All animal procedures were approved by the Institute of Biomedical Sciences Animals Ethics Committee at the University of São Paulo and were performed according to the ethical guidelines adopted by the Brazilian College of Animal Experimentation.
1. Preparation of solutions
2. Brain dissection and slicing
NOTE: Since different brain structures may require cutting in different planes (coronal, sagittal, or horizontal slices), the exact approach for obtaining the slices depends on the brain region of interest. Typically, to study the Kiss1-expressing cells in the AVPV/PeN and ARH (here denominated as AVPV/PeNKisspeptin neurons and ARHkisspeptin neurons; Figure 2A,B), coronal brain slices (200-300 µm) are usually made17,19,20,21,34. The AVPV/PeNKisspeptin neurons are located approximately 0.5 to -0.22 mm from the bregma, whereas ARHkisspeptin neurons are at -1.22 to -2.70 mm. Nuclei location can be determined by using a stereotaxic mouse brain atlas35 or the Allen Mouse Brain Reference Atlas (http://mouse.brain-map.org/). Adult Kiss1-Cre/GFP female (diestrus-stage) and male mice36 were used in this study.
3. Cell sealing for recording
To study the possible effects of human recombinant growth hormone (hGH) on the activity of hypothalamic kisspeptin neurons, we performed whole-cell patch-clamp recordings in brain slices and assessed whether this hormone causes acute changes in the activity of AVPV/PeNKisspeptin and ARHkisspeptin neurons. Adult Kiss1-Cre/GFP female (diestrus-stage) and male mice36 were used in this study. Gonad-intact animals were selected for the experiments, since the properties of their hypothalamic kisspeptin neurons may vary depending on sex steroid levels19,20. While it was beyond the scope of the present study to evaluate differences between sexes, we refer the reader to Croft et al. and Frazão et al.19,20 for more information. Genetically modified animals, such as mice and rats, represent exciting tools for this type of experiment, since cells expressing a specific gene or a selective-induced ablation can be identified by a fluorescent protein such as GFP, among others23,26,36. The use of genetically modified mice represents a breakthrough in understanding kisspeptin neuron activity.
Recorded neurons (26 cells out of 12 animals) were determined according to neuroanatomical features35 and the expression of the endogenous GFP20. In current-clamp mode, neurons were recorded under I = 0 in whole-cell patch-clamp configuration. The AVPV/PeNKisspeptin neurons (12 cells from nine animals) exhibited an average RMP of -59.0 mV ± 3.0 mV (range: -75 mV to -46 mV), input resistance of 0.9 ± 0.1 GΩ, wcc of 12.3 pF ± 1.6 pF, and SR of 19.4 ± 1.9 mΩ. Among the recorded neurons, three out of 12 AVPV/PeNKisspeptin cells showed spontaneous discharges of action potentials (APs) at rest (0.1 Hz ± 0.06 Hz; average RMP of the spontaneously active cells was -50.7 mV ± 2.7 mV). The average RMP of ARHkisspeptin neurons (14 cells from 12 animals) was -50.0 mV ± 1.5 mV (range: -62 mV to -39 mV), the average input resistance was 1.7 ± 0.1 GΩ, wcc was 9.2 pF ± 0.7 pF, and SR of 16.9 ± 1.7 mΩ. Most ARHkisspeptin neurons were quiescent, whereas four out of 14 cells showed spontaneous APs at rest (0.9 Hz ± 0.5 Hz; average RMP of the spontaneously active cells was -52.7 mV ± 1.4 mV).
