Olfaction 1 Flashcards
Billig (2011)
Ano2 highly expressed in MOB / MOE, Ano1 in the vomeronasal organ, but not MOE (western blot). Cre-LoxP (tissue-specific) Ano2 KO: still expressed CNG channel (immunostaining). Patch clamp: KO only had 10% of the Cl- currents that WT had.
R: KO = no change in air-phase olfactograms, - chloride current is not required for physiological levels of olfaction. Functional relevance: go/no-go discrimination: KOs = similar levels of sensitivity to different odourants (CnGa2 KOs showed considerably less sensitivity in task, demonstrating vital importance of CnGa2 in olfactory transduction).
L: NFA (Cl- channel inhibitor) suppressed some of the transduction activity in KO. Another Cl channel in the KO contributing to depolarisation?? OFF-TARGET effects of NFA cannot be ruled out - may be suppressing electrical activity of OSNs by a mechanism independent of Cl- channels. In vitro: IONIC GRADIENTS may not reflect physiological ion gradients generated by in vivo MUCUS.
May be subtle types of discrimination, or other contexts (e.g. lower concentration odorant mixtures), where amplification by the chloride current becomes important.
Kelliher (2003)
CNGA4 KO: go/no-go task: impaired ability to discriminate 10-3M of octanal against a background of 10-4M. Background was reduced to 10-5M, KO mice significantly improved ability to discriminate (90% accuracy).
KO mice had significantly reduced ability to detect octanal on a background of heptanal (they developed cross-adaptation), but were accurately discriminated octanal from a background of cineole.
R: Effect of CNGA4 subunit on adaptation, and CNGA4-mediated adaptation necessary for discriminating odours from background odours, BUT this role is dependent upon the similarity of the odours.
L: KO causes lack of localisation of multiple CNG subunits, to the cilia. BUT KO in this paper still demonstrated olfactory function - if not localised would expect complete lack of ability to discriminate (Billig et al 2011 showed that CNGA2 KOs are unable to discriminate odours). Unclear whether we can trust the findings of this subunit KO, - further experiments with similar methodology (same staining methods, electrophysiology) would be needed to clarify the effects of CNGA2 KOs.
Mombaerts et al (1996)
Cre-LoxP recombination in mice: replace the P2 coding region (OR) with an M12 coding region. Inserted tau-lacZ so that the axonal projections of OSNs could be visualised.
Position of the P2 glomerulus to shifted posteriorly. Suggests that OR type may determine spatial patterning in the MOB. (But when remove transduction with CnGa2 KO - still get same glomerular position, so not dependent on transduction ability?).
However, this cannot be the only determining factor of glomerular positioning, as the glomerulus remained within the same zonal projection, and was nearer to the P2 position than the M12 position.
Imai et al (2006)
T: IRES technology in mice to label a specific (I7) OR with GFP (visualise the associated glomerulus). Changed the DRY sequence of the G protein to ‘RDY’ (shown to eliminate transduction using calcium imaging). Also inserted constitutively active G-protein (caG3).
R: I7(RDY) mutant failed to converge with glomerulus but when caG3 inserted, glomerular convergence restored, and glomerulus shifted posteriorly.
caG3 in WT mouse also caused posterior shift, whereas blocking PKA signalling in WT resulted in an anterior glomerular shift.
Illustrates how altering Gs signalling can determine the anterior-posterior axis of a glomerulus.
Arevian et al (2008)
1) Calcium imaging: more granule cells active when two glomeruli simulatenously activated (rather than individual excitation of each glomerulus) - granule cells are activated by SUMMATION of mitral cell input.
2) Whole cell recordings of pre and post synaptic mitral cells, & evoked lateral IPSPs in the post-synaptic cells, by stimulating nearby mitral cells. Showed activity-dependent lateral inhibition across dendrodendritic reciprocal synapses where lateral inhibition between two neighbouring mitral cells DEPENDS ON ACTIVITY OF CELL BEING INHIBITED
L: in vitro mouse olfactory bulb slices, therefore, it is possible that the process may behave differently in vivo.
