to freely behave and interact with the male. After three pretests, KissICfemales (Cre− and Cre+) were then divided in two groups. On test 4, half of the females received optogenetic stimulation (stimulated), whereas the other half did not (unstimulated). On test 5 (conducted 1 week later), unstimulated females received an optogenetic stimulation while previously stimulated females did not, thus each female acted as her own control. Blue light was delivered through the optic cable at 10 Hz as soon as the male approached the female (sniffing and showing mount attempt). The duration of the stimulation varied as a function of the male, i.e., the time it took him to mount the female, however this was never longer than 15 s. Tests were performed over 10 min and the number of mounts was recorded as well as the number of female lordosis responses. Importantly, in order to observe possible stimulatory effects of photostimulation on lordosis behavior, females were not injected with progesterone before the lordosis test, and were thus only on estradiol treatment (by silastic capsule, previously described).
Transcardial perfusion and OCT embedding for histology
Female mice were anesthetized and perfused transcardially with saline followed immediately by 4% ice-cold paraformaldehyde. Brains were removed and postfixed in 4% paraformaldehyde for 2 h. Brains were then cryoprotected in 30% sucrose68 in PBS and when sunken, were embedded in Optimal Cutting Temperature compound (OCT, Tissue-Tek). A glass box was placed in a slurry of ethanol and dry ice. The glass box was then partially filled with isopentane. The tissue was placed in a plastic cuvette filled with OCT, placed into the isopentane bath and rapidly frozen. Brains were subsequently stored at −80 °C prior to sectioning.
Histological assessment of VNO removal
As previously described65, snouts were removed immediately after perfusion, cleared of all soft tissue, and soaked for 30 min in rapid decalcifier (Apex Engineering Products). Decalcified snouts were then soaked overnight in 30% sucrose68 at which time a 1:1 mixture of 30% sucrose and OCT was suctioned into the nasal passages. Snouts were then incubated for 4 h in the 1:1 solution and were finally frozen in OCT and stored at −80 °C. Snouts were sectioned at 10 µm thickness on a cryostat. One section every 150 µm was transferred directly onto Superfrost Plus glass slides and dried overnight. Sections were rinsed and stained with hemotoxylin and eosin to assess the presence of blood clots in the nasal sinuses and to determine whether the VNO was completely removed. A total of 32 mice were used and upon examination, 8 were excluded because the VNO was not completely removed. No blood clots were detected in any of the animals.
Immunohistochemical detection of c-Fos or kisspeptin
Brain sections (30 µm thick) were cut on a Leica CM3050S cryostat. Forebrains were cut coronally from the rostral telencephalon to the posterior hypothalamus. Sections were saved in four different series, placed in antifreeze solution, and stored at −20 °C.
Immunostaining was carried out on free-floating sections. All incubations were carried out at room temperature, and all washes of brain tissue sections were performed using Tris-buffered saline (TBS 0.05 M) or Tris-buffered saline containing 0.1% Triton X-100 (TBST). Briefly, sections were rinsed and endogenous peroxidase activity was quenched by incubating the sections for 30 min with 0.3% hydrogen peroxide. Non-specific-binding sites were then blocked by incubating sections for 30 min with 5% normal goat serum (NGS) (Dako Cytomation, Denmark). Sections were then incubated either with a rabbit polyclonal antibody (1/5000 in TBST-NGS 5%; anti-kisspeptin-10, AB9754, Chemicon, Millipore) raised against the decapeptide kisspeptin-10 (derived from the Kiss-1 gene product) for 48 h at 4 °C or with a rabbit polyclonal anti-c-Fos antibody (1/2000 in TBST-NGS 5%; c-Fos (4): sc-52R, Santa Cruz Inc.) raised against the N terminus of c-Fos of human origin. Sections were then incubated for 1 h in avidin-biotin complex (1/800, ABC, Vector Laboratory, Burlingam, CA) and then reacted for 5 min with 3,3′-diaminobenzidine tetrahydrochloride (DAB Kit, Vector Laboratory). Sections were then washed, dried overnight, left in xylene (Sigma) for 15 min, and coverslipped using Eukit (Fluka, Steinheim, Germany).
