Lecture 5: Mechanosensation PDF

Mechanosensation Lecture 5  Volunteer Note Takers Needed AccessAbility Services AccessAbility Services is recruiting volunteer note-takers to assist students with disabilities. Volunteers play an essential role in allowing students to access course materials, which the...

Mechanosensation Lecture 5  Volunteer Note Takers Needed AccessAbility Services AccessAbility Services is recruiting volunteer note-takers to assist students with disabilities. Volunteers play an essential role in allowing students to access course materials, which they may otherwise not have access to. Co-Curricular Record (CCR) Approved Position 1.Go to uoft.me/utscnotes and select myAIMS Notetaker Log-In 2.Follow the simple step-by-step process to register. 3.Upload notes les into the AccessAbility Services myAIMS note-taking portal If you have questions please contact AccessAbility Services (416) 208-2662 or [email protected] fi SZ UV Cones Are Light Biased and Have a High Gain and Long Integration Times SyGCaMP6f synaptically tagged fluorescent calcium biosensor mCherry expressed under same promoter non-synaptically localised fluorescent protein  SZ UV Cones Are Light Biased and Have a High Gain and Long Integration Times SyGCaMP6f synaptically tagged fluorescent calcium biosensor mCherry expressed under same promoter non-synaptically localised fluorescent protein Mean and single trial dorsal and SZ single cone 2-photon calcium responses to varying duration light- (6 3 105 photon/s/mm2) and dark-steps (0 photon/ s/mm2) from a constant UV background (2.4 3 104 photon/s/mm2). Fluorescence microscopy bitesizebio.com Fluorescence microscopy bitesizebio.com Fluorescence microscopy bitesizebio.com  GCaMP Genetically encoded calcium indicator Fusion of GFP and Clamodulin Conformational change when bound to Ca causes increased fluorescence Measures increase in internal Ca as a proxy to voltage changes  Methods Z-normalization - correct intensity differences Pre-processing Calcium traces for each ROI were extracted and z- normalized based on the time interval 1-6 s at the beginning of recordings prior to presentation of systematic light stimulation. Imaging as a recording technique Capturing fluorescence signal Mainly Ca, but also voltage, pH, or neurotransmitters (Glutamate). Can be targeted genetically Record from many cells simultaneously C. Elegans or Zebra fish - the entire nervous system/brain Arens lab Imaging as a recording technique Capturing fluorescence signal Mainly Ca, but also voltage, pH, or neurotransmitters (Glutamate). Can be targeted genetically Record from many cells simultaneously C. Elegans or Zebra fish - the entire nervous system/brain Arens lab Mechanosensation Lecture 5 What is this? What is this? Metridium nematocyst firing Metridium nematocyst firing  Nematocyst discharge Thrum, Hydrobiologia, 2004  Nematocyst discharge Thrum, Hydrobiologia, 2004 Nuchter, Curr. Biol., 2006  Nematocyst discharge Thrum, Hydrobiologia, 2004 Nuchter, Curr. Biol., 2006  Nematocyst discharge Hair movement induces response Discharge is extremely costly Regulated by both chemosensory and mechanosensory cue Communication between different units along the same tentacle Mechanotransduction TRP and Piezo channels  TRP Channels Transient Receptor Potential Christensen, Nature Rev. Neurosci., 2007  TRP Channels Transient Receptor Potential Fowler, Life Sciences, 2013 Christensen, Nature Rev. Neurosci., 2007  TRP Channels Transient Receptor Potential Evolutionarily conserved integral membrane proteins 33 TRP genes in mammals 60 in zebrafish, 24 in nematodes, 16 in fruitflies and 1 in yeast Involved in the transduction of a wide variety of other sensations, with roles in vision, olfaction, taste, chemosensation and thermosensation Hehlert, Trends in Neurosci., 2021  Brief nomenclature detour Homo-dimer -dimer -trimer -tetramer -pentamer Hetero-dimer 2 3 4 5 Homo-dimer  Potential gating mechanisms for mechanically sensitive ion channels Membrane model Tether model indirect Tether model direct Secondary signal Christensen, Nature Rev. Neurosci., 2007  Does mechanosensation involve direct activation of a channel? Christensen, Nature Rev. Neurosci., 2007  Does mechanosensation involve direct activation of a channel? Current latency should be faster than second messenger systems Christensen, Nature Rev. Neurosci., 2007  Does mechanosensation involve direct activation of a channel? Current latency should be faster than second messenger systems Channel activation kinetics should depend on stimulus amplitude Christensen, Nature Rev. Neurosci., 2007  Does mechanosensation involve direct activation