3): Computational model of activity-dependent lateral inhibition, based on these electrophysiological findings, where connectivity was ‘all-to-all’, with no spatial structure. Performed contrast enhancement of input patterns, even when the spatial patterning of the input patterns were completely randomised.
Supports the idea that MOB does not act as a spatial topographic map, but acts as a PATTERN SEPARATOR, which uses contrast enhancement to DECORRELATE similar input patterns.
Whitesell et al (2013)
T: patch clamp recordings, sectioned external plexiform layer (where the dendrodendritic synapses between mitral and granule cells are located)
R: Interglomerular lateral inhibition occurred after sectioning EPL - contradicts the idea that granule cells are responsible for lateral inhibition.
Authors argued for functional separation of GABAergic cells in the MOB:
1) GABAergic short axons cells in glomerular layer: mediate inhibition of mitral cells between glomeruli,
2) GABAergic granule cells that project to EPL, which are responsible for synchronising glomeruli.
L: in vitro rat slices
Niessing + Freidrich (2010)
T: Calcium sensitive dyes - image MC activity, in response to different AA odorants (diff concs. Deconvolution matched calcium activity with the underlying AP dynamics, and principle component analysis showed that temporal patterns of activity for different odorants are highly correlated between similar concentrations of an odorant.
R: MIXTURES of SIMILAR odorants represented by patterns that correlated with an INDIVIDUAL.
Mixtures of DISSIMILAR odorants produced patterns of activity that were entirely SEPARATE to those that represented the individual components.
illustrates the role of the MOB as a pattern SEPARATOR, where synthetic patterns are generated to represent odours, rather than representing the proportions of individual components.
L: zebra fish slices, may not translate to activity patterns in mammalian MOBs.
Schusterman et al (2011)
T: studied AWAKE mice (most sniff work on anesthetsied rats) electrophysiological recordings from electrodes implanted in DORSAL & VENTRAL MC layers.
R: INCREASE in MC activity that was more closely aligned with the ONSET OF INHALATION, as opposed to the onset of stimulus presentation. PEAK responses of INDIVIDUAL MCs occurred throughout the course of the sniff cycle, and that ANALYSIS of PEAK firing within the sniff cycle may be used by a COMPUTER to potentially DISCRIMINATE different odours. (highlights how temporal representation of information MOB, in relation to sniff cycle, important in odour discrimination in mammals)
Smear et al (2011)
T: OPTOGENETICs, AWAKE MICE, more precisely characterise the timing of MC input in mice. Inserted channel rhodopsin gene into mature OSNs, and used light stimuli to depolarise the OSNs whilst measuring the sniff cycle.
Go/no-go behavioural task trained to DISCRIMINATE between LIGHT STIMULI delivered at 2 diff points in the SNIFF CYCLE
R: ACTIVELY USE temporal coding of information in the MOB to DISCRIMINATE stimuli.
ELECTROPHYS recordings showed that when light stimuli were delivered at different time points in the sniff cycle, INDIVIDUAL MCs responded to these stimuli with varying AMPLITUDE and varying SPEEDS of activation. illustrates how the intensity and timing of activity in the MOB in relation to the sniff cycle can be used to encode odourant stimuli.
Verhagen et al (2007)
Calcium imaging MOB: AWAKE, head-fixed mice. During HF sniffing: glom. activity LOWER than predicted (suggesting that adaptation occurring). Repeat stimulus presentation = LF sniffing with expected glom. activity.
When ethyl butyrate presented on butanone bckgrnd - if EB was novel, mice showed HF-sniffing + mitral cell activity that reflected the DIFFERENCE between the activity patterns for the individual odourants. If EB was familiar, LF-sniffing occurred, and glomerular activity reflected a SUMMATION of the activity patterns for each component.
R: HF-sniffing is important for adapting out bckgrnd odours, so novel odours can be clearly discriminated from bkgrnd odourants.
However, mechanism by which SNIFF FREQUENCY-DEPENDENT ATTENUATION of glomerular input occurs still somewhat unclear. Primary mechanism: receptor adaptation? higher sniff frequencies allow less time for OSNs to recover from adaptation between each inhalation.
Glomerular activity became attenuated within ~2s during HF-sniffing - supported by Reisert (2001): OR currents decayed to ~20% of their original value within ~2s of first presenting odourant. Supports idea that HF sniffing enhances adaptation at level of OR by preventing recovery from adaptation.
Stettler + Axel (2009)
Over 100 mice: calcium imaging: pyramidal cells. Responses to 16 individual odourants.
R: No spatial patterning across the anterior and posterior PC, not even at coarse level, (like coarse clustering seen in MOB)
Large number of cells responded to octanal or a-pinine - fewer number of cells became active in response to a mixture of both the odourants.
R: Piriform cortex does not respond to mixtures in additive manner - generates new patterns of activity in response to mixtures.
Davidson & Ehlers (2011)
Mice: Glutamate uncaging to optically activate dorsal MOB, recorded mitral cells + pyramidal cells (anterior piriform cortex).
Optically activating single glomerulus not sufficient to activate associated pyramidal cell, but CO-ACTIVATION of 3-4 GLUTAMATE UNCAGING SITES (= ~6-10 GLOMERULI) = sufficient to generate pyramidal cell firing.
Each pyramidal cell responded to different patterns of glomerular activity (where patterns were generated by 16 active glomeruli), which suggests that the anterior PC acts as CO-INCIDENCE DETECTOR, and detects slightly different patterns of glomerular input + therefore different patterns of OR activation.
Chapuis and Wilson (2012)
relevant to role of piriform cortex
Awake rats, forced choice task, then anaesthetised and electrophysiology
Learn to discriminate between an 10c and 10cR1, less able to discriminate between 10c and 10c-1. Patterns of activity in MOB were very different for 10c and 10c-1, but anterior piriform cortex produced similar activity patterns for them.
Highlights role of aPCX as odour object recogniser, as can generate complete pattern of activity from an incomplete pattern of input, in order to generalise similar odourants.
Chapuis + Wilson (2010)
relevant to role of learning
Forced choice task, rats, then anaesthetised and electrophysiology
Trained to discriminate between 10c and either 10c-1 / 10R1. Initially, 10c-1 more challenging than 10R1 (because anterior PC usually generates similar patterns for 10 and 10c-1).
After 8 days of training: anterior piriform cortex showed DECORRELATION of activity patterns for 10c and 10c-1.
When rats retrained to treat odours as the same: increase in correlation of activity patterns for the two different odourants.
Highlights how the process of generalising similar odours can be CHANGED by LEARNING, by directly altering the patterns of activity in the piriform cortex.
Note: recordings were performed under anaesthesia. As anaesthesia shown to change activity patterns in MOB (Kato et al), to further confirm these findings, a similar experiment with a method of recording pyramidal cell activity in awake animals would be desirable.
Haddad et al (2013)
Mice - Optogenetics, OSNs inserted with channel rhodopsin- optically stimulate 2 separate areas of MOB.
Firing rate of some pyr. cells unaffected by timing of the 2 stimuli. Some pyr. cells exhibit SYMMETRICAL TEMPORAL RESPONSE PROFILES (pry. cell firing rate dependent on synchronicity of the 2 MCs stimulated). Some have ASYMMETRICAL temporal response profiles (order in which the 2 MCs are activated determines firing rate of the pyramidal cell).
R: Pyramidal cells in piriform cortex are SENSITIVE to the relative TIMINGS of glomerular activation, within the order of 10s of milliseconds. This is similar to the timing differences that altered the behaviour of mice in Smear et al’s (2011) study.
L: contrasts with Davison + Ehler’s as in this study single stimulations of one glomerulus could activate the pyramidal cells BUT authors admit using larger spots of light so prob activating multiple glomeruli
Wilson (2001)
Prolonged stimulation with odourant causes reduction in response in both the MOB and piriform cortex
After 10-20s, piriform firing rate reduced to ~25% of its initial rate, whereas MOB reduced to ~75% of its initial firing rate. Therefore, HABITUATION in the piriform cortex is much more PROFOUND than adaptation in MOB (partly explained by mGlu2 on MC axon terminals - Glu release causes auto-inhibition - reduces post-synaptic activation of pyramidal cells, even when MC firing maintained)
MOB + piriform cortex also differ in the SELECTIVITY of their adaptation responses. CROSS-adaptation occurs readily in the MOB, (adaptation to one odourant = reduced response to odorants that activate same OR).
