Validation of behavioral phenotypes in the BACHD rat model.

Huntington disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion in the HTT gene. Various HD animal models have been generated to mimic the motor, cognitive and neuropsychiatric disturbances that affect HD patients. Reproducing disease phenotypes within these models is essential to identify reliable readouts for therapy studies. We validated behavioral phenotypes shown earlier by other research groups in the BACHD rat model, using both previously applied and novel tests for motor, cognitive and anxiety-like behaviors. We first confirmed known BACHD rats' phenotypes in rotarod, open field (OF) and elevated plus maze (EPM) tests. We then assessed the reproducibility of key phenotypes in the model using new tests: cliff hanging, passive avoidance (PA), Morris water maze (MWM), light dark box and light spot tests. We confirmed impaired motor coordination in the rotarod test and reduced activity in the OF. In line with earlier results in BACHD rats using different tests, we showed impaired reversal learning in MWM and decreased anxiety-like behavior with the light spot test supporting the validity of BACHD rats as a model of HD. Results in the EPM, light dark box, cliff hanging and PA tests did not confirm earlier findings. This may depend on phenotype inconsistencies or rather be related to differences in environmental variables, test typology, experimental settings, animal age and chosen behavioral parameters.

BACHD rats expressing full-length mutant huntingtin exhibit differences in social behavior compared to wild-type littermates.

BACKGROUND: Huntington disease (HD) is a devastating inherited neurodegenerative disorder characterized by progressive motor, cognitive, and psychiatric symptoms without any cure to slow down or stop the progress of the disease. The BACHD rat model for HD carrying the human full-length mutant huntingtin protein (mHTT) with 97 polyQ repeats has been recently established as a promising model which reproduces several HD-like features. While motor and cognitive functions have been characterized in BACHD rats, little is known about their social phenotype. OBJECTIVE: This study focuses especially on social behavior since evidence for social disturbances exists in human patients. Our objective was to compare social behavior in BACHD and wild-type (WT) rats at different ages, using two different measures of sociability. METHODS: Animals were tested longitudinally at the age of 2, 4 and 8 months in the social interaction test to examine different parameters of sociability. A separate cohort of 7 month old rats was tested in the three chamber social test to measure both sociability and social novelty. Gene expression analyses in 8 months old animals were performed by real time qRT-PCR to evaluate a potential involvement of D1 and D2 dopaminergic receptors and the contribution of Brain-derived neurotrophic factor (BDNF) to the observed behavioral alterations. RESULTS: In the social interaction test, BACHD rats showed age-dependent changes in behaviour when they were-re introduced to their cagemate after a 24 hours-period of individual housing. The time spent on nape attacks increased with aging. Furthermore, a significant higher level of pinning at 2 months of age was shown in the BACHD rats compared to wild-types, followed by a reduction at 4 and 8 months. On the other hand, BACHD rats exhibited a decreased active social behaviour compared to wild-types, reflected by genotype-effects on approaching, following and social nose contact. In the three chamber social test, BACHD rats seemed to show a mild deficit in preference for social novelty, but no changes in social interest. Molecular analyses revealed that BACHD animals exposed to the social interaction test displayed decreased mRNA levels of the total form of BDNF in ventral striatum and unaltered striatal expression of D1 and D2 dopamine receptors. CONCLUSIONS: Taken together, these results indicate deficits in several parameters representative of sociability. Altered BDNF expression in the ventral striatum may contribute to the deficits in sociability in 8 months old BACHD rats. These data support the validity of the BACHD rat model in mimicking features of certain social deficits that could be relevant to symptoms in patients.

Home-cage anxiety levels in a transgenic rat model for Spinocerebellar ataxia type 17 measured by an approach-avoidance task: The light spot test.

