Substrain and light regime effects on integrated anxiety-related behavioral z-scores in male C57BL/6 mice—Hypomagnesaemia has only a small effect on avoidance behavior

Labots, Maaike; Zheng, Xiaochu; Moattari, G.; Klooster, J.G. Lozeman-van’t; Baars, J.M.; Hesseling, Peter; Lavrijsen, Marla; Kirchhoff, Susanne; Ohl, Frauke; Lith, H.A. van

doi:10.1016/j.bbr.2016.01.060

Cited by 6 publications

(10 citation statements)

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“…The animals had ad libitum access to food (Diet # 98341, Harlan Teklad, Madison, WI) and demineralized water. The animals from this study were control animals in an experiment studying the effect of magnesium deficiency on anxiety-related behavior (Labots et al, 2016). As part of the experimental protocol blood samples were taken via tail incision 2 days before behavior testing in order to determine the plasma magnesium level.…”

Section: Animalsmentioning

confidence: 99%

“…However, due to the large number of behaviors observed and the absence of specific predictions regarding substrain and/or light regime effects, we feel thatalthough the observers were not blind to the experimental conditionsit seems unlikely that any consequential bias was introduced. Blood samples were taken via tail incision after 2 weeks of habituation and 2 days before each behavioral test in order to determine the magnesium concentration in plasma (Labots et al, 2016). An incision was made in the tail vein and the blood was collected dropwise (D€ urschlag et al, 1996).…”

Section: Experimental Protocolmentioning

confidence: 99%

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Effects of light regime and substrain on behavioral profiles of male C57BL/6 mice in three tests of unconditioned anxiety

Labots

Zheng

Moattari

et al. 2016

Journal of Neurogenetics

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Substrains of the C57BL/6 inbred mouse are widely used in genetic, behavioral and physiological research, as well as models for human disease. Throughout, the choice of the respective substrain can have a large influence on experimental results. Likewise, the conditions under which experiments are performed, such as the light regime, can significantly affect the outcome of an experiment, especially when aiming at experimental behavior. Here, two commonly used mouse substrains, C57BL/6JOlaHsd and C57BL/6NCrl, were housed under either a conventional or a reverse light regime and were tested in either the light phase or the dark phase, respectively. All animals were exposed to three unconditioned anxiety-related behavior set-ups: the modified Hole Board test, the light-dark box and the elevated plus maze. Significant substrain and light regime effects were found in all three behavioral tests, with some of the latter being substrain and test specific. This signifies the importance of the choice of substrain used in for example, a mouse knockout experiment studying behavior, also in relation to light regime under which the animals are tested.

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Section: Animalsmentioning

confidence: 99%

Section: Experimental Protocolmentioning

confidence: 99%

Effects of light regime and substrain on behavioral profiles of male C57BL/6 mice in three tests of unconditioned anxiety

Labots

Zheng

Moattari

et al. 2016

Journal of Neurogenetics

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“…Through the criteria used, 11.9% of the sampled articles across the behavioral tests were published using appropriate ethological motivational principles (white lowest rectangle) and only n = 50 met the full inclusion criteria, with the Light/Dark Test having better reporting (62.25%), followed by the Open Field (30.09%), then the Elevated Plus Maze (41.41%), and finally the Three Chamber Social Interaction Test with the worst (34.69%) of the respective samples. (Banaskowski et al, 2015;Bentea et al, 2015;Liu et al, 2015;Sauce et al, 2015;Zhang et al, 2016;Acevedo et al, 2016;Christensen et al, 2016;Farajdokht et al, 2016;Fernandez et al, 2016;Labots et al, 2016;Miranda-Morales and Pautassi, 2016;Vogt et al, 2016;Benoit et al, 2017;Borbely et al, 2017;Chandra Sekhar et al, 2017;Khalil and Fendt, 2017;Makinson et al, 2017;Rogers et al, 2017;Sirohi et al, 2017;Heinla et al, 2018;Keenan et al, 2018;Mahmoudi et al, 2018;Wille-Bille et al, 2018;Zhang and Yao, 2018;Laureano-Melo et al, 2019;Morgan et al, 2019;], the Open Field Test [n = 10 (Zhang C. et al, 2015;Estork et al, 2016;Casarrubea et al, 2017;Kawabe, 2017;Khalil and Fendt, 2017;Kuniishi et al, 2017;Blume et al, 2018;Perea-Rodriguez et al, 2018;Struntz and Siegel, 2018;Hetzler et al, 2019)…”

