BISC 421 1st Edition Lecture 26 Outline of Current LectureI. Nociception (Pain and Thermosensation)Current LectureNociception •Fibers coming to the end of the finger-‐ nerve terminals of a cell in the dorsal ganglion •In the face the cells are in the trigeminal ganglion •The cells that mediate pain and temperature are cells that terminate in free nerve endings.•Experiments that find that there are separate nerve fibers that detect different pressure in the nerves that detect pain•First picture is cell responding to light touch•Means there are separate cells for pain and touch•Cells that respond to pain = nociceptorsCongenital insensitivity to painDearborn first reported this condition in 1932describing a man who made a living as a human pincushion act at the circus (1).Mirin Dajo•People who have genetic disorder that makes them insensitive to pain-‐ can feel normal touch just not pain•Circus performer who can't feel pain-‐ sticks pins in his body•Found that if stimulated the alpha beta ones they didn't produce pain but if stimulated the slower fibers pain was produced (sharp or dull)•Pain has its own receptors (the last two)•C are the skinniest-‐ slow aching kind of pain (infection) •Adelta-‐ fast pain (hit by a hammer)•We know a lot about how pain and temperature happen•Detected by ion channels•When the ion channel opens it will depolarize the cell and lead to an action potential•TRPV1-‐ activated by heat•TRPM-‐ cold Method:• Isolate total mRNA from DRG and introduce it into tissue culture cells that do not normally respond to capsaicin. Measure influx of calcium with intracellular calcium imaging (receptor is expected to be a calcium ion channel).• From total mRNA, isolate single mRNA that can generate response to capsaicin and determine receptor amino acid sequence.•Receptive to heat and chili peppers•Chili peppers trick us into thinking we are hot•Because they contain a molecule called capsaicin-‐ so need to identify receptor forcapsaicin which is the same to detect heat•Cloning of TRPV1-‐ cells usually have low levels of calcium, can inject dye that will detectwhen calcium levels go up-‐ meaning cell is responding (Ca2+ imaging)•Remember central dogma (DNARNAProtein)•Idea is: we know that DRGs contain cells that respond to temperature – if we isolatedthe mRNA from DRG we should have mRNA that codes for calcium•Can load this with calcium dye•Add mRNA from DRG cells to regular cells and then apply capsaicin see that the cells now respond•Then started to split up the mRNA and isolated the one that made all the cells respond to capsaicin-‐ we call this TRPV1Experiment: Introduce TRPV1 mRNAinto frog oocytes and measure current at-80 mV in response to extracts of chili peppers. or to warm temperature•Current magnitude associated with the spiciness of the pepper•Same thing happens with warm waterTRPV1 was one of the first members of a now large family of transient receptor potential (TRP) channels•This is what this receptor looks like – it is a receptor ion channel•Opened by capsaicin, heat, acidification •Cold receptor is related to this receptorCapsaicinTemperature•What happens with a knockout•For chili peppers: Shows that TRPV1 is the only sensor for capsaicin• For heat: becomes LESS sensitive but not completely gone-‐ just shows that TRPV1 does act as a temperature sensor•Birds cant taste capsaicin-‐ might eat the peppers and dispersing the seeds-‐ can put capsaicin in your bird seed to keep away other animals•The other heat sensors in the somatosensory system are TRPM8•Menthol acts on the same receptor as cold receptors•Can see the free nerve endings in these pictures•Can map the distribution of the nerve endings in the skin.•TRPA1 is also important for pain•Receptor for mustard and wasabi – different kind of burning sensation•Responds to many things like car exhaust and inflammation Experiment: Inject formalin into the paw of a mouse and measure time spent licking the paw in wt and ko mice.Result: all phases of the pain response are decreased in the knockout.•Animal injected with formalin (hurts) and observe how the animal is in pain•In a wild type animal there is constant licking but in a TRPA1 knockout animal the animal stops lickingWang, Chang and TRPA1 knockout animal find that they do not respond to CO2 (like the feeling when you have a soda)Liman, 2010•Main way in which pain impacts our lives is through inflammation•During inflammation there is a release of a lot of different things-‐ released by blood cells and nerve terminals•They are affecting the nerve terminals sensitizing them to pain•They upregulate the activity of TRPA1 and TRPV1Why does a warmshower feel painful when you have a sunburn?•Example of something that feels hot-‐ involvement of TRPV1Sensitization of TRPV1 by NGF!Nerve fiber response to heat before and after NGF•Record form nerve fibers-‐ found that if they apply a heat ramp the cell doesn't respond,but if they add receptors it responds•Idea that the inflammatory mediators (NGF) are enhancing the response of the cell to stimuli that aren’t very strong.•Organization of the pain and temperature system in the dorsal root ganglion•There are cold fibers, hot fibers, and pain fibers (nociceptors)Dorsal column•The information coming to the brain•The sense of touch involves axons that go directly up the spinal cord without touching but for nociception it synapses and crosses right away in the spinal cord•The nociceptors ascend through a separate anteriolateral tract•Nociceptors synapsing and crossing so if you have a lesion in only one part of the spinalcord-‐ you lose pain on one side of the body and touch on the other side of the body•Somatosensation-‐ same side•Soldier being wounded-‐ soldiers in the field didn't report pain, doctor realized that the perception of the pain depended on the context•The soldier was just relieved to be alive-‐ overcame intense pain•Gave patients a saline injection or a real pain reliever-‐ even patients given the placebo recorded relief from pain•Then did the experiment using a drug that blocked opiate receptors and this made the placebo not work anymoreOpiates bind to opiate receptors on nociceptor and dorsalhorn cells to inhibit activity. Endogenous ligands are peptides: enkephalins, endorphins, dynorphins•This