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Pharmacological characterization of ionotropic glutamate receptors in the zebrafish olfactory bulb
Neuroscience 122 (2003) 1037–1047
PHARMACOLOGICAL CHARACTERIZATION OF IONOTROPIC GLUTAMATE RECEPTORS IN THE ZEBRAFISH OLFACTORY BULB J. G. EDWARDS AND W. C. MICHEL*
are heterogeneously expressed at olfactory sensory neuron (OSN)-mitral/tufted cell, OSN-periglomerular (PG) cell, mitral cell–PG cell and mitral cell– granule cell synapses (Berkowicz et al., 1994; Bardoni et al., 1996; Isaacson, 2001). The NMDA receptors are more uniformly distributed across these cell types than are the AMPA/KA receptors (Petralia et al., 1994b; Montague and Greer, 1999). Differential AMPA/KA receptor distribution has been proposed as one mechanism mediating different roles for glutamate within the mammalian OB (Montague and Greer, 1999). Each iGluR subtype has unique properties including activation/deactivation kinetics, ion permeability, voltage-dependence and kinase regulation. Variations in the subunit composition of each of the three iGluR subtypes further contribute to unique cellular responses elicited by glutamate (Nakanishi et al., 1994). Since iGluR activation has been shown to contribute to other functions such as mediating long-term changes in gene expression, modulating second messenger signaling pathways and differentially affecting long-term potentiation/depression (reviewed by Ziff, 1999) it is of critical importance to correlate the response of different subtypes of iGluRs with their distribution and level of expression. In contrast to the numerous studies in the mammalian OB, much less is known about iGluR function in the fish OB. Several earlier studies hinted to the significance of iGluRs in the fish OB. All three iGluRs were found present in the glomerular and internal cell layers (Flynn et al., 1997; Maler and Monaghan, 1991). Antagonists to either AMPA/KA or NMDA receptors altered olfactory nerve-stimulated activity in the OB (Cinelli and Salzberg, 1990). We recently established the presence of functional AMPA/KA receptors and NMDA receptors in zebrafish OB neurons and demonstrated that odor-stimulated labeling of OB neurons could be completely blocked by a mixture of NMDA and AMPA/KA receptor antagonists (Edwards and Michel, 2002). Application of either NMDA or KA stimulated labeling of most bulbar neurons. However, direct activation and neuronal labeling by either of these iGluR agonists was not isolated from potential indirect activation resulting from glutamate release by agonist-activated neurons. Therefore, in the current study we examined the distribution of iGluR subtypes in greater detail by testing the ability of glutamate agonists, NMDA or KA, to stimulate activitydependent labeling in the presence of either an AMPA/KA receptor antagonist (6-cyano-7-nitroquinoxaline-2,3-dione; CNQX) or an NMDA receptor antagonist (D-2-amino-5phosphono-valeric acid; APV) and an AMPA receptor antagonist (sym 2206), respectively.
University of Utah School of Medicine, Department of Physiology, 410 Chipeta Way, Room 155, Salt Lake City, UT 84108-1297, USA
Abstract—The distribution of N-methyl-D-aspartate- (NMDA) and kainic acid- (KA) sensitive ionotropic glutamate receptors (iGluR) in the zebrafish olfactory bulb was assessed using an activity-dependent labeling method. Olfactory bulbs were incubated with an ion channel permeant probe, agmatine (AGB), and iGluR agonists in vitro, and the labeled neurons containing AGB were visualized immunocytochemically. Preparations exposed to 250 M KA in the presence of a NMDA receptor antagonist (D-2-amino-5-phosphono-valeric acid) and an ␣-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist (sym 2206), revealed KA receptor-mediated labeling of approximately 60 –70% of mitral cells, juxtaglomerular cells, tyrosine hydroxylase-positive cells and granule cells. A higher proportion of ventral olfactory bulb neurons were KA-sensitive. Application of 333 M NMDA in the presence of an AMPA/KA receptor antagonist (6-cyano-7-nitroquinoxaline-2,3-dione) resulted in NMDA receptor-mediated labeling of almost all neurons. The concentrations eliciting 50% of the maximal response (effective concentration: EC50s) for NMDA-stimulated labeling of different cell types were not significantly different and ranged from 148 M to 162 M. These results suggest that while NMDA receptors with similar binding affinities are widely distributed in the neurons of the zebrafish olfactory bulb, KA receptors are heterogeneously expressed among these cells and may serve unique roles in different regions of the olfactory bulb. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: agmatine, NMDA, kainate, sym 2206, activitydependent labeling.
