PCB4843-Chapter 8 - Olfaction PDF

Key concepts for chapter 8 (olfaction) The relationship between the chemical stimulus and perception. Structure of the olfactory epithelium and the different cell types. How odors activate olfactory receptor neurons anatomically (where they bind to), electrically (depolarize) and the transduction...

Key concepts for chapter 8 (olfaction) The relationship between the chemical stimulus and perception. Structure of the olfactory epithelium and the different cell types. How odors activate olfactory receptor neurons anatomically (where they bind to), electrically (depolarize) and the transduction cascade. Odor sensitivity of olfactory receptor neurons (very broadly selective) The number of olfactory receptor genes, and the logic of how different receptors are expressed in each olfactory receptor neuron. What are glomeruli anatomically and what do they represent molecularly? The input-output structure of a glomerular unit. How odor information is encoded in the bulb. Projections from the bulb to the rest of the brain. In humans, olfaction warns of harmful substances, combines with taste for identification and enjoyment of food In animals (possibly humans), some odors are used for communication (pheromones). Although remains controversial in humans. Two animals tracking an odor Although we don’t commonly use olfaction for tracking, humans can learn to do so. Improvements with training Olfactory stimuli are airborne volatile chemical stimuli that are detected by the olfactory system Volatile compounds in popcorn 3-methyl butanal (malty) Many natural compounds contain Furfural (nutty) 2-acetyl pyrrole (rice, bread) Vanillin (vanilla) The same odor presented at different concentrations can evoke different perceptions. Walradt et al., 1970 Indole – pleasant at low concentrations, putrid at high. Perception Enantiomers of carvone. Chemically similar molecules can have different perceptions.  The relationship between olfactory stimuli and perception is complicated. Each tick is a person’s response Descriptors Low concentration High concentration  The relationship between olfactory stimuli and perception is complicated. Each tick is a person’s response Low concentration Descriptors (side by side) High concentration Ethyl-2-methylpentanoate smells similar at different concentrations  The relationship between olfactory stimuli and perception is complicated. Each tick is a person’s response Low concentration Descriptors (side by side) High concentration 3-Hexanol evokes different perceptual experiences  The relationship between olfactory stimuli and perception is complicated. Each tick is a person’s response Low concentration Descriptors (side by side) High concentration Sweeter and more delicious at higher concentrations. Odors have distinct detection thresholds (the lowest concentration that evokes a perceptually detectable response) Ozone is detected at a lower concentration than D-limonene (citrus) Worse Each animal has odors that they are most sensitive to. Better Humans: (from the good scents company) 3-mercapto-3-methylbutyl-formate: Sulfurous, catty, caramelized onion, roast coffee, roast meat. n-octanoic acid: rancid-like n-pentanoic acid: Sweet and fruity McGann 2017 Movie: 0:03-0:14 Olfactory receptor neurons are located in the nasal cavity and respond to odors. Receptor neurons have cilia, which extend into a thick layer of mucus which comes into contact with odor molecules. Bowman’s glands Produces mucus that protects against viruses and helps trap odor molecules. Supporting cells Contain enzymes that protect against potentially harmful chemicals Basal cells: ORNs are generated continuously from dividing stem cells maintained by basal cells. Olfactory receptor cells continually regenerate Basal cells are the source of newborn ORNs Odors generate a receptor potential in the cilia, which triggers an action potential that propagates along its axon Odors are detected by specialized proteins located on the cilia. Puffing odor onto cell body does not evoke a (strong) response. Spiking in axon To the brain (olfactory bulb) Spiking in soma Spiking in the soma is transmitted down the olfactory nerve to the olfactory bulb. Current in cilia In the nose Odor Odor transduction in olfactory receptor neurons 1. Odorants bind to odorant receptor protein. 2. G-protein Golf is stimulated 3. Activates adenylyl cyclase 4. Forms cAMP 5. cAMP binds to cyclic nucleotide-gated cation channel which allows influx of Na+ and Ca2+. 6. Opens Ca2+ activated Cl- channels. Thought to serve as additional amplification step. Odor transduction in olfactory receptor neurons 1. Odorants bind to odorant receptor protein. 