of a channel? Current latency should be faster than second messenger systems Channel activation kinetics should depend on stimulus amplitude Mechanical correlate of gating Christensen, Nature Rev. Neurosci., 2007  Does the candidate protein participate in mechanical transduction? Christensen, Nature Rev. Neurosci., 2007  Does the candidate protein participate in mechanical transduction? Expressed in the receptor cell by the time during development that the mechanically sensitive current is detected Christensen, Nature Rev. Neurosci., 2007  Does the candidate protein participate in mechanical transduction? Expressed in the receptor cell by the time during development that the mechanically sensitive current is detected Located at the site of mechanical transduction within the cell Christensen, Nature Rev. Neurosci., 2007  Does the candidate protein participate in mechanical transduction? Expressed in the receptor cell by the time during development that the mechanically sensitive current is detected Located at the site of mechanical transduction within the cell Blocking candidate protein expression by knockdown or knockout should block the mechanically activated conductance Christensen, Nature Rev. Neurosci., 2007 Is the candidate protein mechanically sensitive?  Is the candidate protein mechanically sensitive? In a heterologous expression system the forces necessary to gate the candidate channel should be comparable to the physiological stimulus.  Is the candidate protein mechanically sensitive? In a heterologous expression system the forces necessary to gate the candidate channel should be comparable to the physiological stimulus. Is the candidate protein a pore- forming subunit?  Is the candidate protein mechanically sensitive? In a heterologous expression system the forces necessary to gate the candidate channel should be comparable to the physiological stimulus. Is the candidate protein a pore- forming subunit? Whether the expression of a candidate channel protein with a mutation in the putative pore region alters the pharmacology or permeation properties of the native conductance in the sensory cell Body touch in nematodes Christensen, Nature Rev. Neurosci., 2007  Body touch in nematodes First group of ion channels to be implicated in mechanosensitivity in eukaryotes Christensen, Nature Rev. Neurosci., 2007  Body touch in nematodes First group of ion channels to be implicated in mechanosensitivity in eukaryotes Heteromultimers of 4–6 subunits Christensen, Nature Rev. Neurosci., 2007  Body touch in nematodes First group of ion channels to be implicated in mechanosensitivity in eukaryotes Heteromultimers of 4–6 subunits Found in mutation screen for insensitivity to light touch Christensen, Nature Rev. Neurosci., 2007  Body touch in nematodes First group of ion channels to be implicated in mechanosensitivity in eukaryotes Heteromultimers of 4–6 subunits Found in mutation screen for insensitivity to light touch Expressed in the six mechanosensitive neurons that innervate the body of the worm, and proteins are appropriately located in these neurons Christensen, Nature Rev. Neurosci., 2007 Body touch in nematodes Yemini, Cell, 2021 Christensen, Nature Rev. Neurosci., 2007 Body touch in nematodes Yemini, Cell, 2021 Response latency can be as short as 0.7ms and it decreases with increasing stimulus amplitude Christensen, Nature Rev. Neurosci., 2007 Body touch in nematodes Yemini, Cell, 2021 Response latency can be as short as 0.7ms and it decreases with increasing stimulus amplitude MEC-4 and MEC-10 by themselves do not form mechanically gated ion channels in heterologous expression system Christensen, Nature Rev. Neurosci., 2007 Body touch in nematodes Yemini, Cell, 2021 Response latency can be as short as 0.7ms and it decreases with increasing stimulus amplitude MEC-4 and MEC-10 by themselves do not form mechanically gated ion channels in heterologous expression system A pore mutation that affects selectivity of the channel has not been identified. Christensen, Nature Rev. Neurosci., 2007 Nociception and bristle touch in Drosophila  Nociception and bristle touch in Drosophila TRPA1 (painless)  Nociception and bristle touch in Drosophila TRPA1 (painless) Found in larval behavioural screen defective in pain response Tracey, Cell, 2003  Nociception and bristle touch in Drosophila TRPA1 (painless) Found in larval behavioural screen defective in pain response Localized to skin multidendritic neurons and associated with their activation  Nociception and bristle touch in Drosophila TRPA1 (painless) Found in larval behavioural screen defective in pain response Localized to skin multidendritic