Immunohistochemical detection of c-Fos and kisspeptin
To determine the distribution of c-Fos and kisspeptin double-labeled neurons, ovary intact females in proestrus (activation of kisspeptin cells following mating) or ovariectomized females (VNOx/MOEx experiment) were perfused with 4% paraformaldehyde in 0.1 M PBS 90 min after the introduction of the male to the female (onset of behavioral testing) or 90 min after being placed in the empty testing arena for the unmated controls. Regarding male bedding exposure (VNOx/MOEx), females were perfused 90 min after the onset of odor exposure (male or clean bedding). For the dual immunohistochemistry, sections were first washed in 0.1 M PBS pH 7.4 (PBS), peroxidase activity was blocked in PBS solution with 0.3% H2O2, and then permeabilized in PBS-0.1% Triton X-100 (PBST) and saturated in 5% NGS in PBST. Immediately after this step, sections were incubated in diluted anti-c-Fos antibody overnight. On the following day, sections were washed in PBST and incubated in a goat anti-rabbit biotinylated secondary antibody (Dako, Prod. Ref. B0432, 0.75 µg ml−1 PBST). Sections were then washed in PBST and incubated in the Vectastain Elite ABC Kit (Vector, Prod. Ref. PK6100). After development with the DAB Substrate Kit (Vector, SK-4100), in a black precipitate (3,3′-diaminobenzidine (DAB) plus Ni2+), sections were washed thoroughly in PBS, and residual peroxidase activity blocked in PBS solution with 0.3% H2O2. Sections were then permeabilized and blocked in 5% NGS-PBST and incubated in anti-kisspeptin-10 antibody for 72 h. Similar secondary antibody and ABC incubation steps were then performed. The developing reaction used in this step was a DAB brown precipitate, using the same kit. Following this, sections were mounted in Eukitt after being air-dried.
Immunohistochemical detection of barley lectin and nNOS
In order to identify cells that are synaptically connected to kisspeptin neurons, KissIC/R26-BIZ mice were transcardially perfused with 4% paraformaldehyde at proestrus or metestrus/diestrus (determined via vaginal cytology). Brains were sectioned at 14 μm and collected in series of five on SuperFrost Plus slides (Roth) and stored at −80 °C. The transsynaptic tracer BL was immunohistologically detected using goat anti-wheat germ agglutinin (1:1000, Vector Laboratories) and has been described previously31. A Tyramide Signal Amplification Plus Biotin kit was used for signal amplification. Briefly, slides were washed 3× for 5 min in PBS, incubated in ice-cold methanol with 0.3% H2O2 for 30 min, washed 3× for 5 min in TNT (0.1 M Tris, 0.15 M NaCl, 0.05% Tween 20), incubated for 10 min in 0.5% Triton X-100 in PBS, washed 3× for 5 min in TNT, blocked with TNB for 30 min, and incubated with anti-wheat germ agglutinin (1:1000) in TNB overnight at 4 °C in a humidified chamber. The next morning, slides were brought to RT for 2 h, washed 3× for 5 min in TNT and incubated with biotinylated anti-goat IgG (1:500) in TNB for 1 h at RT. Slides were then washed 3× for 5 min in TNT and incubated with streptavidin-conjugated horseradish peroxidase (1:100) in TNB for 30 min at RT. Slides were then washed 3× for 5 min in TNT and incubated for 10 min in biotin plus amplification reagent (1:50) in 1× plus amplification diluent at RT. Slides were then washed 3× for 5 min in TNT followed by incubation in streptavidin-conjugated Cy5 (1:500) in TNB for 30 min at RT. Slides were then washed 3× for 5 min with TNT. The slides were incubated overnight at 4 °C followed by 2 h at RT with rabbit anti-nNOS (1:300) in 0.1 M PBS containing 0.5% λ-carrageenan (Sigma) and 0.02% sodium azide. The sections were then treated with Cy3-conjugated donkey anti-rabbit (1:500) in PBS containing 0.5% λ-carrageenan (Sigma) and 0.02% sodium azide in PBS for 1 h at RT. Nuclei were stained with bisbenzimide solution (1:2000 in 0.1 M PBS, 5 min at RT) and coverslipped with Fluoromount-G (Southern Biotech). Images were taken using either a Zeiss Axioskop or a Zeiss Axio Scan Z1 epifluorescence microscope.
