Final Report Summary - P2X IN PAIN (Optochemical control of P2X receptor ion channels: dissecting their role in pain signalling)
Chronic pain affects more than 1.5 billion people worldwide and can be highly debilitating, impacting quality of life, society, and the economy. In the European Union this costs approximately 1.5 to 3% of Gross Domestic Product. Treatments for chronic pain are generally ineffective, as many of the underlying mechanisms remain obscure. Here, we aimed to develop new optical methods to enable functional dissection of the molecules and cells that contribute to pain. The use of advanced tools to understand these processes will enhance the knowledge base across a wide range of fields (neurobiology, physiology, pharmacology, genetics, anatomy, bioengineering, and chemical biology), but will ultimately be beneficial for the health of individuals and our society as a whole.
Light can be manipulated with unparalleled spatial and temporal resolution and there are therefore advantages to controlling channel activity using surrogate optical methods. The fellow was successful in developing a new method to gain optical control of proteins – mammalian P2X receptor ion channels were engineered so that they could be controlled with light. P2X receptors are ligand-gated ion channels opened by extracellular ATP. P2X receptors have been implicated in producing chronic pain and are expressed in high threshold primary afferent neurons, the nociceptors. Optical control of these endogenous ion channels was achieved by bridging a novel chemical close to the ion channel pore so that blue light (440 nm) can rapidly push the channel open by changing the conformation of the chemical. This is reversible since the channel can be pulled closed when the chemical is returned to its initial conformation under UV light (360 nm). Therefore, the activity of the specific ion channel could be controlled rapidly and reversible with exquisite precision. This worked well for both P2X2 and P2X3 receptor ion channels, which are known to be involved in pain signalling but their precise contributions are undefined. To study these roles we aimed to engineer these endogenous receptors in the mouse so that they could be activated with high spatiotemporal precision. We used a genome editing technique, CRISPR/Cas9, to successfully generate a light-gated P2X3 receptor knock-in mouse line. In this mouse line, the gene encoding the P2X3 subunit has a single point mutation (P320C) but is otherwise unaffected. This means that mutant P2X3 receptors are expressed with a normal spatiotemporal expression pattern, and can then be controlled with light, after applying the optical nanotweezer compound. With this mouse line it should now be possible, for the first time, to specifically manipulate the activity of a native-like protein that is expressed at endogenous levels with very high spatiotemporal control to study the role of the P2X3 receptor in health and disease in vivo.
We carried out preliminary experiments using the algae light-activated ion channel, ChR2, as it represents proof-of-principle for in vivo optical control of sensory neurons, some of which express P2X receptors. This optogenetic approach was effective in vivo for controlling nociceptive fibres innervating the skin. This has turned out to provide an unprecedented view of stimulus response relations in the somatosensory system. Nociceptor sensory neurons drive both pain and rapid withdrawal from a noxious stimulus, a reflex described by Sherrington over 100 years ago. The nociceptive withdrawal reflex that Sherrington described has been considered since then as the basic unit of protective pain-related behaviour and is presumed to represent amongst the simplest stereotyped polysynaptic relationship between sensory input and motor output. We have reexamined the relationship between nociceptor activation and behavioural response in freely behaving mice, but at a millisecond timescale - a time domain at which these inputs and outputs operate within the nervous system. ChR2 was targeted to nociceptors innervating the skin for control with high temporal resolution, while a high-speed camera was used to record behaviour. The nociceptive flexion reflex was evoked using a very brief time-locked pulse of light to the hindpaw, while simultaneously recording behaviour at 1,000 frames per second. Careful quantitative analysis of behaviour has revealed that these relationships are not as simple or stereotyped as assumed since Sherrington; they are global, dynamic and organised in distinct sub-second behavioural motifs. These complex behaviours can be initiated by a minimal input – a single action potential volley. We also reveal that this minimal input can wake a sleeping mouse; a single action potential arriving to the spinal cord in a small number of nociceptors can within a fraction of a second change the activity of the entire cortex. Taken together, we have introduced a new way to interrogate behaviour at a millisecond resolution exploiting the temporal and spatial control afforded by optogenetics, a strategy that has divulged completely unsuspected aspects of the way the nervous system operates.
The fellow has published on the optical control of P2X receptors (PNAS) and co-wrote a News and Views (Nature Biotechnology) that is highly related to the theme of the Project. A manuscript has been submitted by the fellow as first author and co-corresponding author to disseminate data from the project to a large audience through a high-impact journal. Additionally, several papers related to the theme of the Project will be submitted in a timely manner.