Very little occurs in piriform- supports idea that MOB recognising individual odourant features (similar structures) because MC will adapt to all of the odourants it responds to, whereas the PC is recognising odour objects.
Reisert (2005)
RT-PCR: mouse OSNs express NKCC1, but not NKCC2
Electrophysiology: KO mice for NKCC1 did not show the outward chloride current that is seen in WT mice.
Suggests that the NKCC1 channel is primarily responsible for generating the electrochemical gradient that is required for the outward chloride current.
note - reisert 2001 showed that there is 80% decay of current after 2 seconds of presenting odourant - different study, supports HF sniffing/adaptation role
Kim et al (2012)
- in terms of transduction
M: Recordings from in vitro mouse slices of VSNs, in response to urine. Also used KO GIRK/SK3 and GIRK/SK3 inhibitors.
In vitro slices: Potassium efflux attenuated TRPC2-mediated / CACC-mediated depolarisation
Extracellular recordings from intact preparations: both channels enhance urine-induced depolarisation in VSNs.
Ion-sensitive electrode in VNO mucus: ↑[potassium]: ∴ in vivo, SK3 / GIRK activation = depolarising, inward current
Kim et al (2012)
- in terms of behaviour..
SK3/GIRK1 KO/inhibitors: channels enhance depolarisation of VSNs in response to urine. Innate behaviours = resident-intruder aggression assays + mating assays.
SK3, GIRK1 and TRPC2 KO = impaired aggressive + sexual behaviour.
TRPC2 KO: MORE REDUCTION in AGGRESSIVE behaviour whereas the SEXUAL behaviour was somewhat LESS IMPAIRED (than SK3/GIRK1 KO)
L: Gene KOs used in this experiment were not restricted the VNO. Therefore, it is possible that gene silencing in other areas of the CNS may have contributed to the behavioural deficits that were measured.
“Alternatively, it is possible that distinct populations of neurons reside in the VNO and that the relative contributions of TPRC2, SK3 and GIRK1 differ in different cells.” then these neurons activate different behaviours.
Nara et al (2011)
Calcium imaging of dissociated OSNs (female mice) - 3000 OSNs, 13 mixtures.
81 OSNs responsed to 36 different sets of aldehydes
Some odourant mixtures activated 3-5x more OSNs than others, and some individual odourants activated 5-10x more OSNs (particularly citrus/fruity).
Most OSNs narrowly tuned (responded to similar structures) but some broadly tuned.
Most odour codes were shown to be unique and combinatorial i.e. individual OR responses are ambiguous but if examine whole population of receptors – unique activation pattern.
However: dissociated OSNs removes sustentacular and microvillar cells (supporting cells), which may influence the firing rates of OSNs - although less relevant to ligand responses.
Recordings from intact preparations result in a 100% response rate to ligands, lower response rate to dissociated neurons - also kept in different solutions.
Dibattista (2012)
Whole cell patch clamp isolated VSNs
In response to calcium uncaging, a large inward current is seen when the cell is held at -50mV, which was partially inhibited by NFA.
Furthermore, if chloride was removed from the extracellular solution and replaced with other membrane impermeable anions, the reversal potential of this current was altered.
Strong evidence that the calcium activated current is a chloride current
Isogai et al (2011)
Egr1 expression
48% of V1R-expressing VSNs = ‘promiscuous’ >1 of: male, female, mammalian non-predator, mammalian predator, reptile, avian predator.
9% of V2Rs = promiscuous
Clades of V2Rs evolved to detect urinary proteins of specific mammalian predators, other clades for non-mammalian predators.
Sulphated steroids: (steroid hormones sulphated by liver then excreted): mixture activated V1Ref and V1Rjk clades - a specific oestrogen, androgen and glucocorticoid were found to activate specific receptors within these clades.
