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. 2009 Sep 23;29(38):11943-53.
doi: 10.1523/JNEUROSCI.0206-09.2009.

Group I mGluR activation enhances Ca(2+)-dependent nonselective cation currents and rhythmic bursting in main olfactory bulb external tufted cells

Hong-Wei Dong  1 , Abdallah Hayar, Joseph Callaway, Xiang-Hong Yang, Qiang Nai, Matthew Ennis
Affiliations

Group I mGluR activation enhances Ca(2+)-dependent nonselective cation currents and rhythmic bursting in main olfactory bulb external tufted cells

Hong-Wei Dong et al. J Neurosci. .

Abstract

In the main olfactory bulb, activation of group I metabotropic glutamate receptors (mGluRs) by olfactory nerve stimulation generates slow (2 Hz) oscillations near the basal respiratory frequency. These oscillations arise in the glomerular layer and may be generated, in part, by the intrinsic neurons, the juxtaglomerular neurons. We investigated the physiological effects of group I mGluR agonists on one population of juxtaglomerular neurons, external tufted (ET) cells, which rhythmically burst at respiratory frequencies and synchronize the intraglomerular network. Electrophysiological studies in rat main olfactory bulb slices demonstrated that the mGluR agonist 3,4-dihydroxyphenylglycine (DHPG) amplified the strength of ET cell spike bursts, principally by increasing the number of spikes per burst. Voltage-clamp and Ca(2+)-imaging studies showed that DHPG elicits a Ca(2+)-dependent nonselective cation current (I(CAN)) in the dendrites of ET cells triggered by Ca(2+) release from internal stores. The DHPG effects on bursting and membrane current were attenuated by flufenamic acid and SKF96365, agents known to antagonize I(CAN) in a variety of neurons. DHPG also elicited slow membrane current oscillations and spikelets in ET cells when synaptic transmission and intrinsic membrane channels were inoperative. These findings indicate that DHPG may passively (by increasing burst strength) or actively (by increasing conductance of gap junctions) enhance the strength of electrical synapses between ET cells. Together, these findings indicate that activation of group I mGluRs on the dendrites of ET cells play a key role in the generation of slow rhythmic oscillation in the glomerular network, which is in turn tuned to sniffing of the animal in vivo.

