Am J Physiol Cell Physiol Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 290: C925-C935, 2006. First published November 2, 2005; doi:10.1152/ajpcell.00392.2005
0363-6143/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/3/C925    most recent
00392.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Blakemore, L. J.
Right arrow Articles by Trombley, P. Q.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blakemore, L. J.
Right arrow Articles by Trombley, P. Q.

RECEPTORS AND SIGNAL TRANSDUCTION

Evidence for Ca2+-permeable AMPA receptors in the olfactory bulb

L. J. Blakemore, M. Resasco, M. A. Mercado, and P. Q. Trombley

Department of Biological Science, Florida State University, Tallahassee, Florida

Submitted 3 August 2005 ; accepted in final form 25 October 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
{alpha}-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs), a subtype of glutamate receptor, contribute to olfactory processing in the olfactory bulb (OB). These ion channels consist of various combinations of the subunits GluR1–GluR4, which bestow certain properties. For example, AMPARs that lack GluR2 are highly permeable to Ca2+ and generate inwardly rectifying currents. Because increased intracellular Ca2+ could trigger a host of Ca2+-dependent odor-encoding processes, we used whole cell recording as well as histological and immunocytochemical (ICC) techniques to investigate whether AMPARs on rat OB neurons flux Ca2+. Application of 1-naphthylacetyl spermine (NAS), a selective antagonist of Ca2+-permeable AMPARs (CP-AMPARs), inhibited AMPAR-mediated currents in subsets of interneurons and principal cells in cultures and slices. The addition of spermine to the electrode yielded inwardly rectifying current-voltage plots in some cells. In OB slices, olfactory nerve stimulation elicited excitatory responses in juxtaglomerular and mitral cells. Bath application of NAS with D,L-2-amino-5-phosphonovaleric acid (AP5) to isolate AMPARs suppressed the amplitudes of these synaptic responses compared with responses obtained using AP5 alone. Co2+ staining, which involves the kainate-stimulated influx of Co2+ through CP-AMPARs, produced diverse patterns of labeling in cultures and slices as did ICC techniques used with a GluR2-selective antibody. These results suggest that subsets of OB neurons express CP-AMPARs, including functional CP-AMPARs at synapses. Ca2+ entry into cells via these receptors could influence odor encoding by modulating K+ channels, N-methyl-D-aspartate receptors, and Ca2+-binding proteins, or it could facilitate synaptic vesicle fusion.

GluR2; polyamines; cobalt; glutamate receptor; olfaction

GLUTAMATE IS THE TRANSMITTER used in most of the brain's excitatory synaptic circuits, including those in the olfactory bulb (OB). Its actions are mediated by three classes of ionotropic receptors: N-methyl-D-aspartate (NMDA) receptors, {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs), and kainate (KA) receptors. AMPARs are heterotetramers formed from the glutamate receptor subunits GluR1–GluR4. Their properties vary in association with multiple factors, including receptor subunit composition, alternative splicing, and RNA editing (11). For example, alternative splicing of the AMPA gene generates "flip" and "flop" splice variants for each of the GluR1–GluR4 subunits, with distinct desensitization kinetics. We recently reported both electrophysiological (3) and molecular (20) data suggesting that flip and flop splice variants are expressed heterogeneously in the OB.

In addition to alternative splicing, some AMPAR subunits undergo RNA editing at the Q/R site. For GluR2, the single-codon, glutamine-to-arginine substitution alters the AMPAR's current-voltage (I-V) relationship (21, 64), reduces its Ca2+ permeability (7, 21), and influences its sensitivity to blockade by internal polyamines and spider toxins (5, 34). The editing of GluR2 is 99.9% effective, so most GluR2-containing AMPARs do not flux Ca2+. However, AMPARs that lack or contain unedited GluR2 generate inwardly rectifying currents and are highly Ca2+ permeable (7, 21, 64). These Ca2+-permeable AMPARs (CP-AMPARs) have been found in a variety of regions (e.g., hippocampus, neocortex, spinal cord) (13, 15, 16, 33, 36, 44) and have been implicated in physiological processes including synaptic transmission (16, 24, 25, 28, 44) and long-term depression (40). Increasing evidence suggests that CP-AMPARs also play a role in some forms of neuropathology (48, 63, 67).

In the OB, Ca2+ entry through CP-AMPARs could activate Ca2+-dependent K+ channels or Ca2+-binding proteins, modulate NMDA receptor function, or mediate a host of second-messenger effects. Accumulating electrophysiological evidence suggests that single neurons express combinations of GluR2-lacking CP-AMPARs and Ca2+-impermeable or GluR2-containing AMPARs that produce a range of Ca2+ permeabilities (37, 60, 73). In the present study, we used immunocytochemistry, histology, and whole cell electrophysiology, combined with several selective antagonists of CP-AMPARs [Joro spider toxin (JSTX) and 1-naphthylacetyl spermine (NAS)], to test two hypotheses. 1) Subsets of principal cells and interneurons in the OB express CP-AMPARs. 2) Some functional CP-AMPARs are expressed at synapses, where they play a role in excitatory synaptic transmission.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The protocols for all procedures that we performed were approved by the Florida State University Institutional Animal Care and Use Committee.

Tissue Culture

The procedure for preparing primary cultures of OB neurons was described in detail elsewhere (61). Briefly, OBs were harvested from postnatal days 1–5 (P1–P5) Sprague-Dawley rat pups, cut into 1-mm cubes, and enzymatically treated in a Ca2+-buffered papain solution for 1 h at 37°C. The tissues were then triturated with a fire-polished pipette until a single-cell suspension was achieved. The cells were plated onto 35-mm-diameter culture dishes on a confluent monolayer of previously prepared OB astrocytes at a density of ~250,000 cells per dish.

Neuronal Identity

The morphological, physiological, and immunohistochemical criteria established by Trombley and Westbrook (62) were used to identify and differentiate presumptive mitral/tufted (M/T) cells and interneurons. Briefly, the OB cultures contained two morphologically distinct populations of neurons: a small number of large-diameter (20–40-µm soma), pyramidal-shaped neurons, as well as a much larger population of small-diameter (5- to 10-µm soma) bipolar neurons. These characteristics correlate with M/T cells and granule/periglomerular cells (OB interneurons), respectively.

Our previous electrophysiological and immunocytochemical (ICC) analyses of these neuron populations further support this notion. In monosynaptically coupled pairs, intracellular stimulation of neurons with M/T cell-like morphology invariably evoked glutamate-mediated excitatory postsynaptic potentials, whereas intracellular stimulation of the small bipolar neurons evoked GABA-mediated inhibitory postsynaptic potentials (62). In addition, the large pyramidal neurons (presumptive M/T cells) were immunoreactive for N-acetylaspartylglutamate, whereas the small bipolar neurons (presumptive interneurons) were immunoreactive for glutamic acid decarboxylase (62).

Preparation of OB Slices for Electrophysiology

OB slices were prepared from 14- to 28-day-old Sprague-Dawley rats that had been anesthetized with halothane and then killed by decapitation. OBs were rapidly removed and placed in ice-cold oxygenated (95% O2-5% CO2) saline solution. Horizontal slices (400 µm) were made using a vibratory microtome (Vibratome, St. Louis, MO) and incubated in a holding chamber for 30 min at 35°C. Slices were then stored at 20–24°C until use. For electrophysiology, slices were placed into a recording chamber and viewed using a Leica microscope (Leica Microsystems, Wetzlar, Germany) equipped with infrared differential interference contrast optics. Mitral cells and juxtaglomerular (JG) cells were discriminated on the basis of morphology and location within the slice (56).