BACKGROUND: Measuring anxiety in a reliable manner is essential for behavioural phenotyping of rodent models such as the rat model for Spinocerebellar ataxia type 17 (SCA17) where anxiety is reported in patients. An automated tool for assessing anxiety within the home cage can minimize human intervention, stress of handling, transportation and novelty. NEW METHOD: We applied the anxiety test "light spot" (LS) (white led directed at the food-hopper) to our transgenic SCA17 rat model in the PhenoTyper 4500® to extend the knowledge of this automated tool for behavioural phenotyping and to verify an anxiety-like phenotype at three different disease stages for use in future therapeutic studies. RESULTS: Locomotor activity was increased in SCA17 rats at 6 and 9 months during the first 15min of the LS, potentially reflecting increased risk assessment. Both genotypes responded to the test with lower duration in the LS zone and higher time spent inside the shelter compared to baseline. COMPARISON WITH EXISTING METHODS: We present the first data of a rat model subjected to the LS. The LS can be considered more biologically relevant than a traditional test as it measures anxiety in a familiar situation. CONCLUSIONS: The LS successfully evoked avoidance and shelter-seeking in rats. SCA17 rats showed a stronger approach-avoidance conflict reflected by increased activity in the area outside the LS. This home cage test, continuously monitoring pre- and post-effects, provides the opportunity for in-depth analysis, making it a potentially useful tool for detecting subtle or complex anxiety-related traits in rodents.

Hippocampal extracellular matrix alterations contribute to cognitive impairment associated with a chronic depressive-like state in rats.

Patients with depression often suffer from cognitive impairments that contribute to disease burden. We used social defeat-induced persistent stress (SDPS) to induce a depressive-like state in rats and then studied long-lasting memory deficits in the absence of acute stressors in these animals. The SDPS rat model showed reduced short-term object location memory and maintenance of long-term potentiation (LTP) in CA1 pyramidal neurons of the dorsal hippocampus. SDPS animals displayed increased expression of synaptic chondroitin sulfate proteoglycans in the dorsal hippocampus. These effects were abrogated by a 3-week treatment with the antidepressant imipramine starting 8 weeks after the last defeat encounter. Next, we observed an increase in the number of perineuronal nets (PNNs) surrounding parvalbumin-expressing interneurons and a decrease in the frequency of inhibitory postsynaptic currents (IPSCs) in the hippocampal CA1 region in SDPS animals. In vivo breakdown of the hippocampus CA1 extracellular matrix by the enzyme chondroitinase ABC administered intracranially restored the number of PNNs, LTP maintenance, hippocampal inhibitory tone, and memory performance on the object place recognition test. Our data reveal a causal link between increased hippocampal extracellular matrix and the cognitive deficits associated with a chronic depressive-like state in rats exposed to SDPS.

The BACHD Rat Model of Huntington Disease Shows Specific Deficits in a Test Battery of Motor Function.

Rationale: Huntington disease (HD) is a progressive neurodegenerative disorder characterized by motor, cognitive and neuropsychiatric symptoms. HD is usually diagnosed by the appearance of motor deficits, resulting in skilled hand use disruption, gait abnormality, muscle wasting and choreatic movements. The BACHD transgenic rat model for HD represents a well-established transgenic rodent model of HD, offering the prospect of an in-depth characterization of the motor phenotype. Objective: The present study aims to characterize different aspects of motor function in BACHD rats, combining classical paradigms with novel high-throughput behavioral phenotyping. Methods: Wild-type (WT) and transgenic animals were tested longitudinally from 2 to 12 months of age. To measure fine motor control, rats were challenged with the pasta handling test and the pellet reaching test. To evaluate gross motor function, animals were assessed by using the holding bar and the grip strength tests. Spontaneous locomotor activity and circadian rhythmicity were assessed in an automated home-cage environment, namely the PhenoTyper. We then integrated existing classical methodologies to test motor function with automated home-cage assessment of motor performance. Results: BACHD rats showed strong impairment in muscle endurance at 2 months of age. Altered circadian rhythmicity and locomotor activity were observed in transgenic animals. On the other hand, reaching behavior, forepaw dexterity and muscle strength were unaffected. Conclusions: The BACHD rat model exhibits certain features of HD patients, like muscle weakness and changes in circadian behavior. We have observed modest but clear-cut deficits in distinct motor phenotypes, thus confirming the validity of this transgenic rat model for treatment and drug discovery purposes.

Short- and long-term behavioral analysis of social interaction, ultrasonic vocalizations and social motivation in a chronic phencyclidine model.

