Neuroscience

Anorexia nervosa (AN) is a psychiatric disorder mainly characterized by extreme hypophagia, severe body weight loss, hyperactivity, and hypothermia. Currently, AN has the highest mortality rate among psychiatric illnesses. Despite decades of research, there is no effective cure for AN nor is there a clear understanding of its etiology. Since a complex interaction between genetic, environmental, social, and cultural factors underlines this disorder, the development of a suitable animal model has been difficult so far. Here, we present our protocol that couples a loss-of-function mouse model to the activity-based anorexia model (ABA), which involves self-imposed starvation in response to exposure to food restriction and exercise. We provide insights into a neural circuit that drives survival in AN and, in contrast to previous protocols, propose a model that mimics the conditions that mainly promote AN in humans, such as increased incidence during adolescence, onset preceded by negative energy balance, and increased compulsive exercise. This protocol will be useful for future studies that aim to identify neuronal populations or brain circuits that promote the onset or long-term maintenance of this devastating eating disorder.

Models of drug addiction in rodents are instrumental in understanding the underlying neurobiology. Intravenous self-administration of drugs in mice is currently the most commonly used model; however, several challenges exist due to complications related to catheter patency. To take full advantage of the genetic tools available to study opioid addiction in mice, we developed a non-invasive mouse model of opioid self-administration using vaporized fentanyl. This model can be used to study various aspects of opioid addiction including self-administration, escalation of drug intake, extinction, reinstatement, and drug seeking despite adversity. Further, this model bypasses the limitations of intravenous self-administration and allows the investigation of drug taking over extended periods of time and in conjunction with cutting-edge techniques such as calcium imaging and in vivo electrophysiology.

Research on exploratory behavior plays a key part in behavioral science. Studying exploratory behavior of laboratory rodents may provide important data about many developmental and neurobiological processes occurring in animal ontogenesis. The proposed protocol for measuring the free (low-stress) exploration behavior in rats is straightforward, requires minimal resources and very little animal training. It can therefore be broadly applied to studying animal cognition, animal behavior in general, the aging processes, and several animal models of various phenomena.

Play is a complex social behavior that is highly conserved across mammals. In most species, males engage in more frequent and vigorous play as juveniles than females, which reflects subtle yet impactful sex differences in brain circuitry and development. In this protocol, we describe a behavioral testing paradigm to assess social play in male and female juvenile rats. We highlight the behavior scoring criteria for distinguishing rough-and-tumble play from other play-related social behaviors. By analyzing both sexes, play behavior can be leveraged as a powerful tool to understand the sex-specific development and expression of social behavior.

Studying social behavior in mouse models empowers the understanding of the neurobiological mechanisms involved, which are affected in neuropsychiatric disorders, allowing the evaluation of therapeutic strategies. Behavioral methods available are time-consuming and reducing the length of behavioral sessions may render more manageable experiments and reduce animal stress. We validated a new reliable and sensitive method to study two features of social behavior (sociability and preference for social novelty) in two strains of male mice, the C57BL/6J inbreed strain and the CD1 (ICR) outbreed strain, using a modified version of the V-shaped maze (Vsoc-maze). The Vsoc-maze for sociability and preference for social novelty improves time performance by shortening the length of the sessions, and reduces variability compared to the classical approach performed in the three-chamber apparatus. Altogether, the Vsoc-maze allows evaluating the specific alterations of social behavior in mice in a time-efficient and reproducible manner.

The alcohol preference model is one of the most widely used animal models relevant to alcoholism. Stressors increase alcohol consumption. Here we present a protocol for a rapid and useful tool to test alcohol preference and stress-induced alcohol consumption in mice. In this model, animals are given two bottles, one with a diluted solution of ethanol in water, and the other with tap water. Consumption from each bottle is monitored over a 24-h period over several days to assess the animal’s relative preference for the ethanol solution over water. In the second phase, animals are stressed by restraining them for an hour daily and their subsequent preference of tap water or the ethanol solution is evaluated. Preference is measured by the volume and/or weight or liquid consumed daily, which is then converted to a preference ratio. The alcohol preference model was combined with the conditioned place preference paradigm to determine alcohol conditioning and preference following the deletion of CB2 cannabinoid receptors in dopaminergic neurons in the DAT-Cnr2 Cre-recombinant conditional knockout (cKO) mice in comparison with the wild-type control mice.