Glutamate acting through metabotropic and ionotropic receptors serves as the primary excitatory neurotransmitter in the brain. Three ionotropic glutamate receptor (iGluR) subtypes, the N-methyl-D-aspartate (NMDA), ␣-amino-3hydroxyl-5-methylisoxazole-4-propionic acid (AMPA) and kainic acid (KA) receptors, mediate glutamate-induced fast synaptic currents. In the olfactory bulb (OB), these iGluRs *Corresponding author. Tel: ⫹1-801-585-5420; fax: ⫹1-801-5813476. E-mail address: [email protected] (W. C. Michel). Abbreviations: ACSF, artificial cerebrospinal fluid; AGB, agmatine; AMPA, ␣-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid; APV, D-2-amino-5-phosphono-valeric acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt; EC50, concentration eliciting 50% of the maximal response; GCL, granule cell layer; GL/MCL, glomerular layer/ mitral cell layer; IR, immunoreactivity; iGluRs, ionotropic glutamate receptors; JG, juxtaglomerular; KA, kainic acid; NMDA, N-methyl-D-aspartate; OB, olfactory bulb; OSN, olfactory sensory neuron; PG, periglomerular; TH, tyrosine hydroxylase; TTX, tetrodotoxin.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00544-X
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The current study advances understanding of the glutamatergic system in the teleost OB by characterizing the distribution of functional NMDA- and KA-stimulated neurons with an activity-dependent labeling technique. The labeling procedure takes advantage of the ability of agmatine (AGB) to permeate active iGluRs, while not permeating other cation channels, such as voltage activated Na⫹ and Ca2⫹ channels, or Na⫹-coupled transporters (Marc, 1999; Edwards and Michel, 2002). Using pharmacological techniques to isolate AGB labeling mediated by specific iGluR agonists, we report on the widespread cellular distribution of NMDA receptors and the more regional expression of KA receptors within the zebrafish OB. The variation in expression of iGluRs may have important functional implications with regards to odor-stimulated activity in the OB.
EXPERIMENTAL PROCEDURES Animal handling Zebrafish (Danio rerio; Hamilton Buchanan), obtained from a commercial supplier (Scientific Hatcheries, Huntington Beach, CA, USA), were housed in recirculating aquaria (40 – 80 L at 28 °C) with fluorescent lighting (12 h-light/dark cycle) and fed (Tetramin: Tetra Werke, Melle, Germany) daily. The University of Utah Institutional Animal Care and Use Committee approved all experimental procedures. Every effort was made to minimize the numbers of animals used and to ensure there was a minimal amount of suffering.
Activity-dependent labeling The OBs and telencephalons were dissected from decapitated zebrafish at room temperature in artificial cerebrospinal fluid (ACSF; see Solutions for composition). To begin an experiment OBs were transferred into 28.5 °C ACSF for 5 min. Then, OBs were incubated with the appropriate iGluR agonists and antagonists in ACSF, together with tetrodotoxin (TTX; 1 M) and 5 mM AGB for 10 min. To isolate NMDA receptor-mediated labeling, NMDA (12 M–3 mM) and CNQX (AMPA/KA receptor antagonist; 50 M) were employed. To isolate kainate (KA) receptor-mediated labeling, KA (2–250 M), APV (NMDA receptor antagonist; 100 M) and sym 2206 (AMPA receptor antagonist; 0.1–100 M) were employed. Finally, the OBs were rinsed (5 min) in ACSF to remove exogenous AGB, fixed (see Solutions for composition) and stored at 4 °C overnight. Control bulbs were exposed to 5 mM AGB in ACSF without iGluR agonists/antagonists. When used, GluR antagonists were included in the ACSF bathing the OBs during all three phases of the experiment.