neurons and associated with their activation Latency unknown  Nociception and bristle touch in Drosophila TRPA1 (painless) Found in larval behavioural screen defective in pain response Localized to skin multidendritic neurons and associated with their activation Latency unknown NOMPC (TRPN1) Tilney, PNAS, 2005  Nociception and bristle touch in Drosophila TRPA1 (painless) Found in larval behavioural screen defective in pain response Localized to skin multidendritic neurons and associated with their activation Latency unknown NOMPC (TRPN1) Probably directly gated since latency is < 200µs Tilney, PNAS, 2005  Nociception and bristle touch in Drosophila TRPA1 (painless) Found in larval behavioural screen defective in pain response Localized to skin multidendritic neurons and associated with their activation Latency unknown NOMPC (TRPN1) Probably directly gated since latency is < 200µs Localised to the bristle complex Christensen, Nature Rev. Neurosci., 2007  Nociception and bristle touch in Drosophila TRPA1 (painless) Found in larval behavioural screen defective in pain response Localized to skin multidendritic neurons and associated with their activation Latency unknown NOMPC (TRPN1) Probably directly gated since latency is < 200µs Localised to the bristle complex Mutations abolish deflection current Christensen, Nature Rev. Neurosci., 2007 Jurgens, Genetics, 2024 Jurgens, Genetics, 2024 Hearing in vertebrates  Hearing in vertebrates TRP family (most likely)  Hearing in vertebrates TRP family (most likely) The most intensely studied mechanoreceptor is the hair cell of the vertebrate inner ear Christensen, Nature Rev. Neurosci., 2007  Hearing in vertebrates TRP family (most likely) The most intensely studied mechanoreceptor is the hair cell of the vertebrate inner ear Non-selective cation channel; large conductance; pharmacology similar to other TRP channels Christensen, Nature Rev. Neurosci., 2007  Hearing in vertebrates TRP family (most likely) The most intensely studied mechanoreceptor is the hair cell of the vertebrate inner ear Non-selective cation channel; large conductance; pharmacology similar to other TRP channels Latency 20-40µs and becomes faster with larger deflections Christensen, Nature Rev. Neurosci., 2007  Mechanosensors Cuticle Sensory neuron Supporting cells Chordotonal Campaniform Bristle Hehlert, Trends in Neurosci., 2021 leg proprioception in Drosophila Agrawal, eLife, 2020 leg proprioception in Drosophila Agrawal, eLife, 2020 leg proprioception in Drosophila Agrawal, eLife, 2020 leg proprioception in Drosophila Agrawal, eLife, 2020 Piezo Channels Hehlert, Trends in Neurosci., 2021  Piezo Channels Expressed in a variety of tissues and organs such as the nervous system, stomach, lungs, bladder, intestines, and blood vessels Hehlert, Trends in Neurosci., 2021  Piezo Channels Expressed in a variety of tissues and organs such as the nervous system, stomach, lungs, bladder, intestines, and blood vessels Built from 3 homomeric subunits. Each with 38 transmembrane helices Hehlert, Trends in Neurosci., 2021  Piezo Channels Expressed in a variety of tissues and organs such as the nervous system, stomach, lungs, bladder, intestines, and blood vessels Built from 3 homomeric subunits. Each with 38 transmembrane helices 36 of the TM helices are in the blades - probably forming the tension sensing mechanism Delmas, Neuron, 2022 Piezo Channels  Piezo Channels Background and knowledge gap  Piezo Channels Methods and results  Piezo Channels Conclusions and importance  PIEZO2 and perineal mechanosensation are essential for sexual function Measuring behavioural sensitivity Reaction of mice to punctate touch (A) wild-type hind-paw, (B) wild-type perineum. (Left) Example responses for individual mice (points and thin lines; four males and four females) and mean (solid lines) to a series of calibrated von Frey filaments (grams, each tested 10 times per mouse). (Middle) Quantitation of von Frey threshold (≥5/10; n = 12 males and 12 females).  