Glomerular input patterns in the mouse olfactory bulb evoked by retronasal odor stimuli.

BACKGROUND: Odorant stimuli can access the olfactory epithelium either orthonasally, by inhalation through the external nares, or retronasally by reverse airflow from the oral cavity. There is evidence that odors perceived through these two routes can differ in quality and intensity. We were curious whether such differences might potentially have a neural basis in the peripheral mechanisms of odor coding. To explore this possibility, we compared olfactory receptor input to glomeruli in the dorsal olfactory bulb evoked by orthonasal and retronasal stimulation. Maps of glomerular response were acquired by optical imaging of transgenic mice expressing synaptopHluorin (spH), a fluorescent reporter of presynaptic activity, in olfactory nerve terminals. RESULTS: We found that retronasally delivered odorants were able to activate inputs to multiple glomeruli in the dorsal olfactory bulb. The retronasal responses were smaller than orthonasal responses to odorants delivered at comparable concentrations and flow rates, and they displayed higher thresholds and right-shifted dose-response curves. Glomerular maps of orthonasal and retronasal responses were usually well overlapped, with fewer total numbers of glomeruli in retronasal maps. However, maps at threshold could be quite distinct with little overlap. Retronasal responses were also more narrowly tuned to homologous series of aliphatic odorants of varying carbon chain length, with longer chain, more hydrophobic compounds evoking little or no response at comparable vapor levels. CONCLUSIONS: Several features of retronasal olfaction are possibly referable to the observed properties of glomerular odorant responses. The finding that retronasal responses are weaker and sparser than orthonasal responses is consistent with psychophysical studies showing lower sensitivity for retronasal olfaction in threshold and suprathreshold tests. The similarity and overlap of orthonasal and retronasal odor maps at suprathreshold concentrations agrees with generally similar perceived qualities for the same odorant stimuli administered by the two routes. However, divergence of maps near threshold is a potential factor in perceptual differences between orthonasal and retronasal olfaction. Narrower tuning of retronasal responses suggests that they may be less influenced by chromatographic adsorption effects.

Visualization of nitric oxide production in the mouse main olfactory bulb by a cell-trappable copper(II) fluorescent probe.

We report the visualization of NO production using fluorescence in tissue slices of the mouse main olfactory bulb. This discovery was possible through the use of a novel, cell-trappable probe for intracellular nitric oxide detection based on a symmetric scaffold with two NO-reactive sites. Ester moieties installed onto the fluorescent probe are cleaved by intracellular esterases to yield the corresponding negatively charged, cell-impermeable acids. The trappable probe Cu(2)(FL2E) and the membrane-impermeable acid derivative Cu(2)(FL2A) respond rapidly and selectively to NO in buffers that simulate biological conditions, and application of Cu(2)(FL2E) leads to detection of endogenously produced NO in cell cultures and olfactory bulb brain slices.

Slice blotting.

Slice blotting is a technique for recording the spatial distribution of extracellular signaling molecules released from thin slices of living tissue. Slices are positioned on the surface of a membrane that can trap secreted substances diffusing from the tissue. The pattern of membrane-bound antigens is subsequently visualized by immunoblotting.

Electrical signaling in the olfactory bulb.

The olfactory bulb employs lateral and feedback inhibitory pathways to distribute odor information across parallel assemblies of mitral and granule cells. The pathways involve dendritic action potentials that can interact with a variety of voltage-dependent conductances and synaptic transmission to produce complex and dynamic patterns of activity. Electrical coupling also helps to ensure proper coordination and synchronization of these patterns. These mechanisms provide numerous options for dynamic modulation and control of signaling in the olfactory bulb.

Action potential backpropagation and multiglomerular signaling in the rat vomeronasal system.

In the accessory olfactory bulb (AOB), sensory neurons expressing a given vomeronasal receptor (VR) gene send divergent projections to many glomeruli, and second-order neurons (mitral cells) link to multiple glomeruli via branched primary dendrites. We used calcium imaging and paired somadendritic patch-clamp recording to track backpropagated action potentials (APs) in rat AOB primary dendrites. In cells loaded with 150 microm Calcium Orange, somatic spikes elicited fluorescence transients over the entire primary dendritic tree, and the relative fluorescence increment DeltaF/F(0) increased along all branches from soma to glomeruli. Backpropagation was reliant on Na+ channels: in 1 microm TTX, somatic AP commands evoked dendritic Ca2+ transients that declined steeply with distance. In paired soma- dendritic whole-cell recordings, backpropagated APs were unattenuated up to approximately 200 microm from the soma, whereas subthreshold voltage transients decayed markedly. Computational modeling indicated that the large distal Ca2+ transients are consistent with active, not passive, backpropagation. Genetic tracing in the AOB has suggested homotypic connectivity with individual mitral cell dendritic arbors projecting only to glomeruli targeted by sensory neurons expressing the same VR gene. Non-decremental, non-dichotomous backpropagation in AOB primary dendrites ensures fast, reliable communication between mitral cells and their homotypic glomeruli, binding them into functional modules in accordance with their VR-coded inputs.

