Browsing by Subject "Nociception"
- Results Per Page
- Sort Options
Item Open Access Circuitry and Genes of Larval Nociception in Drosophila Melanogaster(2009) Hwang, Richard Yi-JenPain is defined by the international association of pain as an "unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage". Most people have experienced one form of pain or another and although such experiences can be unsavory, pain serves the basic need for the detection of dangerous stimuli that can cause bodily harm. Because pain serves such an essential need, it is important to understand how the nervous system processes and encodes noxious or potentially tissue damaging stimuli. This neural processing is called nociception.
In this study, I use Drosophila larvae as a genetic model organism to study nociception. In response to noxious thermal and mechanical stimuli, Drosophila larvae perform a nociceptive defensive behavior (termed nocifensive) where larvae rotate in a corkscrew like fashion along the long axis causing them to move in a lateral direction. Using this behavior and genetic tools which can manipulate neuronal output, we have identified the sensory neurons which serve as larval nociceptors as class IV multidendritic sensory neurons. Further characterization of these larval nociceptors, has also shown that they are both cholinergic and peptidergic.
After the identifying the larval nociceptors, I next identified several molecular components which are required for larval mechanical nociception. I have found that the degenerin epithelial sodium channel (DEG/ENaC) called pickpocket is required for larval mechanical nociception by using genetic mutants and RNAi knockdwon. In addition, after performing a screen using RNAi to knockdown ion channel transcripts in larval nociceptors, I have identified two other DEG/ENaC channels which are required for larval mechanical nociception. DEG/ENaCs are particularly interesting because they have been identified as candidate mechanotransducers in C. elegans for the gentle touch behavior. I propose that DEG/ENaCs may serve as candidate mechanotransducers in larval mechanical nociception because they are not generally required for neuronal excitability. However, future research will be required to establish their true role in mechanical nociceptive signaling.
In addition to DEG/ENaCs, transient receptor potential (TRP) channels also play a role in nociception. painless, a channel that was first identified in a thermal nociception screen on Drosophila larvae, is required for both thermal and mechanical nociception. The last section shows that multiple isoforms of painless exist and that these different isoforms may play different roles in thermal and mechanical nociception.
Taken together, these results have begun to establish Drosophila larva as a model for studying nociception. I have identified the sensory neurons used as larval nociceptors and shown that DEG/ENaC channels play an important role in larval mechanical nociception.
Item Open Access General Anesthetics Activate a Central Pain-Suppression Circuit in the Amygdala(2020) Hua, ThuyGeneral anesthesia (GA) can produce analgesia (loss of pain) independent of inducing loss of consciousness, but the underlying mechanisms remain unclear. We hypothesized that GA suppresses pain in part by activating supraspinal analgesic circuits. We discovered a distinct population of GABAergic neurons activated by GA in the mouse central amygdala (CeAGA neurons). In vivo calcium imaging revealed that different GA drugs activate a shared ensemble of CeAGA neurons. CeAGA neurons also possess basal activity that mostly reflect animals’ internal state rather than external stimuli. Optogenetic activation of CeAGA potently suppressed both pain-elicited reflexive and self-recuperating behaviors across sensory modalities, and abolished neuropathic pain-induced mechanical (hyper-)sensitivity. Conversely, inhibition of CeAGA activity exacerbated pain, produced strong aversion, and cancelled the analgesic effect of low-dose ketamine. CeAGA neurons have widespread inhibitory projections to numerous affective pain-processing centers. Our study points to CeAGA as a potential powerful therapeutic target for alleviating chronic pain.
Item Open Access Genetic Analysis of the Contribution of Ion Channels to "Drosophila" Nociception(2012) Walcott, KiaNociceptors are specialized primary sensory neurons that represent the first line of defense against potentially tissue damaging environmental stimuli, and are involved in pathological pain states caused by nerve damage, inflammation and many chronic diseases. In nociception, these neurons detect harmful stimuli and contribute to the reactions to avoid them. Nociceptors transduce noxious stimuli into membrane depolarization, which in turn, triggers action potentials. These action potentials are conducted to synapses in the central nervous system (CNS), resulting in release of neurotransmitters at the presynaptic terminal. The unifying factor in the progression of nociceptive signaling i.e. transduction, action potential propagation, and neurotransmitter release, is the contribution of ion channels.