Immunohistochemistry The fixed tissue was dehydrated, embedded in Eponate plastic, sectioned and processed for immunohistochemistry (Edwards and Michel, 2002). To minimize inherent variation in immunohistochemical procedures, OBs used in iGluR agonist concentration response experiments were processed as a group (Marc, 1999). Individual plastic-embedded OBs were first horizontally thick-sectioned (130 m) and a single section from the middle of each OB was mounted with up to six sections from other OBs onto a new plastic block. This block of grouped OB tissues was horizontally thin-sectioned (50 –100 nm) and each composite section containing cross-sections through all OBs was placed into a separate well of Teflon-coated spot slides (Erie Scientific, NH, USA). To reconstruct the location of stimulated neurons, KA-exposed prepara-
tions were coronally sectioned at 0.5 m and sections were collected at 12 m steps throughout the entire OB. Rabbit polyclonal anti-AGB, anti-GABA and anti-glutamate (1:100; Signature Immunologics, Salt Lake City, UT, USA) were applied to consecutive sections and visualized by silver intensification after treatment with a goat anti-rabbit nanogold secondary antibody (Amersham, Buckinghamshire, England). Anti-tyrosine hydroxylase (TH; 1:100; Chemicon, Temecula, CA, USA) was visualized with biotinylated goat anti-rabbit secondary antibody and streptavidinconjugated nanogold (1 nm) to improve the signal to noise ratio.
Image analysis Eight-bit digital images of stained material were captured using a Zeiss Axioplan 2 microscope fitted with bright field illumination and a CCD video camera connected to a Pentium-based personal computer using Axiovision software (Zeiss Inc., Germany). The images included a horizontal section of OBs exposed to NMDA and an anterior and posterior coronal section from KA-stimulated preparations (n⫽3 fish per condition). Cell types were identified based on size, location and pattern of glutamate, GABA and TH immunoreactivity (IR) from images (RGB images; Adobe Photoshop 5.5, San Jose, CA, USA) created from consecutive, registered sections using PCI software (Richmond Hill, Ontario, Canada). The mitral cells were found in the glomerular layer/mitral cell layer (GL/MCL) and characterized by large glutamate-positive and GABA-negative somas. Three smaller but similarly sized, GABA-positive and glutamate-positive interneuron types could be distinguished on the basis of TH IR and location. In the GL/MCL, the TH⫹ neurons contained high levels of cytoplasmic TH while the juxtaglomerular (JG) cells did not. The third interneuron type, the granule cells, was also TH-negative but found in the granule cell layer (GCL). Density-scaled images were then inverted, assigned quantifiable pixel-intensity values (0⫽white, 255⫽black) and cell types were independently masked using Image-Pro Plus 4.0 (Media Cybernetics Inc., Silver Spring, MD, USA). Each set of NMDA dose response data was normalized to correct for small variations in immunostaining frequently observed across material processed on different days. Normalization involved determining the average AGB pixel intensity value of the most weakly labeled group of cells, the granule cells exposed to 12 M NMDA. This value was subtracted from the average AGB IR levels for all cell types in all preparations from a given set of grouped OB tissue, so that granule cells exposed to 12 M NMDA were equal to zero. To avoid pseudoreplication effects from the large number of cells sampled in each preparation, statistical comparisons are based on the average AGB IR from all neurons of a given class in each preparation. The NMDA concentration eliciting 50% of the maximal response (EC50) for NMDA receptorstimulated labeling was determined from three separate dose response experiments and then averaged. Average NMDA receptor affinities were compared across cell types using an ANOVA (Tukey’s post hoc test). Cells were considered significantly stimulated by KA if their average AGB IR intensities were greater than three S.D.s above the background intensity. The average background intensity was determined for each image from three areas of interest at different locations in the GL/MCL that did not contain labeled cell bodies. A Z-test (Zar, 1984) was employed to determine significant (P⬍0.05) differences between the proportions of labeled cells in KA stimulated versus control preparations, and to compare between KA-stimulated cells located in the different regions of the OB. The average AGB IR intensities of KA-stimulated cells were compared for significance using an ANOVA (Tukey’s post hoc test).