von Frey stimulation Mice were individually habituated on a mesh floor covered by a transparent glass vessel for 1 hour. Nylon monofilaments (Stoelting) were directly applied to the glabrous skin of the hind-paw or to the perineum until the filament bent slightly. Withdrawal, flinch and jump responses within the following 1 second was recorded. This single force touch was repeated 10 times for each filament with ascending forces of (0.008g, 0.02g, 0.04g, 0.07g, 0.16g, 0.4g, 0.06g, 1.0g, and 1.4g) until the animals responded 100% of the time for two filaments in a row.  von Frey stimulation Mice were individually habituated on a mesh floor covered by a transparent glass vessel for 1 hour. Nylon monofilaments (Stoelting) were directly applied to the glabrous skin of the hind-paw or to the perineum until the filament bent slightly. Withdrawal, flinch and jump responses within the following 1 second was recorded. This single force touch was repeated 10 times for each filament with ascending forces of (0.008g, 0.02g, 0.04g, 0.07g, 0.16g, 0.4g, 0.06g, 1.0g, and 1.4g) until the animals responded 100% of the time for two filaments in a row. PIEZO2 and perineal mechanosensation are essential for sexual function Measuring behavioural sensitivity PIEZO2 and perineal mechanosensation are essential for sexual function Generating Piezo mutant The mechanically activated ion channel PIEZO2 is essential for discriminative touch in mice and humans (11, 12). We anticipated that this mechanoreceptor would also be responsible for the sensitivity of the perineum. Piezo2-null mice die as neonates (19); therefore, we generated conditional genetic deletions using a Hoxb8-Cre line (Piezo2Hoxb8) to target cells below the mid-thoracic region (17). PIEZO2 and perineal mechanosensation are essential for sexual function Generating Piezo mutant The mechanically activated ion channel PIEZO2 is essential for discriminative touch in mice and humans (11, 12). We anticipated that this mechanoreceptor would also be responsible for the sensitivity of the perineum. Piezo2-null mice die as neonates (19); therefore, we generated conditional genetic deletions using a Hoxb8-Cre line (Piezo2Hoxb8) to target cells below the mid-thoracic region (17). Hoxb8-Cre Mice: a Tool for Brain-Sparing Conditional Gene Deletion Witschi, Genesis, 2010 Here, we describe a novel Cre mouse line which expresses the Cre recombinase under the transcriptional control of the Hoxb8 gene. Within the neural axis of these mice, Hoxb8-Cre expression is found in spinal cord neurons and glial cells, and in virtually all neurons of the dorsal root ganglia, but spares the brain PIEZO2 and perineal mechanosensation are essential for sexual function Generating Piezo mutant - Cre lines Cre protein The Jackson Laboratory PIEZO2 and perineal mechanosensation are essential for sexual function Generating Piezo mutant - Cre lines Cre protein The Jackson Laboratory PIEZO2 and perineal mechanosensation are essential for sexual function Generating Piezo mutant - Cre lines Cre protein The Jackson Laboratory PIEZO2 and perineal mechanosensation are essential for sexual function Measuring behavioural sensitivity PIEZO2 and perineal mechanosensation are essential for sexual function Measuring behavioural sensitivity Tracing the relevant neurons  Tracing the relevant neurons cholera toxin subunit-b (CTB) is a bidirectional neuronal tracer  Tracing the relevant neurons cholera toxin subunit-b (CTB) is a bidirectional neuronal tracer CTB-488, CTB-594 reference the fluorophore conjugate Alexa-Fluor 488  Tracing the relevant neurons cholera toxin subunit-b (CTB) is a bidirectional neuronal tracer CTB-488, CTB-594 reference the fluorophore conjugate Imaging cellular responses w GCaMP6f Imaging cellular responses w GCaMP6f PIEZO2 and perineal mechanosensation are essential for sexual function Imaging cellular responses Five representative example GCaMP6f transients from perineal neurons that exhibited differential genital von Frey sensitivity normalized to their maximal response (scale bars, left, ΔF/F, %).  Calculating dF/F Regions of interest (ROI) outlining responding cells were drawn in FIJI/ImageJ and relative change of GCaMP6f fluorescence was calculated as percent ΔF/F. Contaminant signal e.g., from out-of-focus tissue and neighboring cells was removed by subtracting the fluorescence of a donut-shaped area surrounding each ROI using a custom MATLAB script Background fluorescence noise was calculated for each ROI as the standard deviation of the bottom 25% of all data points as described previously (44). A transient rise in fluorescence was considered significant if its peak exceeded 15 times this value.  Calculating dF/F Regions of interest (ROI) outlining responding cells were drawn in FIJI/ImageJ and relative change of GCaMP6f fluorescence was calculated as percent ΔF/F. Contaminant signal e.g., from out-of-focus tissue and neighboring cells was removed by subtracting the fluorescence of a donut-shaped area surrounding each ROI using a custom MATLAB script Background fluorescence noise was calculated for each ROI as the standard deviation of the bottom 25% of all data points as described previously (44). A transient rise in fluorescence was considered significant if its peak exceeded 15 times this value.  