Immunohistochemical detection of mCherry
To immunohistochemically detect mCherry, slides were washed 3× for 5 min in PBS, incubated in 0.3% Triton X-100, 5% donkey serum, and 0.02% sodium azide in PBS for 1 h at RT followed by incubation with anti-ds-red (1:1000, recognizes mCherry) in PBS containing 0.5% λ-carrageenan (Sigma) and 0.02% sodium azide overnight at 4 °C. The next day slides were washed 3× for 5 min with PBS containing 0.5% Tween 20 (PBS+Tween), incubated with Cy3-conjugated donkey anti-rabbit in PBS containing 0.5% λ-carrageenan (Sigma) and 0.02% sodium azide and washed 3× for 5 min in PBS+Tween. Nuclei were stained with bisbenzimide solution (1:2000 in 0.1 M PBS, 5 min at RT) and coverslipped with Fluoromount-G (Southern Biotech). Images were taken using either a Zeiss Axioskop or a Zeiss Axio Scan Z1 epifluorescence microscope.
Quantification and statistical analysis
Kisspeptin-immunoreactive (-ir) cell bodies were counted manually and bilaterally in three to four adjacent brain sections (with an interval of 120 µm between them) delineating the RP3V (anteroventral periventricular area+Periventricular preoptic zone) using a Zeiss Axioskop microscope (×40 objective). Cell counts are expressed as mean number per section for each experimental condition. Analysis of kisspeptin-ir density in the ARC was performed as previously described69. To analyze kisspeptin/c-Fos double labeling, three to four sections were selected from the RP3V and the total number of kisspeptin and kisspeptin/c-Fos co-labeled cells were counted in order to obtain the percentage of kisspeptin cells expressing c-Fos immunoreactivity per section.
nNOS-ir and BL+ nNOS-ir cell bodies were counted unilaterally in eight to ten sections containing the VMHvl (Bregma −1.34 to −1.94 according to ref. 63. Cell counts are expressed as mean number per section for each experimental condition. Statistical significance was determined using Bonferroni’s multiple comparison test.
Randomization was not used in this study and no statistical methods were used to predetermine sample size. Investigators were blinded to the group allocation during experiments or data analysis. Data were analyzed using the GraphPad Prism 7 software. For all statistical comparisons, we first analyzed the data distribution with the Shapiro–Wilk test for normality. Preference score data were analyzed by comparison to hypothesized mean (H0: mean equal 0) using a non-directional one-sample two-tailed t test (Figs. 1b, e, 4a and 6a)70. For comparison of paired samples comparing two groups, statistical analysis was performed by using a paired-sample two-tailed t test (Figs. 2b, d, f, i, 4c and 6d). Comparison of unpaired samples comparing two groups was then performed using an unpaired-sample two-tailed ttest (Figs. 1c and 2a, e). For comparison of more than two groups, an ANOVA test followed by a Tukey’s (Figs. 1e and 6b, c) or Bonferroni’s (Fig. 5) two-tailed multiple comparisons test was used. Comparison of more than two data sets violating the normal distribution, the Kruskal–Wallis ANOVA two-tailed test followed by a Dunn’s multiple comparison two-tailed test was used (Figs. 1a and 3a, b). Comparison of two data sets violating the normal distribution, a one-sample Wilcoxon two-tailed test was used (Figs. 2c and 4b).