Name: hypothalamic kisspeptin neurons as a target for whole-cell patch-clamp recordings
Uploaded: 2023-03-17T14:25:00
Description: Watch this Scientific Journal Video about Hypothalamic Kisspeptin Neurons as a Target for Whole-Cell Patch-Clamp Recordings at JoVE.com

This study was supported by the S&#227;o Paulo Research Foundation [FAPESP grant numbers: 2021/11551-4 (JNS), 2015/20198-5 (TTZ), 2019/21707/1 (RF); and by the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES) - Finance Code 001&#34; (HRV).

All animal procedures were approved by the Institute of Biomedical Sciences Animals Ethics Committee at the University of S&#227;o Paulo and were performed according to the ethical guidelines adopted by the Brazilian College of Animal Experimentation.
1. Preparation of solutions
<ol>
	<li>Preparation of internal solution 
	NOTE: The internal solution fills the patch-clamp micropipette and will contact the cell&#39;s interior (see an example in Figur...

The development of the whole-cell patch-clamp technique had a significant impact on the scientific community, being considered of paramount importance for developing scientific research and enabling several discoveries. Its impact on science was enough to culminate in the Nobel Prize in Medicine in 1991, as this discovery opened the door to a better understanding of how ion channels function under physiological and pathological conditions, as well as the identification of potential targets for therapeutic agents<sup clas...