Phencyclidine (PCP) has been suggested to induce symptoms of schizophrenia. However, animal models using PCP administration have produced ambiguous results thus far. It seems that acute effects are similar to symptoms of schizophrenia, however, it is not clear if PCP can induce permanent behavioral changes that reflect schizophrenic-like symptoms. Therefore, we assessed the ability of chronic PCP administration (3mg/kg, 14 days) to induce short or long lasting behavioral changes in rats. Social behavior, including ultrasonic vocalizations and motivation for social contact were investigated at different time points, up to 29-36 days, after cessation of PCP treatment. During a social separation test, performed at 5 and 36 days, PCP treated rats spent less time near the divider that separates them from their familiar cage mate compared with saline (SAL) treated rats. Further, at short term, PCP was able to induce a decrease in social behavior. In contrast, at long-term, PCP treated animals spent more time in contact when exposed to an unfamiliar partner as compared to SAL treated rats. But, this difference was not observed when exposed to a familiar partner. We did not find any difference in ultrasonic vocalizations at all time points. The results of our study indicate that PCP is unable to induce overt long term deficits in social interaction behavior. Rather, it seems that PCP diminishes motivation for social contact. The long-term consequences of chronic PCP administration on social behavior in rodent models remain complex, and future studies addressing this are still needed.

Novel approach to automatically classify rat social behavior using a video tracking system.

BACKGROUND: In the past, studies in behavioral neuroscience and drug development have relied on simple and quick readout parameters of animal behavior to assess treatment efficacy or to understand underlying brain mechanisms. The predominant use of classical behavioral tests has been repeatedly criticized during the last decades because of their poor reproducibility, poor translational value and the limited explanatory power in functional terms. NEW METHOD: We present a new method to monitor social behavior of rats using automated video tracking. The velocity of moving and the distance between two rats were plotted in frequency distributions. In addition, behavior was manually annotated and related to the automatically obtained parameters for a validated interpretation. RESULTS: Inter-individual distance in combination with velocity of movement provided specific behavioral classes, such as moving with high velocity when "in contact" or "in proximity". Human observations showed that these classes coincide with following (chasing) behavior. In addition, when animals are "in contact", but at low velocity, behaviors such as allogrooming and social investigation were observed. Also, low dose treatment with morphine and short isolation increased the time animals spent in contact or in proximity at high velocity. COMPARISON WITH EXISTING METHODS: Current methods that involve the investigation of social rat behavior are mostly limited to short and relatively simple manual observations. CONCLUSION: A new and automated method for analyzing social behavior in a social interaction test is presented here and shows to be sensitive to drug treatment and housing conditions known to influence social behavior in rats.

Automated quantitative analysis to assess motor function in different rat models of impaired coordination and ataxia.

BACKGROUND: An objective and automated method for assessing alterations in gait and motor coordination in different animal models is important for proper gait analysis. The CatWalk system has been used in pain research, ischemia, arthritis, spinal cord injury and some animal models for neurodegenerative diseases. NEW METHOD: Our goals were to obtain a comprehensive gait analysis of three different rat models and to identify which motor coordination parameters are affected and are the most suitable and sensitive to describe and detect ataxia with a secondary focus on possible training effects. RESULTS: Both static and dynamic parameters showed significant differences in all three models: enriched housed rats show higher walking and swing speed and longer stride length, ethanol-induced ataxia affects mainly the hind part of the body, and the SCA17 rats show coordination disturbances. Coordination changes were revealed only in the case of the ethanol-induced ataxia and the SCA17 rat model. Although training affected some gait parameters, it did not obscure group differences when those were present. COMPARISON WITH EXISTING METHODS: To our knowledge, a comparative gait assessment in rats with enriched housing conditions, ethanol-induced ataxia and SCA17 has not been presented before. CONCLUSIONS: There is no gold standard for the use of CatWalk. Dependent on the specific effects expected, the protocol can be adjusted. By including all sessions in the analysis, any training effect should be detectable and the development of the performance over the sessions can provide insight in effects attributed to intervention, treatment or injury.

Reproducibility and relevance of future behavioral sciences should benefit from a cross fertilization of past recommendations and today's technology: "Back to the future".