Slice blotting.

Slice blotting is a technique for recording the spatial distribution of extracellular signaling molecules released from thin slices of living tissue. Slices are positioned on the surface of a membrane that can trap secreted substances diffusing from the tissue. The pattern of membrane-bound antigens is subsequently visualized by immunoblotting.

From molecule to mind: an integrative perspective on odor intensity.

A fundamental problem in systems neuroscience is mapping the physical properties of a stimulus to perceptual characteristics. In vision, wavelength translates into color; in audition, frequency translates into pitch. Although odorant concentration is a key feature of olfactory stimuli, we do not know how concentration is translated into perceived intensity by the olfactory system. A variety of neural responses at several levels of processing have been reported to vary with odorant concentration, suggesting specific coding models. However, it remains unclear which, if any, of these phenomena underlie the perception of odor intensity. Here, we provide an overview of current models at different stages of olfactory processing, and identify promising avenues for future research.

Neural coding of binary mixtures in a structurally related odorant pair.

The encoding of odorant mixtures by olfactory sensory neurons depends on molecular interactions at peripheral receptors. However, the pharmacological basis of these interactions is not well defined. Both competitive and noncompetitive mechanisms of receptor binding and activation, or suppression, could contribute to coding. We studied this by analyzing responses of olfactory bulb glomeruli evoked by a pair of structurally related odorants, eugenol (EG) and methyl isoeugenol (MIEG). Fluorescence imaging in synaptopHluorin (spH) mice revealed that EG and MIEG evoked highly overlapped glomerular inputs, increasing the likelihood of mixture interactions. Glomerular responses to binary mixtures of EG and MIEG mostly showed hypoadditive interactions at intermediate and high odorant concentrations, with a few near threshold responses showing hyperadditivity. Dose-response profiles were well fitted by a model of two odorants competitively binding and activating a shared receptor linked to a non-linear transduction cascade. We saw no evidence of non-competitive mechanisms.

Cholecystokinin: an excitatory modulator of mitral/tufted cells in the mouse olfactory bulb.

Cholecystokinin (CCK) is widely distributed in the brain as a sulfated octapeptide (CCK-8S). In the olfactory bulb, CCK-8S is concentrated in two laminae: an infraglomerular band in the external plexiform layer, and an inframitral band in the internal plexiform layer (IPL), corresponding to somata and terminals of superficial tufted cells with intrabulbar projections linking duplicate glomerular maps of olfactory receptors. The physiological role of CCK in this circuit is unknown. We made patch clamp recordings of CCK effects on mitral cell spike activity in mouse olfactory bulb slices, and applied immunohistochemistry to localize CCKB receptors. In cell-attached recordings, mitral cells responded to 300 nM-1 µM CCK-8S by spike excitation, suppression, or mixed excitation-suppression. Antagonists of GABAA and ionotropic glutamate receptors blocked suppression, but excitation persisted. Whole-cell recordings revealed that excitation was mediated by a slow inward current, and suppression by spike inactivation or inhibitory synaptic input. Similar responses were elicited by the CCKB receptor-selective agonist CCK-4 (1 µM). Excitation was less frequent but still occurred when CCKB receptors were blocked by LY225910, or disrupted in CCKB knockout mice, and was also observed in CCKA knockouts. CCKB receptor immunoreactivity was detected on mitral and superficial tufted cells, colocalized with Tbx21, and was absent from granule cells and the IPL. Our data indicate that CCK excites mitral cells postsynaptically, via both CCKA and CCKB receptors. We hypothesize that extrasynaptic CCK released from tufted cell terminals in the IPL may diffuse to and directly excite mitral cell bodies, creating a positive feedback loop that can amplify output from pairs of glomeruli receiving sensory inputs encoded by the same olfactory receptor. Dynamic plasticity of intrabulbar projections suggests that this could be an experience-dependent amplification mechanism for tuning and optimizing olfactory bulb signal processing in different odor environments.