2. G-protein Golf is stimulated 3. Activates adenylyl cyclase 4. Forms cAMP 5. cAMP binds to cyclic nucleotide-gated cation channel which allows influx of Na+ and Ca2+. 6. Opens Ca2+ activated Cl- channels. Thought to serve as additional amplification step. Odor transduction in olfactory receptor neurons 1. Odorants bind to odorant receptor protein. 2. G-protein Golf is stimulated 3. Activates adenylyl cyclase 4. Forms cAMP 5. cAMP binds to cyclic nucleotide-gated cation channel which allows influx of Na+ and Ca2+. 6. Opens Ca2+ activated Cl- channels. Thought to serve as additional amplification step. Odor transduction in olfactory receptor neurons 1. Odorants bind to odorant receptor protein. 2. G-protein Golf is stimulated 3. Activates adenylyl cyclase 4. Forms cAMP 5. cAMP binds to cyclic nucleotide-gated cation channel which allows influx of Na+ and Ca2+. 6. Opens Ca2+ activated Cl- channels. Thought to serve as additional amplification step. Odor transduction in olfactory receptor neurons 1. Odorants bind to odorant receptor protein. 2. G-protein Golf is stimulated 3. Activates adenylyl cyclase 4. Forms cAMP 5. cAMP binds to cyclic nucleotide-gated cation channel which allows influx of Na+ and Ca2+. 6. Opens Ca2+ activated Cl- channels. Thought to serve as additional amplification step. Odor transduction in olfactory receptor neurons 1. Odorants bind to odorant receptor protein. 2. G-protein Golf is stimulated 3. Activates adenylyl cyclase 4. Forms cAMP 5. cAMP binds to cyclic nucleotide-gated cation channel which allows influx of Na+ and Ca2+. 6. Opens Ca2+ activated Cl- channels. Thought to serve as additional amplification step. Sensory transduction cascade 1 3 5 6 4 2 Cl- mediates olfactory sensory neuron sensitivity Typical concentrations Cl- normally acts as a hyperpolarizing ion. Since it is typically more concentrated extracellularly. Driving force to flow inside making inside more negative. Cl- is concentrated in ORNs. Active transporter (NKCC) moves sodium, potassium and chloride in the same direction. Opening of Cl- channels causes flow of negative ions outside. Inside of cell becomes more positive. Olfactory receptors are found at the highest concentration in A) Ensheathing cells B) Olfactory cilia C) Cribiform plate D) Bowman’s gland cells E) Olfactory stem cells What would be a consequence of knocking out a protein critical in the olfactory cAMP pathway? A) Odorants diffuse away B) Anosmia occurs C) Cilia channels become sensitized D) Receptor cells degenerate In terms of scent detection and tracking capabilities in humans A) The olfactory epithelium of humans is larger than that of most canines. B) Percentage-wise, humans devote more of their cortex to olfaction than do rats C) Humans, like dogs, can follow scent trails by frequent sniffing and orthogonal digressions D) Humans can detect most chemical compounds at lower thresholds than almost all other organisms Movie: 2:46-4:51 Olfactory sensory neuron receptive fields are complex and respond to many different odors Experiments that recorded individual ORNs in response to odor observed: Many ORNs responded to many odorants. Many responded to some odorants, but not others. Neuron 1 Neuron 2 Neuron 3 Olfactory receptor neurons exhibit broad tuning to different odorants (actual data). Recording from single olfactory receptor neuron Response to different odorants No Odor Odor 1 Odor 2 Odor 3 Odor 4 Odor 5 Odor 6 Odor 7 Odor 8 What is its receptive field? Duchamp-Viret et al., 1999 There are more than 1000 different odorant receptor genes. Mature olfactory sensory neurons Discovered by Linda Buck expressing a green marker and Richard Axel in 1991. 2004 Nobel prize. In mice, nearly 5% of mammalian genome encodes odorant receptors. Olfactory marker protein Fewer in humans (about 400) Still a large number, and many are present as pseudogenes (DNA that cannot be transcribed into a stable mRNA). Each olfactory receptor neuron only I7 receptor expresses one receptor type. Transgenic mouse generated to express GFP in cells that express only one odorant receptor. I7 vs M71 (two different receptor types). M71 receptor This approach allowed a more comprehensive M71 receptor analysis of receptive fields of neurons that express the same receptor type The M71 OSN responds preferentially to odorants containing a mixture of “F”, but not other compounds. That does not mean that F is the optimal ligand for the M71 receptor. (would require testing a nearly infinite number of potential ligands) M71 activation by odors A-F Different odor receptors have specific tuning properties. However, the same receptor type can respond to many different odors. Each odorant receptor is sensitive to a range of ligands. Different ORs The same odor will activate many different receptors (different curves), but at different affinities. Binding They are high affinity for some ligands, low affinity for others. Ligand Concentration High affinity -> Binds at low concentration Low affinity -> Needs higher concentration to bind Explains broad tuning. Predicts that an odorant is encoded as the combination of activated receptors. Olfactory receptor neurons extend long axons that target the olfactory bulb Olfactory receptor neurons expressing the same receptor type converge to a single region of neuropil in the olfactory bulb Olfactory epithelium Olfactory bulb Mapping of olfactory receptor neurons onto the olfactory bulb Olfactory receptors map onto the surface of the olfactory bulb.  The anatomical structure of the olfactory bulb Input: Detecting Odors ↑ Input layer Interneurons Output neurons ↑ ↑ Output neurons ↑ Interneurons Output: To cortex  The neurons surrounding a glomerulus can be divided into many different subtypes Neuronal diversity in the olfactory bulb The input-output organization of the bulb and retina Input Odors coming into nose Input: Detecting Odors ↑ Odors stimulate ORs located on ORN cilia Action potential transmitted to axon terminals located in bulb glomeruli Output Signal from bulb to rest of brain Mitral/tufted cells transmit beyond the bulb. ↑ Output: To cortex Input Light coming into eye Rods and cones primarily carry out phototransduction. Output Signal from retina to rest of brain Ganglion cells provide output to the rest of the brain. How is odor information encoded in the olfactory bulb? Across population of glomeruli Each glomerulus reflects the input from one olfactory receptor type. Glomerular responses are molecularly pure! Odorants evoke activity in distinct spatial patterns of glomeruli (olfactory maps). Odors can evoke different response properties in each glomerulus (time course, adaptation, excitation vs inhibition). Individual olfactory bulb neurons Odorants evoke complex patterns of spiking activity that are influenced by odor and concentration. Neurons are tuned to different phases of the respiratory cycle. Imaging from glomeruli  The GFP protein can be fused to other proteins that can modulate fluorescence Protein calcium Baseline fluorescence sensor (GECI) Calcium (GCaMP6s) Chen et al., 2013 Calcium sensing Akerboom et al., 2012 domain Peak ΔF/F versus [Ca2+] Fluorescence and electrical time course GCaMP6f Badura et al., 2014 Log10 ([Ca2+]/M) * * * ** * Spike Problem: 2-photon microscopy provides significantly better resolution in the lateral (x/y) and axial (z) dimensions. 1-photon 2-photon Chris Xu lab Olfactory bulb glomeruli 1p 2p 1 2 p p Movie of two odorants presented side by side (Deleted from ppt to reduce file size) Peaks of activity in response to odors Isoamyl acetate (Banana) Methyl valerate (Orbitz bubblemint) Different odorants evoke different glomerular patterns Recordings from many glomeruli reveal complex excitatory and inhibitory dynamics Time course from 3 glomeruli Color indicates amplitude of glomerular response Glomerulus  Odorants cause complex changes in (single) neuron spike rate and timing Odor causes delay Not responsive More spikes More spikes Delayed with increase spiking Delayed with increase spiking Olfactory bulb neurons are sensitive to sniff phase Sniffs from 3 of the trials Top: Recording from mitral cell to many odor presentations. Respiration The rows are repeated measurements of Spikes in many trials (black ticks) the same neuron to an odor response. Trials Cell responds to the odor in very confusing way. Time Sniffs from 3 of the trials Respiration occurs at different times Respiration and phases relative to the odor onset. Spikes in many trials Normalize each sniff from 0-2pi Black: Unaligned time vs spikes Red: Time vs spikes when aligning respiration. Neurogenesis also occurs in the olfactory bulb Neurons migrate via the rostral migratory stream continuously throughout life. These neurons rapidly integrate into the olfactory bulb circuit and become functional. Begin responding to odor within hours of arrival. Anatomical pathway beyond the olfactory bulb Anatomical tracing of the projections from a single mitral cell One mitral cell (yellow) can connect to 14 brain areas. Projections from neighboring mitral cells overlapped with one another. No apparent topography in projections out of bulb. What is the organization beyond the bulb? Experiment: Fill neighboring glomeruli with differently colored dyes, examine the projections to cortex. Result: The projections largely overlapped with one another. Output of bulb seems to be random Could reflect that odors may not have innate meaning. This observation is consistent with physiological recordings from the cortex Responses in the olfactory bulb exhibit clear spatial organization. Recordings from piriform cortex reveal a distributed and somewhat random organization of cells responding to different odorants. Olfactory bulb receives feedback from many brain areas Feedback to OB Feedback to OB Olfactory receptors are found outside of the olfactory system The olfactory system is highly enriched with these receptors. Hence “olfactory” receptors. Odor receptors are found in many parts of the body. “Olfactory” receptors may in fact by general chemosensors whose functionality has been adapted by different sensory systems. How many odor receptor genes are there in mice? What does a glomerulus represent? How many odor receptor genes are there in mice? Introduction to microscopy Imaging using brightfield transmitted light Cheap, easy to understand, but many samples don’t have much contrast unless they are stained with something. Brightfield image of Golgi unstained human stained A549 epithelial cells section Lavilla…Vendrell 2018, Chemistry Fluorescence imaging Fluorescence is a phenomena in which the absorption of light results in a near instantaneous (~ 1/1000000 s) emission of a longer wavelength of light. Fluorophore is excited by some wavelength, relaxing causes emission (fluorescence) of a longer wavelength. (Many materials fluoresce in response to ultraviolet light). Abramowitz and Davidson (NHMFL) Fluorescence is not 100% efficient – not all absorbed photons evoke fluorescence. Fluorescence cannot be brighter than the light used to evoke fluorescence. The fluorescence emission of an object can be completely different from any intrinsic absorption or reflectance properties. Green chlorophyll fluoresces red Distinct from bioluminescence which is actually “glowing”. e.g., D-luciferin undergoes a chemical reaction that emits visible light. Wolfs et al., 2014 (Radiolabeling strategies for radionuclide imaging of stem cells) If fluorescence is localized, it can be used to enhance contrast White light 470 nm light Fluorescence stamp Crustacean (Lysiosquilla maculate) Coral (Scolymia) Marshall and Johnsen 2017 (Fluorescence as a means of colour signal enhancement) Molecules exist that absorb and fluorescence in many different colors Absorption and emission spectra of two fluorophores Excitation/emission spectra of the two fluorophores eGFP Absorption Emission Sample DAPI Absorption Emission Cell Bodies The spectra of the two fluorophores are mostly non-overlapping. All cells One Cell Type T  Separating excitation and emission wavelengths using different optical filters in a fluorescence microscope Camera Dichroic mirror Excitation Transmission (%) Light source Emission Emission filter filter filter Dichroic Fluorophore mirror Fluorophore absorption Excitation emission filter Objective Specimen Wavelength (nm) Reflected light fluorescence microscope directs broad Filters spectrum (white) light to the sample through the objective. Separation of wavelengths is accomplished by combining optical filters and mirrors. Filter cube Microscopes can easily change the filter sets to accommodate different fluorophores. In an epifluorescence microscope, which of the following separates the light source and the fluorescence emission? A) Emission filter B) Dichroic mirror C) CCD camera D) Excitation filter This is the absorption and emission spectra of GFP. What would be the best choice for an excitation and emission filter? A) Excitation of 550 nm; emission of 488 nm B) Excitation of 488 nm; emission of 530 nm C) Excitation of 300nm and emission of 488 nm D) Excitation of 515 nm and emission of 470 nm This is the absorption and emission spectra of an Alexa Fluor dye. What would be the best choice for an excitation and emission filter? A) Excitation of 550 nm; emission of 488 nm B) Excitation of 488 nm; emission of 530 nm C) Excitation of 300nm and emission of 488 nm D) Excitation of 550 nm; emission of 575 nm E) Excitation of 600 nm; emission of 525 nm In this epifluorescence microscope diagram, label parts A and B B A) A B) Draw a simple epifluorescence microscope with arrows indicating the light path Conclusions Olfactory system is designed to detect airborne volatile molecules Many different types of sensory neuron receptors, each of which is broadly tuned with a slightly varying affinity. Sensory neurons of the same type converge into the olfactory bulb, forming a topographic mapping of different receptors on the surface. Something happens in the bulb, which transmits signals to many other brain areas. A seemingly highly organized projection to the olfactory bulb is discarded.