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GABAergic periglomerular (PG) cells in the olfactory bulb are proposed to mediate an intraglomerular “high-pass” filter through inhibition targeted onto a glomerulus. With this mechanism, weak stimuli (e.g., an odor with a low affinity for an odorant receptor) mainly produce PG cell-driven inhibition but strong stimuli generate enough excitation to overcome inhibition. PG cells may be particularly susceptible to being activated by weak stimuli due to their intrinsically small size and high input resistance. Here, we used dual-cell patch-clamp recordings and imaging methods in bulb slices obtained from wild-type and transgenic rats with labeled GABAergic cells to test a number of predictions of the high-pass filtering model. We first directly compared the responsiveness of PG cells with that of external tufted cells (eTCs), which are a class of excitatory interneurons that reside in a parallel but opposing position in the glomerular circuitry. PG cells were in fact found to be no more responsive than eTCs at low levels of sensory neuron activity. While PG cells required smaller currents to be excited, this advantage was offset by the fact that a given level of sensory neuron activity produced much smaller synaptic currents. We did however identify other factors that shaped the excitation/inhibition balance in a manner that would support a high-pass filter, including glial glutamate transporters and presynaptic metabotropic glutamate receptors. We conclude that a single glomerulus may act as a high-pass filter to enhance the contrast between different olfactory stimuli through mechanisms that are largely independent cell intrinsic properties.Key Points SummaryGABAergic periglomerular (PG) cells in the olfactory bulb are proposed to mediate a “high-pass” filter at a single glomerulus that selectively blocks weak stimulus signals. Their efficacy may reflect their intrinsically small size and high input resistance, which allows them to be easily excited.We found that PG cells were in fact no more likely to be activated by weak stimuli than excitatory neurons. PG cells spiked more readily in response to a fixed current input, but this advantage for excitability was offset by small synaptic currents.Glomeruli nevertheless display an excitation/inhibition balance that can support a high pass filter, becoming much more favorable with increasing stimulus strength. Factors shaping the filter include glial glutamate transporters and presynaptic metabotropic glutamate receptors.We conclude that a single glomerulus may act as a high-pass filter to enhance stimulus contrast through mechanisms that are largely independent of cell-intrinsic properties.

The local circuitry within olfactory bulb glomeruli filters, transforms, and facilitates information transfer from olfactory sensory neurons to bulb output neurons. Two key elements of this circuit are glutamatergic tufted cells (TCs) and GABAergic periglomerular (PG) cells, both of which actively shape mitral cell activity and bulb output. A subtype of TCs, the external tufted cells (eTCs), can synaptically excite PG cells, but there are unresolved questions about other aspects of the glomerular connections, including the extent of connectivity between eTCs and the precise nature of reciprocal interactions between eTCs and PG cells. We combined patch-clamp recordings in OB slices and optophysiological tools to investigate local functional connections within glomeruli. When TCs were optically suppressed, we found a large decrease in excitatory post-synaptic currents (EPSCs) in “uniglomerular” PG cells that extend dendrites to one glomerulus, indicating that TC activation was required for most excitation of these PG cells. However, TC suppression had no effect on EPSCs in eTCs, arguing that TCs make few, if any, direct excitatory synaptic connections onto eTCs. The absence of synaptic connections between eTCs was also supported by recordings in eTC pairs. Lastly, we show using similar optical suppression methods that PG cells that express GAD65, mainly uniglomerular PG cells, provide strong inhibition onto eTCs. Our results indicate that the local network of TCs form potent reciprocal synaptic connections with GAD65-expressing uniglomerular PG cells but not other TCs. This configuration favors local inhibition over recurrent excitation within a glomerulus, limiting information transfer to downstream cortical regions.