Preparation of OB Slices for Histology

OB slices were prepared from 28- to 180-day-old Sprague-Dawley rats. For Co2+ staining, slices were prepared as described above for electrophysiological recording. For GluR2 immunocytochemistry, animals were anesthetized deeply by administration of chlornembutal (0.3 ml/100 g) and then transcardially perfused with 100 ml of 0.1 M PBS (pH 7.2) containing 0.2% heparin, followed by 250 ml of a 4% formaldehyde solution.

Co2+ Staining

Co2+ staining is a histological method of identifying CP-AMPARs (43, 47, 53) that has been used successfully to demonstrate the presence of CP-AMPARs in other brain regions (e.g., hippocampus, cortex) (69, 71). It was originally reported that Co2+ staining reflects Ca2+ influx through CP-AMPARs and/or KA receptors and not through voltage-gated Ca2+ channels or NMDA receptors (53). More recent data have suggested that Co2+ staining reflects Ca2+ influx specifically through CP-AMPARs and not through KA receptors (30, 43).

To identify OB neurons that express CP-AMPARs, brain slices (400 µm) and primary cultures were exposed to 100 µM KA together with 5 mM CoCl2 in physiological buffer for 5 min at room temperature. The samples were then washed with buffer containing 3 mM EDTA to remove extracellular Co2+. Next, the cell samples were incubated in 0.12% (NH4)2S in buffer for 5 min to precipitate intracellular Co2+ and then were washed three times with buffer. The cells were subsequently fixed with 4% paraformaldehyde for 30 min. After fixation, the cells were washed three times in buffer and incubated in a developmental solution containing 5 parts 0.1 M AgNO3, 20 parts 2% hydroquinone and 5% citric acid, and 100 parts 20% gum arabic. This solution was changed every 15 min, and silver-staining enhancement was monitored using microscopy. Once enhancement was complete (~45–50 min), the reaction was terminated by washing the solution three times in water and then samples were mounted and photographed for analysis.

Immunocytochemistry

Our procedures were modified from previously described protocols (62). Brain slices were examined for immunoreactivity against the GluR2 subunit using GluR2 antibodies (Chemicon International, Temecula, CA). Parallel experiments were conducted in culture. OB slices (permeabilized with 0.1% Triton X-100) and cultures were incubated in blocking serum (5% goat serum) for 45 min, washed three times with PBS, and incubated for 24 h at 4°C in rabbit anti-rat GluR2 antibody (1:500 dilution). After being washed in PBS, the tissue was incubated in a goat anti-rabbit Cy3-labeled secondary antibody (1:800 dilution) for 45 min. The tissue was washed three times with PBS and mounted using Gel/Mount (Biomeda, Foster City, CA). Light microscopy was used to evaluate the labeling of individual neurons by GluR2 antibodies to determine cellular and laminar patterns of GluR2 expression.

Electrophysiology

Primary culture. OBs were prepared for primary dissociated cultures using the methods described above (61). Whole cell voltage-clamp recording in OB neurons was performed at room temperature after the OB neurons had spent 7–21 days in culture. The 35-mm-diameter culture dish was used as the recording chamber and was perfused at 0.5–2.0 ml/min with a bath solution containing (in mM) 162.5 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.3 with NaOH. The final osmolarity was 325 mosmol/l. Patch-clamp electrodes were pulled from borosilicate glass to a final electrode resistance of 4–6 M{Omega}. These electrodes were filled with a solution containing (in mM) 145 CsCl or KMeSO4, 1 MgCl2, 10 HEPES, 4 Mg2+-ATP, 0.5 Mg2+-GTP, and 1.1 EGTA (pH 7.2; 310 mosmol/l). In a subset of experiments, 100 µM spermine was included in the intracellular solution for analysis of current rectification.

Drugs were diluted in the bath solution and applied via a gravity-fed flow pipe perfusion system assembled from an array of 600-µm-diameter square glass barrels. An electronic manipulator (Warner Instrument, Hamden, CT) was used to position the flow pipes near the neuron, and pinch clamps were used to control drug flow. The speed of the solution changes allowed peak drug responses to occur within 100 ms. Neurons were perfused continuously with bath solution (control), except during drug application. The applied drugs were 100 µM KA, 10 µM NAS, 0.5 µM JSTX, 3–10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 100 µM D,L-2-amino-5-phosphonovaleric acid (AP5), 1 µM TTX, and 100 µM spermine (Sigma, St. Louis, MO).

OB slices. Whole cell patch-clamp recordings were obtained from JG and mitral cells in OB slices using methods similar to those that we and others have described previously (2, 55). For all experiments, the extracellular solution was oxygenated (95% O2-5% CO2) and contained (in mM) 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 25 glucose, 2.5 KCl, 1.0 MgCl2, and 2 CaCl2, pH 7.3. Patch pipettes were pulled to a resistance of 1–3 M{Omega} in mitral cell recordings and to a resistance of 3–10 M{Omega} in JG cell recordings. Excitatory postsynaptic current (EPSC) measurements were performed using a pipette solution containing (in mM) 125 CsMeSO4 or KMeSO4, 2 MgCl2, 0.025 CaCl2, 1 EGTA, 2 Na+-ATP, 0.5 Na+-GTP, and 10 HEPES, pH 7.3.

Electrical stimulation of OB sensory neuron axons was conducted using a bipolar tungsten electrode (125-µm tip separation; Frederick Haer, Brunswick, ME) to stimulate the olfactory nerve layer (ONL). In some experiments, an extracellular patch pipette filled with extracellular solution was used to stimulate the ONL. Stimulatory pulses were generated by the computer, which triggered a stimulus isolation unit (stimulation ranged from 200 to 500 µA). In these experiments, 10 mM lidocaine N-ethyl bromide was added to the electrode solution to prevent action currents. Extracellular drugs were delivered using the flow pipe or bath perfusion.

Use of selective antagonists of CP-AMPARs. In whole cell electrophysiological studies, we examined the effects of two selective antagonists, JSTX and NAS, of CP-AMPARs on AMPAR-mediated, KA-evoked currents recorded in OB neurons. JSTX, the toxin of the Nephila clavata spider, has been shown to selectively block recombinant (4) and native (16, 22, 45) AMPARs that lack GluR2. For example, Iino et al. (22) reported that JSTX-3, a synthetic form of JSTX, specifically suppressed responses mediated by CP-AMPARs in rat hippocampal neurons.

NAS is another synthetic analog of JSTX that has been reported to have effects similar to those of JSTX (37). In another study, NAS selectively suppressed the inwardly rectifying currents and CP-AMPARs expressed by one type of hippocampal neuron, with no effect on AMPARs observed with slight outward rectification and little Ca2+ permeability expressed by another type of hippocampal neuron (37). Thus evidence exists that both of these drugs are selective antagonists of CP-AMPARs.

Experimental Procedures

Presumptive M/T cells and interneurons in primary culture were identified on the basis of previously established morphological, physiological, and immunohistochemical criteria (see above) (62). To examine membrane currents evoked by KA (100 µM), whole cell recordings were obtained from the cell bodies of randomly selected M/T cells and interneurons using an AxoClamp 2B amplifier (Axon Instruments, Sunnyvale, CA) in discontinuous (switch frequency of 10–15 kHz) or continuous voltage-clamp mode. Membrane currents were filtered at 1–3 kHz, digitized at 5–10 kHz, and analyzed using AxoGraph software (Axon Instruments).

