We are grateful to Raymond Sanchez at the University of Washington for his valuable help in manuscript editing and to M.Sc. Georgina Cort&#233;s and M.Sc. Cintia Javier for their technical support in the standardization of this technique. We are also grateful to the members of the vet services at Facultad de Estudios Superiores Zaragoza: MVZ. Adriana Altamirano, MVZ. Roman Hern&#225;ndez and MVZ. Dolores-Elizabeth Guzm&#225;n for the excellent maintenance and care of experimental animals. The experiments described in this protocol were supported by DGAPA-PAPIIT grant number: IN216015 and by CONACyT grant number: 236908 to Roberto Dom&#237;nguez. Carlos-Camilo Silva is a doctoral student from Programa de Doctorado en Ciencias Biom&#233;dicas, Universidad Nacional Aut&#243;noma de M&#233;xico (UNAM) and is supported by the Consejo Nacional de Ciencia y Tecnolog&#237;a (Grant number: 294555).

Procedures involving animals were approved by the Ethics Committee of Facultad de Estudios Superiores Zaragoza, UNAM. This institution operates in strict accordance with the Mexican rules for animal handling, Official Norm: NOM-062-ZOO-1999, which agrees with international guidelines.
1. Construction of bilateral cannulas
<ol>
	<li>Extract the stainless-steel shaft from the hub of two 23 G hypodermic needles using pressure tweezers and then remove any remaining glue using a scalpel blade.</l...

The authors have nothing to disclose relative to this article.

This article describes a method to transiently inactivate, at any given time, a discrete region in the brain of awake and unrestrained rats. A simple method to track their estrous cycle and assess ovulation is also provided. This protocol allows a straightforward analysis of the contribution of specific brain regions to the mechanisms that drive ovulation by comparing the ovulatory outcome of TTX-treated animals with that of vehicle-treated ones. With the exception of the stereotaxic instrument and the microinjection pum...

Ovulation is the process by which one or more mature oocytes are released from the ovaries once every estral/menstrual cycle. As all mammalian species depend on the production of gametes to breed, the understanding of the mechanisms that regulate ovulation has a huge impact in areas ranging from biomedicine, the livestock industry and the maintenance of endangered species. Ovulation is regulated by the hypothalamic-pituitary-ovarian axis, which involves several hypothalamic and extra-hypothalamic areas, the gonadotropes in the anterior pituitary and the theca and granulosa cells that, along with the oocytes, form the ovarian follicles inside the ovaries1.
Ovarian follicles grow, develop and eventually ovulate in response to the tonic and phasic secretion of the follicle-stimulating hormone and the luteinizing hormone, the two gonadotropins secreted by the gonadotropes. The pattern of gonadotropin secretion is pivotal for proper follicular development and ovulation and it is regulated by the gonadotropin-releasing hormone (GnRH)1,2. This neuropeptide is synthesized by neurons scattered throughout the basal diencephalon and then secreted to the portal vasculature that links the hypothalamus and the anterior pituitary. The secretory activity of the GnRH-neurons is in turn modulated by synaptic input arising from diverse brain structures. These structures convey information about the state of the external and internal environment of the organism including the availability of food, the length of the photoperiod and the concentration of hormones in the blood. In this sense, they shape the reproductive pattern of each species and the specific roles of such structures must be determined in order to properly understand the mechanisms governing ovulation. As an example, it has been shown that the fluctuation in estradiol levels during the estrous cycle regulates the secretion of GnRH; however, GnRH-neurons does not express the estradiol receptor isoform needed to detect such changes. Two populations of neurons expressing these receptors are located in the rostral periventricular region of the third ventricle and in the arcuate nucleus, respectively, and stablish synapses with GnRH-neurons. There is evidence to suggest that these neurons interpret the concentration of estradiol and then stimulate the activity of GnRH-neurons by releasing kisspeptin, a potent inductor of GnRH secretion3.
Experiments involving thermic or chemical lesions, as well as mechanical deafferentation, allowed researchers to determine the involvement of several brain structures in the regulation of ovulation4,5,6,7,8,9,10,11,12. These experiments, however, have the disadvantage of being invasive and traumatic, requiring several days of recovery before evaluating the effects of the treatment, impeding the analysis of the acute effects of the treatment. In addition, they permanently affect the targeted areas and disrupt other physiological processes in the long term. Due to these problems, the results of these experiments are usually obscured by the homeostatic compensatory mechanisms in the animal&#180;s body and extracting accurate information about the temporal regulatory dynamics in which the area is involved is rather difficult.
The microinjection of drugs that transiently disrupt the activity of neurons through guide cannulas is a suitable alternative that surpasses the disadvantages mentioned above. The cannulas can be placed in any brain region by a stereotaxic surgery, allowing the researcher to start the drug treatment after the confounding effects of the surgery disappear. The timed microinjection of the drugs allows researchers to test hypotheses regarding the contribution of the region to a particular step of the process and can be performed in awake restrained or free-moving animals. A variety of drugs including local anesthetics, agonists, antagonists, inverse agonists and biological toxins such as tetrodotoxin (TTX) can be microinjected into the region of interest at specific times.
TTX is a biological toxin synthesized by bacteria living in the body of the pufferfish as well as other vertebrates and invertebrates. TTX silences neural activity through the selective and transient blockade of sodium channels, which results in the inhibition of sodium-dependent action potentials. In the presence of TTX, cells experience an alteration in the depolarization phase and thus are not excitable but remain alive. The blocking effect of TTX is explained by its molecular composition: a guanidinium group is able to pass through the extracellular aspect of the sodium channel, but the rest of the molecule cannot pass due to its size, so it is stuck and blocks the channel13,14,15,16,17. The mechanism of action of the TTX allowed its use as a tool to study the nervous system both in vitro and in vivo. Intracerebral injection of this toxin has been used to study the role of discrete brain areas in several processes like memory retention18, sleep and arousal19, place recognition20, spatial navigation21, drug abuse22, thermoregulation23, development of schizophrenia24, sexual behavior25 and regulation of ovulation26 among others. In this protocol we describe the effects on ovulation of the transient inactivation of hypothalamic nuclei by TTX microinjection in awake and unrestrained rats.

