HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 95/4/2417    most recent 00964.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Yuan, Q. Articles by Knöpfel, T. Search for Related Content PubMed PubMed Citation Articles by Yuan, Q. Articles by Knöpfel, T.

Olfactory Nerve Stimulation-Induced Calcium Signaling in the Mitral Cell Distal Dendritic Tuft

¹Laboratory for Neuronal Circuit Dynamics, Brain Science Institute, RIKEN, Saitama, Japan

Submitted 13 September 2005; accepted in final form 26 November 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Olfactory receptor neuron axons form the olfactory nerve (ON) and project to the glomerular layer of the olfactory bulb, where they form excitatory synapses with terminal arborizations of the mitral cell (MC) tufted primary dendrite. Clusters of MC dendritic tufts define olfactory glomeruli, where they involve in complex synaptic interactions. The computational function of these cellular interactions is not clear. We used patch-clamp electrophysiology combined with whole field or two-photon Ca²⁺ imaging to study ON stimulation-induced Ca²⁺ signaling at the level of individual terminal branches of the MC primary dendrite in mice. ON-evoked subthreshold excitatory postsnaptic potentials induced Ca²⁺ transients in the MC tuft dendrites that were spatially inhomogeneous, exhibiting discrete "hot spots." In contrast, Ca²⁺ transients induced by backpropagating action potentials occurred throughout the dendritic tuft, being larger in the thin terminal dendrites than in the base of the tuft. Single ON stimulation-induced Ca²⁺ transients were depressed by the NMDA receptor antagonist D-aminophosphonovaleric acid (D-APV), increased with increasing stimulation intensity, and typically showed a prolonged rising phase. The synaptically induced Ca²⁺ signals reflect, at least in part, dendrodendritic interactions that support intraglomerular coupling of MCs and generation of an output that is common to all MCs associated with one glomerulus.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Mitral cells (MCs) are the principal output neurons of the olfactory bulb and represent the first stage of central odor processing. The distally tufted primary dendrites of rodent MCs outline complex structures termed olfactory glomeruli. A given glomerulus is innervated by axons of olfactory receptor neurons (ORNs), each of which expresses the same odor receptor protein (Vassar et al. 1994). The convergence of ORN axons onto topographically defined glomeruli creates a spatial map of the odor's molecular features in the glomerular layer of the olfactory bulb (Mombaerts et al. 1996; Vassar et al. 1994; Wachowiak and Cohen 2001).

The glomeruli of rodent MCs exhibit complex synaptic interactions between each other and with periglomerular cells (Isaacson 1999; Schoppa and Westbrook 2001, 2002; Urban and Sakmann 2002). Because a given glomerulus receives inputs essentially from the same type of ORNs, glomerular processing of odor information may primarily serve to average over ORN inputs (approximately 11,000 in rodents; Shepherd et al. 2004), and to produce a uniform signal carried by each of the approximately 25 (Allison 1953) MCs that are associated with a given glomerulus. Averaging of ORN inputs increases sensitivity far beyond the sensitivity of isolated ORNs (Van Drongelen et al. 1978), while coupling and synchronization of MCs likely also increase fidelity of the output signal (Isaacson 1999; Schoppa and Westbrook 2001 2002). In this concept, the MC firing rate from approximately 1,800 glomeruli in the mouse olfactory bulb (Royet et al. 1988) represent the combinatorial odor code. Interglomerular signaling may, in addition, enhance odor discrimination (Aungst et al. 2003). Glomerular interactions likely also determine the exact temporal structure of the oscillatory synchronization between MCs, which may be important for entraining network oscillations within the olfactory bulb (Adrian 1950; Zochowski and Cohen 2005) and for enhancing the signal-to-noise ratio when the MC activity is encoded in the olfactory cortex (Friedrich et al. 2004; Laurent et al. 2001). Conceptually, MC firing may represent independent rate and temporal coding (Huxter et al. 2003).

The plausibility of any of these concepts is constrained by the detailed features of glomerular signal processing. Direct electrophysiological measurement of the activity of terminal dendritic branches in the tuft is not feasible because these structures are too small to tolerate intracellular electrode recordings. A powerful method to access subcelluar signaling is provided by combining somatic patch clamp and dendritic Ca²⁺ imaging techniques. Ca²⁺ signals in the glomerular tuft of the primary dendrite of the MC have been used to access backpropagating action potentials (bAPs), presynaptic inputs, and postsynaptic signaling (Debarbieux et al. 2003; Ma and Lowe 2004; Urban and Castro 2005). There is controversial experimental evidence about some characteristics of these Ca²⁺ signals (Debarbieux et al. 2003; Ma and Lowe 2004; Urban and Castro 2005), and little information is available as to the mechanism associated with the intercellular Ca²⁺ concentration ([Ca²⁺]_i) increase of bAP and local, subthreshold excitatory postsynaptic potentials (EPSPs). The bAP-induced Ca²⁺ signals of MC dendritic tufts are likely mediated by voltage-gated Ca²⁺ channels including N- and P/Q-type, but not dihydropyridine-sensitive L-type channels (Isaacson and Strowbridge 1998; Yuan et al. 2004). In addition, olfactory nerve (ON) synapses that release glutamate might mediate Ca²⁺ signals via N-methyl-D-aspartate (NMDA) receptor or mGluR1 (Aroniadou-Anderjaska et al. 1999; Carlson et al. 2000; Ennis et al. 1996; Friedman and Strowbridge 2000; Hayar et al. 2004; Heinbockel et al. 2004; Isaacson 1999; Trombley and Westbrook 1990).