The effects of JSTX or NAS on AMPAR-mediated currents were evaluated by comparing the steady-state amplitudes of currents evoked by 100 µM KA alone and 100 µM KA with NAS (or JSTX). The resulting degree of current inhibition was expressed as a percentage of the control (KA alone) current and estimated using the following formula: [amplitude of KA + NAS (or JSTX) current/amplitude of KA-alone current] x 100. Similarly to methods used in previous studies (28), KA rather than AMPA was used as an agonist to avoid the effects of receptor desensitization. Because KA receptors mediate relatively small currents and desensitize rapidly, measurement of the steady-state amplitudes of these nondesensitizing currents likely reflects currents mediated by AMPARs.

In addition to fluxing Ca2+, CP-AMPARs generate inwardly rectifying currents (64). To further test our hypothesis that OB neurons express CP-AMPARs, we investigated the rectification properties of currents recorded in M/T cells and interneurons in culture. I-V plots were generated from ramps run during application of control, KA, or KA + NAS, and currents were leak subtracted. Ramp I-V plots were obtained by holding the membrane potential at +50 mV and ramping the cell membrane potential to –100 mV for 2 s. Cells were ramped from positive membrane potentials to negative membrane potentials to reduce contributions from voltage-gated channels (66).

Data from several studies suggest that current rectification, rather than being an intrinsic property of the CP-AMPAR channel, is generated by a polyamine block (5, 34, 35). Whole cell recording tends to dialyze the cell, removing intracellular polyamines responsible for rectification (5). Inclusion of polyamines (e.g., spermine, spermidine) in the electrode restores the inward rectification, which is lost with the diffusion of cytoplasmic factors (5, 34). Therefore, 100 µM spermine was added to the intracellular solution in studies of rectification. Rectification indexes (RIs) were determined by comparing the conductance ratios at +40 and –60 mV (RI = G+40/G–60) (32, 60).

To investigate the presence of synaptic CP-AMPARs, NAS was applied during synaptic events (EPSCs) evoked in JG and mitral cells using ONL stimulation. In these experiments, AMPAR-mediated events were isolated by blocking NMDA receptors with 100 µM AP5.

Data Analysis

Averaged values are expressed as means ± SE. Student's t-test and contingency tables in StatView software (SAS Institute, Cary, NC) were used to establish the statistical significance (P < 0.05) of neuron population-based differences.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
GluR2 Is Heterogeneously Expressed in OB Cultures and Slices

To test our first hypothesis that subsets of OB principal cells and interneurons express GluR2-lacking CP-AMPARs, we first performed immunohistochemistry in OB cultures and slices with a GluR2-specific antibody. In OB slices from 28- to 180-day-old rats, widespread GluR2 immunoreactivity was observed among mitral cells, granule cells, and JG cells (Fig. 1, A–C), but subsets of each population remained weakly labeled or unlabeled. In primary culture, AMPARs on somas and dendrites of most but not all M/T cells and interneurons expressed GluR2 (Fig. 1, D and E).


Figure 1
View larger version (123K):
[in this window]
[in a new window]
  		Fig. 1. GluR2 immunoreactivity. In olfactory bulb (OB) slices from adult rats, widespread GluR2 immunoreactivity was observed among mitral cells (A), granule cells (B), and juxtaglomerular (JG) cells (C), but subsets of each population remained weakly labeled or unlabeled. In primary culture, {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) on somas and dendrites of most but not all mitral/tufted (M/T) cells (D) and interneurons (E) expressed GluR2. This heterogeneous expression of GluR2 supports the hypothesis that subsets of OB neurons express GluR2-lacking, Ca2+-permeable AMPARs (CP-AMPARs).

 
Co2+ Labeling Supports Diverse Expression of CP-AMPARs in OBs

Co2+ staining, which involves the KA-stimulated influx of Co2+ through CP-AMPARs, is a histological means of identifying these receptors (30, 43, 53). Studies conducted in both 28- and 180-day-old rat OB slices (Fig. 2, A and B) and primary culture (P1–P5 tissue samples) (Fig. 2, E and F) revealed diverse patterns of Co2+ staining of OB neurons. Labeling in each bulb layer in OB slices (Fig. 2A) suggested that subsets of most OB neuron subtypes (e.g., mitral cells, tufted cells, juxtaglomerular cells, granule cells) express CP-AMPARs. Also consistent with this hypothesis is that in control experiments, coapplication of 10–30 µM NAS with KA and Co2+ blocked KA-stimulated Co2+ labeling (Fig. 2C). No staining was observed in other control experiments, which included treating cells with KA alone (data not shown), Co2+ alone (data not shown), and KA + Co2+ + 3–10 µM CNQX (an AMPAR antagonist) (Fig. 2D).


Figure 2
View larger version (122K):
[in this window]
[in a new window]
  		Fig. 2. Co2+ staining revealed diverse patterns of silver enhancement, representing the kainate (KA)-stimulated influx of Co2+ ions through CP-AMPARs in OB slices and culture preparations. In OB slices, labeling in each OB layer (A), including the glomerular layer (B), suggests that subsets of most OB neuron subtypes express CP-AMPARs. White arrowheads in A and B indicate a mitral cell in the mitral cell layer (MCL) and a tufted cell in the external plexiform layer (EPL) (A) and JG cells (B). Also consistent with this observation is that in control experiments, coapplication of 1-naphthylacetyl spermine (NAS; 10 µM cultures, 30 µM slices) with the KA + Co2+ blocked the KA-stimulated Co2+ labeling (C). No staining was observed in other control experiments, which included treating cells with KA alone (data not shown), Co2+ alone (data not shown), and KA+ Co2+ + 3–10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (D). In OB primary cultures, subsets of M/T cells (E) and interneurons (F) were labeled (solid arrowhead in F), although others remained unlabeled or weakly labeled (F, white arrowhead). ONL, olfactory nerve layer; GL, glomerular layer; GCL, granule cell layer.

 
Selective CP-AMPAR Antagonists Inhibit AMPAR-Mediated Currents in the Majority of Cultured OB Neurons

The recent commercial availability of selective antagonists of CP-AMPARs affords investigators another means of identifying these receptors. In whole cell electrophysiological studies, we examined the effects of two selective antagonists, JSTX and NAS, on AMPAR-mediated currents recorded in OB neurons in primary culture. To avoid effects of AMPAR desensitization, KA rather than AMPA was used as an agonist.

In voltage-clamp mode at a holding potential of –60 mV, flow pipe application of 100 µM KA evoked currents in all OB neurons examined (n = 124, including 76 M/T cells and 48 interneurons). In 87 (70.2%) of 124 of these cells, coapplication of 10 µM NAS during AMPAR-mediated (KA-evoked) currents produced some degree of current inhibition (Fig. 3A, traces 2–4). No effect of NAS was observed in the remainder of the cells [37 (29.8%) of 124 cells] (Fig. 3A, trace 1). In comparing neuronal subtypes, current inhibition was observed in 51 (67.1%) of 76 of M/T cells vs. 36 (75.0%) of 48 of interneurons, but these differences were not statistically significant (P = 0.35). JSTX (0.5 µM) produced qualitatively similar results, inhibiting AMPAR-mediated currents in 10 (62.5%) of 16 cells examined (Fig. 3B).