In this study, I use Drosophila melanogaster larvae as a model system to study the contribution of ion channels to nociception. Larvae stimulated with a noxious thermal or mechanical stimulus perform a stereotyped and quantifiable escape behavior. Larvae exhibiting this nocifensive behavior rotate around their long body axis in a corkscrew-like manner thus escaping the damage of the noxious stimulus. This behavior is triggered by the Class IV multidendritic (md) neurons, which are the main larval nociceptors. I describe here, the results of my systematic screen for ion channels required for larval thermal nociception. To perform this screen, I utilized RNAi to knock down the expression of 98% of the predicted ion channels in the Drosophila genome. I observed the effects of ion channel knockdown in the thermal nociception behavioral assay.
In addition, I present detailed characterization of an ion channel that I found to be critical for inhibition of nociceptor excitability, the small conductance calcium-activated potassium channel, SK. This channel inhibits both thermal and mechanical nociception. Results of calcium imaging studies show enhanced excitability of larval nociceptors in SK mutant animals. My findings support a role for SK function at the sensory afferents, cell body, and axon.
Another candidate ion channel gene, shadrach, encodes a Degenerin/Epithelial Na+ channel (DEG/ENaC) that I found to be required for thermal nociception. DEG/ENaCs are conserved in flies, nematodes, and several vertebrates including humans. These channels are expressed in a variety of tissues including kidney epithelia, muscle, and neurons. Members of this superfamily play a role in a host of biological processes including salt homeostasis, neurodegeneration, proprioception, touch transduction, and nociception. RNAi knockdown of shadrach results in increased thermal nociceptive threshold. Optogenetic experiments suggest that shadrach functions downstream of transduction.
Furthermore, I identified seven ion channel genes in the thermal nociception screen, which affect nociceptor dendrite morphology. It is possible that thermal nociception behavioral phenotypes in these RNAi mutants are a consequence of the altered dendritic field. Reduction in segmental coverage by the nociceptors may influence the ability to detect noxious stimuli. Future research in our laboratory will establish the relationship between these ion channels, nociceptor development, and nociceptive behavioral output.
Drosophila melanogaster is emerging as a powerful model for the study of pain signaling. I have uncovered several candidate ion channel genes that contribute to thermal nociception; of these, SK and shadrach are required for the response to noxious heat. I have shown that dendritic field coverage is important for the detection of noxious stimuli, and I have identified many candidate genes that are required for normal dendrite morphology.
Item Open Access Microtubule Severing Protein Regulation of Sensory Neuron Form and Function in Drosophila melanogaster(2011) Stewart, AndreaDendrite shape is a defining component of neuronal function. Yet, the mechanisms specifying diverse dendritic morphologies, and the extent to which their functioning depends on these morphologies, remain unclear. Here, we demonstrate a dendrite-specific requirement for the microtubule severing protein Katanin p60-like 1 (Kat-60L1) in regulating the elaborate branch morphology and nocifensive functions of Drosophila melanogaster larval class IV dendritic arborization (da) neuron dendrites. Through genetic loss of function analysis we show that loss of kat-60L1 reduced dendrite branching and process length, particularly during a period of normally extensive growth. This morphological defect was paralleled by a reduction in nocifensive responsiveness mediated by these neurons, indicating a tight correlation between neuronal function and the full extent of the dendritic arbor. To understand the mechanism underlying Kat-60L1's effects, we used in vivo imaging of the microtubule plus-end binding protein EB1, and found fewer polymerizing microtubules within mutant dendrites. Kat-60L1 thus promotes microtubule growth within class IV dendrites to establish the full arbor complexity and nocifensive functions of these neurons.