Section: Refined Search and Inclusion/exclusionary Criteriamentioning

confidence: 99%

Under or Absent Reporting of Light Stimuli in Testing of Anxiety-Like Behaviors in Rodents: The Need for Standardization

Neuwirth

Verrengia

Harikinish-Murrary

et al. 2022

Front. Mol. Neurosci.

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Behavioral neuroscience tests such as the Light/Dark Test, the Open Field Test, the Elevated Plus Maze Test, and the Three Chamber Social Interaction Test have become both essential and widely used behavioral tests for transgenic and pre-clinical models for drug screening and testing. However, as fast as the field has evolved and the contemporaneous involvement of technology, little assessment of the literature has been done to ensure that these behavioral neuroscience tests that are crucial to pre-clinical testing have well-controlled ethological motivation by the use of lighting (i.e., Lux). In the present review paper, N = 420 manuscripts were examined from 2015 to 2019 as a sample set (i.e., n = ~20–22 publications per year) and it was found that only a meager n = 50 publications (i.e., 11.9% of the publications sampled) met the criteria for proper anxiogenic and anxiolytic Lux reported. These findings illustrate a serious concern that behavioral neuroscience papers are not being vetted properly at the journal review level and are being released into the literature and public domain making it difficult to assess the quality of the science being reported. This creates a real need for standardizing the use of Lux in all publications on behavioral neuroscience techniques within the field to ensure that contributions are meaningful, avoid unnecessary duplication, and ultimately would serve to create a more efficient process within the pre-clinical screening/testing for drugs that serve as anxiolytic compounds that would prove more useful than what prior decades of work have produced. It is suggested that improving the standardization of the use and reporting of Lux in behavioral neuroscience tests and the standardization of peer-review processes overseeing the proper documentation of these methodological approaches in manuscripts could serve to advance pre-clinical testing for effective anxiolytic drugs. This report serves to highlight this concern and proposes strategies to proactively remedy them as the field moves forward for decades to come.

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“…= Anti-Nociception and Side Effect Z-Score Calculation Z-scores are a mathematical tool used to normalise the means from multiple related data sets allowing direct comparisons between experiments. The Z-scores for the anti-nociceptive and side effects of morphine, kurkinol, and kurkinorin were generated for male and female mice individually and combined as previously described (Guilloux et al, 2011, Labots et al, 2016. In brief, Z-scores were generated for individual mice using the test value from each mouse (χ) compared to the mean (μ) and standard deviation (σ) of the control group as follows;…”

Section: ( ) = ( ) × ( ) × (( ( − )) ÷ ( ( − )) − ( − ))mentioning

confidence: 99%

Investigating the Role of G-protein Bias in the Anti-Nociceptive and Side Effect Profiles of Two Novel Mu Opioid Receptor Agonists Kurkinol and Kurkinorin