Figure 3
View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3. A: coapplication of the selective antagonist of Ca2+-fluxing AMPARs (CP-AMPARs), 10 µM NAS, during AMPAR-mediated currents (evoked by 100 µM KA) produced varying degrees of current inhibition in cultured OB M/T neurons (traces A2–A4). Trace A1 shows a cell with no block. Consistent with findings in other brain regions (22), traces A2 and A3 show the use dependence of the NAS-induced block of AMPAR-mediated currents. Trace A4 shows the time course of recovery from NAS application. B: coapplication of 0.5 µM Joro spider toxin (JSTX), another selective antagonist of CP-AMPARs, during AMPAR-mediated currents produced a qualitatively similar range of current block. C: coapplication of 10 µM NAS during a current evoked by KA in an interneuron revealing ~50% block (trace C1). Currents evoked by KA alone and KA + NAS (traces C2 and C3). Trace C2 shows use-dependent block by NAS, and trace C3 shows the time course of recovery from NAS application. Trace C4 shows that KA-evoked currents were blocked completely by 3–10 µM CNQX. In all current traces, neurons were perfused with control solution where no drug is indicated. Holding potential, –60 mV.

 
Degree of NAS-Induced Block of AMPAR-Mediated Currents Varies Within and Between Neuron Populations

Among the interneurons with NAS-induced blocks (n = 36), NAS decreased the amplitudes of AMPAR-mediated (KA-evoked) currents to between 13% and 91% of the control current. Among the M/T cells with NAS-induced blocks (n = 51), NAS decreased the amplitudes of AMPAR-mediated currents to 33–90% of the control current. Thus the degree of inhibition caused by NAS varied widely within neuron populations as observed in a previous study in cultured hippocampal cells (37).

We also observed differences between neuron populations regarding the degree to which NAS inhibited AMPAR-mediated currents. In interneurons with NAS-induced blocks (n = 36), NAS (10 µM) decreased the steady-state amplitudes of AMPAR-mediated currents to 57.6 ± 3.8% of the control current (Fig. 3C). In M/T cells in which there was an inhibitory effect of NAS (n = 51), NAS (10 µM) decreased the steady-state amplitudes of AMPAR-mediated currents to 81.6 ± 2.2% of the control current (Fig. 3A). Thus NAS produced a greater degree of current inhibition in interneurons than in M/T cells (57.6% vs. 81.6%; P < 0.0001).

Other Characteristics of CP-AMPARs Are Found in OB Neurons

In addition to fluxing Ca2+, CP-AMPARs generate inwardly rectifying currents (64). Prior data from cultured hippocampal neurons indicate that the degree of Ca2+ permeability and inward rectification are well correlated; that is, more inward rectification corresponds to higher Ca2+ permeability (41). Thus, as another means of confirming our findings that OB neurons express CP-AMPARs, we generated I-V plots from ramps run during application of control, KA, or KA + NAS as described in METHODS. To restore intracellular polyamines that could be depleted during whole cell recording, spermine (100 µM) was added to the intracellular solution. RIs were determined by comparing the conductance ratios at +40 and –60 mV (RI = G+40/G–60) (32, 60). Lower RI indicates greater inward rectification.

Consistent with the expression of CP-AMPARs, inwardly rectifying currents were observed in 14 (50%) of 28 of the cultured OB neurons examined (15 M/T cells and 13 interneurons) (Fig. 4). Figure 4A shows an example of currents evoked by applying KA or KA + NAS and demonstrates ~50% NAS-induced block of the current. Figure 4, trace B1, is an I-V plot showing inward rectification of the current evoked by KA in the same cell, indicating the presence of CP-AMPARs; the NAS-sensitive portion of the current (KA-only current – KA + NAS current) demonstrates even greater rectification (Fig. 4, trace B2). In contrast, Fig. 4C shows an example of a cell with no NAS-induced block of KA-evoked currents. The I-V plot in the same cell demonstrates no rectification of the current evoked by KA (Fig. 4, trace D1), indicating the presence of GluR2-containing AMPARs. Consistent with this finding, virtually no NAS-sensitive current was observed in this cell (Fig. 4, trace D2).


Figure 4
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 4. Consistent with the expression of CP-AMPARs, some rat OB neurons generated inwardly rectifying currents. Current-voltage (I-V) plots shown were recorded from ramps run during application of control, KA, or KA + NAS; currents are leak subtracted. A: currents evoked by applying KA or KA + NAS to a rat OB interneuron in culture showing ~50% block of the current. Trace B1: I-V plot showing inward rectification of the current evoked by 100 µM KA in the same cell indicating the presence of CP-AMPARs. The NAS-sensitive portion of the current (KA-only current – KA + NAS current) shows even greater rectification (trace B2). C: currents evoked by applying KA or KA + NAS to rat OB M/T cell in culture showing no block. Trace D1: I-V plot showing no rectification of the current evoked by 100 µM KA in the same cell indicating the presence of GluR2-containing AMPARs. Consistent with this finding, virtually no NAS-sensitive current was present in this cell (trace D2). Cells with and without rectification were observed within both neuron populations.

 
The mean RI in cells with inwardly rectifying currents was 0.67 ± 0.06 vs. 1.25 ± 0.06 in cells with linear or outwardly rectifying I-V plots. The difference between these I-V plot groups was statistically significant (P < 0.0001). The rectification was most evident when the NAS-sensitive portion of the current (KA alone current – KA + NAS current) was isolated (Fig. 4).

Regarding cell type, the mean RI in M/T cells with inwardly rectifying currents (n = 6) was 0.75 ± 0.08 vs. 1.26 ± 0.07 in M/T cells with linear or outwardly rectifying I-V plots (n = 9). The difference between these values was statistically significant (P = 0.001). Among M/T cells with inwardly rectifying currents, 10 µM NAS decreased the amplitudes of AMPAR-mediated currents to 68.8 ± 8.1% of the control current compared with 90.8 ± 2.4% of the control current in cells without inward rectification. This difference was statistically significant (P = 0.005).

The mean RI in interneurons with inwardly rectifying currents (n = 8) was 0.58 ± 0.06 vs. 1.21 ± 0.15 in interneurons with linear or outwardly rectifying I-V plots (n = 5). The difference between these groups was statistically significant (P = 0.004). In interneurons with inwardly rectifying currents, 10 µM NAS decreased the amplitudes of AMPAR-mediated currents to 59.5 ± 7.8% of the control current compared with 94.1 ± 3.2% of the control current in cells with linear or outwardly rectifying I-V plots. These differences were statistically significant (P < 0.01).

Some granule cells, PG cells, and external tufted cells in previous studies showed anomalous rectification activated by hyperpolarization to less than –80 mV (8, 52). This phenomenon was attributed to the superimposition of two components: 1) a Ba2+-insensitive current blocked by 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288; Zeneca Pharmaceuticals, Macclesfield, UK), a blocker of hyperpolarization-activated currents (8, 14, 52, 70); and 2) a Ba2+-sensitive current, suggesting the presence of inwardly rectifying K+ currents (52). These currents, which are sensitive to extracellular Cs+, would not be blocked under our experimental conditions (internal Cs+). However, because these currents activate at potentials more negative (–80 mV) than our RI based on –60 mV as a negative reference, this condition should not have affected our results.