In this work, we studied subthreshold synaptic stimulation and bAP-induced signals using combined patch clamp and Ca²⁺ imaging techniques. We characterized Ca²⁺ transients induced by bAPs in the primary dendrite of the MC and used these signals as a reference for synaptically induced signals. We found that focal ON stimulation induced patch-like Ca²⁺ signals that were NMDA receptor dependent and exhibited a prolonged rising phase that, at least in part, reflect dendrodendritic interactions (Isaacson 1999). Our results are consistent with the concept that intraglomerular signaling supports coupling of MCs and generation of an output that is common to all MCs associated with one glomerulus.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Slice preparation and electrophysiology

Horizontal slices (350 µm) of olfactory bulbs were obtained from 18- to 26-day-old ICR mice of both sexes. Slices were cut by a Vibroslicer (VT 1000S, Leica), recovered at 32°C for the 1st hour and afterwards at room temperature (23–25°C). After more than 1 hour of recovery, slices were transferred into a recording chamber and perfused with artificial cerebrospinal fluid (ACSF) containing (in mM) 118 NaCl, 25 NaHCO₃, 1 NaH₂PO₄, 3 KCl, 1 MgCl₂, 2 CaCl₂, and 10 glucose, equilibrated with 95% O₂-5% CO₂. Experiments were performed at either room temperature or 32°C. For electrophysiological and fluorescence recordings, slices were placed in an immersion-type perfusion chamber mounted on the stage of an upright microscope (Leica, DMLFS or Nikon, E600FN) and visualized using Nikon 63X water immersion lenses (NA = 0.9). The procedures had approval from the animal ethical committee of the RIKEN Brain Science Institute.

Patch-clamp recordings were carried out in whole cell configuration. Glass pipettes (3.5–5 M resistance) were pulled from borosilicate glass using a two-stage vertical puller (Narishige, Tokyo). Pipettes contained (in mM) 0.2 Oregon green BAPTA-1, 125 K-gluconate, 2 MgCl₂, 0.025 CaCl₂, 10 HEPES, 1 EGTA, 2 Na₂ATP, and 0.5 Na₃GTP, pH 7.25. In one set of experiments, the Na⁺ channel blocker QX-314 (10 mM) was included in the intracellular solution.

Whole cell patch-clamp recordings were made from the somata of MCs using an Axon Instrument Axonpatch 200B (Axon Instruments, Sunnyvale, CA). Under differential interference contrast visual guide, MCs were identified and selected by their location in the MC layer, the size and shape of the somata (around 25 µm diam), and the distinct single primary dendrite that project into a glomerulus. Cells that had resting membrane potential more negative than –45 mV at zero holding current and without correction for junction potentials were selected for recording. Holding current of 300pA was used to hold the membrane potential at rest between –60 and –65 mV. Backpropagating APs were generated by current injection (0.7–1.2 nA, 2 ms) through the patch pipette (AP amplitude 128.1 ± 2.1 mV, overshoot 65.8 ± 2.1 mV; mean ± SE, n = 15 cells). Size and shape of bAPs remained stable throughout the experiments.

The EPSPs were elicited by extracellular stimulation of the olfactory nerve using either a glass pipette (0.8–1 M) containing normal ACSF solution or a miniature bipolar concentric electrode (tip diam 25 µm, Model MCE-100; Rhodes Medical Instruments, Woodland Hills, CA). The electrode was positioned, under visual control, on a small bundle of presynaptic axons just outside the imaged glomerulus. Both types of electrodes produced identical results (in particular, focal Ca²⁺ signals were seen with both types of electrodes).

Drugs applied in the bath were made from aqueous stock solutions. For recording of bAP-induced Ca²⁺ signals in the absence of synaptic transmission, the ACSF contained 50 µM D-aminophosphonovaleric acid (D-APV, an NMDA receptor antagonist), 40 µM 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), and 40 µM bicuculline. For recording of EPSPs in the absence of inhibition, the ACSF contained 40 µM bicuculline and, in a series of experiments, also contained 2–4 µM NBQX (to prevent synaptically driven oscillatory activity). To test the contribution of NMDA and mGluRs in generating local Ca²⁺ signals, D-APV (100 µM) and methyl-4-carboxyphenylglycine (MCPG, 1 mM, a group I and II mGluR antagonist) were used through bath application. All drugs were purchased from Tocris Cookson (Bristol, United Kingdom).

Loading of olfactory nerve axons with Calcium Green-1 dextran

For Ca²⁺ imaging in olfactory nerve axons, mice (16 to 18 days old) were anesthetized by pentobarbital sodium (50 mg/kg, s.c.). Olfactory receptor neurons in nasal cavities were exposed to 2 µl of 0.25% Triton X-100 in physiological saline for 5 min. Subsequently, nasal cavities were injected with 8 µl of 4% Calcium Green-1 dextran (10 kDa; Molecular Probes, Eugene, OR) in physiological saline, and mice were recovered from anesthesia. After 2–5 days, dye-injected mice were used for slice preparation (Mutoh et al. 2005).

Ca²⁺ Imaging

Fluorescence of Oregon green BAPTA-1 or Calcium Green-1 dextran was excited by a two-photon laser (810 nm, Verdi; Coherent, Palo Alto, CA) using a multifocal scanner (frame rate 7Hz) (Straub et al. 2000) or by whole field epi-illumination (488 nm; frame rate 20Hz), with light provided by a monochromator (Polychrome IV; TILL photonics, Gräfelfing) and detected by a cooled CCD camera (PCO Sensicam, PCO imaging, Kelheim) with spatial resolution of 0.226 µm/pixel (x63 objective, 520x680 pixels, 2x2 binning) under the control of ImagePro software (Media Cybernetics, Silver Spring, MD). Optical filters for whole field epifluorescence consisted of a dichroic beam splitter (DCLP 505 LP; Chroma Technology, Brattleboro, VT) and an emission filter (535 ± 25 nm). Fluorescence from intracellularly loaded dye equilibrated throughout the cell within 30–40 min of commencing whole cell configuration. Changes of [Ca²⁺] were expressed as relative fluorescence changes (F/F, where F is the baseline fluorescence before a stimulus and F is the evoked change in fluorescence). F/F was measured in areas of interest in the shaft and in the terminal tuft of the MC primary dendrite. Fluorescence values were determined by subtracting pixel values averaged over regions outside the stained cell from each pixel of the series of images (i.e., fluorescence values are "background subtracted"). The F/F images were spatially low-pass filtered (Gaussian kernel with half-width of 0.5–5 µm). Ca²⁺ maps represent the average of two to five series of images (trials) unless stated otherwise. For recordings under two-photon excitation, Ca²⁺ images from different layers of the tufts were added to yield projection images. Responsive areas within the dendritic tuft were identified as "hot spots" if their peak amplitude exceeded the response averaged over the whole tuft ("global tuft response") by a factor of 2. The border of these hot spots was defined by the half-maximal value of the hot spot amplitude relative to the global tuft response. Color-coded maps of F/F were obtained using custom-made macros in ImagePro Plus. Color-coded images show the maps of the peak Ca²⁺ transient if not otherwise stated.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Ca²⁺ signals induced by bAPs in the MC primary dendrite glomerular tuft