The local circuitry within olfactory bulb glomeruli filters, transforms, and facilitates information transfer from olfactory sensory neurons to bulb output neurons. Two key elements of this circuit are glutamatergic tufted cells (TCs) and GABAergic periglomerular (PG) cells, both of which actively shape mitral cell activity and bulb output. A subtype of TCs, the external tufted cells (eTCs), can synaptically excite PG cells, but there are unresolved questions about other aspects of the glomerular connections, including the extent of connectivity between eTCs and the precise nature of reciprocal interactions between TCs and PG cells. We combined patch-clamp recordings in OB slices and optophysiological tools to investigate local functional connections within glomeruli of mice and rats. When TCs that express cholecystokinin (CCK) were optically suppressed, excitatory post-synaptic currents (EPSCs) in “uniglomerular” PG cells that extend dendrites to one glomerulus were decreased, consistent with TC activation being required for most excitation of these PG cells. However, TC suppression had no effect on EPSCs in eTCs, arguing that TCs make few, if any, direct glutamatergic synaptic connections onto eTCs. The absence of synaptic connections between eTCs was also supported by recordings in eTC pairs. Lastly, we show using similar optical suppression methods that GAD65-expressing PG cells, mainly uniglomerular cells, provide strong inhibition onto eTCs. Our results imply that the local network of CCK-expressing TCs form potent reciprocal chemical synaptic connections with GAD65-expressing uniglomerular PG cells but not eTCs. This configuration favors local inhibition over recurrent excitation within a glomerulus, limiting its output. Significance Statement The brain circuits involved with the initial processing of sensory information are comprised of networks of excitatory and inhibitory neurons. The strength of local connections can impact the weighting of excitation versus inhibition and determine whether sensory signals are passed on to higher-order brain structures. We investigated the connectivity between excitatory and inhibitory neurons in discrete glomerular structures of the rodent olfactory bulb. We found that excitatory neurons make potent chemical synaptic connections with a class of inhibitory cells with dendrites confined to one glomerulus. However, excitatory cells do not form chemical synaptic connections with each other. This circuit configuration suggests that sensory input is generally biased toward driving more local inhibition than recurrent excitation, thus suppressing bulbar output.

A common feature of the primary processing structures of sensory systems is the presence of parallel output “channels” that convey different information about a stimulus. In the mammalian olfactory bulb, this is reflected in the mitral cells (MCs) and tufted cells (TCs) that have differing sensitivities to odors, with TCs being more sensitive than MCs. In this study, we examined potential mechanisms underlying the different responses of MCs vs. TCs. For TCs, we focused on superficial TCs (sTCs), which are a population of output TCs that reside in the superficial-most portion of the external plexiform layer, along with external tufted cells (eTCs), which are glutamatergic interneurons in the glomerular layer. Using whole-cell patch-clamp recordings in mouse bulb slices, we first measured excitatory currents in MCs, sTCs, and eTCs following olfactory sensory neuron (OSN) stimulation, separating the responses into a fast, monosynaptic component reflecting direct inputs from OSNs and a prolonged component partially reflecting eTC-mediated feedforward excitation. Responses were measured to a wide range of OSN stimulation intensities, simulating the different levels of OSN activity that would be expected to be produced by varying odor concentrationsin vivo. Over a range of stimulation intensities, we found that the monosynaptic current varied significantly between the cell types, in the order of eTC > sTC > MC. The prolonged component was smaller in sTCs vs. both MCs and eTCs. sTCs also had much higher whole-cell input resistances than MCs, reflecting their smaller size and greater membrane resistivity. To evaluate how these different electrophysiological aspects contributed to spiking of the output MCs and sTCs, we used computational modeling. By exchanging the different cell properties in our modeled MCs and sTCs, we could evaluate each property's contribution to spiking differences between these cell types. This analysis suggested that the higher sensitivity of spiking in sTCs vs. MCs reflected both their larger monosynaptic OSN signal as well as their higher input resistance, while their smaller prolonged currents had a modest opposing effect. Taken together, our results indicate that both synaptic and intrinsic cellular features contribute to the production of parallel output channels in the olfactory bulb.

The mammalian olfactory bulb has presented a challenging system for understanding information processing, in part because the bulb largely lacks the topographical ordering of neurons that promotes processes such as lateral inhibition. Here we have used dual and triple-cell recordings in rodent bulb slices combined with ultrastructural methods to provide the first experimental evidence for a processing mechanism circumventing this problem that operates at the level of single glomeruli, the bulb’s odorant receptor-specific modules. A key feature is non-traditional, extrasynaptic glutamatergic signaling derived from excitatory interneurons and what it means for the local balance between excitation (E) and inhibition (I). We found that the distinct dynamic properties of extrasynaptic excitation versus synaptic inhibition create a thresholding effect whereby only strong stimuli produce a favorable E/I balance enabling an output. This single-glomerulus threshold could have a number of important functions during natural odor responses, for example enhancing stimulus tuning.

Recent studies have provided evidence that corticofugal feedback (CFF) from the olfactory cortex to the olfactory bulb (OB) can significantly impact the state of excitation of output mitral cells (MCs) and tufted cells (TCs) and also modulate neural synchrony. Interpreting these effects however has been complicated by the large number of cell targets of CFF axons in the bulb. Within the granule cell layer (GCL) alone, CFF axons target both GABAergic granule cells (GCs) as well as GABAergic deep short-axon cells (dSACs) that inhibit GCs. Because GCs are a major source of inhibition of MCs/TCs, CFF could be inhibitory to MCs (by exciting GCs) or disinhibitory (by exciting dSACs that inhibit GCs). In this study, we used patch-clamp recordings combined with optogenetic and electrical stimulation methods to investigate the role of presynaptic cannabinoid receptors in regulating CFF pathways, which could alter the weights of inhibition and disinhibition. Recording first from dSACs, we found that the cannabinoid receptor (CB-R) agonist WIN-55212.2 (WIN) reduced excitatory post-synaptic currents (CFF-EPSCs) driven by stimulation of CFF axons. The effects were reversed by the Type 1 CB-R (CB1-R)-specific antagonist SR-141716A. Furthermore, prolonged 5-s depolarizations applied to postsynaptic dSACs effectively reduced CFF-EPSCs in a CB1-R-dependent fashion, providing evidence for depolarization-induced suppression of excitation (DSE) at CFF-to-dSAC synapses. Further analysis indicated that CB1-Rs mediate widespread suppressive effects on synaptic transmission, occurring at CFF synapses onto different dSAC subtypes and CFF synapses onto GCs. Feedforward excitation of dSACs, mediated by MCs/TCs, however, was not impacted by CB1-Rs. In recordings from MCs, performed to examine the net effect of CB1-R activation on GC-to-MC transmission, we found that WIN could both increase and decrease disynaptic inhibition evoked by CFF axon stimulation. The exact effect depended on the size of the inhibitory response, reflecting the local balance of dSAC vs. GC activation. Our results taken together indicate that CB1-Rs can bidirectionally alter the weighting of inhibition and disinhibition of MCs through their effects on CFF pathways.

Glutamatergic transmission in the brain typically occurs at well-defined synaptic connections, but increasing evidence indicates that neural excitation can also occur through activation of “extrasynaptic” glutamate receptors. Here, we investigated the underlying mechanisms and functional properties of extrasynaptic signals that are part of a feedforward path of information flow in the olfactory bulb. This pathway involves glutamatergic interneurons, external tufted cells (eTCs), that are excited by olfactory sensory neurons (OSNs) and in turn excite output mitral cells (MCs) extrasynaptically. Using pair-cell and triple-cell recordings in rat bulb slices (of either sex), combined with ultrastructural approaches, we first present evidence that eTC-to-MC signaling results from “spillover” of glutamate released at eTC synapses onto GABAergic periglomerular (PG) cells in glomeruli. Thus, feedforward excitation is an indirect result of and must cooccur with activation of inhibitory circuitry. Next, to examine the dynamics of the competing signals, we assayed the relationship between the number of spikes in eTCs and excitation of MCs or PG cells in pair-cell recordings. This showed that extrasynaptic excitation in MCs is very weak due to single spikes but rises sharply and supralinearly with increasing spikes, differing from sublinear behavior for synaptic excitation of PG cells. Similar dynamics leading to a preference for extrasynaptic excitation were also observed during recordings of extrasynaptic and inhibitory currents in response to OSN input of increasing magnitude. The observed alterations in the balance between extrasynaptic excitation and inhibition in glomeruli with stimulus strength could underlie an intraglomerular mechanism for olfactory contrast enhancement.

The fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane requires two classes of molecules-SNAP receptor (SNARE) and Sec1/Munc18 (SM) protein. Reconstitution studies suggest that the SM protein Munc18-1 promotes the zippering of trans-SNARE complexes and accelerates the kinetics of SNARE-dependent membrane fusion. However, the physiological role of this trans-SNARE-regulating function in synaptic exocytosis remains to be established. Here we first demonstrate that two mutations in the vesicle-anchored v-SNARE selectively impair the ability of Munc18-1 to promote trans-SNARE zippering, whereas other known Munc18-1/SNARE-binding modes are unaffected. In cultured neurons, these v-SNARE mutations strongly inhibit spontaneous as well as evoked neurotransmitter release, providing genetic evidence for the trans-SNARE-regulating function of Munc18-1 in synaptic exocytosis. Finally, we show that the trans-SNARE-regulating function of Munc18-1 is compromised by a mutation associated with Ohtahara Syndrome, a severe form of epilepsy.

Increasing evidence indicates that the neural circuitry within glomeruli of the olfactory bulb plays a major role in affecting information flow between olfactory sensory neurons (OSNs) and output mitral cells (MCs). Glutamatergic external tufted (ET) cells, located at glomeruli, can act as intermediary cells in excitation between OSNs and MCs, whereas activation of MCs by OSNs is, in turn, suppressed by inhibitory synapses onto ET cells. In this study, we used patch-clamp recordings in rat olfactory bulb slices to examine the function of metabotropic glutamate receptors (mGluRs) in altering these glomerular signaling mechanisms. We found that activation of group II mGluRs profoundly reduced inhibition onto ET cells evoked by OSN stimulation. The mGluRs that mediated disinhibition were located on presynaptic GABAergic periglomerular cells and appeared to be activated by glutamate transients derived from dendrites in glomeruli. In terms of glomerular output, the mGluR-mediated reduction in GABA release led to a robust increase in the number of action potentials evoked by OSN stimulation in both ET cells and MCs. Importantly, however, the enhanced excitation was specific to when a glomerulus was strongly activated by OSN inputs. By being selective for strong vs. weak glomerular activation, mGluR-mediated disinhibition provides a mechanism to enhance the contrast in odor signals that activate OSN inputs into a single glomerulus at varying intensities.

Mitral and tufted cells constitute the primary output cells of the olfactory bulb. While tufted cells are often considered as “displaced” mitral cells, their actual role in olfactory bulb processing has been little explored. We examined dendrodendritic inhibition between tufted cells and interneurons using whole cell voltage-clamp recording. Dendrodendritic inhibitory postsynaptic currents (IPSCs) generated by depolarizing voltage steps in tufted cells were completely blocked by the N-methyl-d-aspartate (NMDA) receptor antagonistd,l-2amino-5-phosphonopentanoic acid (d,l-AP5), whereas the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 2-3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f] quinoxaline-7-sulfonamide (NBQX) had no effect. Tufted cells in the external plexiform layer (EPL) and in the periglomerular region (PGR) showed similar behavior. These results indicate that NMDA receptor–mediated excitation of interneurons drives inhibition of tufted cells at dendrodendritic synapses as it does in mitral cells. However, the spatial extent of lateral inhibition in tufted cells was much more limited than in mitral cells. We suggest that the sphere of influence of tufted cells, while qualitatively similar to mitral cells, is centered on only one or a few glomeruli.

At many central excitatory synapses, AMPA receptors relay the electrical signal, whereas activation of NMDA receptors is conditional and serves a modulatory function. We show here quite a different role for NMDA receptors at dendrodendritic synapses between mitral and granule cells in the rat olfactory bulb. In whole-cell patch-clamp recordings in bulb slices, stimulation of mitral cells elicited slowly decaying, GABAA receptor-mediated reciprocal IPSCs that reflected prolonged GABA release from granule cells. Although granule cells had a normal complement of AMPA and NMDA receptors, the IPSC was completely blocked by the NMDA receptor antagonist D,L-AP-5, suggesting that NMDA receptor activation is an absolute requirement for dendrodendritic inhibition. The AMPA receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX) had no effect on IPSCs in the absence of extracellular magnesium but modestly reduced IPSCs in 1 mM magnesium, indicating that the primary effect of the AMPA receptor-mediated depolarization was to facilitate the unblocking of NMDA receptors. Granule cell voltage recordings indicated that effective spike stimulation in granule cells depended on the slow NMDA receptor kinetics. Granule cells also showed a pronounced delay between synaptic stimulation and action potential generation, suggesting that their intrinsic membrane properties underlie the ineffectiveness of brief AMPA receptor-mediated EPSPs. NMDA receptors also seem to have a central role in dendrodendritic inhibition in vivo, because intraperitoneal dizocilpine maleate (MK-801) injection in young adult rats resulted in disinhibition of mitral cells as measured by the generation of c-fos mRNA. The unique dependence of dendrodendritic inhibition on slow EPSPs generated by NMDA receptors suggests that olfactory information processing depends on long-lasting reciprocal and lateral inhibition.

A functional kinetic model is developed to describe the activation gating process of the Shaker potassium channel. The modeling in this paper is constrained by measurements described in the preceding two papers, including macroscopic ionic and gating currents and single channel ionic currents. These data were obtained from the normally activating wild-type channel as well as a mutant channel V2, in which the leucine at position 382 has been mutated to a valine. Different classes of models that incorporate Shaker 9s symmetrical tetrameric structure are systematically examined. Many simple gating models are clearly inadequate, but a model that can account for all of the qualitative features of the data has the channel open after its four subunits undergo three transitions in sequence, and two final transitions that reflect the concerted action of the four subunits. In this model, which we call Scheme 3+2′, the channel can also close to several states that are not part of the activation path. Channel opening involves a large total charge movement (10.8 e 0 ), which is distributed among a large number of small steps each with rather small charge movements (between 0.6 and 1.05 e 0 ). The final two transitions are different from earlier steps by having slow backward rates. These steps confer a cooperative mechanism of channel opening at Shaker 9s activation voltages. In the context of Scheme 3+2′, significant effects of the V2 mutation are limited to the backward rates of the final two transitions, implying that L382 plays an important role in the conformational stability of the final two states.

Schoppa, N. E. and G. L. Westbrook. Modulation of mEPSCs in olfactory bulb mitral cells by metabotropic glutamate receptors. J. Neurophysiol. 78: 1468–1475, 1997. Olfactory bulb mitral cells express group I (mGluR1), group II (mGluR2), and group III (mGluR7 and mGluR8) metabotropic glutamate receptors. We examined the role of these mGluRs on excitatory synaptic transmission in cultured mitral cells with the use of whole cell patch-clamp recordings. The effects of group-selective mGluR agonists and antagonists were tested on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-receptor-mediated miniature excitatory postsynaptic currents (mEPSCs). (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylate (ACPD) or the group-I-selective agonist 3,5-dihydroxyphenylglycine evoked an inward current accompanied by a decrease in membrane conductance, consistent with the previously described closure of potassium channels by group I agonists. The increased cellular excitability was accompanied by an increase in mEPSC frequency in some cells. When calcium entry was blocked by cadmium, ACPD or the group-II-selective agonist 2-(2,3-dicarboxycyclopropyl)-glycine reduced the mEPSC frequency. l-2-amino-4-phosphonobutyric acid (l-AP4), a group-III-selective agonist, caused a similar decrease. The concentration-dependence ofl-AP4-mediated inhibition was most consistent with activation of mGluR8. We investigated two possible effector mechanisms for the group III presynaptic receptor. Bath application of forskolin or 3-isobutyl-1-methylxantine had no effect on mEPSC frequency. Increasing calcium influx by raising extracellular K+caused a large increase in the mEPSC frequency but did not enhance l-AP4-mediated inhibition. Thus inhibition of mEPSCs involves a mechanism downstream of calcium entry and appears to be independent of adenosine 3′,5′-cyclic monophosphate. Our results indicate that both group II and III receptors can inhibit glutamate release at mitral cell terminals. Although group II/III receptors had a similar effect on mEPSCs, differences in location on nerve terminals and in glutamate sensitivity suggest that each mGluR may have discrete actions on mitral cell activity.

