Elimination of Serotonergic Neurons by Stereotaxic Injection of 5,7-Dihydroxytryptamine in the Dorsal Raphe Nuclei of Mice

Summary

This protocol describes a dorsal raphe nucleus (DRN)-lesioned mouse model (>90% survival rate in experimental mice) with stable loss of dorsal raphe serotonergic neurons by stereotaxic injection of 5,7-dihydroxytryptamine into the DRN using an angled approach to prevent injury to the superior sagittal sinus.

Abstract

Stereotaxic injection has been widely used for direct delivery of compounds or viruses to targeted brain areas in rodents. Direct targeting of serotonergic neurons in the dorsal raphe nucleus (DRN) can cause excessive bleeding and animal death, due to its location below the superior sagittal sinus (SSS). This protocol describes the generation of a DRN serotonergic neuron-lesioned mouse model (>90% survival rate) with stable loss of >70% 5-HT-positive cells in the DRN. The lesion is induced by stereotaxic injection of a selective serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into the DRN using an angled approach (30° in the anterior/posterior direction) to avoid injury to the SSS. DRN serotonergic neuron-lesioned mice display anxiety-associated behavior alterations, which helps to confirm success of the DRN lesion surgery. This method is used here for DRN lesions, but it can also be used for other stereotaxic injections that require angular injections to avoid midline structures. This DRN serotonergic neuron-lesioned mouse model provides a valuable tool for understanding the role of serotonergic neurons in the pathogenesis of psychiatric disorders, such as generalized anxiety disorder and major depressive disorder.

Introduction

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter mainly produced in the intestines and brain and impacts a variety of psychological functions. In the central nervous system (CNS), the serotonergic system plays a central role in the regulation of mood and social behavior, sleep and waking, appetite, memory, and sexual desire. In the CNS, serotonin is synthesized by serotonergic neurons, which can be separated into the following two groups: the rostral group, which has ascending projections innervating virtually the whole brain; and the caudal group, which mainly projects to the spinal cord¹. The rostral group, which contains about 85% of serotonergic neurons in the brain, is composed of the caudal linear nucleus, median raphe nucleus, and DRN, in which the largest population of serotonergic neurons in the brain is located.

Dysregulation of the serotonergic system is generally believed to be linked with the pathogenesis of major depressive disorder (MDD) and generalized anxiety disorders (GAD)². This is due to the fact that selective serotonin reuptake inhibitors (SSRIs) are effective pharmacological treatment for these psychiatric disorders^3,4. In addition, accumulative evidences suggest that mania⁵ and suicidal behavior⁶ may be associated with lower levels of serotonin functioning in the DRN. It has also been reported that Pet1-Cre;Lmx1b^flox/flox mice and hTM-DTA^iPet1 mice (genetic mouse models lacking most central serotonergic neurons from late embryonic stage⁷ and adulthood⁸, respectively) display enhanced contextual fear memory. However, despite extensive research, the exact involvement of DRN serotonergic neurons in these psychiatric disorders remains to be elucidated.

In order to explore the mechanisms by which DRN serotonergic neurons regulate the pathogenesis of the serotonin-associated psychiatric disorders, animal models have been generated. Optogenetic tools have been applied to inhibit serotonergic neurons in rat DRN, and these animals display increased anxiety-like behaviors⁹. However, optogenetics has limitations. For example, a light-delivery device must be implanted into the targeted region deep within the brain, and the surrounding tissue may be injured during implantation surgery or by heat emitted from the light device. Even if temperature alteration may not cause detectable brain tissue damage, it can still induce remarkable physiological and behavioral effects¹⁰.

Pharmacological manipulation may be an easier approach to create DRN serotonergic neuron-lesioned animal models. Some groups have generated DRN serotonergic neuron-lesioned rats by stereotaxic microinjection of serotonin neurotoxin 5,7-DHT in the DRN. However, these rat models display different behavioral alterations, such as anxiolytic behavior¹¹, increased anxiety-like behavior¹², and impaired object memory¹³. Despite many studies in rats, fewer studies have been performed on the influences of 5,7-DHT on mice. One group reported excessive mortality (>50%) and limited serotonin depletion in experimental mice that received stereotaxic microinjections of 5,7-DHT in the DRN¹⁴. Another group reported that unpredictable chronic mild stress (UCMS) can induce significant attack latency alteration in 5,7-DHT-induced DRN-lesioned mice. However, no histological results were provided to confirm the exact serotonergic neuron loss in the DRN¹⁵. Stereotaxic injection in the DRN using standard procedures may lead to massive bleeding and high mortality to mice, given the fact that the anatomical location of DRN is below the SSS¹⁶.