NAS Inhibits AMPAR-Mediated Synaptic Events

As is true of many ionotropic receptors, AMPARs can be synaptic and extrasynaptic, and both populations may be important to function. To test the hypothesis that CP-AMPARs are synaptically activated, we examined the effects of 10 µM NAS on AMPAR-mediated synaptic events (EPSCs) evoked in JG and mitral cells using ONL stimulation. AMPAR-mediated synaptic events were isolated from NMDA receptor-mediated events by coapplication of AP5.

In 7 (77.8%) of 9 JG cells and 6 (60%) of 10 of mitral cells, bath application of 10 µM NAS + 100 µM AP5 suppressed the amplitudes of the synaptic responses (EPSCs) compared with responses obtained using AP5 alone (Fig. 5). These percentages were similar to the percentages of cultured interneurons (75%) and M/T cells (67%) with NAS-induced inhibition of AMPAR-mediated currents. When coapplied with 100 µM AP5, 10 µM NAS decreased the mean amplitude of EPSCs in JG cells from 264.2 ± 83.0 pA to 168.8 ± 47.6 pA (63.8% of control current; P < 0.001) and the mean amplitude of EPSCs in mitral cells from 80.7 ± 15.9 pA to 58.2 ± 12.5 pA (72.1% of control current; P = 0.004) (Fig. 5).


Figure 5
View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5. Consistent with the expression of synaptic CP-AMPARs, NAS also inhibited AMPAR-mediated excitatory postsynaptic currents (EPSCs) evoked by ONL stimulation. In 7 (77.8%) of 9 JG cells (trace A1) and 6 (60%) of 10 mitral cells (B), bath application of 10 µM NAS + 100 µM D,L-2-amino-5-phosphonovaleric acid (AP5) suppressed the amplitudes of the EPSCs compared with responses obtained using AP5 alone. AP5 was used to block NMDA receptors and isolate AMPAR-mediated events. Trace A2 shows the effect of NAS on AMPAR-mediated currents evoked by flow pipe application of KA + NAS or KA alone (each applied with 1 µM TTX) and recorded in the above JG cell (trace A1) from an OB slice. Trace A3 shows that coapplication of 10 µM CNQX with KA completely blocked the KA-evoked current.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Collectively, our ICC, histological, and electrophysiological data support the hypothesis that the majority of OB neurons express CP-AMPARs, including some at synapses. These data represent the first evidence that OB neurons express AMPARs that flux Ca2+, which supports our initial observations (2a).

Heterogeneous GluR2 and Co2+ Labeling in the OB

ICC data from the present study and RT-PCR data from our previous work (20) suggest that although GluR2 is expressed widely and heterogeneously in the OB, subsets of neurons in both culture and slice remained unlabeled or weakly labeled. These results are consistent with previous reports that the degree of GluR2 labeling varies between and within hippocampal and cortical neuron populations (50, 63, 68), as well as between the soma and dendrites of individual pyramidal cells (65, 72). Labeling was evident in several olfactory structures in a previous study in which a selective GluR2 antibody was used to investigate GluR2 expression in the OB, including granule cells of the main OB and M/T cells of the accessory OB (50).

Similarly to our ICC data, Co2+ staining produced diverse patterns of the degree of labeling in both culture and slice preparations among individual cell types. These findings are consistent with previous reports that KA-activated Co2+ uptake, a marker for CP-AMPARs (30, 53), is present in subsets of cortical (71) and hippocampal (69) neurons expressing CP-AMPARs, including GABAergic interneurons and principal cells.

Effects of NAS on AMPAR-Mediated Currents Vary Within and Between Cell Types in Culture

Although NAS produced some degree of current inhibition in the majority (70.2%) of OB neurons examined, the degree to which NAS blocked AMPAR-mediated currents varied widely within each neuron population. One interpretation of these electrophysiological and pharmacological data is that single OB neurons express various combinations of Ca2+-permeable and Ca2+-impermeable AMPARs. In a previous study (37) conducted in cultured hippocampal cells, the degree of NAS-induced inhibition of AMPAR-mediated (KA-evoked) currents also varied widely among neurons with a mean RI similar to that reported in our present study.

In the present study, the degree of inhibition was greatest in interneurons. This finding is similar to results demonstrated in other brain regions suggesting that CP-AMPARs are principally expressed by GABAergic interneurons (23, 28, 33, 36, 44, 71). For example, AMPARs expressed by dentate gyrus basket cells in the hippocampus, which are thought to be GABAergic interneurons, are highly Ca2+ permeable (36).

Most electrophysiological studies have produced little evidence that principal cells express CP-AMPARs (15, 33). However, our finding that NAS inhibited AMPAR-mediated currents in a subset of M/T cells suggests that some, if not most, principal cells of the OB do express CP-AMPARs. Using a combination of techniques, Yin et al. (72) showed that many hippocampal pyramidal neurons expressed CP-AMPARs on their dendrites. In the OB, M/T cells showed both dendritic and somatic labeling. The expression of CP-AMPARs on the dendrites of M/T cells suggests that they could play a role at both axodendritic and dendrodendritic synapses.

Intracellular Spermine Demonstrates Inward Rectification of AMPAR-Mediated Currents

Although data from a previous study (29) suggested that OB interneurons express mainly AMPARs with low Ca2+ permeability, the researchers in that study did not investigate principal cells. Jardemark et al. (29) found that current responses to KA in acutely isolated and cultured OB interneurons showed a linear/outwardly rectifying I-V relationship in contrast to the inwardly rectifying I-V plots associated with expression of CP-AMPARs.

One explanation for this different finding is the diffusion of polyamines during whole cell recording (5, 34). It also is possible that a significant proportion of the CP-AMPARs could be lost through the dendritic pruning that occurs with acute isolation, because evidence from other brain regions suggests that CP-AMPARs may be expressed primarily on the dendrites of some neurons.

Our observations regarding rectification are similar to those in some previous studies. In one previous study (37) conducted in cultured hippocampal neurons, cells with a mean RI similar to that in the present study (mean RI in our study 0.67 ± 0.06 vs. 0.69 in their study) had degrees of NAS-induced block comparable to that in the cells in the present study (mean NAS-induced block in our study 64.1 ± 5.5% vs. 58.4 ± 6.8% in their study). In contrast, cells in which NAS had no effect on AMPAR-mediated (KA-evoked) currents in the previous study had an RI of 1.05 compared with 1.25 ± 0.06 in the present study.

Possible Roles of AMPARs in the OB

Increasing evidence suggests that CP-AMPARs can be synaptic or extrasynaptic (38) and that both populations may be important to function. There are a number of possible roles for CP-AMPARs in the OB. In dorsal horn neurons, Ca2+ influx into cells has been shown to modify synaptic strength (16). Ca2+ entry into postsynaptic cells of the OB via CP-AMPARs could affect synaptic strength through several mechanisms. For example, an intracellular rise in Ca2+ could (via dephosphorylation) inactivate AMPA/KA receptors, thus altering fast components of synaptic transmission (42, 57).

It also has been shown in rat dorsal horn neurons that Ca2+ entry via CP-AMPARs can inhibit adjacent NMDA receptors (39). In the OB, the release of GABA from the granule cell spines at reciprocal dendrodendritic synapses depends on the activation of NMDA receptors (27, 55). Thus the presence of CP-AMPARs at these synapses could influence reciprocal inhibition.

