Final Report Summary - DHISP (Dorsal Horn Interneurons in Sensory Processing)
Chronic pain affects about 20% of the European population. In many cases, efficacy and tolerability of currently available medications are far from being satisfactory. It is widely accepted that neuroplastic changes in the spinal dorsal horn circuitry contribute to the transition from acute to chronic pain and to the maintenance of chronic pain states. Despite this key role, the dorsal horn circuitry is not well understood. This gap in our knowledge originates from the absence of a comprehensive classification of dorsal horn neurons, from the lack of genetic markers that would permit the analysis of these neurons both in terms of their integration in the dorsal horn circuit and the lack of tools to specifically interfere with their activity in vivo with high spatial and temporal control. This project aimed to tackle these challenges through the development of tools and techniques that allow an unbiased genome-wide search for marker genes of dorsal horn interneuron populations, and through the generation of genetically modified mice and viruses that allow the specific manipulation and control of such neurons.
With the use of four lines of BAC-TRAP transgenic mice generated in this project and of two lines of mice that lack transcription factors required for the development of inhibitory dorsal horn neurons, we identified genes that are highly enriched either in excitatory or inhibitory neurons of the dorsal horn. Capitalizing on this knowledge, we generated several lines of cre transgenic mice that can be used to manipulated different subtypes of dorsal horn neurons with high spatial and temporal resolution. Because some neuron populations cannot be targeted specifically using only a single marker gene, we have also developed tools for so called intersectional approaches and identified means to systematically spare peripheral sensory neurons whose central terminals end in the spinal dorsal horn. These tools for intersectional intervention include mice expressing the dre recombinase and mice and viruses carrying reporter gene expression cassettes which get translated only when both cre and dre are expressed in a given neuron population.
Proof-of-principle evidence verifying the suitability of these tools was obtained in a study that addressed the functional role of glycinergic dorsal horn neurons (Foster et al., Neuron 2015). In this study, we have used adenoassociated virus-mediated gene transfer to locally control the activity of glycinergic dorsal horn neurons. We found that ablation and silencing of glycinergic neurons sensitizes mice to noxious heat, cold and mechanical stimuli and induces spontaneous aversive behaviors. Activation of the same neuron population through a pharmacogenetic approach reduced responses to noxious heat, cold and mechanical stimuli in naïve mice, reduced hyperalgesia in neuropathic mice and inhibited histamine-dependent and histamine-independent itch. Subsequent experiments employed an intersectional approach to address the function of the parvalbumin-expressing subpopulation of glycinergic neurons. This intersectional approach made use of the two recombinases cre and dre respectively driven by the GlyT2 and parvalbumin genes. While activation and inhibition of glycinergic neurons had produced very wide-spread phenotypes, restricting inhibition to the parvalbumin-positive subpopulation selectively increased the responses of mice to light mechanical stimulation but had no effect on responses elicited by noxious thermal stimulation.
Our work on the role of inhibitory interneurons in spinal pain control indicates that spinal GABA and glycine receptors should be considered promising targets for the development of novel analgesics. We have therefore extended our work to inhibitory neurotransmitter receptors. We found that targeting of inhibitory GABA-A and glycine receptors in the spinal dorsal horn can alleviate pathological pain states (Ralvenius et al., Nature Commun, 2015; Acuña et al., J Clin Invest, 2016).
The work done in the context of this project has led to the development of highly versatile tools that will greatly foster our understanding of sensory processing in the spinal dorsal horn. In addition, our results on the specific function of glycine receptors in pain and itch control have led to the initiation of drug discovery programs in both academia and industry that aim at the development of glycine receptor modulators as novel analgesic drugs.
With the use of four lines of BAC-TRAP transgenic mice generated in this project and of two lines of mice that lack transcription factors required for the development of inhibitory dorsal horn neurons, we identified genes that are highly enriched either in excitatory or inhibitory neurons of the dorsal horn. Capitalizing on this knowledge, we generated several lines of cre transgenic mice that can be used to manipulated different subtypes of dorsal horn neurons with high spatial and temporal resolution. Because some neuron populations cannot be targeted specifically using only a single marker gene, we have also developed tools for so called intersectional approaches and identified means to systematically spare peripheral sensory neurons whose central terminals end in the spinal dorsal horn. These tools for intersectional intervention include mice expressing the dre recombinase and mice and viruses carrying reporter gene expression cassettes which get translated only when both cre and dre are expressed in a given neuron population.
Proof-of-principle evidence verifying the suitability of these tools was obtained in a study that addressed the functional role of glycinergic dorsal horn neurons (Foster et al., Neuron 2015). In this study, we have used adenoassociated virus-mediated gene transfer to locally control the activity of glycinergic dorsal horn neurons. We found that ablation and silencing of glycinergic neurons sensitizes mice to noxious heat, cold and mechanical stimuli and induces spontaneous aversive behaviors. Activation of the same neuron population through a pharmacogenetic approach reduced responses to noxious heat, cold and mechanical stimuli in naïve mice, reduced hyperalgesia in neuropathic mice and inhibited histamine-dependent and histamine-independent itch. Subsequent experiments employed an intersectional approach to address the function of the parvalbumin-expressing subpopulation of glycinergic neurons. This intersectional approach made use of the two recombinases cre and dre respectively driven by the GlyT2 and parvalbumin genes. While activation and inhibition of glycinergic neurons had produced very wide-spread phenotypes, restricting inhibition to the parvalbumin-positive subpopulation selectively increased the responses of mice to light mechanical stimulation but had no effect on responses elicited by noxious thermal stimulation.
Our work on the role of inhibitory interneurons in spinal pain control indicates that spinal GABA and glycine receptors should be considered promising targets for the development of novel analgesics. We have therefore extended our work to inhibitory neurotransmitter receptors. We found that targeting of inhibitory GABA-A and glycine receptors in the spinal dorsal horn can alleviate pathological pain states (Ralvenius et al., Nature Commun, 2015; Acuña et al., J Clin Invest, 2016).
The work done in the context of this project has led to the development of highly versatile tools that will greatly foster our understanding of sensory processing in the spinal dorsal horn. In addition, our results on the specific function of glycine receptors in pain and itch control have led to the initiation of drug discovery programs in both academia and industry that aim at the development of glycine receptor modulators as novel analgesic drugs.