MCs were loaded with the fluorescent Ca²⁺ indicator Oregon green BAPTA-1 (200 µM) from the patch-pipette attached to the soma. Brief intrasomatic depolarizing pulses (2 ms, 0.7–1.2 nA) were used to evoke single bAPs, and related Ca²⁺ transients were imaged from the glomerular tuft of the primary dendrite (Fig. 1).

View larger version (64K): [in this window] [in a new window]   FIG. 1. Backpropagating action potentials (bAP) induce Ca2+ transients throughout the dendritic tuft. A–E: bAP induced Ca2+ transients under whole-field fluorescence excitation. A: fluorescence image of a mitral cell (MC) dendritic tuft filled with Oregon green BAPTA-1 via a somatic recording pipette. B: color-coded map of the peak Ca2+ transient induced by a single bAP evoked by short current pulse (2 ms, 0.7–1.2 nA) injected into the cell body. Blue to red indicates relative fluorescence change F/F from 0 to 19%. C: time-courses of Ca2+ transients in four regions of interest (indicated by numbers in A). Note that the bAP invaded all parts of the dendritic tree, being larger in fine terminal branches. D: Ca2+ transients induced by 1, 2 and 4 bAPs. E: peak amplitudes of Ca2+ transients normalized to that induced by one bAP (n = 3 cells). Doted line indicates linear relationship. F–J: bAP induced Ca2+ transients under two-photon laser excitation. F: fluorescence image of a MC dendritic tuft. G: color-coded map of the peak Ca2+ transient induced by a single bAP. Blue to red indicates relative fluorescence change F/F from 0 to 8%. H: selected areas of interest outlining shaft (1, blue), first order branches (2 and 3, green), and second order branches (4, 5, and 6, red). I: voltage traces of bAP and Ca2+ transients from areas of interest in C. bAP shape is shown as inset. J: peak amplitudes of Ca2+ transients measured in different regions as the example in C. Averaged values from four cells. Note that largest Ca2+ transients were observed in higher order terminal branches of the dendritic tuft. *, P < 0.01. Black squares in C, D, and I indicate time of current injection. Bars in A and F indicate 50 µm.

An alternative explanation for the gradient could be that auto-excitation of tuft branches or inhibitory mechanisms at more proximal sites are involved (Aroniadou-Anderjaska et al. 1999; Carlson et al. 2000; Chen et al. 2000; Didier et al. 2001; Isaacson 1999; Lowe 2002; Salin et al. 2001). These mechanisms may produce inhomogeneities in the bAP-Ca²⁺ maps that cannot be resolved by whole field fluorescence excitation (Debarbieux et al. 2003).

To clarify this point, we performed an additional series of measurements using two-photon laser scanning excitation to improve spatial resolution. The two-photon system employed uses full frame integration; therefore it gives an accurate measure of the Ca²⁺ signal over the integration period (100 ms) (Straub et al. 2000). The advantage of two-photon excitation, as employed here, lies in the high spatial resolution, and the complementary whole field fluorescence recording allowed a high temporal resolution. In the experiments employing two-photon excitation, both excitatory and inhibitory synaptic transmissions were blocked by 40 µM NBQX, 50 µM D-APV, and 40 µM bicuculline. The results obtained under these conditions were consistent with those obtained with whole field imaging in control ACSF: bAPs invaded all parts of the MC dendritic tuft, and with synaptic transmission blocked, Ca²⁺ transients still increased in amplitude with the decrease in dendritic diameter (Fig. 1, F–J). We therefore measured and compared Ca²⁺ transients from different locations. Data from n = 4 cells were pooled in Fig. 1J. The peak amplitude F/F values were 4.2 ± 1.3% at the base of the dendritic tuft,; 6.4 ± 1% at first order branch, and 12 ± 1% at second order branch (mean ± SE; P = 0.0005, one-way ANOVA, post hoc Tukey test). We concluded that larger Ca²⁺ transients in more distal branches were based on differences in the surface-to-volume ratio and were not due to auto-excitation or dendrodendritic interactions.

Ca²⁺ signals induced by activation of ON synapses

The subthreshold EPSPs were evoked by stimulating bundles of ON axons that innervate the glomerular tuft of the dye-filled MC. Optical and electrical recordings of the evoked EPSP were interleaved with recordings of bAPs under either whole field illumination (Fig. 2) or two-photon excitation (Fig. 3). Synaptic stimulation induced Ca²⁺ maps that were distinct in several respects from those induced by bAP. First, Ca²⁺ transients associated with subthreshold EPSPs were pronounced in the tuft, whereas changes in the primary dendrite proximal to the tuft were either small or absent (Fig. 2, B–E). Second, synaptically induced Ca²⁺ transients were clearly localized to one or several hot spots (Fig. 2, C and D; n = 12). The localization of Ca²⁺ hot spots was robust throughout single trials, with only small variation in peak amplitudes (Fig. 2C1–C3, ).

