Lecture 5 Part 2 Mechanosenstions PDF

Mechanosenstions Lecture 5 - Part 2 A few comments on a confusing question Piezo Channels  Piezo Channels Background and knowledge gap  Piezo Channels Methods and results  Piezo Channels Conclusions and importance  PIEZO2 and perineal mechanos...

Mechanosenstions Lecture 5 - Part 2 A few comments on a confusing question 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  How to read a figure? Heatmaps representing calcium (GCaMP6f) responses to (left) repetitive application of naturalistic stimuli and (right) graded von Frey stimulation. LTMRs and HTMRs are separated, and relative fluorescence changes (DF/F) are colored as indicated. Scale bar, 10 s.  Which axis is time? A B C A B C 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 … 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? 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 No quiz this week  Questions? Any topic from previous lectures