This protocol describes the protocol to generate a DRN serotonergic neuron-lesioned mouse model (>90% survival rate of the experimental mice) with stable loss of DRN serotonergic neurons by stereotaxic injection of 5,7-DHT. The injection in DRN uses an angled approach to prevent the injury to the SSS. This surgery consistently causes >70% loss of serotonergic neuron in the DRN of mice, and it produces anxiety-associated behavior alterations. The protocol used here is for inducing DRN lesions, but it can also be useful to researchers who want to perform stereotaxic injections in other midline structures. In addition, this DRN serotonergic neuron-lesioned mouse model provides a valuable tool for understanding the role of serotonergic neurons in psychiatric disorders (i.e., MDD and GAD) and assessing potential neuroprotective agents or therapeutic strategies for these conditions.

Protocol

All surgical interventions and animal care procedures have been approved by the Animal Committee of School of Life Sciences and Technology, Tongji University, Shanghai, China. 1. Housing of animals Maintain male C57BL/6NCrl mice (10 weeks old, 25 g, n = 21) in standard conditions (24 °C temperature; 55% humidity) under a 12 h/12 h light/dark cycle. Provide food and water ad libitum. NOTE: Here, three of the mice are used for confirming the needle trac…

Representative Results

In a coronal section, the location of the DRN is just below the SSS and aqueduct (Figure 1B,C); thus, targeting the DRN using standard procedures can lead to massive bleeding and high mortality in mice16. Therefore, stereotaxic injections were performed here using an angled approach instead of the standard vertical approach to avoid damage to the SSS (Figure 1A,B). To confirm the location of the needle en…

Discussion

This protocol successfully describes production of a reliable DRN serotonergic neuron-lesioned mouse model with high lesion reproducibility and low mortality rate. Targeting the DRN is a complex task, since it can damage the SSS located just above the DRN¹⁶ and lead to excessive bleeding and even death¹⁴. Therefore, stereotaxic injections were performed by setting the manipulation arm at 30° in the AP direction to avoid injury to the SSS (Figure 1<…

Materials

0.22micron syringe filter	Millipore	SLGPRB
3% hydrogen peroxide	Caoshanhu Co.,Ltd, Jiangxi, China
5,7-Dihydroxytryptamine	Sigma-Aldrich	SML2058	3ug/ul, 2ul
Compact small animal anesthesia machine	RWD Life Science Co., Ltd	R500 series
Cryostat	Leica Biosystems, Wetzlar, Germany	CM1950
Cy 3 AffiniPure Donkey Anti-Goat IgG (H+L)	Jackson ImmunoResearch	705-165-003	1:2,000
dapi	Sigma-Aldrich	D8417
desipramine hydrochloride	Sigma-Aldrich	PHR1723	25mg/kg
Eppendorf tube	Quality Scientific Plastics	509-GRD-Q
goat anti-5-HT antibody	Abcam	ab66047	1:800
GraphPad Prism	Graphpad Software Inc, CA, US
Hamilton Microliter syringe	Hamilton	87943
Ketoprofen	Sigma-Aldrich	K1751-1G	5mg/kg
L-ascorbic acid	BBI Life Sciences	A610021-0500	0.10%
lidocaine ointment	Tsinghua Tongfang Pharmaceutical Co. Ltd	H20063466
ofloxacin eye ointment	Shenyang Xingqi Pharmaceutical Co.Ltd, China	H10940177
peristaltic pump	Huxi Analytical Instrument Factory Co., Ltd, Shanghai, China	HL-1D
stereotaxic apparatus	RWD Life Science Co., Ltd	68018
ultra-low temperature freezer	Haier	DW-86L388
Vortex	Kylin-bell	VORTEX-5