Thanks to the discovery of novel technologies and sophisticated analysis tools we can now 'see' molecules, genes and even patterns of gene expression, which have resulted in major advances in many areas of biology. Recently, similar technologies have been developed for behavioral studies. However, the wide implementation of such technological progress in behavioral research remains behind, as if there are inhibiting factors for accepting and adopting available innovations. The methods of the majority of studies measuring and interpreting behavior of laboratory animals seem to have frozen in time somewhere in the last century. As an example of the so-called classical tests, we will present the history and shortcomings of one of the most frequently used tests, the open field. Similar objections and critical remarks, however, can be made with regard to the elevated plus maze, light-dark box, various other mazes, object recognition tests, etc. Possible solutions and recommendations on how progress in behavioral neuroscience can be achieved and accelerated will be discussed in the second part of this review.

A sustained depressive state promotes a guanfacine reversible susceptibility to alcohol seeking in rats.

High rates of comorbidity between alcohol use disorder (AUD) and major depressive disorder (MDD) are reported. Preclinical models examining effects of primary depression on secondary AUD are currently absent, preventing adequate testing of drug treatment. Here, we combined social defeat-induced persistent stress (SDPS) and operant alcohol self-administration (SA) paradigms to assess causality between these two neuropsychiatric disorders. We then exploited guanfacine, an FDA-approved adrenergic agent reported to reduce drug craving in humans, against SDPS-induced modulation of operant alcohol SA. Wistar rats were socially defeated and isolated for a period of ≥9 weeks, during which depression-like symptomatology (cognitive and social behavioral symptoms) was assessed. Subsequently, animals were subjected to a 5-month operant alcohol SA paradigm, examining acquisition, motivation, extinction, and cue-induced reinstatement of alcohol seeking. The effects of guanfacine on motivation and relapse were measured at >6 months following defeat. SDPS rats exhibited significant disruption of social and cognitive behavior, including short-term spatial and long-term social memory, several months following defeat. Notably, SDPS increased motivation to obtain alcohol, and cue-induced relapse vulnerability. Guanfacine reversed the SDPS-induced effects on motivation and relapse. Together, our model mimics core symptomatology of a sustained depressive-like state and a subsequent vulnerability to alcohol abuse. We show that SDPS is strongly associated with an enhanced motivation for alcohol intake and relapse. Finally, we show that the clinically employed drug guanfacine has potential as a novel treatment option in comorbid patients, as it effectively reduced the enhanced sensitivity to alcohol and alcohol-associated stimuli.

An automated system for the recognition of various specific rat behaviours.

The automated measurement of rodent behaviour is crucial to advance research in neuroscience and pharmacology. Rats and mice are used as models for human diseases; their behaviour is studied to discover and develop new drugs for psychiatric and neurological disorders and to establish the effect of genetic variation on behavioural changes. Such behaviour is primarily labelled by humans. Manual annotation is labour intensive, error-prone and subject to individual interpretation. We present a system for automated behaviour recognition (ABR) that recognises the rat behaviours 'drink', 'eat', 'sniff', 'groom', 'jump', 'rear unsupported', 'rear wall', 'rest', 'twitch' and 'walk'. The ABR system needs no on-site training; the only inputs needed are the sizes of the cage and the animal. This is a major advantage over other systems that need to be trained with hand-labelled data before they can be used in a new experimental setup. Furthermore, ABR uses an overhead camera view, which is more practical in lab situations and facilitates high-throughput testing more easily than a side-view setup. ABR has been validated by comparison with manual behavioural scoring by an expert. For this, animals were treated with two types of psychopharmaca: a stimulant drug (Amphetamine) and a sedative drug (Diazepam). The effects of drug treatment on certain behavioural categories were measured and compared for both analysis methods. Statistical analysis showed that ABR found similar behavioural effects as the human observer. We conclude that our ABR system represents a significant step forward in the automated observation of rodent behaviour.

Announced reward counteracts the effects of chronic social stress on anticipatory behavior and hippocampal synaptic plasticity in rats.

Chronic stress causes insensitivity to rewards (anhedonia) in rats, reflected by the absence of anticipatory behavior for a sucrose-reward, which can be reversed by antidepressant treatment or repeated announced transfer to an enriched cage. It was, however, not clear whether the highly rewarding properties of the enriched cage alone caused this reversal or whether the anticipation of this reward as such had an additional effect. Therefore, the present study compared the consequences of the announcement of a reward to the mere effect of a reward alone with respect to their efficacy to counteract the consequences of chronic stress. Two forms of synaptic plasticity, long-term potentiation and long-term depression were investigated in area CA1 of the hippocampus. This was done in socially stressed rats (induced by defeat and subsequent long-term individual housing), socially stressed rats that received a reward (short-term enriched housing) and socially stressed rats to which this reward was announced by means of a stimulus that was repeatedly paired to the reward. The results were compared to corresponding control rats. We show that announcement of enriched housing appeared to have had an additional effect compared to the enriched housing per se as indicated by a significant higher amount of LTP. In conclusion, announced short-term enriched housing has a high and long-lasting counteracting efficacy on stress-induced alterations of hippocampal synaptic plasticity. This information is important for counteracting the consequences of chronic stress in both human and captive rats.

Announced rewards counteract the impairment of anticipatory behaviour in socially stressed rats.

It is known that stress can influence the sensitivity to rewarding stimuli. Previous observations revealed that socially stressed rats do not display an appetitive behavioural response in anticipation of a reward. A previous study showed that this insensitivity to rewards (anhedonia) could be restored by chronic administration of an antidepressant. Several lines of evidence exist for the role of dopamine in the mechanism of action of antidepressant treatments concerning their therapeutic effect on anhedonia. Therefore, it was hypothesized that regular activation of the reward system, that involves mesolimbic dopaminergic systems, could counteract the effect of social stress on reward-sensitivity. For this, it was investigated whether a treatment of regular reward announcements could prevent the development of anhedonia. This was confirmed by the result that socially stressed rats that received this treatment were able to display anticipatory behaviour which is characterized by increased activity after presentation of a stimulus that was previously associated with a sucrose reward. Surprisingly, a non-treated socially stressed group, that did not show an anticipatory response for sucrose, did display anticipatory behaviour for another type of reward (enriched cage). It seems that, although one might assume the existence of an anhedonic state based upon the absence of anticipatory activity towards a sucrose reward, this assumption cannot be generalised to other types of reward. It will be discussed whether this might be caused by the highly rewarding properties of the enriched cage which probably has a therapeutical efficacy of its own.

Standard housed rats are more sensitive to rewards than enriched housed rats as reflected by their anticipatory behaviour.

The present study was designed to investigate the effects of potentially stressful standard housing conditions for laboratory rats on the sensitivity to rewards as reflected by their anticipatory behaviour for sucrose. This anticipatory response is evoked in a conditioning paradigm in which a sucrose reward is repeatedly announced by a stimulus. The underlying neurocircuitry of this anticipatory response in expectation of a reward involves mesolimbic dopaminergic systems of which it is known that they can be sensitised by stressors. The results show that the anticipatory response for the sucrose reward is stronger in the standard housed animals which indicates that these animals are more sensitive to the reward than the enriched animals. From this, it is concluded that standard housed rats are stressed which is likely to be caused by deprivation of the ability to satisfy behavioural needs in these impoverished housing conditions.

Anticipation is differently expressed in rats (Rattus norvegicus) and domestic cats (Felis silvestris catus) in the same Pavlovian conditioning paradigm.

In rats (Rattus norvegicus) anticipation to an oncoming food reward in an appetitive Pavlovian conditioning procedure is expressed as an increase of behavioural transitions, i.e. hyperactivity. This behaviour might be related to the spontaneous appetitive behaviour of animals in relation to oncoming food rewards. To deepen our insight into anticipatory behaviour we decided to study anticipation in rats and cats (Felis silvestris catus) using the same paradigm, as they show different types of spontaneous appetitive behaviour in relation to oncoming food rewards: 'search behaviour' and 'sit-and-wait behaviour' respectively. Using exactly the same Pavlovian conditioning paradigm in rats and cats it turned out that individuals of both species learned the association between conditioned stimulus (CS) (a tone) and unconditioned stimulus (US) (a food reward) as judged by their conditioned approach to the food dispenser. However, rats showed an increase in behavioural transitions whereas as cats a decrease during the 3 min interval between the offset of the CS and the onset of the US. Under extinction conditions the number of transitions of the rats decreased towards that of controls, whereas that of cats increased towards that of controls. This suggests that the same internal psychological process-anticipation to a coming reward-leads to different anticipatory behaviour in different species.