systematic-assessment-well-being-mice-for-procedures-using-general

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

Freie Universität Berlin

German Federal Institute for Risk Assessment (BfR)

Sudden darkness is a non-invasive behavioural analysis tool which increases motor activity and decreases anxiety in rats. It has been shown in previous studies that in rats, dark test conditions can also modify behavioural responses to drugs acting on the dopaminergic system. The increasing use of transgenic mice in behavioural research has raised interest in developing new tests for phenotyping mice. Hence, the aim of the present study was to adapt the sudden darkness paradigm for mice. In the first part of this study, effects of sudden darkness on the performance of mice in the elevated plus maze test were evaluated. Both genders of two mouse strains (Swiss and Balb/c) were tested either in high light intensity conditions or were exposed to sudden darkness. In the second part, responses to the 5-HT(1A) receptor agonist 8-OH-DPAT (0.5 and 1.0 mg/kg) and 5-HT(2C) receptor agonist mCPP (1.0 and 2.0 mg/kg) were investigated in male Swiss mice. Sudden darkness induced a clear anxiolytic effect in male and female Swiss mice. In Balb/c mice, anxiety-related behaviour was only decreased in females, whereas in males the anxiety state remained unchanged. An increase in motor activity was only observed in male Swiss mice; in the other groups, sudden darkness did not affect locomotion. Depending on the light conditions used, the behavioural response to receptor agonists was more evident: 8-OH-DPAT (1.0 mg/kg) only significantly decreased the anxiety state when mice were tested under high levels of illumination, whereas the anxiogenic effect of mCPP (1.0 and 2.0 mg/kg) was only evident in the dark. This study suggests that the sudden darkness paradigm is also a useful tool for the analysis of mice and can be used to modulate the anxiety level without administering drugs. Depending on the mouse strain tested, the same effects on anxiety and motor activity were observed as have been shown for rats.

The use of sudden darkness in mice: a behavioural and pharmacological approach.

The serotonin 1A (5-HT(1A)) receptor is one of the best described receptor subtypes of the serotonergic system. Due to the complex distribution pattern, the pre- and postsynaptic localisation, the impact on various monoamines, as well as the influence on a wide range of physiological functions, the contribution of 5-HT(1A) receptors to behavioural outcomes is difficult to define. In this study, we present a new transgenic mouse model with a prominent over-expression of the 5-HT(1A) receptor in the outer cortical layers (I-III) and the dentate gyrus. Behavioural studies revealed a slight decrease in baseline motor activity of homozygous mice during the open field test. Moreover, core body temperature of male transgenic mice was significantly lower than that of wild-type mice. Pharmacological studies with the 5-HT(1A) receptor agonist 8-OH-DPAT (0.1-2.5 mg/kg, i.p.) revealed an exaggerated drug response in mutant mice. 8-OH-DPAT led to a drastic decrease in motor activity in the open field and elevated plus maze test. This significant effect on motor activity became more apparent by investigating the serotonergic syndrome induced by 8-OH-DPAT. Concentration as low as 0.5 mg/kg 8-OH-DPAT caused immobility in transgenic mice for 30 min, head weaving behaviour, and backward walking, whereas in wild-type animals, typical behaviours of the serotonin syndrome were first observed at concentrations of 1.5 mg/kg and more. In addition, the 8-OH-DPAT induced hypothermia was more pronounced in mutant mice than in wild-type animals. Therefore, these genetically modified mice represent a promising model for further investigations of the role of 5-HT(1A) receptors.

Mice over-expressing the 5-HT(1A) receptor in cortex and dentate gyrus display exaggerated locomotor and hypothermic response to 8-OH-DPAT.

Sprague-Dawley rats are often the background stock for transgenic rats, and rats from various sources may differ in their physical development and behavior. In 1990, the Institute of Cytology and Genetics in Novosibirsk obtained Sprague-Dawley rats from a commercial vendor and bred them in a closed colony. To study various aspects of the physical development and behavior of male F1 progeny of the Novosibirsk colony (Nov:SD) and commercial colony (Crl:SD) raised in identical environments, we evaluated body weight; food and water consumption; behavior in the elevated-plus maze (X maze), open field, free exploration paradigm, hole board, and the rotarod; and serotonin content in brain regions. Crl:SD rats were heavier and consumed more food than did the Nov:SD rats, which displayed a higher level of motor activity in all tests without displaying differences in anxiety-related behavior in the X maze or open-field test. In the free exploration paradigm, more Nov:SD rats explored the outside and started exploration earlier; they also were more active and showed less habituation in the hole-board test. Brain serotonin content was higher in the Crl:SD rats. In conclusion, prolonged isolated breeding of 2 stocks of Sprague-Dawley rats led to populations that differed in their exploratory and anxiety-related behavior, physical development, and serotonergic neurotransmission. Therefore, rats of the same stock but obtained from different breeders should be used with caution in research involving these measures.

