Getting a handle on rat familiarization: The impact of handling protocols on classic tests of stress in Rattus norvegicus.

Experimenter familiarization with laboratory rodents through handling prior to experimentation is an important practice in neurobehavioral research and is implicated in stress, study variability, and replicability. Unfortunately, different handling protocols have not been thoroughly examined. Determining optimal experimenter familiarization protocols is expected to reduce animal stress and thus improve welfare and data consistency. The impact of different handling protocols was determined through behavioral assessments (i.e. elevated plus maze, light/dark box, open field) as well as via analysis of fecal boli counts, ultrasonic vocalizations, and blood corticosterone. Male and female Sprague Dawley rats were distributed among three groups: never handled, picked-up, and handled for 5 min once daily over five days. Handled and picked-up rats spent more time in open arms and less time in closed arms of the elevated plus maze and more time in the center and less time at the perimeter of the open field compared to rats that were never handled, indicating that handled and picked-up rats were less anxious than those that were never handled. Male rats consistently defecated more frequently throughout the handling process and throughout behavioral testing, whereas females showed greater concentrations of blood corticosterone. Female rats were found to emit more 50-kHz calls and fewer 22-kHz calls compared to males. The results observed suggest that picking animals up may suffice as a handling method compared to time-intensive handling procedures, and that there are significant sex differences in response to handling.

The Effects of Rearing in a Shelved Environment on Behavioral and Physiological Markers of Welfare in Rats (Rattus norvegicus).

Early-life experiences are critical modifiers of development. An important component of early-life experience is the nature of maternal interactions, which can be modified by stress. During rearing, mothers are typically allocated to single-level cages where they are readily accessible to pups, a potentially stressful scenario not reflective of nature. Accordingly, mothers regularly removed from the rearing environment interact differently with their offspring, leading to long-term changes in offspring physiology and behavior. Such changes commonly include modifications within the hypothalamic-pituitary-adrenal axis, of which corticosterone is a major component. Modifications in the hypothalamic-pituitary-adrenal axis may also be manifested through changes in affective behavior and assessed via tests such as the open field and elevated plus maze as well as via ultrasonic vocalization (USV) analysis. As a means of assessing the impact of rearing in a shelved environment, we allocated mothers to standard single-level cages or cages with an integrated shelf, which allowed the mother to temporarily escape pups. While there were no differences in fecal cortico-sterone, behavior in the elevated plus maze, or USVs, male rats reared in standard cages weighed more, and all standard single-level housed rats spent more time in the center of the open field. The observed differences indicate that allocating nursing mothers to shelved environments throughout the postnatal period has long-lasting effects on offspring behavior that must be considered when establishing dam enrichment protocols.

Longitudinal Assessment of Behaviour and Associated Bio-Markers Following Chronic Consumption of ß-Sitosterol ß-D-Glucoside in Rats: A Putative Model of Parkinson's Disease.

The consumption of cycad (Cycas circinalis) seeds has been linked to the development of Amyotrophic Lateral Sclerosis-Parkinsonism Dementia Complex (ALS-PDC) in humans. ALS-PDC is a clinically variable disease presenting as a combination of symptoms typical of PD and/or ALS. Chronic consumption of ß-sitosterol ß-D-glucoside (BSSG), a component of the cycad seed, by rats (Rattus norvegicus) has been previously reported to initiate a progressive pathology that develops over several months and manifests as behavioural and histopathological changes that resemble characteristic features of Parkinson's disease. As part of an independent multi-site validation study, we have tried to replicate and further characterize the BSSG model with a focus on motor function, and associated immunohistochemical markers. Beginning at 3 months of age, male CD® (Sprague Dawley) rats (N = 80) were dosed orally with either a flour pellet or a flour pellet containing BSSG (3 mg) daily (5×/week) for 16 weeks consistent with previous reports of the model. Following BSSG intoxication, separate cohorts of animals (n = 10/treatment) were exposed to a behavioural test battery at 16, 24, 32, or 40 weeks post-initial BSSG feeding. The test battery consisted of the open field test, cylinder test, and ultrasonic vocalization (USV) assessment. No changes in behaviour were observed at any time point. Following behavioural testing, animals were processed for immunohistochemical markers of substantia nigra integrity. Immunohistochemistry of brain tissue revealed no differences in the microglial marker, Iba1, or the dopaminergic integrity marker, tyrosine hydroxylase (TH), in the substantia nigra at any assessment point. The absence of any group differences in behaviour and immunhistochemistry indicates an inability to replicate previous reports. Further investigation into the sources of variability in the model is necessary prior to further utilization of the BSSG model in preclinical studies.

Ultrasonic Vocalization Analysis as a Novel Metric to Assess Cage Enrichment in Rats.

Laboratory rodent housing conditions vary significantly across laboratories and facilities. Variation in housing can be associated with animal stress leading to study variability and the subsequent inability to replicate experimental findings. Optimization and standardization of animal housing are necessary to promote animal welfare and data consistency, thereby reducing the number of animals necessary to detect treatment effects. While interest in environmental enrichment is increasing, many studies do not examine the behavior of animals in the home cage, neglecting important aspects of enrichment. To determine how increased vertical home cage area affects animal welfare, double-decker cages (enriched), which allow rats to upright stand, were compared with standard single-level cages, which impede the ability to upright stand. Home cage welfare was assessed by analyzing ultrasonic vocalizations, fecal corticosterone, upright standing, and fighting. Ultrasonic vocalization was further explored by analyses of call type as defined by a 14 call-type schematic. Rats housed in enriched cages spent more time fighting, produced fewer 50 kHz calls, and had higher levels of fecal corticosterone. Rats in standard cages attempted to upright stand more often but remained upright for a shorter amount of time due to the height limitation imposed by standard cages. In addition, standard cages restrict some naturalistic behaviors such as upright standing and reduce fighting, which may be attributable to their single-tier organization and floor space. Enriched cages permit rats to engage in normal ethological behavior but also increase fighting. This study demonstrates that housing conditions have a meaningful impact on multiple measures of animal affect. When considering study design, researchers should be aware of how housing conditions affect animal subjects.

Early-life seizures modify behavioral response to ultrasonic vocalization playback in adult rats.

Early-life seizures (ELS) are associated with autism spectrum disorder (ASD); however, due to a lack of effective treatments for ELS, it is not clear whether ELS plays a causal role, potentiates the ASD phenotype, or is the result of a common pathophysiology. Deficits in communications are a core feature of ASD. To isolate the impact of ELS on communication, we probed the behavioral consequences of a single episode of kainic acid-induced early-life seizures (KA-ELS) in male and female Sprague-Dawley (CD) rats. Deficits in auditory communication were observed in adult male rats as assessed by behavioral response to ultrasonic vocalization (USV) playback. Ultrasonic vocalizations are classified into two major categories - 50-kHz (positive) calls and 22-kHz (aversive) calls. Behavioral response was assessed via rat preference for different USV playback in a radial arm maze. Response to 22-kHz calls was not impacted by ELS while response to 50-kHz calls was impacted. All rats demonstrated positional preference for the arms adjacent to where 50-kHz calls were playing compared to background noise; however, male ELS rats demonstrated a greater positional preference for the arms adjacent to where 50-kHz calls were playing compared to male control rats. These studies demonstrate that responses to socially relevant auditory cues are chronically altered in adult male rats following a single episode of ELS. We speculate that these changes contribute to previously reported social deficits associated with ELS.