<table>
	<tbody>
		<tr>
			<td>10 &#956;L Hamilton syringes</td>
			<td>Hamilton</td>
			<td>80314</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G x 1&#34; stainless steel hypdermic needle</td>
			<td>BD</td>
			<td>305165</td>
			<td></td>
		</tr>
		<tr>
			<td>23 G x 1&#34; stainless steel hypdermic needle</td>
			<td>BD</td>
			<td>305145</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G x 1/2&#34; stainless steel hypdermic needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial cerebrospinal fluid</td>
			<td>BASi</td>
			<td>MD-2400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone trimer</td>
			<td>Fine Science Tools</td>
			<td>16152-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Burr for micro drill</td>
			<td>Fine Science Tools</td>
			<td>19007-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Clipper</td>
			<td>Wahl</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cut-off disc</td>
			<td>Dremel</td>
			<td>SM5010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting tweezers</td>
			<td>Truper</td>
			<td>17367</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanocrylate glue</td>
			<td>Kola loka</td>
			<td>K-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Nic Tone</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Enrofloxasin</td>
			<td>Senosiain</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Sigma</td>
			<td>E4009</td>
			<td></td>
		</tr>
		<tr>
			<td>Estereoscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Extra fine Bonn scissors</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Face mask</td>
			<td>Lanceta HG</td>
			<td>60036</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11050-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxilin</td>
			<td>Sigma</td>
			<td>H3136</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostats</td>
			<td>Fine Science Tools</td>
			<td>13008-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma</td>
			<td>320331</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypromelose artificial tears</td>
			<td>Sophia Labs</td>
			<td>8950015</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Pisa Agropecuaria</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Aranda</td>
			<td>1183</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection pump</td>
			<td>KD Scientific</td>
			<td>788380</td>
			<td></td>
		</tr>
		<tr>
			<td>Monomer</td>
			<td>Nic Tone</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mototool</td>
			<td>Dremel</td>
			<td>3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td>Lanceta HG</td>
			<td>69028</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Rupture Ear Bars</td>
			<td>David Kopf Instruments</td>
			<td>855</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L lysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Dermo Dine</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine with soap</td>
			<td>Germisin espuma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure tweezers</td>
			<td>Truper</td>
			<td>17371</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anesthesia mask</td>
			<td>David Kopf Instruments</td>
			<td>Model 906</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline solution</td>
			<td>PISA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10004-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Fine Science Tools</td>
			<td>10015-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Pisa Agropecuaria</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard electrode holder</td>
			<td>David Kopf Instruments</td>
			<td>1770</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel wire</td>
			<td>American Orthodontic</td>
			<td>856-612</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>David Kopf Instruments</td>
			<td>Model 900LS</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Sissors</td>
			<td>Fine Science Tools</td>
			<td>14001-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon connectors</td>
			<td>Basi</td>
			<td>MD-1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon tubing</td>
			<td>Basi</td>
			<td>MF-5164</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Alomone labs</td>
			<td>T-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>Kent scientific</td>
			<td>VetFlo</td>
			<td></td>
		</tr>
	</tbody>
</table>

unraveling the role of discrete areas of the rat brain in the regulation of ovulation through reversible inactivation by tetrodotoxin microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

The protocol described above was tested by evaluating the effects of a single TTX or vehicle (artificial cerebrospinal fluid; ACSF) microinjection into one of two different nuclei known to be involved in the regulation of ovulation in the rat: the suprachiasmatic and the arcuate nucleus. The suprachiasmatic nucleus was chosen since it contains the central circadian pacemaker in mammals. It is involved in the regulation of cyclic events as the secretion of gonadotropins. The arcuate nucleus was chosen because it contains ...

Watch this Scientific Journal Video about Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections at JoVE.

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

This protocol describes the construction of a low-cost microinjection system, its stereotaxic implantation into deep-brain structures and the procedure for timed microinjections of tetrodotoxin in awake and unrestrained rats. The goal is to reveal the participation of hypothalamic structures in the regulation of ovulation by inhibiting their neural activity.

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and ...

This protocol allows researchers to explore the nuclei of the neuronal circuits that regulates ovulation in female mammals at specific times of their estrous cycle.This technique can be adapted to study any process on the regulation of the brain in any species.Also they can less art fabricator in the lab lowering the overall cost.Demonstrating this procedure will be Montserrat Bola

unraveling-role-discrete-areas-rat-brain-regulation-ovulation-through

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JoVE Journal

Neuroscience

2.9K Görüntüleme.  Universidad Nacional Autónoma de México. This protocol allows researchers to explore the nuclei of the neuronal circuits that regulates ovulation in female mammals at specific times of their estrous cycle.This technique can be adapted to study any process on the regulation of the brain in any species.Also they can less art fabricator in the lab lowering the overall cost.Demonstrating this procedure will be Montserrat Bola

Video: Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections