Can social buffering pheromones be used to reduce stress?

Stress is a major factor affecting the health and welfare of captive animals. In addition to the ethical issues surrounding animal welfare, stress also decreases the quality of data obtained by the use of laboratory animals such as mice, increasing the number of animals used due to increased variability and decreased reliability. It is well established that the presence of other animals of the same species has beneficial welfare effects to reduce stress. Moreover, this social buffering effect of other individuals is largely mediated by chemical signals that are detected by the sense of smell. A mixture of three such chemicals has recently been shown to activate a specific type of smell receptor, known as OR37 receptors. Moreover, this mix of social buffering chemicals dramatically reduces activation of the hormonal stress response of mice placed in a novel environment. This occurs via activation of a direct brain pathway that inhibits cells that control the release of stress hormones. The overall aim of this project is to determine whether these social buffering chemicals have a general stress reducing effect in mice that could be utilized to reduce the stress experienced in the laboratory environment. We will firstly determine the optimum ratio and concentration of the three chemicals to reduce novelty-induced stress. We will go on to determine whether the mixture reduces the stress to other stressful situations, including learned anxiety responses and social isolation.

A major focus of the project will be to use our established experimental protocols to determine whether these chemical signals reduce the stress and anxiety observed in mice in response to carbon dioxide exposure. This is particularly important, as millions of laboratory mice are killed each year by means of carbon dioxide euthanasia. In the absence of a more humane alternative to carbon dioxide, the discovery of an agent that could reduce stress associated with carbon dioxide euthanasia would have a major welfare impact. We will also study whether the social buffering mix of chemicals are effective against the stress-promoting effects of exposure to a mouse alarm pheromone, which is released during carbon dioxide euthanasia. In the last part of this study we aim to apply this knowledge to develop a less stressful carbon dioxide euthanasia procedure, as well as improvements to husbandry procedures to reduce the stress experienced by laboratory mice in other experimental contexts. A range of behavioural measures of anxiety will be recorded, in addition to measuring activation of stress-related brain regions and stress hormone levels. This will not only give us a more complete picture of the stress response, but will enable us to determine whether the social buffering mix of chemicals has differential affects on the different behavioural/hormonal components of the stress response.

Most excitingly, the same class of OR37 smell receptors that respond to these stress hormone-reducing chemicals are found across most other mammalian species. Therefore, if we can show that these chemicals have a general stress reducing effect in mice, it will open up a new field of research to look for similar chemicals that have stress reducing effects in other mammals including humans. This would have enormous potential impact, to not only improve the welfare, of farmed, zoo and companion animals, but would also have multiple potential medical applications that could enhance both animal and human health and wellbeing.

Recently identified ligands of mouse OR37 olfactory receptors, pentadecanal, hexadecanal and heptadecanal, have been shown to inhibit activation of corticotrophin releasing hormone (CRH) containing neurons, in the paraventricular hypothalamus (PVN), in response to a novel environment in mice. We aim to optimise the mixture of OR37 ligands, and determine whether its effectiveness generalises to other stressful contexts, such as fear conditioning and social isolation. We will use established behavioural measures of anxiety and learned fear, along with physiological measures of the endocrine stress response including corticosterone levels and fluorescent in situ hybridization for heteronuclear CRH mRNA and/or c-Fos immunohistochemistry in genetically-labelled PVN CRH neurons. cFos expression will also be used to assess activation of amygdala nuclei and periaqueductal grey regions involved in active and passive coping behaviours. These multiple measures will not only enable us to assess the integrated stress response, but also to detect differential effectiveness of OR37 ligands on different components of the response. In particular we will determine whether OR37 ligands alter the concentration-response relationship to CO2 exposure and the stress response to the mouse alarm chemosignal SBT, which is potently released during CO2 euthanasia. We will go on to determine the potential for OR37 ligands to be applied to enhance the welfare of laboratory mice in the contexts of social isolation, cage cleaning and CO2 euthanasia. Importantly, the OR37 olfactory receptors are evolutionarily conserved across mammalian species. Therefore, this work potentially provides a basis for identifying ligands for the highly similar OR37 receptors found in other mammalian species, with enormous potential application to the enhancement of both animal and human health and well being.

Millions of rodents are killed worldwide each year using CO2 euthanasia, despite the abundant evidence that CO2 exposure is stressful to rodents at concentrations much lower than those used for euthanasia. Although there are clear welfare concerns, there currently appear to be no better alternatives to humanely killing the large numbers of animals involved, as exposure to inert gases, such as argon or to gaseous anaesthetics are also stressful to rodents. Much of the recent research on rodent euthanasia has therefore focused on procedural refinements to minimise the time to loss of consciousness and therefore the duration of the period during which distress and pain may be perceived. We propose a novel approach to tackle this welfare issue based on the dramatic effects of the recently identified OR37 ligands to inhibit activation of the hypothalamic neurons that control the hormonal stress response in mice. These compounds are safe, effective at low concentrations, commercially available and relatively inexpensive. If we find that exposure to these OR37 ligands has a biologically significant effect to reduce the activation of the stress response to CO2 exposure, they could readily be introduced into the gas stream entering a euthanasia apparatus, without changing the euthanasia procedure itself. This could be exploited commercially by companies to incorporate OR37 ligand exposure into existing CO2 euthanasia equipment and there would be a low barrier to prevent its adoption in mouse euthanasia procedures worldwide, within a timescale of a few years. Given the large number of mice involved, this refinement of the euthanasia procedure would result in a considerable improvement in welfare for laboratory mice. It would also emotionally benefit both those directly involved with conducting the euthanasia procedure, and the scientists conducting the research, by knowing that stress experienced by mice during euthanasia was being minimised. It would also help to partly mitigate concerns in the general public regarding the large number of mice being euthanized in the process of scientific research.

A greater understanding of the effects of stress promoting effects of mouse alarm pheromones has the potential to inform current best practice, in the housing conditions of animals exposed to stressors, such as following surgery. If the OR37 ligands are also found to have a general stress reducing effect to a variety of stressors, then they could easily and immediately be introduced into the cages of mice. This could potentially have a major impact in refining scientific procedures that result in unavoidable stress and distress, such as surgery and social isolation, which could improve the quality and reliability of experimental data. Furthermore, by reducing stress and associated variability in experimental measures, such an approach could also reduce the number of animals required to achieve meaningful results. This would both have a positive impact on research productivity and a societal impact around concerns about the number of animals used in scientific research.
Moreover, as the OR37 olfactory receptor family that mediates the effects of the OR37 ligands is unusually conserved across species, there is the potential to open a new area of research to identify similar ligand mixtures that are effective for other mammalian species. This has potential ethical and productivity impacts for food production, the welfare of companion animal and captive species, as well as numerous potential applications in human and veterinary medicine.