View larger version (80K): [in this window] [in a new window]   FIG. 2. Olfactory nerve (ON) stimulation-induced localized Ca2+ transients. A: fluorescence image using whole field fluorescence excitation of a dendritic tuft with areas of interest (shaft, blue; local tuft area, red). Yellow rectangle indicates area shown in D and E. B: bAPs and EPSPs recorded with the somatic pipette (bottom traces) and Ca2+ transients (top traces) measured from the two areas of interest indicated in A. Time of bAP is indicated by black square; time of synaptic stimulation is indicated by black arrow. C: maps (C1–C3) of synaptically induced Ca2+ transients in three sequential trials. Note reproducibility of pattern of yellow/red hot spots. D: average of maps shown in C1–C3. E: color-coded map of bAP-induced Ca2+ elevation. Synaptic recordings were interleaved with bAP recordings, with a 2-min interval. Blue to red indicates F/F from 0 to 5% (C1–C3 and D) and 0 to 15% F: bar in A indicates 50 µm.

View larger version (54K): [in this window] [in a new window]   FIG. 3. Synaptically induced local Ca2+ transients imaged with two-photon laser scanning microscopy. A: fluorescence image of a MC dendritic tuft. Yellow rectangle indicates area shown in D-G. B: bAPs (bottom) and associated Ca2+ transients (top), from regions of interest outlined in D. Time of bAP is indicated by black square. C: ON-EPSPs (lower traces) and associated Ca2+ transients (top traces) from regions of interest outlined in D. Time of synaptic stimulation is indicated by black arrow. D: regions of interest. E–F: maps of Ca2+ transients induced by bAP (E) and ON stimulation (F). G: ratio of fluorescence during synaptically and bAP-induced Ca2+ transients. Bar in A indicates 50 µm.

For cells recorded under whole-field illumination (n = 8), quantitative analysis was applied to compare the Ca²⁺ signals over the hot spot regions with the average response over the entire tufts ("global responses"). We defined "hot spot(s)" as the region(s) in the EPSP-Ca²⁺ map where the F/F values exceeded the global response by a factor of two. Eight out of twelve cells exhibited one to three clearly defined hot spots according to this definition. The border of hot spots was defined by a threshold value calculated as half-maximal amplitude within the hot spot region relative to the global response. The average size for hot spots was 7.8 ± 0.7 µm in diameter (mean ± SE, n = 15 hot spots from n = 8 cells). The Ca²⁺ signals averaged over the whole tuft and over hot spots as well as the ratio between these values were calculated for bAP and EPSP-induced signals in each cell. The average signals (F/F; mean ± SE, n = 8) were 8.16 ± 2.26% (bAP at hot spot), 6.53 ± 1.55% (bAP over the whole tuft; P > 0.08, compared with bAP at hot spot), 5.14 ± 0.60% (EPSP at hot spot), and 1.35 ± 0.22% (EPSP over the whole tuft; P < 0.0005, compared with EPSP at hot spot). The mean ratio between the EPSP-induced signals over the hot spots and the signals measured over the whole tuft was 4.8 ± 1.2 (SE) and is significantly larger than the corresponding value for the bAPs (1.2 ± 0.1, mean ± SE; P < 0.02, paired t-test).

To better understand the input-output relationship between ON axons and MC terminal tufts, we estimated the spread of activated fibers in our slice preparation by imaging Ca²⁺ signals in the presynaptic terminals of ON axons. Ca²⁺ staining in the ON axons was achieved by nose injection of Calcium Green-1 dextran 2–5 days before slice physiology and imaging (n = 5). Figure 3 shows examples of presynaptic Ca²⁺ signals that were obtained by the same protocol used to image postsynaptic responses. These examples indicate activation of a subset of dye-loaded ON fibers with relatively low stimulation intensity (Fig. 4, A2 and B1), and under higher stimulation intensity, a more widespread activation of ON fibers with subregions that exhibit larger than average signals (Fig. 4, A3 and B2). The subset of activated ON fibers depended on the positioning of the stimulation electrode, as illustrated by the extreme case, in which the stimulation electrode was placed onto the lower border of a glomerulus (Fig. 4B3). The presynaptic Ca²⁺ signals were graded with stimulation intensities (Fig. 4, A2–B2) and thus represented compound responses, as does the ON-EPSP. This result suggests that postsynaptic hot spots may be recruited at locations where the density of activated synapses is high.

View larger version (83K): [in this window] [in a new window]   FIG. 4. ON stimulation induced Ca2+ transients in ON axon terminals. A1: fluorescence image of dye-loaded ON, outline of two glomeruli (dotted circles), and position of the stimulation electrode (white lines) in the ON layer (ONL). A2–A3: color-coded maps of Ca2+ transients evoked at low (30 µA, A2) and higher (60 µA, A3) stimulation intensities. Note that higher stimulation intensity recruited more nerve fibers (yellow arrow indicated ON branch that was recruited only at higher stimulation intensity). B1–B3: example maps of ON Ca2+ transients at different stimulation intensities (60 µA, B1 and 90 µA, B2) and after moving the stimulation electrode to the deeper portion of the glomerulus (B3; stimulation intensity 90 µA).

The localized Ca²⁺ changes in the MC dendritic tuft could be due to Ca²⁺ influx through NMDA receptors or voltage-gated Ca²⁺ channels, or may reflect the release of Ca²⁺ from internal stores following activation of group I mGluRs. To test the involvement of NMDA receptors, we compared EPSP-induced Ca²⁺ transients under control conditions and after bath application of 100 µM of the NMDA receptor antagonist D-APV.

D-APV did not affect the initial fast phase of the ON-EPSP that is mediated by AMPA receptors but did block the slow EPSP component (n = 11, Fig. 5A, Carlson et al. 2000; Ennis et al. 1996; Trombley and Westbrook 1990). Single bAPs that consistently generated larger Ca²⁺ signals than subthreshold EPSPs (Fig. 2) did not induce any detectable D-APV-sensitive potential (Fig. 5 A3).

