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

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.