Activation of glutamate receptors generally increases neuronal excitability. However, Isaacson and Murphy show in olfactory bulb granule cells that NMDA receptor-mediated calcium influx couples to large conductance (BK) calcium-activated potassium channels. The resulting inhibition is long lasting, which may be critical to the operation of the dynamic circuitry of the bulb.

Background It is generally thought that granule-mitral cell synapses in the olfactory bulb function to inhibit mitral cell firing, and that this inhibition can underlie such functionally important phenomena as lateral inhibition and synchronization [1]. Recent electrophysiology [2] and imaging [3] studies indicate that the location of the dendrodendritic synapse must be close to the soma to impact the mitral cell's firing.

The high sensitivity of voltage-gated ion channels to changes in membrane potential implies that the process of channel opening is accompanied by large charge movements. Previous estimates of the total charge displacement, q, have been deduced from the voltage dependence of channel activation and have ranged from 4 to 8 elementary charges (e0). A more direct measurement of q in Drosophila melanogaster Shaker 29-4 potassium channels yields a q value of 12.3 e0. A similar q value is obtained from mutated Shaker channels having reduced voltage sensitivity. These results can be explained by a model for channel activation in which the equilibria of voltage-dependent steps are altered in the mutant channels.

Chloride ions (Cl−) are concentrated in airway epithelial cells and subsequently secreted into the tracheal lumen by downhill flux through apical Cl− channels. We have studied Cl− currents in cultured canine tracheal cells using the whole-cell voltage-clamp technique. Ultrastructural techniques demonstrated that the cells used in the electrophysiological experiments possessed apical membrane specializations known to be present in the intact, transporting cell type. Cultured cells 2–6 days old were characterized by an input resistance of 3.4±0.8 GΩ (n=11) and a capacitance of 63.8±10.8 pF (n=26). A comparison of 3 and 4 day-old cells with 5 and 6 day-old cells showed that the input resistance decreased almost 50%, and the cell capacitance and the inward and outward currents increased concomitantly approximately 200%. Cultured cells 3–4 days old held at −40 mV produced currents of 196±22 pA at 50 mV and −246±27 pA at −90 mV (n=212) with pipette and bath solutions containing primarily 140 KCl and 140 NaCl, respectively. The chloride channel blocker diphenylamine-2-carboxylate (DPC, 100 μm) suppressed whole-cell currents by 76.8% at 60 mV; however, currents were unaffected by the stilbenes SITS (1mm) and DNDS (1–30 μm). Replacement of K+ with Cs+ in the pipette solution did not affect the outward current, the current reversal potential, or the input resistance of the cells, indicating that the current was not significantly K+ dependent when the intrapipette solution was buffered to a Ca2+ concentration of 20nm. The Cl−/Na+ permeability ratio was estimated to be greater than 11 as calculated from reversal potential measurements in the presence of an internal to external NaCl concentration ratio of 1∶2. Current equilibrium permeabilities, relative to Cl− were: I− (2.9)≫NO 3 − (1.1)≥Br− (1.1)≥Cl− (1.0)≥F− (0.93)≫MeSO 4 − (0.19)≥gluconate (0.18)≥aspartate (0.14). Depolarizations to potentials greater than 20 mV elicited a time-dependent component in the outward current in 71% of the cells studied. Currents inactivated with a double exponential time course at the most depolarized voltages. Recovery from inactivation was fast, holding potential-dependent, and followed a double exponential time course. Current amplitude was increased via a cAMP-dependent pathway as has been demonstrated for single Cl-selective channels in cell-attached patches from cultured canine and human tracheal epithelial cells. Forskolin, an activator of adenylate cyclase, produced a 260% increase in the outward current at +50 mV. In summary, cultured canine tracheal cells have a single resting conductance that is Cl− selective, voltage-dependent, and modulated by a cAMP-dependent mechanism. This preparation appears to be appropriate for analysis of cellular modulation of airway Cl− channels and Cl− secretion.