Choosing the right wild type: behavioral and neurochemical differences between 2 populations of Sprague-Dawley rats from the same source but maintained at different sites.

Even though the role of the serotonin1A (5-HT(1A))-receptor for cognitive processes is undisputed, the exact involvement of pre- and postsynaptic sites remains unexplained. Recently, we introduced a mouse line overexpressing the 5-HT(1A)-receptor in the hippocampus and cortex. In this study we investigated in comparison to wild-type mice their cognitive abilities using the Morris water-maze task and inhibitory avoidance test. Acute effects of pre- and posttraining administered 8-OH-DPAT (0.03-0.3 mg/kg i.p.) were examined in the inhibitory avoidance test. Additionally, habituation learning was studied in the hole-board test. Transgenic mice showed no overall learning deficit. Spatial learning and memory revealed in the Morris water-maze task was comparable to wild-type mice, and both genotypes habituated to the hole-board arena in a similar manner. Comparing the performance of both genotypes in the inhibitory avoidance test, cognitive functions of transgenic mice seemed to be slightly impaired. When 8-OH-DPAT was administered pretraining an amnesic effect was produced only in transgenic mice and only at the highest dose (0.3 mg/kg). Posttraining administered 0.3 mg/kg 8-OH-DPAT did not affect the performance of both genotypes. Overall, the cortical and hippocampal overexpression of the 5-HT(1A)-receptor had no major effect on cognitive functions in mice, suggesting that changes in the 5-HT(1A)-receptor density are not necessarily accompanied with alterations of learning and memory processes.

Increasing the number of 5-HT(1A)-receptors in cortex and hippocampus does not induce mnemonic deficits in mice.

The serotonin syndrome (SS) is a potentially life-threatening disorder in humans which is induced by ingestion of an overdose or by combination of two or more serotonin (5-HT)-enhancing drugs. In animals, acute administration of direct and indirect 5-HT agonists also leads to a set of behavioral and autonomic responses. In the current review, we provide an overview of the existing versions of the animal model of the SS. With a focus on studies in rats and mice, we analyze the frequency of behavioral and autonomic responses following administration of 5-HT-enhancing drugs and direct 5-HT agonists administered alone or in combination, and we briefly discuss the receptor mediation of these responses. Considering species differences, we identify a distinct set of behavioral and autonomic responses that are consistently observed following administration of direct and indirect 5-HT agonists. Finally, we discuss the importance of a standardized assessment of SS responses in rodents and the utility of animal models of the SS in translational studies, and provide suggestions for future research.

Animal models of the serotonin syndrome: a systematic review.

In humans, the ingestion of the combination of two or more serotonin (5-HT)-enhancing drugs but also of a single drug in overdose can induce serious adverse effects, which are characteristics of the serotonin syndrome (SS). In mice, acute administration of direct and indirect 5-HT agonists also leads to behavioral and autonomic responses, but in literature different responses are thought to be essential. In order to detect common behavioral SS responses induced by 5-HT-enhancing drugs with different mechanisms of action, we investigated the effects of the 5-HT precursor 5-hydroxy-l-tryptophan (5-HTP), the selective serotonin reuptake inhibitor (SSRI) fluoxetine (FLX), and the monoaminooxidase (MAO) inhibitor tranylcypromine (TCP) in male NMRI mice. The drugs were administered alone or in combination to investigate additive effects or drug potentiation. Moreover, we compared the 5-HT responses to the effects induced by the dopamine, noradrenaline, and cholinergic agonists, apomorphine (APO), atomoxetine (ATO), and oxotremorine (OXO). Our results show that the studied 5-HT-enhancing drugs induced a different number of concomitant responses. The following five responses consistently and dose-dependently occurred in NMRI mice: flat body posture, hindlimb abduction, piloerection, tremor, and decreased rearings. Like in humans, the combination of 5-HT-enhancing drugs leads to a potentiation of drug effects. With the exception of flat body posture the responses are not specific for serotonergic hyperactivity. The findings demonstrate that the SS in NMRI mice is a suitable animal model for preclinical research, if it is taken into account that the spectrum of typical responses to 5-HT enhancing drugs may differ depending on drug and mouse strain and that some responses might be evoked by activation of other transmission systems, too.

The murine serotonin syndrome - evaluation of responses to 5-HT-enhancing drugs in NMRI mice.