Although reduction of the related microtubule severing protein Spastin also compromised class IV dendrite arborization and nocifensive responses, microtubule polymerization in dendrites was unchanged in spastin mutants, and behavioral defects arose from generally compromised neuronal excitation. Kat-60L1 and Spastin thus function in distinct neuronal compartments to establish the complex dendritic morphology and sensory functions of class IV da neurons via distinct mechanisms of microtubule regulation. Whereas Spastin regulates stable microtubules affecting both pre- and post-synaptic compartments of these neurons, Kat-60L1 function is required specifically in dendrites to promote their complex arborization through the addition of growing microtubule numbers. Double mutant analysis demonstrated that Kat-60L1 and Spastin function antagonistically to promote dendritic aborization, likely involving other molecular players involved in regulating the microtubule cytoskeleton. Lastly, we identified Mi-2 as a transcriptional regulator of both kat-60L1 and spastin and show a genetic interaction between mi-2 and kat-60L1 in the class IV dendritic arbor, demonstrating that Mi-2 antagonizes Kat-60L1 function, possibly through the parallel upregulation of spastin. These data support a key role for the differential utilization of microtubule severing in generating distinct neuronal morphologies and subsequent function.
Item Open Access Nociceptor-Enriched Genes Required for Normal Thermal Nociception.(Cell reports, 2016-07) Honjo, Ken; Mauthner, Stephanie E; Wang, Yu; Skene, JH Pate; Tracey, W DanielHere, we describe a targeted reverse genetic screen for thermal nociception genes in Drosophila larvae. Using laser capture microdissection and microarray analyses of nociceptive and non-nociceptive neurons, we identified 275 nociceptor-enriched genes. We then tested the function of the enriched genes with nociceptor-specific RNAi and thermal nociception assays. Tissue-specific RNAi targeted against 14 genes caused insensitive thermal nociception while targeting of 22 genes caused hypersensitive thermal nociception. Previously uncategorized genes were named for heat resistance (i.e., boilerman, fire dancer, oven mitt, trivet, thawb, and bunker gear) or heat sensitivity (firelighter, black match, eucalyptus, primacord, jet fuel, detonator, gasoline, smoke alarm, and jetboil). Insensitive nociception phenotypes were often associated with severely reduced branching of nociceptor neurites and hyperbranched dendrites were seen in two of the hypersensitive cases. Many genes that we identified are conserved in mammals.Item Open Access TRPV channel-mediated calcium transients in nociceptor neurons are dispensable for avoidance behaviour.(Nat Commun, 2014-09-02) Lindy, Amanda S; Parekh, Puja K; Zhu, Richard; Kanju, Patrick; Chintapalli, Sree V; Tsvilovskyy, Volodymyr; Patterson, Randen L; Anishkin, Andriy; van Rossum, Damian B; Liedtke, Wolfgang BAnimals need to sense and react to potentially dangerous environments. TRP ion channels participate in nociception, presumably via Ca(2+) influx, in most animal species. However, the relationship between ion permeation and animals' nocifensive behaviour is unknown. Here we use an invertebrate animal model with relevance for mammalian pain. We analyse the putative selectivity filter of OSM-9, a TRPV channel, in osmotic avoidance behaviour of Caenorhabditis elegans. Using mutagenized OSM-9 expressed in the head nociceptor neuron, ASH, we study nocifensive behaviour and Ca(2+) influx. Within the selectivity filter, M(601)-F(609), Y604G strongly reduces avoidance behaviour and eliminates Ca(2+) transients. Y604F also abolishes Ca(2+) transients in ASH, while sustaining avoidance behaviour, yet it disrupts behavioral plasticity. Homology modelling of the OSM-9 pore suggests that Y(604) may assume a scaffolding role. Thus, aromatic residues in the OSM-9 selectivity filter are critical for pain behaviour and ion permeation. These findings have relevance for understanding evolutionary roots of mammalian nociception.