Inhibition of backpropagating action potentials in mitral cell secondary dendrites.

The mammalian olfactory bulb is a geometrically organized signal-processing array that utilizes lateral inhibitory circuits to transform spatially patterned inputs. A major part of the lateral circuitry consists of extensively radiating secondary dendrites of mitral cells. These dendrites are bidirectional cables: they convey granule cell inhibitory input to the mitral soma, and they conduct backpropagating action potentials that trigger glutamate release at dendrodendritic synapses. This study examined how mitral cell firing is affected by inhibitory inputs at different distances along the secondary dendrite and what happens to backpropagating action potentials when they encounter inhibition. These are key questions for understanding the range and spatial dependence of lateral signaling between mitral cells. Backpropagating action potentials were monitored in vitro by simultaneous somatic and dendritic whole cell recording from individual mitral cells in rat olfactory bulb slices, and inhibition was applied focally to dendrites by laser flash photolysis of caged GABA (2.5-microm spot). Photolysis was calibrated to activate conductances similar in magnitude to GABA(A)-mediated inhibition from granule cell spines. Under somatic voltage-clamp with CsCl dialysis, uncaging GABA onto the soma, axon initial segment, primary and secondary dendrites evoked bicuculline-sensitive currents (up to -1.4 nA at -60 mV; reversal at approximatety 0 mV). The currents exhibited a patchy distribution along the axon and dendrites. In current-clamp recordings, repetitive firing driven by somatic current injection was blocked by uncaging GABA on the secondary dendrite approximately 140 microm from the soma, and the blocking distance decreased with increasing current. In the secondary dendrites, backpropagated action potentials were measured 93-152 microm from the soma, where they were attenuated by a factor of 0.75 +/- 0.07 (mean +/- SD) and slightly broadened (1.19 +/- 0.10), independent of activity (35-107 Hz). Uncaging GABA on the distal dendrite had little effect on somatic spikes but attenuated backpropagating action potentials by a factor of 0.68 +/- 0.15 (0.45-0.60 microJ flash with 1-mM caged GABA); attenuation was localized to a zone of width 16.3 +/- 4.2 microm around the point of GABA release. These results reveal the contrasting actions of inhibition at different locations along the dendrite: proximal inhibition blocks firing by shunting somatic current, whereas distal inhibition can impose spatial patterns of dendrodendritic transmission by locally attenuating backpropagating action potentials. The secondary dendrites are designed with a high safety factor for backpropagation, to facilitate reliable transmission of the outgoing spike-coded data stream, in parallel with the integration of inhibitory inputs.

Flash photolysis reveals a diversity of ionotropic glutamate receptors on the mitral cell somatodendritic membrane.

It is widely held that the soma and basal dendrites of olfactory bulb mitral cells receive exclusively inhibitory synaptic input from local interneurons. However, the mitral somatodendritic membrane exhibits immunoreactivity for a variety of glutamate receptors, and blocking GABA receptors unmasks mitral cell self-excitation. This excitation is proposed to be mediated either by diffuse spillover of the mitral cells' own released glutamate, or by punctate transmission from glutamate-releasing granule cells. This study examined the pharmacology and kinetics of glutamate sensitivity of mitral cells by flash photolysis of nitroindoline caged glutamates, which facilitate reliable activation of receptors in the synaptic cleft. Wide-field laser uncaging (3.5-ms flash) of approximately 0.5-1 mM glutamate onto the soma activated large currents with fast (3.4-ms rise, 7.5-ms decay) and slow (64-ms rise, >10-s decay) components. In 100 microM APV, slow currents were reduced to 53% of control (257-ms rise, 2-s decay), displayed outward rectification in 1.3 mM Mg2+, and blocked by 15 microM 5,7-dichlorokynurenate. Responses to less, similar 100 microM glutamate were fully antagonized by 100 microM APV, consistent with competitive inhibition at high-affinity NMDA receptors. An APV-resistant NMDA receptor was not observed, refuting the punctate transmission model. Fast currents were blocked by 10 microM NBQX, boosted 3.28-fold by 100 microM cyclothiazide, and resolved into AMPA (40%) and kainate (60%) receptor components by 100 microM SYM2206. The results suggest that self-excitation depends on AMPA, kainite, and conventional NMDA autoreceptors on the mitral cell.