Calculating dF/F Regions of interest (ROI) outlining responding cells were drawn in FIJI/ImageJ and relative change of GCaMP6f fluorescence was calculated as percent ΔF/F. Contaminant signal e.g., from out-of-focus tissue and neighboring cells was removed by subtracting the fluorescence of a donut-shaped area surrounding each ROI using a custom MATLAB script Background fluorescence noise was calculated for each ROI as the standard deviation of the bottom 25% of all data points as described previously (44). A transient rise in fluorescence was considered significant if its peak exceeded 15 times this value.  Calculating dF/F Regions of interest (ROI) outlining responding cells were drawn in FIJI/ImageJ and relative change of GCaMP6f fluorescence was calculated as percent ΔF/F. Contaminant signal e.g., from out-of-focus tissue and neighboring cells was removed by subtracting the fluorescence of a donut-shaped area surrounding each ROI using a custom MATLAB script Background fluorescence noise was calculated for each ROI as the standard deviation of the bottom 25% of all data points as described previously (44). A transient rise in fluorescence was considered significant if its peak exceeded 15 times this value.  Calculating dF/F F(t) − F0 δF/F = F Signal F0 F(t) Realtime fluorescence F0 Stimulus Baseline fluorescence  Calculating dF/F F(t) − F0 δF/F = F Signal F0 F0 F(t) Realtime fluorescence F0 Stimulus Baseline fluorescence  Calculating dF/F F(t) − F0 F(t) δF/F = F Signal F0 F0 F(t) Realtime fluorescence F0 Stimulus Baseline fluorescence  Calculating dF/F F(t) − F0 F(t) δF/F = F Signal F0 F0 F(t) Realtime fluorescence F0 Stimulus Baseline fluorescence  Calculating dF/F F(t) − F0 F(t) δF/F = F Signal F0 F0 F(t) Realtime fluorescence F0 Stimulus Baseline fluorescence PIEZO2 and perineal mechanosensation are essential for sexual function Imaging cellular responses PIEZO2 and perineal mechanosensation are essential for sexual function Imaging cellular responses PIEZO2 and perineal mechanosensation are essential for sexual function Imaging cellular responses Fig. 2. Functional characterization of perineal mechanoreceptors and role of PIEZO2. What is the figure trying to tell us? Fig. 2. Functional characterization of perineal mechanoreceptors and role of PIEZO2. What is the figure trying to tell us? Cells were defined as High Threshold Mechanosensory Neurons (HTMRs) if they responded primarily only to pinch and/or high force von Frey filaments and as LTMRs if they were activated by any or all gentle stimuli. To do this computationally (34), cells were classified as air-puff cells if they had significant responses to air-puff that were at least 2 times as strong as their brush response. Vibration cells had significant vibration responses and a ratio of vibration responses to all other responses greater than 3 … PIEZO2 and perineal mechanosensation are essential for sexual function Imaging cellular responses PIEZO2 and perineal mechanosensation are essential for sexual function Behavioural implications PIEZO2 and perineal mechanosensation are essential for sexual function Behavioural implications Penile Protrusion Test Animals, restrained by scruffing, were held in a supine position allowing clear tubing (Tygon S3™ E-3603 F ACFUN007) to be applied around the base of the penis immediately over the external prepuce. Each animal was custom fitted with tubing of optimal inner diameter for gentle prepuce retraction over 5 test trials. Once the correctly sized tubing was determined, 10 tests were performed in succession. The protraction of the internal prepuce as the penis extended into the tube away from the body wall was scored. The test assay was repeated the following day for an additional 10 trials and results pooled. PIEZO2 and perineal mechanosensation are essential for sexual function Behavioural implications PIEZO2 and perineal mechanosensation are essential for sexual function Behavioural implications Relevance to humans  Conclusions Deficits in PIEZO2-dependent mechanosensation interfere with: perigenital sensation physiological response copulation The crucial role of PIEZO2 for perineal touch in mice and humans may have therapeutic potential Both for Hypo and hypersensitivity  Limitations PIEZO2 deficiency is extremely rare, and we were unable to carry out detailed quantitative sensory testing in a larger group of human subjects Functional imaging experiments were carried out in anesthetized mice Additional specialized roles for mechanosensory neurons in mating that were not revealed in this study  Reading assignment painless, a Drosophila Gene Essential for Nociception Read only up to Figure 3