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Figures

Figure 1.
Figure 1.
DHPG increases the strength of ET cell bursting. A, Sample extracellular recordings (cell-attached, voltage-clamp mode) from an ET cell show that bath-applied DHPG (10 μm) and l-CGG-I (3 μm), but not l-AP4, increase properties of rhythmic spike bursts; all experiments in this figure were performed with ACSF containing CNQX-APV-gabazine. B, Running average showing the effects of DHPG, CCGI, and l-AP4 on bursting parameters. C1, Whole-cell current-clamp trace showing that DHPG (30 μm) depolarized and increased the discharge of this ET cell; the depolarization is more clearly seen in the lower filtered (0.5 Hz) trace. C2, Faster timescale records for the cell shown in C1. Note increase in the number of spikes per burst. C3, DHPG increased the amplitude and duration of the slow depolarizing envelope underlying ET cell spike bursts; same cell as in C1 and C2. Dashed horizontal lines denote AMP; PP, peak potential (see Materials and Methods for details).
Figure 2.
Figure 2.
DHPG evokes an inward current in ET cells. A, An ET cell recorded in whole-cell voltage-clamp mode was initially identified on the basis of extracellularly recorded spike bursts (A1). In voltage-clamp mode, sEPSCs occurred randomly (A2). A3, Lucifer yellow staining shows the morphology of this typical ET cell. B, A focal puff of DHPG (1 mm) adjacent to this ET cell evoked an inward current at a HP of −60 mV. C1, C2, Membrane currents (C1) produced by slow voltage ramps (C2, command voltages) were used to generate I–V curves before and during bath-applied DHPG (30 μm), and again after washout; at right, hyperpolarizing pulses monitored changes in input resistance. C3, The inward current elicited by bath-applied DHPG is accompanied by decreased membrane resistance (HP = −60 mV). C4, The DHPG-evoked current for a single ET cell, obtained by subtraction of the control and DHPG I–V curves in C1, was linear and reversed polarity near 0 mV. D, Mean I–V plot of the DHPG current in 27 ET cells; the mean reversal potential was −5.2 ± 2.3 mV. E, Dose–response relationship of DHPG-evoked currents in ET cells. The magnitude of the inward current elicited by cumulative doses of 3, 10, and 30 μm DHPG were measured in individual ET cells (open bars, n = 6, HP = −60 mV); 10 and 30 μm DHPG significantly increased the amplitude of the inward current relative to baseline (*p < 0.05 one-way repeated-measures ANOVA followed by Newman–Keuls tests), and the currents evoked by 30 μm were significantly greater than that evoked by 3 μm (p < 0.05, Newman–Keuls test). The amplitude of the inward current elicited by a single 30 μm application of DHPG (black bar, n = 27) did not differ from that elicited in the cumulative condition (p > 0.05, unpaired t test).
Figure 3.
Figure 3.
IDHPG requires intracellular Ca2+ and is largely mediated by Na+ influx. A, I–V curves generated from recordings with a BAPTA-based internal solution (BAPTA, see text) before and during application of DHPG (30 μm, +DHPG); the subtraction I–V curve (Diff) shows that DHPG-evoked currents were nil with BAPTA. B, I–V curves generated from recordings with the standard pipette solution in the presence of the Ca2+ store-depleting agent thapsigargin (1 μm) before and during application of DHPG; the subtraction I–V curve (Diff) shows that DHPG did not evoke an appreciable current in the presence of thapsigargin. C, I–V curves generated in the presence of low-Na+ ACSF (reduction from 152 to 51 mm) before and after bath application of DHPG (30 μm); the subtraction I–V curve (Diff) shows that DHPG did not evoke an appreciable current in low-Na+ ACSF. D, Group data subtraction I–V curves showing the effect of DHPG (30 μm) in control, BAPTA, and thapsigargin conditions. E, Group data illustrating the magnitude of IDHPG (HP = −60 mV) in the above three conditions. Note that BAPTA (n = 6), thapsigargin (n = 4), and low Na+ (n = 8) replacement significantly and nearly completely abolished IDHPG; *p < 0.05 compared with control (n = 27), unpaired t tests.
Figure 4.
Figure 4.
IDHPG is associated with a rise in [Ca2+]i. A1, Fluorescence image of an ET cell recorded with a pipette containing 100 μm fura-2 and voltage clamped at −60 mV; the position of the puffer pipette is shown and the arrow denotes flow of ACSF. A2, [Ca2+]i transients in the soma and dendrites (red and blue traces respectively, from the regions indicated by rectangles in A1), and current (black trace) evoked by a DHPG puff (1 mm) 30 μm upstream from the soma. Experiments in A conducted with modified ACSF containing APV-CNQX-gabazine-TTX-TEA-Cd-Ni. B1, B2, The protocol is identical to A1 and A2 above except that Ca2+-free ACSF (no antagonists or channel blockers) was used. Scale bars: A1, B1, 10 μm. C, The magnitude of IDHPG was linearly related to the rise in dendritic [Ca2+]i in modified ACSF (R2 = 0.60, p < 0.05, n = 7) or Ca2+-free ACSF (R2 = 0.84, p < 0.05, n = 5).
Figure 5.
Figure 5.
DHPG-evoked [Ca2+]i transients originate in ET cell dendrites. A, Fluorescence image of an ET cell recorded with a pipette containing 100 μm fura-2 and voltage clamped at −60 mV. B, Time course of changes in [Ca2+]i in the soma (red trace), proximal (green trace) and distal (blue trace) dendritic regions (corresponding to the rectangles in A1), and inward current (black trace) elicited by a focal puff of DHPG (1 mm) 30 μm upstream from the soma; note that the time course of the inward current (inverted trace shown in gray) closely corresponds to that of the Ca2+ transient in the distal dendrite. Experiment was in conducted in normal ACSF (no antagonists). C, Schematic diagram showing the position of six sampled ET cells with respect to the puffer pipette. D, Individual cell and group data (mean ± SEM, black circles) showing that the latencies of [Ca2+]i transients in the soma were longer than those in the dendrites (*p < 0.001, n = 6).
Figure 6.
Figure 6.
IDHPG is attenuated by ICAN antagonists. A, B, Subtraction I–V curves showing responses to DHPG (30 μm) before and during application of 100 μm SKF96365 (SKF, A) or 100 μm FFA (B); data from different ET cells are shown in A and B. C, Group data show the magnitude of IDHPG (HP = −60 mV) before (control) and during SKF (n = 6) or FFA (n = 4) application (*p < 0.05, paired t tests).
Figure 7.
Figure 7.
ICAN antagonists dampen ET cell rhythmic bursting. A, Current-clamp recordings showing that ET cell bursting is enhanced by DHPG (30 μm). After DHPG washout, SKF96365 (100 μm) dampened basal and DHPG-evoked bursting; experiment performed in the presence of CNQX-APV-gabazine (control). B, Group data summarizing the effects of DHPG and SKF on properties of ET cell bursting. n = 6, *p < 0.05 versus control, #p < 0.05 DHPG versus DHPG + SKF; one-way repeated-measures ANOVA followed by Newman–Keuls tests.
Figure 8.
Figure 8.
DHPG induces slow rhythmic oscillations in ET cells. A, Recordings from an ET cell were made in voltage-clamp mode (HP = −60 mV) using a pipette containing CsCl and QX-314 in the presence of CNQX + APV + gabazine. Top trace, Under these recording conditions, small infrequent slow membrane current oscillations (arrow) and spikelets (asterisks) occurred. Bottom three traces, DHPG dose-dependently increased the oscillation frequency from <1 Hz (control) before to 2.0 Hz (30 μm DHPG). Note also that DHPG increased the amplitude of slow oscillations in a concentration-dependent manner. B, Faster time scale records showing oscillations and spikelets before and during DHPG application. C, Autocorrelograms of membrane current oscillations corresponding to the four conditions shown in A. Note that DHPG increased the regularity of the oscillations as shown by an enhanced sideband in the autocorrelograms.
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