The serotonin (5-HT) syndrome (SS) in man covers side effects of drugs in over dose that increase synaptic 5-HT concentration or directly activate 5-HT receptors. The SS is characterized by mental state alterations, neuromuscular excitation, and autonomic dysregulation. In mice, a set of behavioral and autonomic responses can be induced by the same serotonergic drugs as in man. The role of the 5-HT1A receptor for the murine SS has been extensively studied and several responses have been attributed to 5-HT1A receptor activation. So far, 5-HT2A receptor activation is thought to induce head twitches and hypothermia. The aim of this study is to define the impact of the 5-HT2A and the 5-HT1A receptor for different SS-like responses.

Role of 5-HT(1A)- and 5-HT(2A) receptors for the murine model of the serotonin syndrome.

Brain serotonin (5-HT) is involved in the control of food intake. The ingestive effects of 5-HT are mediated by various receptor subtypes, among others the 5-HT1A receptor. While the involvement of presynaptic 5-HT1A receptors is regarded as certain, the role of postsynaptic 5-HT1A receptors is rather vague. Here, we studied the role of the 5-HT1A receptor on feeding in non-food-deprived and food-deprived (young adult and adult, both sexes) wild-type NMRI mice as well as transgenic NMRI mice, which are characterized by a distinct overexpression of postsynaptic 5-HT1A receptors. The known hyperphagic effect of the 5-HT1A receptor full agonist 8-OH-DPAT ((±)-8-hydroxy-N,N-dipropyl-2-aminotetralin) in non-food-deprived animals was demonstrated in male NMRI wild-type mice and could be antagonized by the selective 5-HT1A receptor antagonist WAY100635. In transgenic mice, this hyperphagic response was induced at lower doses, with an earlier onset and even in females. However, in adult male transgenic mice, the hyperphagic effect did not occur. In food-deprived NMRI wild-type as well as transgenic mice, 8-OH-DPAT first induced a hypophagic and subsequently a hyperphagic effect. Again, in transgenic animals most responses occurred at lower doses and with an earlier onset. The results indicate that postsynaptic 5-HT1A receptors exert a modulatory function in food intake in free-feeding and fasted mice, which for the first time shows an involvement of postsynaptic 5-HT1A receptors in feeding behavior. Understanding the function of pre- and postsynaptic 5-HT1A receptors may help to achieve new insights into the regulation of food intake and foster prospective treatment strategies for eating disorders.

Modulatory Role of Postsynaptic 5-Hydroxytryptamine Type 1A Receptors in (±)-8-Hydroxy-N,N-dipropyl-2-aminotetralin-Induced Hyperphagia in Mice.

Editorial: 5-HT2A/2B/2C Receptors, Memory, and Neuropsychiatric Disorders.

According to the EU Directive 2010/63, the severity of a procedure has to be classified as mild, moderate or severe. General anesthesia is thought to be mild, but the Directive does not differentiate between single and repeated anesthesia. Therefore, we investigated the impact of repeated administration of isoflurane, the most commonly used inhalation anesthetic, on the well-being of adult C57BL/6JRj mice, in comparison to single administrations and to untreated animals, when applied six times for 45 min at an interval of 3-4 days. For the animals anesthetized, excitations, phases of anesthesia, and vital parameters were monitored. Well-being after anesthesia was assessed using a behavioral test battery including luxury behavior like burrowing and nest building behavior, the Mouse Grimace Scale (MGS), the free exploratory paradigm for anxiety-related behavior, home cage activity and the rotarod test for activity, as well as food intake and body weight. Additionally, hair corticosterone and fecal corticosterone metabolites were measured. Our results show that nest building behavior, home cage activity, body weight, and corticosterone concentrations were not influenced by anesthesia, whereas changes in burrowing behavior, the MGS, food intake, and the free exploratory behavior indicated that the well-being of the mice was more affected by repeated than single isoflurane anesthesia. This effect depended on the sex of the animals, with female mice being more susceptible than male mice. However, repeated isoflurane anesthesia caused only short-term mild distress and impairment of well-being, mainly in the immediate postanesthetic period. Well-being stabilized at 8 days after the last anesthesia, at the latest. Therefore, we conclude that when using our anesthesia protocol, the severity of both single and repeated isoflurane anesthesia in C57BL/6JRj mice can be classified as mild. However, within the mild severity category, repeated isoflurane anesthesia ranks higher than single isoflurane anesthesia. Additionally, our results imply that male and female mice can differently perceive the severity of a procedure.

Severity classification of repeated isoflurane anesthesia in C57BL/6JRj mice-Assessing the degree of distress.