Recent data has suggested that CaMKII is highly expressed in the external plexiform layer (EPL) and the granule cell layer of the OB and is localized to granule cell spines and dendrites at sites of reciprocal dendrodendritic synapses with M/T cells (74). At these synapses, the influx of Ca2+ via NMDA receptors may activate CaMKII, which may phosphorylate multiple substrates important for synaptic transmission and neural plasticity (6, 19, 74). Because the present report is the first to describe CP-AMPARs in the OB, the potential for CP-AMPARs in the OB to activate CaMKII at these synapses has not been examined specifically. However, Ca2+ influx through CP-AMPARs in the retina activates CaMKII and mediates dendritic plasticity of retinal horizontal cells (47), and similar mechanisms could exist in the OB.

Another possible Ca2+/calmodulin pathway in the OB that could be influenced by CP-AMPARs involves the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Halpain and Greengard (18) reported that activation of NMDA receptors in the hippocampus induced rapid dephosphorylation of microtubule-associated protein 2 (MAP2). Other brain regions, including the OB, also showed dephosphorylation of MAP2 in response to NMDA. On the basis of these and other results, Halpain and Greengard hypothesized that NMDA receptor activation induces the dephosphorylation of MAP2 by stimulating a protein phosphatase, possibly calcineurin. Because CP-AMPARs have been shown to inhibit NMDA receptors in dorsal horn neurons (39), the presence of CP-AMPARs at glutamatergic synapses in the OB could provide a source of Ca2+ for the dephosphorylation of MAP2 under conditions in which NMDA receptors could be inhibited (see also below).

A recent focus in studying the OB has been the role of dendritic Ca2+ influx at OB reciprocal synapses and its main sources in the dendrites of interneurons. One proposed mechanism of Ca2+ entry involves NMDA receptors (9, 17, 26). For example, Chen et al. (9) found that feedback inhibition elicited by photorelease of caged Ca2+ in mitral cell secondary dendrites persisted when Cd2+ and Ni2+ were used to block voltage-gated Ca2+ channels. On the basis of these results, Chen et al. concluded that Ca2+ influx through NMDA receptors can directly trigger presynaptic GABA release for local dendrodendritic feedback.

Other proposed sources of dendritic Ca2+ at reciprocal synapses include low- and high-threshold voltage-gated Ca2+ channels (12, 46, 51) and Ca2+-induced Ca2+ release from internal stores (12). Using two-photon imaging in acute rat brain slices and glomerular stimulation of M/T cells, Egger et al. (12) found that weak activation of M/T cells produced stochastic Ca2+ transients in individual granule cell spines. Ca2+ sources for these local synaptic events included NMDA receptors, voltage-dependent Ca2+ channels, and Ca2+-induced Ca2+ release from internal stores. Although CP-AMPARs are not likely to be as effective as NMDA receptors as a source of Ca2+ influx or voltage-gated Ca2+ channels, they may be an important source under certain conditions. For example, under physiological conditions, NMDA receptors are subject to voltage-dependent block by Mg2+, whereas CP-AMPARs are not. Consequently, Ca2+ influx though CP-AMPARs is significant at negative membrane potentials; under these conditions, the driving force on Ca2+ is high but NMDA receptors remain blocked and most voltage-gated Ca2+ are not activated. This situation could produce significant elevations of intracellular Ca2+, particularly in small microdomain compartments such as the dendritic spines that are the targets of excitatory synapses in the OB.

OB neurons also express other Ca2+-binding proteins, including calbindin, calretinin, and parvalbumin (54). These intracellular proteins, which act as buffers to modulate intracellular free Ca2+ levels (1, 10, 31, 49), show cell-type and layer-specific expression (31). For example, a subset of interneurons expressing parvalbumin has been localized to reciprocal synapses with M/T cells in the EPL (58). Parvalbumin tends to be localized to neurons that are selectively deficient in GluR2 (i.e., CP-AMPARs) (59), which provides additional support for the notion that CP-AMPARs are present at reciprocal synapses.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institute on Deafness and Other Communication Disorders Grant DC-04320 (to P. Q. Trombley).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Q. Trombley, Dept. of Biological Science, Florida State Univ., Tallahassee, FL 32306-4340 (e-mail: trombley{at}neuro.fsu.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Baimbridge KG, Celio MR, and Rogers JH. Calcium-binding proteins in the nervous system. Trends Neurosci 15: 303–308, 1992.[CrossRef][ISI][Medline]

2. Berkowicz DA, Trombley PQ, and Shepherd GM. Evidence for glutamate as the olfactory receptor cell neurotransmitter. J Neurophysiol 71: 2557–2561, 1994.[Abstract/Free Full Text]

2a. Blakemore LJ, Mercado MA, Resasco M, and Trombley PQ. Electrophysiological and histological evidence of functional calcium-fluxing AMPA receptors on olfactory bulb neurons (Abstract 276.4). Abstr Soc Neurosci (Washington, DC: Society for Neuroscience, 2004, Online Abstract Viewer/Itinerary Planner).

3. Blakemore LJ and Trombley PQ. Kinetic variability of AMPA receptors among olfactory bulb neurons in culture. Neuroreport 14: 965–970, 2003.[CrossRef][ISI][Medline]

4. Blaschke M, Keller BU, Rivosecchi R, Hollmann M, Heinemann S, and Konnerth A. A single amino acid determines the subunit-specific spider toxin block of {alpha}-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor channels. Proc Natl Acad Sci USA 90: 6528–6532, 1993.[Abstract/Free Full Text]

5. Bowie D and Mayer ML. Inward rectification of both AMPA and kainate subtype glutamate receptors generated by polyamine-mediated ion channel block. Neuron 15: 453–462, 1995.[CrossRef][ISI][Medline]

6. Braun AP and Schulman H. The multifunctional calcium/calmodulin-dependent protein kinase: from form to function. Annu Rev Physiol 57: 417–445, 1995.[CrossRef][ISI][Medline]

7. Burnashev N, Zhou Z, Neher E, and Sakmann B. Fractional calcium currents through recombinant GluR channels of the NMDA, AMPA and kainate receptor subtypes. J Physiol 485: 403–418, 1995.[ISI][Medline]

8. Cadetti L and Belluzzi O. Hyperpolarisation-activated current in glomerular cells of the rat olfactory bulb. Neuroreport 12: 3117–3120, 2001.[CrossRef][ISI][Medline]

9. 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]

10. Clapham DE. Calcium signaling. Cell 80: 259–268, 1995.[CrossRef][ISI][Medline]

11. Dingledine R, Borges K, Bowie D, and Traynelis SF. The glutamate receptor ion channels. Pharmacol Rev 51: 7–61, 1999.[Abstract/Free Full Text]

12. Egger V, Svoboda K, and Mainen ZF. Dendrodendritic synaptic signals in olfactory bulb granule cells: local spine boost and global low-threshold spike. J Neurosci 25: 3521–3530, 2005.[Abstract/Free Full Text]

13. Engelman HS, Allen TB, and MacDermott AB. The distribution of neurons expressing calcium-permeable AMPA receptors in the superficial laminae of the spinal cord dorsal horn. J Neurosci 19: 2081–2089, 1999.[Abstract/Free Full Text]