In summary, these results should impact many aspects of neuroscience and pain biology. The tools and findings will complement an on-going effort to understand the mechanisms underlying chronic pain, which has a large impact on society and the economy. In the long-term, it is hoped that these studies will contribute to the development of new therapies for the treatment of chronic pain.
Light can be manipulated with unparalleled spatial and temporal resolution and there are therefore advantages to controlling channel activity using surrogate optical methods. The fellow was successful in developing a new method to gain optical control of proteins – mammalian P2X receptor ion channels were engineered so that they could be controlled with light. P2X receptors are ligand-gated ion channels opened by extracellular ATP. P2X receptors have been implicated in producing chronic pain and are expressed in high threshold primary afferent neurons, the nociceptors. Optical control of these endogenous ion channels was achieved by bridging a novel chemical close to the ion channel pore so that blue light (440 nm) can rapidly push the channel open by changing the conformation of the chemical. This is reversible since the channel can be pulled closed when the chemical is returned to its initial conformation under UV light (360 nm). Therefore, the activity of the specific ion channel could be controlled rapidly and reversible with exquisite precision. This worked well for both P2X2 and P2X3 receptor ion channels, which are known to be involved in pain signalling but their precise contributions are undefined. To study these roles we aimed to engineer these endogenous receptors in the mouse so that they could be activated with high spatiotemporal precision. We used a genome editing technique, CRISPR/Cas9, to successfully generate a light-gated P2X3 receptor knock-in mouse line. In this mouse line, the gene encoding the P2X3 subunit has a single point mutation (P320C) but is otherwise unaffected. This means that mutant P2X3 receptors are expressed with a normal spatiotemporal expression pattern, and can then be controlled with light, after applying the optical nanotweezer compound. With this mouse line it should now be possible, for the first time, to specifically manipulate the activity of a native-like protein that is expressed at endogenous levels with very high spatiotemporal control to study the role of the P2X3 receptor in health and disease in vivo.
We carried out preliminary experiments using the algae light-activated ion channel, ChR2, as it represents proof-of-principle for in vivo optical control of sensory neurons, some of which express P2X receptors. This optogenetic approach was effective in vivo for controlling nociceptive fibres innervating the skin. This has turned out to provide an unprecedented view of stimulus response relations in the somatosensory system. Nociceptor sensory neurons drive both pain and rapid withdrawal from a noxious stimulus, a reflex described by Sherrington over 100 years ago. The nociceptive withdrawal reflex that Sherrington described has been considered since then as the basic unit of protective pain-related behaviour and is presumed to represent amongst the simplest stereotyped polysynaptic relationship between sensory input and motor output. We have reexamined the relationship between nociceptor activation and behavioural response in freely behaving mice, but at a millisecond timescale - a time domain at which these inputs and outputs operate within the nervous system. ChR2 was targeted to nociceptors innervating the skin for control with high temporal resolution, while a high-speed camera was used to record behaviour. The nociceptive flexion reflex was evoked using a very brief time-locked pulse of light to the hindpaw, while simultaneously recording behaviour at 1,000 frames per second. Careful quantitative analysis of behaviour has revealed that these relationships are not as simple or stereotyped as assumed since Sherrington; they are global, dynamic and organised in distinct sub-second behavioural motifs. These complex behaviours can be initiated by a minimal input – a single action potential volley. We also reveal that this minimal input can wake a sleeping mouse; a single action potential arriving to the spinal cord in a small number of nociceptors can within a fraction of a second change the activity of the entire cortex. Taken together, we have introduced a new way to interrogate behaviour at a millisecond resolution exploiting the temporal and spatial control afforded by optogenetics, a strategy that has divulged completely unsuspected aspects of the way the nervous system operates.
The fellow has published on the optical control of P2X receptors (PNAS) and co-wrote a News and Views (Nature Biotechnology) that is highly related to the theme of the Project. A manuscript has been submitted by the fellow as first author and co-corresponding author to disseminate data from the project to a large audience through a high-impact journal. Additionally, several papers related to the theme of the Project will be submitted in a timely manner.
In summary, these results should impact many aspects of neuroscience and pain biology. The tools and findings will complement an on-going effort to understand the mechanisms underlying chronic pain, which has a large impact on society and the economy. In the long-term, it is hoped that these studies will contribute to the development of new therapies for the treatment of chronic pain.