View larger version (47K): [in this window] [in a new window]   FIG. 5. Synaptically induced Ca2+ transients are mediated by NMDA receptors. A1–A3: ON-EPSPs and bAPs induced before, during D-APV application, and after D-APV washout. A1: representative EPSP. A2: average EPSP over nine cells. Note that D-APV blocked the late component of the EPSP but did not affect EPSP peak amplitude. A3: membrane voltage during induction of bAP (Arrow indicates truncated AP that is followed by AP afterhyperpolarization; average over 9 cells). Note that the membrane potential following the bAP was not affected by the blockage of the NMDA receptor. B: synaptically induced Ca2+ transients before, during D-APV application, and after D-APV washout. Color-coded maps of Ca2+ show hot spots (yellow arrows) before and after application of D-APV. Bottom panel shows time course of Ca2+ transients (region of interest indicated by yellow rectangle in C). Note that D-APV application abolished the local Ca2+ increase induced by synaptic stimulation of the ON. Blue to red indicates F/F from 0 to 3%. C: bAP-induced Ca2+ transients before, during D-APV application, and after D-APV washout. Color-coded map (top) and time course of Ca2+ transients (bottom) induced by bAPs. Blue to red indicates F/F from 0 to 12%. Bar indicates 50 µm. Black arrows and squares indicate time of synaptic stimulation and bAP induction, respectively.

View larger version (12K): [in this window] [in a new window]   FIG. 6. Involvement of NMDA receptors but not mGluRs in synaptic responses. A: Ca2+ increase (% F/F) in local tuft responsive areas due to bAP and synaptic stimulation either under control condition or in the presence of D-APV (n = 8). B: ON-EPSP recorded in control ACSF and in the presence of MCPG average over six cells. MCPG did not affect the peak amplitude of ON-EPSPs, but slightly (statistically not significantly) reduced the initial part of a slow EPSP component (open arrows at 100 ms after stimulation). C: summary data showing the effects of D-APV (n = 8) and MCPG (n = 6) on synaptically induced Ca2+ signals. Values (mean ± SE) represent the ratios of bAP-induced Ca2+ signals (control), and of synaptically induced Ca2+ signals before and in the presence of MCPG. The ratios of D-APV and MCPG were compared with control. **, P < 0.001.

Ca²⁺ signals induced by activation of ON synapses are graded

The ON stimulation-induced local Ca²⁺ signals may represent all-or-none "Ca²⁺ spikes" (Urban and Castro 2005) or may be graded with recruitment of increasing numbers of ORN axons. To investigate this issue, we determined the minimal ON stimulation intensity needed to evoke an AP in the patch-clamped MC and then measured the responses evoked by fractions of this reference intensity. Compound EPSP amplitude varied with stimulation intensity, as did the evoked Ca²⁺ signals (Fig. 7). The peak amplitude of the EPSP and the initial portion of the slower EPSP component (EPSP area, between 50 and 200 ms after ON stimulation) varied in near-linear relation to stimulation intensity (Fig. 7C). However, larger minimal stimulation intensities are required to evoke the Ca²⁺ transients and the later NMDA receptor-mediated EPSP component (EPSP area, between 200 and 500 ms after ON stimulation; Fig. 7C, n = 5 cells). To test the effect of larger stimulation intensities without AP firing in the recorded cell, we performed the next set of experiments with pipettes containing the Na⁺ channel blocker QX-314 (10 mM). Higher stimulation intensities induced large stimulus-locked EPSPs and a fast rise in Ca²⁺, followed by unsynchronized synaptic events and a further increase in Ca²⁺ (Fig. 8, n = 3 cells). A prolonged or delayed rise in Ca²⁺ and unsynchronized synaptic events were also seen in cells with standard pipettes (see Fig. 7B) and were efficiently depressed by D-APV (see Fig. 5, A and B).

View larger version (30K): [in this window] [in a new window]   FIG. 7. Synaptic EPSPs and Ca2+ transients increase with increasing stimulation intensities. A: fluorescence image of a MC dendritic tuft (top panel, left) and color-coded Ca2+ maps representing Ca2+-transients induced by synaptic stimulations with increasing intensities, as indicated. The time of imaging is indicated by the black rectangle in (B) left top panel. B: Ca2+ transients and synaptic EPSPs induced by synaptic stimulation at different intensities. Top panels show Ca2+ transients obtained from two regions of interest as indicated in the fluorescence image in A. The transients shown in the left and the right were obtained from the regions outlined in yellow and green colors, respectively. Bottom panels show sub- and suprathreshold EPSPs induced by synaptic stimulation at different intensities at the time scale of the Ca2+ transients (left) and at faster time scale (right). C: plots showing the relationship between ON stimulation intensity (fraction of intensity threshold for induction of an AP) and EPSP components or Ca2+ transients. Note that the peak amplitude of the EPSP (top left) and the early EPSP component (EPSP area, between 50 to 200 ms after ON stimulation, top right) varied close to linearly with stimulation intensity whereas larger minimal stimulation intensities are required to evoke the late EPSP component (EPSP area, between 200 to 500 ms after ON stimulation, bottom left) and the Ca2+ transients (bottom right). Early and late NMDA components are indicated by black rectangles in B left bottom.