14. Gasparini S and DiFrancesco D. Action of the hyperpolarization-activated current (Ih) blocker ZD7288 in hippocampal CA1 neurons. Pflügers Arch 435: 99–106, 1997.[CrossRef][ISI][Medline]

15. Geiger JR, Melcher T, Koh DS, Sakmann B, Seeburg PH, Jonas P, and Monyer H. Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS. Neuron 15: 193–204, 1995.[CrossRef][ISI][Medline]

16. Gu JG, Albuquerque C, Lee CJ, and MacDermott AB. Synaptic strengthening through activation of Ca2+-permeable AMPA receptors. Nature 381: 793–796, 1996.[CrossRef][Medline]

17. Halabisky B, Friedman D, Radojicic M, and Strowbridge BW. Calcium influx through NMDA receptors directly evokes GABA release in olfactory bulb granule cells. J Neurosci 20: 5124–5134, 2000.[Abstract/Free Full Text]

18. Halpain S and Greengard P. Activation of NMDA receptors induces rapid dephosphorylation of the cytoskeletal protein MAP2. Neuron 5: 237–246, 1990.[CrossRef][ISI][Medline]

19. Hanson PI and Schulman H. Neuronal Ca2+/calmodulin-dependent protein kinases. Annu Rev Biochem 61: 559–601, 1992.[CrossRef][ISI][Medline]

20. Horning MS, Kwon B, Blakemore LJ, Spencer CM, Goltz M, Houpt TA, and Trombley PQ. {alpha}-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor subunit expression in rat olfactory bulb. Neurosci Lett 372: 230–234, 2004.[CrossRef][ISI][Medline]

21. Hume RI, Dingledine R, and Heinemann SF. Identification of a site in glutamate receptor subunits that controls calcium permeability. Science 253: 1028–1031, 1991.[Abstract/Free Full Text]

22. Iino M, Koike M, Isa T, and Ozawa S. Voltage-dependent blockage of Ca2+-permeable AMPA receptors by Joro spider toxin in cultured rat hippocampal neurones. J Physiol 496: 431–437, 1996.[ISI]

23. Iino M, Ozawa S, and Tsuzuki K. Permeation of calcium through excitatory amino acid receptor channels in cultured rat hippocampal neurones. J Physiol 424: 151–165, 1990.[Abstract/Free Full Text]

24. Isa T, Iino M, and Ozawa S. Spermine blocks synaptic transmission mediated by Ca2+-permeable AMPA receptors. Neuroreport 7: 689–692, 1996.[ISI][Medline]

25. Isa T, Itazawa S, Iino M, Tsuzuki K, and Ozawa S. Distribution of neurones expressing inwardly rectifying and Ca2+-permeable AMPA receptors in rat hippocampal slices. J Physiol 491: 719–733, 1996.[ISI][Medline]

26. Isaacson JS and Murphy GJ. Glutamate-mediated extrasynaptic inhibition: direct coupling of NMDA receptors to Ca2+-activated K+ channels. Neuron 31: 1027–1034, 2001.[CrossRef][ISI][Medline]

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

28. Itazawa SI, Isa T, and Ozawa S. Inwardly rectifying and Ca2+-permeable AMPA-type glutamate receptor channels in rat neocortical neurons. J Neurophysiol 78: 2592–2601, 1997.[Abstract/Free Full Text]

29. Jardemark K, Nilsson M, Muyderman H, and Jacobson I. Ca2+ ion permeability properties of (R,S){alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in isolated interneurons from the olfactory bulb of the rat. J Neurophysiol 77: 702–708, 1997.[Abstract/Free Full Text]

30. Jensen JB, Schousboe A, and Pickering DS. Development of calcium-permeable {alpha}-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptors in cultured neocortical neurons visualized by cobalt staining. J Neurosci Res 54: 273–281, 1998.[CrossRef][ISI][Medline]

31. Jia C and Halpern M. Calbindin D28k, parvalbumin, and calretinin immunoreactivity in the main and accessory olfactory bulbs of the gray short-tailed opossum, Monodelphis domestica. J Morphol 259: 271–280, 2004.[CrossRef][ISI][Medline]

32. Jia Y, Jeng JM, Sensi SL, and Weiss JH. Zn2+ currents are mediated by calcium-permeable AMPA/kainate channels in cultured murine hippocampal neurones. J Physiol 543: 35–48, 2002.[Abstract/Free Full Text]

33. Jonas P, Racca C, Sakmann B, Seeburg PH, and Monyer H. Differences in Ca2+ permeability of AMPA-type glutamate receptor channels in neocortical neurons caused by differential GluR-B subunit expression. Neuron 12: 1281–1289, 1994.[CrossRef][ISI][Medline]

34. Kamboj SK, Swanson GT, and Cull-Candy SG. Intracellular spermine confers rectification on rat calcium-permeable AMPA and kainate receptors. J Physiol 486: 297–303, 1995.[ISI][Medline]

35. Koh DS, Burnashev N, and Jonas P. Block of native Ca2+-permeable AMPA receptors in rat brain by intracellular polyamines generates double rectification. J Physiol 486: 305–312, 1995.[ISI][Medline]

36. Koh DS, Geiger JR, Jonas P, and Sakmann B. Ca2+-permeable AMPA and NMDA receptor channels in basket cells of rat hippocampal dentate gyrus. J Physiol 485: 383–402, 1995.[ISI][Medline]

37. Koike M, Iino M, and Ozawa S. Blocking effect of 1-naphthyl acetyl spermine on Ca2+-permeable AMPA receptors in cultured rat hippocampal neurons. Neurosci Res 29: 27–36, 1997.[CrossRef][ISI][Medline]

38. Konig N, Poluch S, Estabel J, Durand M, Drian MJ, and Exbrayat JM. Synaptic and non-synaptic AMPA receptors permeable to calcium. Jpn J Pharmacol 86: 1–17, 2001.[CrossRef][Medline]

39. Kyrozis A, Goldstein PA, Heath MJ, and MacDermott AB. Calcium entry through a subpopulation of AMPA receptors desensitized neighbouring NMDA receptors in rat dorsal horn neurons. J Physiol 485: 373–381, 1995.[ISI][Medline]

40. Laezza F, Doherty JJ, and Dingledine R. Long-term depression in hippocampal interneurons: joint requirement for pre- and postsynaptic events. Science 285: 1411–1414, 1999.[Abstract/Free Full Text]

41. Lerma J, Morales M, Ibarz JM, and Somohano F. Rectification properties and Ca2+ permeability of glutamate receptor channels in hippocampal cells. Eur J Neurosci 6: 1080–1088, 1994.[CrossRef][ISI][Medline]

42. Lisman J. A mechanism for the Hebb and the anti-Hebb processes underlying learning and memory. Proc Natl Acad Sci USA 86: 9574–9578, 1989.[Abstract/Free Full Text]

43. Malva JO, Vieira AP, Ambrosio AF, and Oliveira CR. Cobalt staining of hippocampal neurons mediated by non-desensitizing activation of AMPA but not kainate receptors. Neuroreport 14: 847–850, 2003.[CrossRef][ISI][Medline]

44. McBain CJ and Dingledine R. Heterogeneity of synaptic glutamate receptors on CA3 stratum radiatum interneurones of rat hippocampus. J Physiol 462: 373–392, 1993.[Abstract/Free Full Text]

