Matching of feedback inhibition with excitation ensures fidelity of information flow in the anterior piriform cortex.

Odor-evoked responses in mitral cells of the olfactory bulb are characterized by prolonged patterns of action potential (spike) activity. If downstream neurons are to respond to each spike in these patterns, the duration of the excitatory response to one spike should be limited, enabling cells to respond to subsequent spikes. To test for such mechanisms, we performed patch-clamp recordings in slices of the mouse anterior piriform cortex. Mitral cell axons in the lateral olfactory tract (LOT) were stimulated electrically at different intensities and with various frequency patterns to mimic changing input conditions that the piriform cortex likely encounters in vivo. We found with cell-attached measurements that superficial pyramidal (SP) cells in layer 2 consistently responded to LOT stimulation across conditions with a limited number (1-2) of spikes per stimulus pulse. The key synaptic feature accounting for the limited spike number appeared to be somatic inhibition derived from layer 3 fast-spiking cells. This inhibition tracked the timing of the first spike in SP cells across conditions, which naturally limited the spike number to 1-2. These response features to LOT stimulation were, moreover, not unique to SP cells, also occurring in a population of fluorescently labeled interneurons in glutamic acid decarboxylase 65-eGFP mice. That these different cortical cells respond to incoming inputs with 1-2 spikes per stimulus may be especially critical for relaying bulbar information contained in synchronized oscillations at beta (15-30Hz) or gamma (30-80Hz) frequencies.

Inhibition acts globally to shape olfactory cortical tuning.

Lateral inhibition between near-neighbor neurons has long been thought to be important for narrowing the receptive fields of neurons in many sensory systems. A new study by Poo and Isaacson in this issue of Neuron examining olfactory processing finds that "global" inhibition within the primary olfactory cortex might accomplish a similar end.

A novel local circuit in the olfactory bulb involving an old short-axon cell.

Many local circuit interactions in the olfactory bulb involve atypical dendrodendritic synapses. In this issue of Neuron, Pressler and Strowbridge report a functional analysis of a class of short-axon interneurons in the bulb called Blanes cells. Blanes cells make GABAergic axonal contacts onto granule cells and may mediate a form of local feedforward disinhibition.

NMDA receptors turn to another channel for inhibition.

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.

Glomerulus-specific synchronization of mitral cells in the olfactory bulb.

Odor elicits a well-organized pattern of glomerular activation in the olfactory bulb. However, the mechanisms by which this spatial map is transformed into an odor code remain unclear. We examined this question in rat olfactory bulb slices in recordings from output mitral cells. Electrical stimulation of incoming afferents elicited slow ( approximately 2 Hz) oscillations that originated in glomeruli and were highly synchronized for mitral cells projecting to the same glomerulus. Cyclical depolarizations were generated by glutamate activation of dendritic autoreceptors, while the slow frequency was determined primarily by the duration of regenerative glutamate release. Patterned stimuli elicited stimulus-entrained oscillations that amplified weak and variable inputs. We suggest that these oscillations maintain the fidelity of the spatial map by ensuring that all mitral cells within a glomerulus-specific network respond to odor as a functional unit.

Tufted cell dendrodendritic inhibition in the olfactory bulb is dependent on NMDA receptor activity.

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 antagonist D,L-2amino-5-phosphonopentanoic acid (D,L-AP5), whereas the alpha-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.

Regulation of synaptic timing in the olfactory bulb by an A-type potassium current.

Although rapid synaptic transmission confers signal fidelity, the activity of some neuronal circuits depends on prolonged excitation or inhibition. Here we demonstrate that GABAergic granule cells in the rat olfactory bulb produce prolonged inhibition of mitral cells through a precise kinetic matching between transmitter-gated and voltage-gated channels in their dendritic membrane. A transient A-type potassium current (IA) specifically attenuated dendrodendritic inputs mediated by fast-acting AMPA receptors such that the excitation and subsequent inhibitory output of granule cells followed the prolonged kinetics of their NMDA receptors. Altering the weights of the AMPA and NMDA receptor-mediated inputs by modulating IA provides a mechanism to regulate the timing of inhibition according to the demands on the bulb network.

Dendrodendritic inhibition in the olfactory bulb is driven by NMDA receptors.

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.

Activation of shaker potassium channels. I. Characterization of voltage-dependent transitions.

The conformational changes associated with activation gating in Shaker potassium channels are functionally characterized in patch-clamp recordings made from Xenopus laevis oocytes expressing Shaker channels with fast inactivation removed. Estimates of the forward and backward rates for transitions are obtained by fitting exponentials to macroscopic ionic and gating current relaxations at voltage extremes, where we assume that transitions are unidirectional. The assignment of different rates is facilitated by using voltage protocols that incorporate prepulses to preload channels into different distributions of states, yielding test currents that reflect different subsets of transitions. These data yield direct estimates of the rate constants and partial charges associated with three forward and three backward transitions, as well as estimates of the partial charges associated with other transitions. The partial charges correspond to an average charge movement of 0.5 e0 during each transition in the activation process. This value implies that activation gating involves a large number of transitions to account for the total gating charge displacement of 13 e0. The characterization of the gating transitions here forms the basis for constraining a detailed gating model to be described in a subsequent paper of this series.

Activation of Shaker potassium channels. II. Kinetics of the V2 mutant channel.

This second of three papers, in which we functionally characterize activation gating in Shaker potassium channels, focuses on the properties of a mutant channel (called V2), in which the leucine at position 382 (in the Shaker B sequence) is mutated to valine. The general properties of V2's ionic and gating currents are consistent with changes in late gating transitions, in particular, with V2 disrupting the positively cooperative gating process of the normally activating wild type (WT) channel. An analysis of forward and backward rate constants, analogous to that used for WT in the previous paper, indicates that V2 causes little change in the rates for most of the transitions in the activation path, but causes large changes in the backward rates of the final two transitions. Single channel data indicate that the V2 mutation causes moderate changes in the rates of transitions to states that are not in the activation path, but little change in the rates from these states. V2's data also yield insights into the general properties of the activation gating process that could not be readily obtained from the WT channel, including evidence that intermediate transitions have rapid backward rates, and an estimate of a total charge 2 e0 for the final two transitions. Taken together, these data will help constrain an activation gating model in the third paper of this series, while also providing an explanation for V2's effects.

Activation of Shaker potassium channels. III. An activation gating model for wild-type and V2 mutant channels.

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's 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 e0), which is distributed among a large number of small steps each with rather small charge movements (between 0.6 and 1.05 e0). 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's 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.

Modulation of mEPSCs in olfactory bulb mitral cells by metabotropic glutamate receptors.

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 alpha-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 of L-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.

Functional expression and purification of a homomeric human alpha 1 glycine receptor in baculovirus-infected insect cells.

The human alpha 1 glycine receptor (GlyR) was expressed in Sf9 insect cells infected with a recombinant Autographa californica nuclear polyhedrosis baculovirus. Previous studies had indicated that transient expression of this subunit in Xenopus oocytes or human kidney cell lines is sufficient to form active agonist-gated chloride channels. Expression of the alpha 1 GlyR protein resulted in functional channels present on the cell surface of infected Sf9 cells as evidenced by whole-cell patch-clamping and single-channel recordings. These channels were gated by glycine, but not in the presence of strychnine. An immunoreactive 48-kDa protein could be easily visualized on Coomassie-stained sodium dodecyl sulfate-polyacrylamide gels of whole-cell lysates with maximal expression 3 days postinfection. The alpha 1 GlyR protein was solubilized from a membrane fraction of infected Sf9 cells in 1% digitonin and 0.1% deoxycholate and purified by affinity chromatography using aminostrychnine agarose, yielding 0.33 mg/liter of cells. Given the low natural abundance of the native channel, the development of this expression system now provides sufficient purified channel protein for future biochemical and biophysical characterization. Since the glycine receptor shares sequence and structural homology with other members of a ligand-gated channel superfamily, further characterization may establish general rules governing the structure and mechanism of these membrane protein channels.

The size of gating charge in wild-type and mutant Shaker potassium channels.

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.