Maladaptive avoidance behaviors are seen in many stress-related psychiatric illnesses. Patients with these illnesses favor passive, avoidant coping strategies rather than adaptive, active coping strategies. Preclinically, coping strategy can be measured in rats using the shock-probe defensive burying test, wherein rats receive a shock from an electrified probe inserted into a test cage that mimics their home cage environment, and behavioral output (immobility or burying) is recorded for 15 min following the shock. Immobility in response to the perceived threat of the shock-probe, associated with elevated stress hormone levels, is regarded as a passive, maladaptive coping strategy. In opposition, burying the probe is associated with lower stress hormone levels and is considered an active, adaptive coping style. In rats, chronic stress induces a shift from active to passive coping in this test (i.e., proportionally less burying and more immobility), modeling the avoidant symptoms presented across many stress-related psychiatric illnesses. The stress-induced shifts in coping style and overall behavioral reactivity to the shock-probe provide a unique and well-validated measure of not only an anxiety-like behavioral response but also coping strategy selection in rat models of psychiatric illness.

The fundamental of neuroscience is to connect the firing of neurons to physiological and behavioral outcomes. Chemogenetics enables researchers to control the activity of a genetically defined population of neurons in vivo through the expression of designer receptor exclusively activated by designer drug (DREADD) in specific neurons and the administration of its synthetic ligand clozapine N-oxide (CNO) (Sternson and Roth, 2014). Using stimulatory Gq-coupled DREADD (hM3Dq) in mice, we showed that leptin receptor (LepRb)-expressing neurons in the preoptic area (POA) of the hypothalamus are warm-sensitive neurons that mediate warm-responsive metabolic and behavioral adaptations by reducing energy expenditure and food intake (Yu et al., 2016). We also used DREADD technology to test effects of chronic stimulation of POA LepRb neurons on energy expenditure, food intake, and body weight with the TSE indirect calorimetry system. Here we describe the detailed protocol of how we used indirect calorimetry to study the outcome of chronic stimulation of POA LepRb neurons. This protocol can be adapted to study long-term metabolic and behavioral consequences of other neuronal modulations, with possible modifications to the type of DREADD, duration of CNO treatment, or method of CNO delivery.

Experimental animal models are unique tools (i) to study pain transmission and pathophysiology of neuropathic pain, (ii) to identify novel molecular targets and (iii) to test the potential analgesic effect of specific molecules. The chronic constriction injury (CCI) model of neuropathic pain is the first model of post-traumatic painful peripheral neuropathy, originally developed by Bennett and Xie in the late 1980s. The chronic constriction is performed in the sciatic nerve and induces a partial denervation involving myelinated afferent axons and unmyelinated axons. Damage to unmyelinated axons is much more severe than myelinated afferents. As the model induces a partial denervation, it is very useful for the analysis of pain behaviours. Stimulation of the hind paw, a target of the sciatic nerve, induces pain which can be quantitated. Thus, mechanical allodynia is usually assessed 7, 14 and 21 days after CCI of the sciatic nerve by measuring the hind paw withdrawal response to von Frey filament stimulation. Here, we describe in detail the protocol allowing a reliable and reproducible CCI model in mice. Overall, researchers most commonly use this surgical model to discover more efficacious drugs for the pharmacological control of chronic pain states.

Pain research is mostly based on experimental assays that use animal models, which may allow deciphering the physiopathology of this condition and to propel drug discovery. The formalin nociception test is considered one of the most predictive approaches to study acute pain in rodents. This test permits monitoring pain-related responses (i.e., itch) caused by a subcutaneous injection of an inflammatory agent, namely 2.5% formalin solution, in the hind paw. After the injection, two distinct time periods or phases of licking/biting behaviour occur, which are separated by a quiescent period. Importantly, these phases differ in duration and underlying mechanisms. Hence, the initial acute phase (phase I), commonly recorded for 5 min just after formalin administration, reflects acute peripheral pain, probably due to direct activation of nociceptors through TRPA1 channels. On the other hand, the phase II, which starts after the quiescent period (5-15 min) and is commonly recorded for 15-30 min, is due to the ongoing inflammatory input and central nociceptive sensitization. Here, we describe in detail the protocol used to perform a reliable and reproducible formalin test in mice.