45. Meucci O, Fatatis A, Holzwarth JA, and Miller RJ. Developmental regulation of the toxin sensitivity of Ca2+-permeable AMPA receptors in cortical glia. J Neurosci 16: 519–530, 1996.[Abstract/Free Full Text]

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

47. Okada T, Schultz K, Geurtz W, Hatt H, and Weiler R. AMPA-preferring receptors with high Ca2+ permeability mediate dendritic plasticity of retinal horizontal cells. Eur J Neurosci 11: 1085–1095, 1999.[CrossRef][ISI][Medline]

48. Pellegrini-Giampietro DE, Zukin RS, Bennett MV, Cho S, and Pulsinelli WA. Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats. Proc Natl Acad Sci USA 89: 10499–10503, 1992.[Abstract/Free Full Text]

49. Persechini A, Moncrief ND, and Kretsinger RH. The EF-hand family of calcium-modulated proteins. Trends Neurosci 12: 462–467, 1989.[CrossRef][ISI][Medline]

50. Petralia RS, Wang YX, Mayat E, and Wenthold RJ. Glutamate receptor subunit 2-selective antibody shows a differential distribution of calcium-impermeable AMPA receptors among populations of neurons. J Comp Neurol 385: 456–476, 1997.[CrossRef][ISI][Medline]

51. Pinato G and Midtgaard J. Dendritic sodium spikelets and low-threshold calcium spikes in turtle olfactory bulb granule cells. J Neurophysiol 93: 1285–1294, 2005.[Abstract/Free Full Text]

52. Pinato G and Midtgaard J. Regulation of granule cell excitability by a low-threshold calcium spike in turtle olfactory bulb. J Neurophysiol 90: 3341–3351, 2003.[Abstract/Free Full Text]

53. Pruss RM, Akeson RL, Racke MM, and Wilburn JL. Agonist-activated cobalt uptake identifies divalent cation-permeable kainate receptors on neurons and glial cells. Neuron 7: 509–518, 1991.[CrossRef][ISI][Medline]

54. Qin ZP, Ye SM, Du JZ, and Shen GY. Postnatal developmental expression of calbindin, calretinin and parvalbumin in mouse main olfactory bulb. Acta Biochim Biophys Sin (Shanghai) 37: 276–282, 2005.

55. Schoppa NE, Kinzie JM, Sahara Y, Segerson TP, and Westbrook GL. Dendrodendritic inhibition in the olfactory bulb is driven by NMDA receptors. J Neurosci 18: 6790–6802, 1998.[Abstract/Free Full Text]

56. Shepherd GM and Greer CA. The olfactory bulb. In: The Synaptic Organization of the Brain, edited by Shepherd GM. New York: Oxford, 1998, p. 133–169.

57. Smeraski CA, Dunwiddie TV, Diao L, and Finger TE. NMDA and non-NMDA receptors mediate responses in the primary gustatory nucleus in goldfish. Chem Senses 24: 37–46, 1999.

58. Toida K, Kosaka K, Heizmann CW, and Kosaka T. Synaptic contacts between mitral/tufted cells and GABAergic neurons containing calcium-binding protein parvalbumin in the rat olfactory bulb, with special reference to reciprocal synapses between them. Brain Res 650: 347–352, 1994.[CrossRef][ISI][Medline]

59. Toledo CA, Britto LR, Pires RS, Veenman CL, and Reiner A. Interspecific differences in the expression of the AMPA-type glutamate receptors and parvalbumin in the nucleus of Edinger-Westphal of chicks and pigeons. Brain Res 947: 122–130, 2002.[CrossRef][ISI][Medline]

60. Tóth K and McBain CJ. Afferent-specific innervation of two distinct AMPA receptor subtypes on single hippocampal interneurons. Nat Neurosci 1: 572–578, 1998.[CrossRef][ISI][Medline]

61. Trombley PQ and Blakemore LJ. Mammalian olfactory bulb neurons. In: The Neuron in Tissue Culture, edited by Haynes LW. Chichester, UK: Wiley, 1999, p. 585–592.

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

63. Van Den Bosch L, Vandenberghe W, Klaassen H, Van Houtte E, and Robberecht W. Ca2+-permeable AMPA receptors and selective vulnerability of motor neurons. J Neurol Sci 180: 29–34, 2000.[CrossRef][ISI][Medline]

64. Verdoorn TA, Burnashev N, Monyer H, Seeburg PH, and Sakmann B. Structural determinants of ion flow through recombinant glutamate receptor channels. Science 252: 1715–1718, 1991.[Abstract/Free Full Text]

65. Vickers JC, Huntley GW, Edwards AM, Moran T, Rogers SW, Heinemann SF, and Morrison JH. Quantitative localization of AMPA/kainate and kainate glutamate receptor subunit immunoreactivity in neurochemically identified subpopulations of neurons in the prefrontal cortex of the macaque monkey. J Neurosci 13: 2982–2992, 1993.[Abstract]

66. Washburn MS, Numberger M, Zhang S, and Dingledine R. Differential dependence on GluR2 expression of three characteristic features of AMPA receptors. J Neurosci 17: 9393–9406, 1997.[Abstract/Free Full Text]

67. Weiss JH and Sensi SL. Ca2+-Zn2+ permeable AMPA or kainate receptors: possible key factors in selective neurodegeneration. Trends Neurosci 23: 365–371, 2000.[CrossRef][ISI][Medline]

68. Wenthold RJ, Petralia RS, Blahos J II, and Niedzielski AS. Evidence for multiple AMPA receptor complexes in hippocampal CA1/CA2 neurons. J Neurosci 16: 1982–1989., 1996.[Abstract/Free Full Text]

69. Williams LR, Pregenzer JF, and Oostveen JA. Induction of cobalt accumulation by excitatory amino acids within neurons of the hippocampal slice. Brain Res 581: 181–189, 1992.[CrossRef][ISI][Medline]

70. Williams SR, Turner JP, Hughes SW, and Crunelli V. On the nature of anomalous rectification in thalamocortical neurones of the cat ventrobasal thalamus in vitro. J Physiol 505: 727–747, 1997.[CrossRef][ISI][Medline]

71. Yin H, Turetsky D, Choi DW, and Weiss JH. Cortical neurones with Ca2+ permeable AMPA/kainate channels display distinct receptor immunoreactivity and are GABAergic. Neurobiol Dis 1: 43–49, 1994.[CrossRef][Medline]

72. Yin HZ, Sensi SL, Carriedo SG, and Weiss JH. Dendritic localization of Ca2+-permeable AMPA/kainate channels in hippocampal pyramidal neurons. J Comp Neurol 409: 250–260, 1999.[CrossRef][ISI][Medline]

73. Zhang D, Sucher NJ, and Lipton SA. Co-expression of AMPA/kainate receptor-operated channels with high and low Ca2+ permeability in single rat retinal ganglion cells. Neuroscience 67: 177–188, 1995.[CrossRef][ISI][Medline]

74. Zou DJ, Greer CA, and Firestein S. Expression pattern of {alpha}CaMKII in the mouse main olfactory bulb. J Comp Neurol 443: 226–236, 2002.[CrossRef][ISI][Medline]




This article has been cited by other articles:


Home page
 J. Physiol.Home page
D. O. Pimentel and T. W. Margrie
Glutamatergic transmission and plasticity between olfactory bulb mitral cells
J. Physiol., April 15, 2008; 586(8): 2107 - 2119.
[Abstract] [Full Text] [PDF]