Postsynaptic 5-HTreceptors (5-HTR) play an important role in anxiety and stress, although their contribution is still controversial. Previous studies report that mice overexpressing postsynaptic 5-HTRs show no changes in basal anxiety, though the influence of stress conditions has not been addressed yet. In this study, we used this animal model to evaluate the role of 5-HTRs in anxiety response after pre-exposure to an acute stressor. Under basal conditions, 5-HTR overexpressing animals presented high corticosterone levels and a lower mineralocorticoid/glucocorticoid receptor ratio. After pre-exposure to a single stressor, they showed a high anxiety-like response, associated with a blunted increase in corticosterone levels and higher c-Fos activation in the prefrontal cortex. Moreover, these mice also presented a lack of downregulation of hippocampal long-term potentiation after stress exposure. Therefore, higher postsynaptic 5-HTR activation might predispose to a high anxious phenotype and an impaired stress coping behavior.

Enhanced Stress Response in 5-HTR Overexpressing Mice: Altered HPA Function and Hippocampal Long-Term Potentiation.

In the European Union (EU), animal welfare is seen as a matter of great importance. However, with respect to animal experimentation, European citizens feel quite uninformed. The European Directive 2010/63/EU for the protection of laboratory animals aims for greater transparency and requires that a comprehensible, nontechnical summary (NTS) of each authorised research project involving animals is published by the respective Member State. However, the NTSs remain sleeping beauties if their contents are not easily and systematically accessible. The German web-based NTS database AnimalTestInfo is a unique channel for scientists to communicate their work, and provides the opportunity for large-scale analyses of planned animal studies to inform researchers and the public. For an in-depth meta-analysis, we classified the duly completed NTSs submitted to AnimalTestInfo in 2014 and 2015 according to the International Classification of Diseases and Related Health Problems (ICD) system. Indexing the NTSs with ICD codes provided a fine-grained overview of the prospective uses of experimental animals. Using this approach, transparency, especially for highly controversial animal research involving, for example, nonhuman primates, is fostered, as it enables pinpointing the envisaged beneficiary down to the level of the addressed disease. Moreover, research areas with many planned projects involving animals can be specified in detail. The development of 3R (replacement, reduction, and refinement) measures in these research areas may be most efficient, as a large number of experimental animals would benefit from it. Indexing NTSs with ICD codes can support governments and funding agencies in advancing target-oriented funding of 3R research. Data drawn from NTSs can provide a basis for the development, validation, and implementation of directed 3R strategies as well as guidance for rethinking the role of animal research models.

Article	Year
The use of sudden darkness in mice: a behavioural and pharmacological approach. Psychopharmacology\| PubMed ID: 15619112	2005
Mice over-expressing the 5-HT(1A) receptor in cortex and dentate gyrus display exaggerated locomotor and hypothermic response to 8-OH-DPAT. Behavioural brain research\| PubMed ID: 16256213	2006
Choosing the right wild type: behavioral and neurochemical differences between 2 populations of Sprague-Dawley rats from the same source but maintained at different sites. Journal of the American Association for Laboratory Animal Science : JAALAS\| PubMed ID: 17877322	2007
Increasing the number of 5-HT(1A)-receptors in cortex and hippocampus does not induce mnemonic deficits in mice. Pharmacology, biochemistry, and behavior\| PubMed ID: 19010345	2009
Animal models of the serotonin syndrome: a systematic review. Behavioural brain research\| PubMed ID: 24004848	2013
The murine serotonin syndrome - evaluation of responses to 5-HT-enhancing drugs in NMRI mice. Behavioural brain research\| PubMed ID: 24780865	2015
Role of 5-HT(1A)- and 5-HT(2A) receptors for the murine model of the serotonin syndrome. Journal of pharmacological and toxicological methods\| PubMed ID: 25087754	2014
Modulatory Role of Postsynaptic 5-Hydroxytryptamine Type 1A Receptors in (±)-8-Hydroxy-N,N-dipropyl-2-aminotetralin-Induced Hyperphagia in Mice. ACS chemical neuroscience\| PubMed ID: 25781502	2015
Editorial: 5-HT2A/2B/2C Receptors, Memory, and Neuropsychiatric Disorders. Frontiers in pharmacology\| PubMed ID: 26869926	2016
Severity classification of repeated isoflurane anesthesia in C57BL/6JRj mice-Assessing the degree of distress. PloS one\| PubMed ID: 28617851	2017
Enhanced Stress Response in 5-HTR Overexpressing Mice: Altered HPA Function and Hippocampal Long-Term Potentiation. ACS chemical neuroscience\| PubMed ID: 28777913	2017
Rethinking 3R strategies: Digging deeper into AnimalTestInfo promotes transparency in in vivo biomedical research. PLoS biology\| PubMed ID: 29240762	2017

Bettina Bert

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