View larger version (40K): [in this window] [in a new window]   FIG. 8. Synaptic EPSPs and Ca2+ transients recorded with pipettes containing QX-314. A: fluorescence image of a MC dendritic tuft (top, left) and color-coded Ca2+ maps representing ON stimulation-induced Ca2+ transients. B: Ca2+ transients and synaptic responses induced by ON stimulation at different intensities in the region of interest indicated in A. Note prolonged rising phase of Ca2+ transient (arrow). Inset illustrates an enlarged view of a voltage trace (100 µA) with asynchronous synaptic events.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

Ca²⁺ signals induced by bAP in the glomerular tuft

Ca²⁺ signals evoked by bAPs in the MC glomerular dendritic tuft have attracted much attention because terminal dendritic branches in the tuft behave both as postsynaptic structures that receive olfactory receptor cell terminals and as presynaptic structures that release transmitter (Bischofberger and Jonas 1997; Murphy et al. 2005; Rall et al. 1966). It has been shown previously by indirect measurements using Ca²⁺ imaging (Charpak et al. 2001; Debarbieux et al. 2003), as well as by direct measurement using voltage imaging (Djurisic et al. 2004), that bAPs propagate in a nondecremental manner into the dendritic tuft of MCs. Our results are in agreement with previous reports showing that single bAPs trigger significant Ca²⁺ transients in the distal branches of the dendritic tuft. We did not, however, find failures of bAPs in triggering Ca²⁺ signals in selected dendritic tuft branches as reported in earlier in vivo measurements (Debarbieux et al. 2003). This discrepancy might be due to differences in intraglomerular inhibitory processes between in vitro, slice preparation, and in vivo recording from an anesthetized animal. Also in contrast to one earlier report showing that the amplitude of Ca²⁺ transients did not depend on the diameter of the dendrite (Debarbieux et al. 2003), our current results clearly demonstrated Ca²⁺ signals that were consistently larger in thinner dendrites, in agreement with concurrent observations made in the accessory olfactory bulb (Ma and Lowe 2004; Urban and Castro 2005). The reason for this discrepancy between the in vitro and in vivo data is not clear. Our results also demonstrated that bAP-induced Ca²⁺ transients were not influenced by autoexcitation (Figs. 1 and 5C) or GABAergic local inhibition (Fig. 1). We also found no direct evidence for NMDA receptor-dependent autoexcitation in the voltage trace following a single bAP (Aroniadou-Anderjaska et al. 1999; Isaacson 1999; Salin et al. 2001).

Ca²⁺ signals induced by ON-EPSPs in the glomerular tuft

In contrast to the widespread Ca²⁺ signals induced by bAPs, dendritic Ca²⁺ signals following subthreshold ON stimulation were pronounced in the tuft, whereas changes in the primary dendrite proximal to the tuft were either small or absent. Also, these synaptically induced Ca²⁺ transients were clearly localized to one or several hot spots.

The EPSP-associated dendritic Ca²⁺ transients could theoretically be caused by mobilization of Ca²⁺ from internal stores. Our experiments excluded the role of mGluRs because the mGluR antagonist MCPG did not significantly affect the Ca²⁺ transient associated with the ON-EPSP. The mGluR antagonist had also only a minor effect on the ON-EPSP itself, consistent with a previous report (De Saint and Westbrook 2005). The Ca²⁺ signals were, however, either blocked completely or greatly attenuated by the NMDA receptor antagonist D-APV. Another potential entry pathway is via voltage-gated Ca²⁺ channels that are activated by the NMDA receptor-mediated depolarization. Because the peak amplitude of the ON stimulation-induced EPSP was not dependent on NMDA receptors, it is unlikely that voltage-gated Ca²⁺ channels are the major source for the NMDA receptor-dependent Ca²⁺ transients. The source of the D-APV-insensitive residual component of the Ca²⁺ signal following ON stimulation (approximately 25% of the control response), however, has not been clarified. It is, therefore possible that a part of the Ca²⁺ signal is mediated by low-threshold voltage-gated Ca²⁺ current, as suggested by previous experiments of Charpak et al. 2001. However, on the basis of indirect evidence, these authors concluded that Ca²⁺ signals are completely independent of NMDA receptor activation, which is markedly different from our results.

NMDA receptors may be activated directly by glutamate released from the ON, and by glutamate released from MC dendrites following an initial activation of dendritic glutamate release by the ON input. Our data provide evidence for additional dendritically released glutamate. Thus Ca²⁺ signals and slower synaptic components increase supralinearly with stimulation intensity, while the EPSP peak amplitudes (which is a measure of recruited ON fibers) increase more linearly with stimulation intensity (Fig. 7C). Moreover, ON stimulation was often followed by barrages of unsynchronized synaptic events and a prolonged rising phase of the Ca²⁺ transient. Indeed, the slow NMDA receptor-dependent component has previously been attributed to recurrent, intraglomerular dendrodendritic interactions (Carlson et al. 2000).

The most likely scenario for glomerular integration of ORN inputs is that dendrodendritic mutual excitation of dendrites of neighboring MCs can become self-supportive after an initial ON input. The hot spots in our Ca²⁺ maps would then reflect regenerative glutamate release in chemical glomerular compartments, as proposed by Schoppa and Westbrook (2001). The mechanisms for termination of this regenerative mutual excitation of dendritic tufts is unclear but may involve depletion of releasable dendritic glutamate, Ca²⁺ activated outward currents, or GABAergic inhibition. It should be noted that natural stimulation most likely would activate ORN axon terminals that are more homogenously distributed over the glomeruli than was the case under our experimental focal stimulation conditions. It is therefore likely that the local hot spots of slow NMDA receptor-dependent process studied here will fuse to a more homogenous pattern under natural conditions. In the absence of hot spots, the dendrodendritic interactions that are reflected by the hot spots seen with focal ON activation will, however, still contribute to the coupling of MCs. This scenario is in agreement with previous proposals that glutamate released from MC dendrites is regenerative and contributes to synchronization of glomerular responses to ON activity (Christie et al. 2005; Isaacson 1999; Schoppa and Westbrook 2001).