The administration of hGH (20 µg/g) to the bath induced a significant hyperpolarization of the RMP of many of the recorded neurons, five out of 12 AVPV/PeNKisspeptin neurons (≈55% of cells from 9 mice), and nine out of 14 recorded ARHkisspeptin neurons (≈65% of cells from 12 mice, p = 0.0006, Fisher's exact test; Figure 3). The AVPV/PeNKisspeptin and ARHkisspeptin hyperpolarized neurons significantly changed the RMP compared to the unresponsive cells (Figure 3B,E; Mann-Whitney test). The effects on RMP (Figure 3C,F; repeated measures ANOVA and Tukey's post-test) were followed by a significant reduction of the whole-cell input resistance (IR) on AVPV/PeNKisspeptin (0.9 ± 0.1 GΩ to 0.7 ± 0.1 GΩ during hGH application, p = 0.02; Figure 3D), and on ARHkisspeptin (1.7 ± 0.1 GΩ to 1.0 ± 0.1 GΩ during hGH application, p < 0.0001; repeated measures ANOVA and Tukey's post-test; Figure 3G) neurons. Additionally, the frequency of spontaneous APs (fAPs) of hGH-hyperpolarized neurons decreased in both populations of cells (0.1 Hz ± 0.06 Hz to 0.0 Hz ± 0.0 Hz in AVPV/PeNKisspeptin and 1.0 Hz ± 0.5 Hz to 0.2 Hz ± 0.1 Hz in ARHkisspeptin neurons). However, the extent of this decrease failed to reach a level of statistical significance (p > 0.05, Mann-Whitney test). After the hGH washout, the RMP and IR were restored to baseline (Figure 3A,C,D,F,G). The remaining kisspeptin neurons were unresponsive to hGH administration.
We have previously demonstrated that pGH induces no effects on hypothalamic kisspeptin neuron activity (please refer to Silveira et al.25; Figure 3H). Of note, it is known that hGH can activate prolactin (PRL) receptors in addition to GH receptors37,38. Besides, PRL indirectly depolarizes only ≈20% of AVPV/PeNKisspeptin neurons in mice24. In contrast, PRL does not modulate the fast synaptic transmission of the ARHkisspeptin cells24. Therefore, the hGH-induced hyperpolarization effect reported here seems to be nonspecific. The observed differences may depend on variables such as drug concentration, species difference (human vs. mouse), or even the presence of salts in the composition of the used drugs, as previously reported28.
Figure 1: Schematic diagram summarizing the whole-cell patch-clamp technique's contribution to the knowledge of the kisspeptin neurons' activity. Kisspeptin neurons (shown in green) are located in the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) and arcuate nucleus of the hypothalamus (ARH). The AVPV/PeNKisspeptin and ARHkisspeptin cells send direct connections to gonadotrophin-releasing hormone (GnRH) neurons' soma located in the preoptic area (POA) and their terminals at the median eminence (ME), culminating in the modulation of the hypothalamus-pituitary-axis (HPG). Different neuromodulators, such as hormones, have been shown to differentially modulate the activity of the AVPV/PeNKisspeptin and ARHkisspeptin neurons. Possible effects on the resting membrane potential are schematically demonstrated by representative tracings obtained using the whole-cell path-clamp technique and current-clamp recordings. The red color indicates that a specific neurotransmitter induces the depolarization of the resting membrane potential (RMP)17,22,24,26,28,29,30,31,32; the blue color indicates no effect on RMP24,25,26,27,30. The dashed line indicates the RMP. Please click here to view a larger version of this figure.
Figure 2: Basic steps to obtain the sealing of the cell of interest by the whole-cell patch-clamp technique. (A,B) Representative photomicrographs of brain slices (250 µm) containing kisspeptin cells at the anteroventral periventricular nucleus (AVPV) and arcuate nucleus of the hypothalamus (ARH). Kisspeptin neurons were identified by green fluorescent protein (GFP) expression. (C) Photomicrograph demonstrating a micropipette (containing electrolyte solution [internal solution]) close enough to the cell to create a dimple in the plasma membrane to perform the seal. (D,E) Mild negative pressure (mouth suction performed on a tube attached to the headstage and micropipette) is required to seal the cell membrane to the micropipette (D). A second application of negative pressure (mild and brief) is necessary to induce the plasma membrane rupture (E). (F) The registration of the cell activity is performed by a mechanical setup used for patch-clamp experiments. After breaking the plasma membrane, currents flowing through the ionic channels in the patched cell can be recorded by an electrode connected to a highly sensitive amplifier. A feedback resistor generates the current needed for voltage-clamp (G) or current-clamp (H) recordings. Abbreviations: 3V = third ventricle; ME = median eminence. Scale bars: A = 130 µm, B = 145 µm, C = 20 µm, D = 15 µm. Please click here to view a larger version of this figure.