Alder¹

2021

Preprint

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Chronic pain is a major problem worldwide, affecting 1 in 5 New Zealanders resulting in a decreased quality of life for the patient and a large socioeconomic problem costing an estimated $13-$14.5 billion a year. Current therapeutics target the mu opioid receptor (μ receptor) and include drugs such as morphine and fentanyl. While these drugs are highly effective in the treatment of strong acute pain, their long-term use is associated with tolerance to the analgesic effects and increasing rates of side effects such as respiratory depression and constipation. Due to their high abuse liability, they are also known to cause dependence and addiction when prescribed for extended periods. This is believed to have played a role in the opioid crisis in the United States and highlights the need for improved therapeutics for the treatment of chronic pain. One mechanism that has been proposed to generate μ receptor agonists for the treatment of chronic pain with reduced analgesic tolerance and safer side effects is the development of G-protein biased agonists. Such agonists selectively activate the canonical G-protein signalling to a greater extent than the non-canonical β-arrestin2 pathway. This is based on previous work in β-arrestin2 knockout mice where the antinociceptive effects were increased, while side effects, including respiratory depression, tolerance, and constipation are reduced, increasing the therapeutic window. In this thesis, we aimed to assess the anti-nociceptive and side effect behavioural profiles of two novel μ receptor agonists, kurkinol (bias = 0.14) and kurkinorin (bias = 0.57), with a varying bias for the G-protein pathway to assess the role of this paradigm. Evaluation of the behavioural profile of kurkinol and kurkinorin revealed that G-protein bias was correlated to increased anti-nociceptive potency and reduced tolerance in wildtype C57BL/6J mice. Furthermore, the anti-nociceptive potency of morphine was increased, and tolerance decreased in in β-arrestin2 knockout mice. While the level of tolerance was reduced for kurkinorin. However, in the chemotherapy-induced model of neuropathic pain, tolerance to kurkinol and kurkinorin developed at the same rate as morphine. Overall this work showed a poor correlation between G-protein bias and therapeutic window. With the G-protein selective kurkinol inducing worse respiratory depression, constipation, and motor coordination impairment compared to kurkinorin. Interestingly, respiratory depressive and constipation effects of kurkinol were not prevented in the β-arrestin2 knockout mice indicating that they are induced through the G-protein pathway. These results highlight the change that has occurred in the biased agonism field over the last 4 years, with the lack of reproducibility of key experiments and poor translation of G-protein biased μ receptor agonists resulting in improved therapeutic windows both clinically and pre-clinically. Moreover, recent research has shown that pathway efficacy (i.e. partial agonism) and not G-protein bias is responsible for the behavioural profiles of compounds previously identified as G-protein biased. We, therefore, decided to further investigate the cell signalling profiles of our two novel agonists to assess them for partial agonism and to assess downstream signalling molecules activated by G-protein and β-arrestin2. <div> </div> This revealed cell-specific inhibition of membrane hyperpolarisation in Hek293 and CHO cells stably expressing the human μ receptor, with kurkinol found to be the most potent in both cell lines, followed by kurkinorin, morphine, and [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO). However, no differences were identified between the μ receptor agonists in the activation of the inwardly rectifying channels in the CHO cell line. The assessment of pCREB (phosphorylated cAMP response-element binding protein) as a β-arrestin2 dependent pathway revealed poor activation by kurkinorin while kurkinol was a potent activator. Bias factors generated from this data showed poor correlations to therapeutic windows. While the differences om CREB phosphorylation was shown to have a stronger correlation to therapeutic windows generated from the behavioural data. Overall this thesis has identified kurkinorin as a μ receptor agonist that induces potent anti-nociception with reduced side effects, without strong-G-protein bias. We also show that the highly selective μ receptor agonist kurkinol has improved anti-nociception with a worse side effect profile adding to the growing body of literature showing bias is not a good predictor in its current state. Furthermore, the discrepancies between cell lines, differential activation of subcellular pathways, and lack of reproducibility between bias equations indicate that the field has massively oversimplified a complex system. Which has, most likely, resulted in the poor translation of in vitro bias factors to clinically available μ receptor agonists for chronic pain.

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Substrain and light regime effects on integrated anxiety-related behavioral z-scores in male C57BL/6 mice—Hypomagnesaemia has only a small effect on avoidance behavior

Cited by 6 publications

References 53 publications

Effects of light regime and substrain on behavioral profiles of male C57BL/6 mice in three tests of unconditioned anxiety

Effects of light regime and substrain on behavioral profiles of male C57BL/6 mice in three tests of unconditioned anxiety

Under or Absent Reporting of Light Stimuli in Testing of Anxiety-Like Behaviors in Rodents: The Need for Standardization

Investigating the Role of G-protein Bias in the Anti-Nociceptive and Side Effect Profiles of Two Novel Mu Opioid Receptor Agonists Kurkinol and Kurkinorin

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