Functional implications

The elaborated MC dendritic tufts and multifaceted dendro-dentric interactions are suggestive of specific dendritic information processing (as opposed to simply relaying ON inputs) in olfactory glomeruli. All signaling mechanisms that are known to occur at the level of the olfactory glomeruli can be attributed to one specific computational outcome, namely, to increase sensitivity for the specific class of ORNs that provide input to that given glomerulus. Increase in sensitivity is achieved by sampling of and averaging over all ORN inputs (Van Drongelen et al. 1978). This process requires coupling of MCs so that the average ORN inputs can be represented by the population of MCs that are associated with one given glomerulus. In addition to sampling and averaging, dendrodendritic interaction can boost the ON inputs by regenerative mutual excitation. In this picture, inhibitory mechanism is required to dampen sensitivity for strong ON inputs. Faster interaction such as gap-junction coupling (Christie et al. 2005) may synchronize spike timing of MCs. This synchronization of spiking may further enhance sensitivity via stochastic resonance, or may provide information that is encoded by OB network oscillations or the MC target cells in the olfactory cortex.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We thank H. Mutoh for help with Ca²⁺ imaging in the olfactory nerve and technical support. We also thank all members of the Knöpfel lab for many helpful discussions and encouragement and J. Yokoyama for editorial help.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. Knöpfel, Laboratory for Neuronal Circuit Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan. (E-mail: tknopfel{at}brain.riken.jp)

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Adrian ED. The electrical activity of the mammalian olfactory bulb. Electroencephalogr Clin Neurophysiol 2: 377–388, 1950.[CrossRef][ISI][Medline]

Allison AC. The structure of the olfactory bulb and its relationship to the olfactory pathways in the rabbit and the rat. J Comp Neurol 98: 309–353, 1953.[CrossRef][ISI][Medline]

Aroniadou-Anderjaska V, Ennis M, and Shipley MT. Dendrodendritic recurrent excitation in mitral cells of the rat olfactory bulb. J Neurophysiol 82: 489–494, 1999.[Abstract/Free Full Text]

Aungst JL, Heyward PM, Puche AC, Karnup SV, Hayer A, Szabo G, and Shipley MT. Centre-surround inhibition among olfactory bulb glomeruli. Nature 426: 623–629, 2003.[CrossRef][Medline]

Bischofberger J and Jonas P. Action potential propagation into the presynaptic dendrites of rat mitral cells. J Physiol 504: 239–265, 1997.

Carlson GC, Shipley MT, and Keller A. Long-lasting depolarizations in mitral cells of the rat olfactory bulb. J Neurosci 20: 2011–2021, 2000.[Abstract/Free Full Text]

Charpak S, Mertz J, Beaurepaire E, Moreaux L, and Delaney K. Odor-evoked calcium signals in dendrites of rat mitral cells. Proc Natl Acad Sci USA 98: 1230–1234, 2001.[Abstract/Free Full Text]

Chen WR, Xiong W, and Shepherd GM. Analysis of relations between NMDA receptors and GABA release at olfactory bulb reciprocal synapses. Neuron 25: 625–633, 2000.[CrossRef][ISI][Medline]

Christie JM, Bark C, Hormuzdi SG, Helbig I, Monyer H, and Westbrook GL. Connexin 36 mediates spike synchrony in olfactory bulb glomeruli. Neuron 46: 761–772, 2005.[CrossRef][ISI][Medline]

Coutinho V and Knöpfel T. Metabotropic glutamate receptors: electrical and chemical signaling properties. Neuroscientist 8: 551–561, 2002.[CrossRef][ISI][Medline]

Debarbieux F, Audinat E, and Charpak S. Action potential propagation in dendrites of rat mitral cells in vivo. J Neurosci 23: 5553–5560, 2003.[Abstract/Free Full Text]

De Saint Jan D, and Westbrook GL. Detecting activity in olfactory bulb glomeruli with astrocyte recording. J Neurosci 25: 2917–2924, 2005.[Abstract/Free Full Text]

Didier A, Carleton A, Bjaalie JG, Vincent JD, Ottersen OP, Storm-Mathisen J, and Lledo PM. A dendrodendritic reciprocal synapse provides a recurrent excitatory connection in the olfactory bulb. Proc Natl Acad Sci USA 98: 6441–6446, 2001.[Abstract/Free Full Text]

Djurisic M, Antic S, Chen WR, and Zecevic D. Voltage imaging from dendrites of mitral cells: EPSP attenuation and spike trigger zones. J Neurosci 24: 6703–6714, 2004.[Abstract/Free Full Text]

Ennis M, Zimmer LA, and Shipley MT. Olfactory nerve stimulation activates rat mitral cells via NMDA and non-NMDA receptors in vitro. NeuroReport 7: 989–992, 1996.[ISI][Medline]

Friedman D and Strowbridge BW. Functional role of NMDA autoreceptors in olfactory mitral cells. J Neurophysiol 84: 39–50, 2000.[Abstract/Free Full Text]

Friedrich RW, Habermann CJ, and Laurent G. Multiplexing using synchrony in the zebrafish olfactory bulb. Nat Neurosci 7: 862–871, 2004.[CrossRef][ISI][Medline]

Hayar A, Karnup S, Ennis M, and Shipley MT. External tufted cells: a major excitatory element that coordinates glomerular activity. J Neurosci 24: 6676–6685, 2004.[Abstract/Free Full Text]

Heinbockel T, Heyward P, Conquet F, and Ennis M. Regulation of main olfactory bulb mitral cell excitability by metabotropic glutamate receptor mGluR1. J Neurophysiol 92: 3085–3096, 2004.[Abstract/Free Full Text]

Huxter J, Burgess N, and O'Keefe J. Independent rate and temporal coding in hippocampal pyramidal cells. Nature 425: 828–832, 2003.[CrossRef][Medline]

Isaacson JS. Glutamate spillover mediates excitatory transmission in the rat olfactory bulb. Neuron 23: 377–384, 1999.[CrossRef][ISI][Medline]

Isaacson JS and Strowbridge BW. Olfactory reciprocal synapses: dendritic signalling in the CNS. Neuron 20: 749–761, 1998.[CrossRef][ISI][Medline]