Figure 3: Testing drug specificity. (A) Representative current-clamp recording demonstrating that human recombinant growth hormone (hGH) induced a hyperpolarization of the resting membrane potential (RMP) of the kisspeptin neurons located at the arcuate nucleus of the hypothalamus (ARHkisspeptin). (B–G) Bar graphs demonstrating the average change in the resting membrane potential (RMP) (B,C,E,F) and average input resistance (IR) (D,G) of hGH-responsive kisspeptin neurons located at the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeNKisspeptin) (B–D) or ARH (E–G). Representative current-clamp recording demonstrating that porcine growth hormone (pGH) induced no effect on the RMP of ARHkisspeptin neurons, as previously reported25 (H). The significance tests used are the Mann-Whitney test for (B) and (E), and repeated measures ANOVA and Tukey's post-test for (C,D,F,G). The dashed line indicates the RMP. *p = 0.02; **p = 0.004,***p = 0.0003; ****p < 0.0001. Please click here to view a larger version of this figure.
Internal Solution (100 mL) | |||
Salt | FW (g/mol) | Concentration | Weight (g) |
K-gluconate | 234.2 | 120 mM | 2.81 |
NaCl | 58.4 | 1.0 mM | 0.006 |
KCl | 74.5 | 10 mM | 0.074 |
HEPES | 238.3 | 10 mM | 0.24 |
EGTA | 380.3 | 5.0 mM | 0.19 |
CaCl2 | 147.0 | 1.0 mM | 0.015 |
MgCl2 | 203.0 | 1.0 mM | 0.02 |
KOH | 56.11 | 3.0 mM | 0.017 |
ATP | 507.18 | 4.0 mM | 0.20 |
pH =7.3 / osmolarity = 275 – 280 mOsm |
Table 1: Reagents for the preparation of the internal solution. The table contains the molecular weight (FW), desired concentrations, and the calculated weight of the salts for the preparation of 100 mL of solution.
aCSF/Slicing Solution (250 mL) | |||
Salt | FW | Concentration | Weight (g) |
Sucrose | 342.3 | 238 mM | 18.5 |
KCL | 74.5 | 2.5 mM | 0.046 |
NaHCO3 | 84.0 | 26 mM | 0.546 |
NaH2PO4 | 120.0 | 1.0 mM | 0.03 |
MgCl2 | 203.0 | 5 mM | 0.254 |
D-glucose | 180.2 | 10 mM | 0.450 |
CaCl2 | 147.0 | 1.0 mM | 0.037 |
pH = 7.3 / osmolarity = 290 – 295 mOsm |
Table 2: Reagents to prepare the slicing solution. The table contains the molecular weight (FW), desired concentrations, and calculated weight of the salts for the preparation of 250 mL of solution. The brain is submerged in this solution to be sliced.
aCSF for recording (1 L) | |||
Salt | FW | Concentration | Weight (g) |
NaCl | 58.4 | 135 mM | 7.88 |
KCL | 74.5 | 3.5 mM | 0.261 |
NaHCO3 | 84.0 | 26 mM | 2,184 |
NaH2PO4 | 120.0 | 1.25 mM | 0.150 |
MgSO4 | 246.5 | 1.2 mM | 0.296 |
D-glucose | 180.2 | 10 mM | 1,802 |
CaCl2 | 147.0 | 1.0 mM | 0.148 |
pH = 7.3 / osmolarity = 290-300 mOsm |
Table 3: Reagents to prepare aCSF for recordings. The table contains the molecular weight (FW), desired concentrations, and calculated weight of the salts for the preparation of 1 L of solution.
Supplementary Figure 1: Example of an in-house made recovery chamber. (A,B) An in-house recovery chamber can be fabricated as follows: cut a 24-well plate so that nine wells are available. Glue a nylon screen to the base of the nine wells. With the remainder of the well plate, make a base so that the lower part of the nylon is free. (C) This adapted base can be placed inside a 500 mL beaker containing artificial cerebrospinal spinal fluid (aCSF) constantly saturated with carbogen (95% O2 and 5% CO2). (D) The beaker holding the recovery chamber is kept in the water bath during experimentation. (E,F) An acrylic transfer pipette is used to transfer the brain slices to the recovery chamber. Please click here to download this File.
Compounds for aCSF, internal and slicing solutions | |||
ATP | Sigma Aldrich/various | A9187 | |
CaCl2 | Sigma Aldrich/various | C7902 | |
D-(+)-Glucose | Sigma Aldrich/various | G7021 | |
EGTA | Sigma Aldrich/various | O3777 | |
HEPES | Sigma Aldrich/various | H3375 | |
KCL | Sigma Aldrich/various | P5405 | |
K-gluconate | Sigma Aldrich/various | G4500 | |
KOH | Sigma Aldrich/various | P5958 | |
MgCl2 | Sigma Aldrich/various | M9272 | |
MgSO4 | Sigma Aldrich/various | 230391 | |
NaCl | Sigma Aldrich/various | S5886 | |
NaH2PO4 | Sigma Aldrich/various | S5011 | |
NaHCO3 | Sigma Aldrich/various | S5761 | |
nitric acid | Sigma Aldrich/various | 225711 | CAUTION |
Sucrose | Sigma Aldrich/various | S1888 | |
Equipments | |||
Air table | TMC | 63-534 | |
Amplifier | Molecular Devices | Multiclamp 700B | |
Computer | various | – | |
DIGIDATA 1440 LOW-NOISE DATA ACQUISITION SYSTEM | Molecular Devices | DD1440 | |
Digital peristaltic pump | Ismatec | ISM833C | |
Faraday cage | TMC | 81-333-03 | |
Imaging Camera | Leica | DFC 365 FX | |
Micromanipulator | Sutter Instruments | Roe-200 | |
Micropipette Puller | Narishige | PC-10 | |
Microscope | Leica | DM6000 FS | |
Osteotome | Bonther equipamentos & Tecnologia/various | 128 | |
Recovery chamber | Warner Instruments/Harvard apparatus | – | can be made in-house |
Recording chamber | Warner Instruments | 640277 | |
Spatula | Fisher Scientific /various | FISH-14-375-10; FISH-21-401-20 | |
Vibratome | Leica | VT1000 S | |
Water Bath | Fisher Scientific /various | Isotemp | |
Software and systems | |||
AxoScope 10 software | Molecular Devices | – | Commander Software |
LAS X wide field system | Leica | – | Image acquisition and analysis |
MultiClamp 700B | Molecular Devices | MULTICLAMP 700B | Commander Software |
PCLAMP 10 SOFTWARE FOR WINDOWS | Molecular Devices | Pclamp 10 Standard | |
Tools | |||
Ag/AgCl electrode, pellet, 1.0 mm | Warner Instruments | 64-1309 | |
Curved hemostatic forcep | various | – | |
cyanoacrylate glue | LOCTITE/various | – | |
Decapitation scissors | various | – | |
Filter paper | various | – | |
Glass capillaries (micropipette) | World Precision Instruments, Inc | TW150F-4 | |
Iris scissors | Bonther equipamentos & Tecnologia/various | 65-66 | |
Pasteur glass pipette | Sigma Aldrich/various | CLS7095B9-1000EA | |
Petri dish | various | – | |
Polyethylene tubing | Warner Instruments | 64-0756 | |
Razor blade for brain dissection | TED PELLA | TEDP-121-1 | |
Razor blade for the vibratome | TED PELLA | TEDP-121-9 | |
Scissors | Bonther equipamentos & Tecnologia/various | 71-72, 48,49; | |
silicone teat | various | – | |
Slice Anchor | Warner Instruments | 64-0246 | |
Syringe filters | Merck Millipore Ltda | SLGVR13SL | Millex-GV 0.22 μm |
Tweezers | Bonther equipamentos & Tecnologia/various | 131, 1518 |
Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in the anteroventral periventricular nucleus and rostral periventricular nucleus, as well as the arcuate nucleus of the hypothalamus, project to gonadotrophin-releasing hormone (GnRH) neurons, among other cells. Previous studies have demonstrated that kisspeptin signaling occurs through the Kiss1 receptor (Kiss1r), ultimately exciting GnRH neuron activity. In humans and experimental animal models, kisspeptins are sufficient for inducing GnRH secretion and, consequently, luteinizing hormone (LH) and follicle stimulant hormone (FSH) release. Since kisspeptins play an essential role in reproductive functions, researchers are working to assess how the intrinsic activity of hypothalamic kisspeptin neurons contributes to reproduction-related actions and identify the primary neurotransmitters/neuromodulators capable of changing these properties. The whole-cell patch-clamp technique has become a valuable tool for investigating kisspeptin neuron activity in rodent cells. This experimental technique allows researchers to record and measure spontaneous excitatory and inhibitory ionic currents, resting membrane potential, action potential firing, and other electrophysiological properties of cell membranes. In the present study, crucial aspects of the whole-cell patch-clamp technique, known as electrophysiological measurements that define hypothalamic kisspeptin neurons, and a discussion of relevant issues about the technique, are reviewed.
Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in the anteroventral periventricular nucleus and rostral periventricular nucleus, as well as the arcuate nucleus of the hypothalamus, project to gonadotrophin-releasing hormone (GnRH) neurons, among other cells. Previous studies have demonstrated that kisspeptin signaling occurs through the Kiss1 receptor (Kiss1r), ultimately exciting GnRH neuron activity. In humans and experimental animal models, kisspeptins are sufficient for inducing GnRH secretion and, consequently, luteinizing hormone (LH) and follicle stimulant hormone (FSH) release. Since kisspeptins play an essential role in reproductive functions, researchers are working to assess how the intrinsic activity of hypothalamic kisspeptin neurons contributes to reproduction-related actions and identify the primary neurotransmitters/neuromodulators capable of changing these properties. The whole-cell patch-clamp technique has become a valuable tool for investigating kisspeptin neuron activity in rodent cells. This experimental technique allows researchers to record and measure spontaneous excitatory and inhibitory ionic currents, resting membrane potential, action potential firing, and other electrophysiological properties of cell membranes. In the present study, crucial aspects of the whole-cell patch-clamp technique, known as electrophysiological measurements that define hypothalamic kisspeptin neurons, and a discussion of relevant issues about the technique, are reviewed.
Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in the anteroventral periventricular nucleus and rostral periventricular nucleus, as well as the arcuate nucleus of the hypothalamus, project to gonadotrophin-releasing hormone (GnRH) neurons, among other cells. Previous studies have demonstrated that kisspeptin signaling occurs through the Kiss1 receptor (Kiss1r), ultimately exciting GnRH neuron activity. In humans and experimental animal models, kisspeptins are sufficient for inducing GnRH secretion and, consequently, luteinizing hormone (LH) and follicle stimulant hormone (FSH) release. Since kisspeptins play an essential role in reproductive functions, researchers are working to assess how the intrinsic activity of hypothalamic kisspeptin neurons contributes to reproduction-related actions and identify the primary neurotransmitters/neuromodulators capable of changing these properties. The whole-cell patch-clamp technique has become a valuable tool for investigating kisspeptin neuron activity in rodent cells. This experimental technique allows researchers to record and measure spontaneous excitatory and inhibitory ionic currents, resting membrane potential, action potential firing, and other electrophysiological properties of cell membranes. In the present study, crucial aspects of the whole-cell patch-clamp technique, known as electrophysiological measurements that define hypothalamic kisspeptin neurons, and a discussion of relevant issues about the technique, are reviewed.