Hodgkin and Huxley made the first intracellular record of an action potential described in several scientific studies. This recording was performed on the squid axon, which has a large diameter (~500 &#181;m), allowing a microelectrode to be placed inside the axon. This work provided great possibilities for scientific research, later culminating in the creation of the voltage-clamp mode, which was used to study the ionic basis of action potential generation1,2,3,4,5,6,7,8. Over the years, the technique has been improved, and it has become widely applied in scientific research6,9. The invention of the patch-clamp technique, which took place in the late 1970s through studies initiated by Erwin Neher and Bert Sakmann, allowed researchers to record single ion channels and intracellular membrane potentials or currents in virtually every type of cell using only a single electrode9,10,11,12. Patch-clamp recordings can be made on a variety of tissue preparations, such as cultured cells or tissue slices, in either voltage-clamp mode (holding the cell membrane at a set voltage allowing the recording of, for example, voltage-dependent currents and synaptic currents) or current-clamp mode (allowing the recording of, for example, changes in resting membrane potential induced by ion currents, action potentials, and postsynaptic potential frequency).
The use of the patch-clamp technique made several notable discoveries possible. Indeed, the seminal findings on the electrophysiological properties of hypothalamic kisspeptin neurons located at the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeNKisspeptin), also known as the rostral periventricular area of the third ventricle (RP3V), and the arcuate nucleus of the hypothalamus (ARHkisspeptin)13,14,15 are of particular interest. In 2010, Ducret et al. performed the first recordings of AVPV/PeNKisspeptinneurons in mice using another electrophysiological tool, the loose-cell patch-clamp technique. These studies provided an electrical description of AVPV/PeNKisspeptin neurons and demonstrated that their firing patterns are estrous cycle-dependent16. In 2011, Qiu et al. used the whole cell patch-clamp technique to demonstrate that ARHkisspeptin neurons express endogenous pacemaker currents17. Subsequently, Gottsch et al. showed that kisspeptin neurons exhibit spontaneous activity and express both h-type (pacemaker) and T-type calcium currents, suggesting that ARHkisspeptin neurons share electrophysiological properties with other central nervous system pacemaker neurons18. Additionally, it has been demonstrated that ARHkisspeptin neurons exhibit sexually dimorphic firing rates and that AVPV/PeNKisspeptin neurons exhibit a bimodal resting membrane potential (RMP) influenced by ATP-sensitive potassium channels (KATP)19,20. Furthermore, it was established that gonadal steroids positively affect the spontaneous electrical activity of the kisspeptin neurons in mice19,20,21. The first works that study kisspeptin neurons&#39; electrophysiological properties are mentioned16,17,18,19,20. Since then, many studies have used the whole-cell patch-clamp technique to demonstrate which factors/neuromodulators are sufficient to modulate the electrical activity of kisspeptin neurons (Figure 1)17,21,22,23,24,25,26,27,28,29,30,31,32.
Given the importance of this technique for the study of neurons that are required for reproduction, among other cell types not covered here, this article describes the basic steps for the development of the whole-cell patch-clamp technique, such as preparing the solutions, dissecting and slicing the brain, and performing the seal of the cell membrane for recordings. Moreover, relevant issues about the technique are discussed, such as its advantages, technical limitations, and important variables that must be controlled for optimal experimental performance.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compounds for aCSF, internal and slicing solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma Aldrich/various</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma Aldrich/various</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma Aldrich/various</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich/various</td>
			<td>O3777</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich/various</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>KCL</td>
			<td>Sigma Aldrich/various</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Sigma Aldrich/various</td>
			<td>G4500</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma Aldrich/various</td>
			<td>P5958</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma Aldrich/various</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma Aldrich/various</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich/various</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4&#160;</td>
			<td>Sigma Aldrich/various</td>
			<td>S5011</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich/various</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>nitric acid</td>
			<td>Sigma Aldrich/various</td>
			<td>225711</td>
			<td>CAUTION</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich/various</td>
			<td>S1888</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Air table</td>
			<td>TMC</td>
			<td>63-534</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Molecular Devices</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>various</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>DIGIDATA 1440 LOW-NOISE DATA ACQUISITION SYSTEM</td>
			<td>Molecular Devices</td>
			<td>DD1440</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital peristaltic pump</td>
			<td>Ismatec</td>
			<td>ISM833C&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>TMC</td>
			<td>81-333-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Camera</td>
			<td>Leica</td>
			<td>DFC 365 FX</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>Roe-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM6000 FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Osteotome</td>
			<td>Bonther equipamentos &#38; Tecnologia/various</td>
			<td>128</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery chamber</td>
			<td>Warner Instruments/Harvard apparatus</td>
			<td>-</td>
			<td>can be made in-house</td>
		</tr>
		<tr>
			<td>Recording chamber</td>
			<td>Warner Instruments</td>
			<td>640277</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Fisher Scientific /various</td>
			<td>FISH-14-375-10; FISH-21-401-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome&#160;</td>
			<td>Leica</td>
			<td>VT1000 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath&#160;</td>
			<td>Fisher Scientific /various</td>
			<td>Isotemp</td>
			<td></td>
		</tr>
		<tr>
			<td>Software and systems</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AxoScope 10 software</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>Commander Software</td>
		</tr>
		<tr>
			<td>LAS X wide field system</td>
			<td>Leica</td>
			<td>-</td>
			<td>Image acquisition and analysis</td>
		</tr>
		<tr>
			<td>MultiClamp 700B</td>
			<td>Molecular Devices</td>
			<td>MULTICLAMP 700B</td>
			<td>Commander Software</td>
		</tr>
		<tr>
			<td>PCLAMP 10 SOFTWARE FOR WINDOWS</td>
			<td>Molecular Devices</td>
			<td>Pclamp 10 Standard</td>
			<td></td>
		</tr>
		<tr>
			<td>Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ag/AgCl electrode, pellet, 1.0 mm</td>
			<td>Warner Instruments</td>
			<td>64-1309</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved hemostatic forcep</td>
			<td>various</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>cyanoacrylate glue</td>
			<td>LOCTITE/various</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Decapitation scissors</td>
			<td>various</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>various</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillaries (micropipette)</td>
			<td>World Precision Instruments, Inc</td>
			<td>TW150F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris scissors</td>
			<td>Bonther equipamentos &#38; Tecnologia/various</td>
			<td>65-66</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur glass pipette&#160;</td>
			<td>Sigma Aldrich/various</td>
			<td>CLS7095B9-1000EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>various</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing&#160;</td>
			<td>Warner Instruments</td>
			<td>64-0756</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade for brain dissection</td>
			<td>TED PELLA</td>
			<td>TEDP-121-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade for the vibratome</td>
			<td>TED PELLA</td>
			<td>TEDP-121-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Bonther equipamentos &#38; Tecnologia/various</td>
			<td>71-72, 48,49;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone teat</td>
			<td>various</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Slice Anchor&#160;</td>
			<td>Warner Instruments</td>
			<td>64-0246</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filters</td>
			<td>Merck Millipore Ltda</td>
			<td>SLGVR13SL</td>
			<td>Millex-GV 0.22 &#956;m</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Bonther equipamentos &#38; Tecnologia/various</td>
			<td>131, 1518</td>
			<td></td>
		</tr>
	</tbody>
</table>

hypothalamic kisspeptin neurons as a target for whole-cell patch-clamp recordings

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.

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.&#160;Gonad-intact animals were selected for the experiments, since the properties of the...

Watch this Scientific Journal Video about Hypothalamic Kisspeptin Neurons as a Target for Whole-Cell Patch-Clamp Recordings at JoVE.com

Hypothalamic Kisspeptin Neurons as a Target for Whole-Cell Patch-Clamp Recordings

Here, we present a protocol to perform a whole-cell patch-clamp on brain slices containing kisspeptin neurons, the primary modulator of gonadotrophin-releasing hormone (GnRH) cells. By adding knowledge about kisspeptin neuron activity, this electrophysiological tool has served as the basis for significant advancements in the neuroendocrinology field over the last 20 years.

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in ...

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