Kasowski HJ, Kim H, and Greer CA. Compartmental organization of the olfactory bulb glomerulus. J Comp Neurol 407: 261–274, 1999.[CrossRef][ISI][Medline]

Laurent G, Stopfer M, Friedrich RW, Rabinovich MI, Volkovskii A, and Abarbanel HD. Odor encoding as an active, dynamical process: experiments, computation, and theory. Annu Rev Neurosci 24: 263–297, 2001.[CrossRef][ISI][Medline]

Lowe G. Inhibition of backpropagating action potentials in mitral cell secondary dendrites. J Neurophysiol 88: 64–85, 2002.[Abstract/Free Full Text]

Ma J and Lowe G. Action potential backpropagation and multiglomerular signaling in the rat vomeronasal system. J Neurosci 24: 9341–9352, 2004.[Abstract/Free Full Text]

Mombaerts P, Wang F, Dulac C, Chao SK, Nemes A, Mendelsohn M, Edmondson J, and Axel R. Visualizing an olfactory sensory map. Cell 87: 675–686, 1996.[CrossRef][ISI][Medline]

Murphy GJ, Darcy DP, and Isaacson JS. Intraglomerular inhibition: signaling mechanisms of an olfactory microcircuit. Nat Neurosci 8: 354–364, 2005.[CrossRef][ISI][Medline]

Mutoh H, Yuan Q, and Knöpfel T. Long-term depression at olfactory nerve synapses. J Neurosci 25: 4252–4259, 2005.[Abstract/Free Full Text]

Rall W, Shepherd GM, Reese TS, and Brightman MW. Dendrodendritic synaptic pathway for inhibition in the olfactory bulb. Exp Neurol 14: 44–56, 1966.[CrossRef][ISI][Medline]

Royet JP, Souchier C, Jourdan F, and Gervais R. Morphometric study of the glomerular population in the mouse olfactory bulb: numerical density and size distribution along the rostrocaudal axis. J Comp Neurology 270: 559–568, 1988.[CrossRef][ISI][Medline]

Sahara Y, Kubota T, and Ichikawa M. Cellular localization of metabotropic glutamate receptors mGluR1, 2/3, 5 and 7 in the main and accessory olfactory bulb of the rat. Neurosci Lett 312: 59–62, 2001.[CrossRef][ISI][Medline]

Salin PA, Lledo PM, Vincent JD, and Charpak S. Dendritic glutamate autoreceptors modulate signal processing in rat mitral cells. J Neurophysiol 85: 1275–1282, 2001.[Abstract/Free Full Text]

Schoppa NE and Westbrook GL. Glomerulus-specific synchronization of mitral cells in the olfactory bulb. Neuron 31: 639–651, 2001.[CrossRef][ISI][Medline]

Schoppa NE and Westbrook GL. AMPA autoreceptors drive correlated spiking in olfactory bulb glomeruli. Nat Neurosci 5: 1194–1202, 2002.[CrossRef][ISI][Medline]

Shepherd GM, Chen WR, and Greer CA. Olfactory bulb. In: The synaptic organization of the brain, edited by Shepherd GM. New York: Oxford, 2004.

Shigemoto R, Nakanishi S, and Mizuno N. Distribution of the mRNA for a metabotropic glutamate receptor (mGluR1) in the central nervous system: an in situ hybridization study in adult and developing rat. J Comp Neurol 322: 121–135, 1992.[CrossRef][ISI][Medline]

Straub M, Lodemann P, Holroyd P, Jahn R, and Hell SW. Live cell imaging by multifocal multiphoton microscopy. Eur J Cell Biol 79: 726–734, 2000.[CrossRef][ISI][Medline]

Trombley PQ and Westbrook GL. Excitatory synaptic transmission in cultures of rat olfactory bulb. J Neurophysiol 64: 598–606, 1990.[Abstract/Free Full Text]

Urban NN and Sakmann B. Reciprocal intraglomerular excitation and intra- and interglomerular lateral inhibition between mouse olfactory bulb mitral cells. J Physiol 542: 355–367, 2002.[Abstract/Free Full Text]

Urban NN and Castro JB. Tuft calcium spikes in accessory olfactory bulb mitral cells. J Neurosci 25: 5024–5028, 2005.[Abstract/Free Full Text]

Van den Pol AN. Presynaptic metabotropic glutamate receptors in adult and developing neurons: autoexcitation in the olfactory bulb. J Comp Neurol 359: 253–271, 1995.[CrossRef][ISI][Medline]

Van Drongelen W, Holley A, and Doving KB. Convergence in the olfactory system: quantitative aspects of odour sensitivity. J Theor Biol 71: 39–48, 1978.[CrossRef][ISI][Medline]

Vassar R, Chao SK, Sitcheran R, Nunez JM, Vosshall LB, and Axel R. Topographic organization of sensory projections to the olfactory bulb. Cell 79: 981–991, 1994.[CrossRef][ISI][Medline]

Wachowiak M and Cohen LB. Representation of odorants by receptor neuron input to the mouse olfactory bulb. Neuron 32: 723–735, 2001.[CrossRef][ISI][Medline]

Yuan Q, Mutoh H, Debarbieux F, and Knöpfel T. Calcium signaling in mitral cell dendrites of olfactory bulbs of neonatal rats and mice during olfactory nerve stimulation and beta-adrenoceptor activation. Learn Mem 11: 406–411, 2004.[Abstract/Free Full Text]

Zochowski MR and Cohen LB. Oscillations in the olfactory bulb carry information about odorant history. J Neurophysiol 94: 2667–2675, 2005.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. Djurisic, M. Popovic, N. Carnevale, and D. Zecevic Functional Structure of the Mitral Cell Dendritic Tuft in the Rat Olfactory Bulb J. Neurosci., April 9, 2008; 28(15): 4057 - 4068. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 95/4/2417    most recent 00964.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal