Measurements and neural mechanisms of conscious states in rodents
The aim of the proposed experiments is to i) establish methods for the measurement of conscious states in rodents (rats and mice), and ii) explore neural mechanisms that are involved in the generation of consciousness on both cellular and circuit levels. In a previous pilot-project (FOTS 9781), we have established methods and equipment for making the types of measurements (EEG, ECoG) required to assess brain activity in rodents. These methods now permit us to correlate different conscious states (normal or pharmacologically induced) with activity in large brain networks. We will further develop these methods to permit simultaneous investigation of specific circuit and cellular elements in the brain, using single-cell electrophysiology and optical imaging.
Animals (rats and mice) will undergo general surgical anesthesia for the placement of neural measurement probes and devices, and emphasis will be placed on proper post-operative procedures to alleviate suffering. Electrodes will be placed over the cranium, directly on the dural surface or intra-cranially. Cannulae and optic fibers will be implanted intra-cranially. Brain activity will be measured electrically and optically (single and multi-photon), during natural states (awake, sleeping) or while receiving different anesthetics (propofol, midazolam, ketamine, pentobarbital, thiopental, sevoflurane, xenon). Brain activity will be also modulated optically (by the combination of optic fibers and optogenetic tools) or chemically (by local delivering of drugs or photo-switchable molecules).
Methods to measure conscious states are being developed in multiple laboratories around the world, with the intention of being translatable to the investigation of consciousness in humans, particularly in patients with consciousness disorders following brain trauma or patients undergoing general anesthesia that may occasionally consciously experience pain during surgical procedures. Current methods cannot reliably detect consciousness in humans on a single trial basis, causing uncertainty about the proper treatment and hence increased risk of suffering in patients with disorders of consciousness. The aims and expected value of the proposed experiments is to further our understanding of the neural circuits involved in the generation of conscious states to permit the continued development of methodologies, experimental and statistical, that permit real-time assessment of conscious states in humans.
We propose to use up to 120 rats (Sprague Dawley), adults males, minimum 300 grams, and 160 mice (wildtype and transgenic C57bl), male adults, minimum 20 grams over a period of 4 years in the proposed experiments. Additionally, 800 mice will be required for the breeding of transgenic strains for the lifetime of the experiments. We have used power analysis to estimate the number of animals required for these experiments, and will conduct experiments that adhere to the aims of the three R’s in animal research: Replacement, Reduction and Refinement.
Animals (rats and mice) will undergo general surgical anesthesia for the placement of neural measurement probes and devices, and emphasis will be placed on proper post-operative procedures to alleviate suffering. Electrodes will be placed over the cranium, directly on the dural surface or intra-cranially. Cannulae and optic fibers will be implanted intra-cranially. Brain activity will be measured electrically and optically (single and multi-photon), during natural states (awake, sleeping) or while receiving different anesthetics (propofol, midazolam, ketamine, pentobarbital, thiopental, sevoflurane, xenon). Brain activity will be also modulated optically (by the combination of optic fibers and optogenetic tools) or chemically (by local delivering of drugs or photo-switchable molecules).
Methods to measure conscious states are being developed in multiple laboratories around the world, with the intention of being translatable to the investigation of consciousness in humans, particularly in patients with consciousness disorders following brain trauma or patients undergoing general anesthesia that may occasionally consciously experience pain during surgical procedures. Current methods cannot reliably detect consciousness in humans on a single trial basis, causing uncertainty about the proper treatment and hence increased risk of suffering in patients with disorders of consciousness. The aims and expected value of the proposed experiments is to further our understanding of the neural circuits involved in the generation of conscious states to permit the continued development of methodologies, experimental and statistical, that permit real-time assessment of conscious states in humans.
We propose to use up to 120 rats (Sprague Dawley), adults males, minimum 300 grams, and 160 mice (wildtype and transgenic C57bl), male adults, minimum 20 grams over a period of 4 years in the proposed experiments. Additionally, 800 mice will be required for the breeding of transgenic strains for the lifetime of the experiments. We have used power analysis to estimate the number of animals required for these experiments, and will conduct experiments that adhere to the aims of the three R’s in animal research: Replacement, Reduction and Refinement.
Etterevaluering
The need for single housing of rats with implanted probes and electrodes increases the severity of the project from moderate to severe. All projects involving severe procedures must be retrospectively assessed by the Norwegian Food Safety Authority.
Begrunnelse for etterevalueringen
In the period between 2017 and 2020 the experimenters performed experiments using 76 rats and 17 mice. The severity of the procedures was considered to be mild or moderate for the majority of the animals. To prevent damage to the chronically implanted electrodes, thus avoiding an increase in the number of animals needed for the experiments, a minority of the animals (~16% of the total) have been single housed. The procedure of single housing increased the severity from moderate to severe and was approved 16/7-2019. Single housing was accompanied by additional enriched environment in the housing cage (big cage with 2 floors, paper tubes, wooden bricks and cotton stripes) and it was well tolerated by adult rats. The rats did not show specific signs of distress throughout the experiments. The experimenters judge the single housing beneficial for the experimental project.
The experiments have been aimed to understand the neuronal mechanisms of consciousness and transitions towards unconscious states. The results contribute to increasing our current knowledge and may help improve the treatment of disorders of consciousness in humans. Because the scientific goals demand the study of brain functioning as a whole, it is necessary to use living rodents for the experiments. The experimenters actively contribute to reduction/replacement by sharing their data through the EBRAIN platform, allowing the community to freely reuse already existing data.
The experimenters have showed for the first time in literature (Arena et al., 2020) that the rodent brain exhibits the same complex spatiotemporal dynamics that in humans have been associated with consciousness. They have developed an experimental method to replicate the PCI method in rats, thus measuring these dynamics independently from behaviour.
The experiments have contributed to obtaining a valuable, objective method for assessing the capacity for consciousness in rodents, without behavioural reports.
Not all the planned experimental procedures were performed, for different reasons. For example, the surgical procedure that involved the disconnection of brain areas (fiber tract lesion) was not reliable and more difficult than expected. That part of the experiments was therefore interrupted. A revised version of these experiments that should ensure a higher reliability has been approved in a new FOTS application, ID 24068. The exploration of the neural correlates of consciousness at the single cell level has been only initiated with pilot experiments. The pilot experiments gave promising results, even if not conclusive, and are continued in FOTS ID 24068.
The experiments have been aimed to understand the neuronal mechanisms of consciousness and transitions towards unconscious states. The results contribute to increasing our current knowledge and may help improve the treatment of disorders of consciousness in humans. Because the scientific goals demand the study of brain functioning as a whole, it is necessary to use living rodents for the experiments. The experimenters actively contribute to reduction/replacement by sharing their data through the EBRAIN platform, allowing the community to freely reuse already existing data.
The experimenters have showed for the first time in literature (Arena et al., 2020) that the rodent brain exhibits the same complex spatiotemporal dynamics that in humans have been associated with consciousness. They have developed an experimental method to replicate the PCI method in rats, thus measuring these dynamics independently from behaviour.
The experiments have contributed to obtaining a valuable, objective method for assessing the capacity for consciousness in rodents, without behavioural reports.
Not all the planned experimental procedures were performed, for different reasons. For example, the surgical procedure that involved the disconnection of brain areas (fiber tract lesion) was not reliable and more difficult than expected. That part of the experiments was therefore interrupted. A revised version of these experiments that should ensure a higher reliability has been approved in a new FOTS application, ID 24068. The exploration of the neural correlates of consciousness at the single cell level has been only initiated with pilot experiments. The pilot experiments gave promising results, even if not conclusive, and are continued in FOTS ID 24068.