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Fig. 1. (Caption overleaf).
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Solutions ACSF was composed of the following (in mM): NaCl, 131; NaHCO3, 20; KCl, 2; KH2PO4, 1.25; MgSO4, 2; CaCl2, 2.5; glucose, 10, adjusted to pH 7.4 after equilibration for at least 1 h with 95% O2/5% CO2. PB contained (in mM): NaH2PO4, 64; Na2HPO4, 15; CaCl2, 24; glucose, 167, with a pH of 7.4. Fixative contained 2.5% glutaraldehyde, 1% paraformaldehyde, 3% sucrose and 0.01% CaCl2 in 0.1 M PB, adjusted to pH 7.4. NMDA, NMDA receptor antagonist APV, kainate and AGB were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). The AMPA/KA receptor antagonist CNQX was purchased from Sigma-RBI Corporation (Natick, MA, USA). The AMPA receptor antagonist sym 2206 was purchased from Tocris (Ballwin, MO, USA). Sym 2206 and CNQX were dissolved in DMSO and ddH2O to 50 mM and 21 mM then diluted serially in ACSF to final working concentrations of 0.1–100 M and 50 M, respectively, on the day of use. APV was dissolved in 0.5 M NaOH to 10 mM, diluted in ACSF to a final working concentration of 100 M and adjusted to pH 7.4 on the day of use. NMDA and KA were dissolved in ACSF containing 5 mM AGB to a concentration of 3 mM and 250 M, respectively.
RESULTS NMDA receptor distribution and sensitivity Using AGB labeling as a measure of NMDA receptor activation we previously suggested that NMDA receptors were expressed by virtually all neurons within the OB (Edwards and Michel, 2002). In the current study we confirmed the expression of NMDA receptors in the interneuron populations and determined the EC50s for NMDA receptor-stimulated labeling of mitral cells, granule cells and pooled TH⫹ and JG cells. To isolate NMDA-stimulated labeling, experiments were conducted in the presence of TTX to block neuronal firing and the AMPA/KA receptor antagonist CNQX (50 M). NMDA stimulated the labeling of bulbar neurons in dose-dependent fashion (Fig. 1). At higher doses of NMDA (111 M–3 mM), virtually all neurons were labeled. However, NMDA stimulation significantly reduced TH IR making discrimination of JG and TH⫹ cells impossible. Consequently, data for interneurons located in the glomerular layer were pooled for all NMDA concentrations. Quantification of AGB IR revealed the dose dependent increase in NMDA-stimulated labeling of mitral cells, granule cells and JG/TH⫹ cells from 12 M to 333 M (Fig. 2). Neuronal labeling decreased at 1 mM and 3 mM NMDA relative to the peak labeling observed at 333 M (see Figs. 1 and 2), suggesting potential excitotoxic effects of NMDA stimulation at these higher concentrations. NMDA binding affinities were estimated using the EC50 as measured from raw data with 333 M taken as the maximum response. The EC50s for NMDA-stimulated labeling of mitral cells, granule cells and JG/TH⫹ cells were 161.6⫾12.6 M,
Fig. 2. Neurons in the zebrafish OB have similar EC50s for NMDA stimulated labeling. The data plotted represent the average AGB IR from three separate concentration response experiments (⫾S.E.M.). For each concentration response series, the average AGB pixel intensity was determined as the average AGB IR of all neurons from a given cell class in a representative section. Average AGB intensities (⫾S.E.M.) for mitral cells (■), JG cells/TH-positive cells (Œ) and granule cells (F) are graphed and were used to measure EC50s.
162.1⫾16.2 M and 148⫾24 M, respectively (⫾ 1 S.D., n⫽3 concentration response series each), and not significantly different from one another (P⬎0.05). At 333 M, NMDA stimulated a range of labeling intensities that varied across the cell types. The peak of AGB intensity was highest in mitral cells but the range of labeling intensities was broader for the granule cells and JG cells (Fig. 3). Kainate receptor distribution Our previous study had demonstrated that at high concentrations, KA stimulates labeling of virtually all bulbar neurons (Edwards and Michel, 2002). This earlier study did not, however, attempt to discriminate between KA-stimulated labeling via KA receptors versus AMPA receptors. In the current study, KA receptor activation was isolated from AMPA receptor activation using the AMPA receptor antagonist, sym 2206. An appropriate dose of sym 2206 was established by testing concentrations of KA (100 M) and AMPA (50 M) known to elicit near saturating responses (Hollmann et al., 1989; Marc, 1999; Edwards and Michel, 2002; data not shown) in the presence of several sym 2206 doses. All AMPA- (n⫽4) and KA- (n⫽2) stimulated labeling was blocked by 100 M sym 2206 (Fig. 4). In the presence of 25 M sym 2206, KA-stimulated labeling persisted (n⫽2) but AMPA-stimulated labeling was completely blocked in one of two preparations with only a few, lightlylabeled mitral cells observed in the other (see Fig. 4).
Fig. 1. NMDA stimulates agmatine (AGB) labeling of bulbar neurons in a dose-dependent fashion. (A) OB incubated with AGB (5 mM), CNQX (50 M), TTX (1 M) and increasing concentrations of NMDA (12 M–3 mM). (B) High magnification images of enclosed areas in A illustrate labeled cells. The corresponding RGB image of registered and inverted AGB (red), glutamate (green) and GABA (blue) IR was used to identify cell types. Mitral cells (M) ranged in color from heavily labeled yellow cells to unlabeled green cells. Granule cells are present in the GCL and JG and TH⫹ cells are present in the GL/MCL. Interneurons are either pink (heavily labeled; arrow) or purple (lightly labeled; arrowhead). Note differential labeling within the granule cell population in B (333 M and 3 mM). Lines in A (3 mM) denote GCL, GL/MCL and nerve layer (NL) boundaries. A representative control image is shown in Fig. 6A. Abbreviations: C, caudal; L, lateral; M, medial; R, rostral. Scale bars⫽100 m (A); B⫽25 m.
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to visualize KA-stimulated labeling, while assuring AMPA receptor blockade, we used 25 M sym 2206 for subsequent experiments. Kainate also stimulated labeling of OB neurons in a dose-dependent fashion (Fig. 5). The lowest concentration of KA that stimulated labeling was 4 M; maximal labeling was noted at 125–250 M KA. In contrast to NMDAstimulated preparations, many cells in each cell class in KA-stimulated preparations were unlabeled, even at high KA concentrations. In addition, TH IR persisted during stimulation with higher KA concentrations allowing us to examine TH⫹ and JG cell responses separately. Perhaps of greater interest was the finding that KA-mediated labeling was not distributed uniformly in the OB. The majority of labeled cells and labeled dendritic neuropil were present in the ventral region throughout the OB (Fig. 6). To explore the spatial distribution of KA-stimulated neurons we serial sectioned each entire OB and analyzed labeling on one anterior coronal section and one posterior coronal section from each preparation (n⫽3). To examine labeling in the dorsal and ventral aspects of the bulb, the bulb was divided in half at the point of maximum height. There was a significantly (P⬍0.01) higher percentage of neurons labeled in the ventral and dorsal regions of KAstimulated preparations than in control preparations (Fig. 7A). Similar distributions of KA-stimulated neurons in both these regions were noted in each of the planes of section; therefore, the data from both anterior and posterior sections were pooled. The proportion of KA-stimulated cells ranged from approximately 75–95% in the ventral OB versus 45–58% in the dorsal OB. The proportion of labeled TH⫹ cells was greater in the ventral OB but not significantly so. The average AGB intensity of mitral cells and JG cells that were considered KA-stimulated was also significantly (P⬍0.05) greater for those in the ventral versus the dorsal OB (Fig. 7B). Granule cells and TH⫹ cells showed a similar but non-significant trend in average AGB intensity.
DISCUSSION
Fig. 3. Histograms of AGB intensity reveal the range of NMDA-stimulated labeling in the different cell populations. Data are plotted for all cells analyzed in the 333 M NMDA-stimulated preparations (n⫽3), previously summarized in Fig. 2. (A) Mitral cells (n⫽87 cells) were the most intensely labeled neuron population but had the narrowest distribution. The maximum pixel intensity value for mitral cells was 240 (255⫽black) and therefore were not saturated. (B) Granule cell (n⫽670 cells) labeling intensity was more widely distributed showing a peak at approximately 150 and a left shoulder indicating a wide range of weakly stimulated cells. (C) Pooled TH⫹/JG cells (n⫽137 cells) also had a wide distribution of labeling intensities. The AGB intensities for each preparation exposed to 333 M NMDA were normalized by either adding or subtracting a constant offset such that the overall average intensities of granule cells exposed to 12 M NMDA were equal.
Lower sym 2206 concentrations (5 M, 1 M or 0.1 M) failed to block AMPA-mediated labeling (n⫽2). Therefore,
Although iGluRs have been studied in the fish locomotor regions (Ali et al., 2000), retina (O’Dell and Christensen, 1986; Ayoub et al., 1998; Maguire et al., 1998; Connaughton and Nelson, 2000; Van Epps et al., 2001), spinal cord (Brodin and Grillner, 1985) and vagal lobe (Smeraski et al., 1999), little is known about their cellular distribution or role in the OB. The current report describes the functional labeling of neurons in the zebrafish OB following NMDA receptor activation and demonstrates that stimulated neurons are distributed throughout the OB. On the other hand, neurons labeled via KA receptor activation were most abundant in the ventral OB. Our observation that NMDA receptor but not KA receptor activation reduced TH IR is a further indication that each receptor mediates unique cellular functions. NMDA receptor expression Several earlier studies hinted to the significance of NMDA receptors in the fish OB. The NR1 subunit of the NMDA
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Fig. 4. The AMPA receptor antagonist, sym 2206, was used to isolate kainate (KA) receptor-mediated labeling of bulbar neurons. (A, B) Sym 2206 at 100 M blocked both KA- (100 M) and AMPA- (50 M) stimulated AGB labeling. (C, D) At 25 M, sym 2206 continued to block AMPA-mediated labeling but not KA-mediated labeling. (E, F) Low concentrations of sym 2206 (5 M) failed to block either KA- or AMPA-mediated labeling. All sections are oriented coronally. Abbreviations: D, dorsal; L, lateral; M, medial; V, ventral. Scale bar⫽100 m.
receptor has been cloned from the electric fish (Bottai et al., 1997) and antibodies for it distinguished a few unidentified cells containing NMDA receptors in the platyfish OB (Flynn et al., 1997). NMDA receptors were also identified in the glomerular layer and internal cell layer of fish using autoradiography (Maler and Monaghan, 1991), suggesting
their expression by one or more cell types in the glomerular layer and by granule cells. Using optical imaging, NMDA receptor function in the OB was ascertained when the NMDA receptor antagonist APV increased olfactory nervestimulated activity (Cinelli and Salzberg, 1990). This was suggested to be a direct effect on mitral cells, although an
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Fig. 5. Kainate stimulates a dose-dependent increase in OB neuron labeling. The OBs incubated with AGB (5 mM), APV (100 M), TTX (1 M), sym 2206 (25 M) and (A) 4 M, (B) 67 M or (C) 250 M KA revealed a dose-dependent increase in neuronal labeling. Corresponding high magnification images of the enclosed areas, illustrate the labeling within some mitral cells (M; arrows). All sections are oriented transversely. Scale bars⫽100 m for low magnification; 25 m for high magnification images. Asterisks in the AGB IR images in A illustrate debris.
indirect effect mediated by inhibitory neurons is possible. We more recently demonstrated that NMDA receptor activation was required during odor stimulation of the OB and that all the neuron types of both the glomerular layer and internal cell layer of the zebrafish OB expressed NMDA receptors (Edwards and Michel, 2002). The current study confirms that NMDA receptors are widely expressed in fish OB neurons, including all interneurons. Based on affinity (EC50), all cell types seemingly express similar NMDA receptors. The EC50 values we report of approximately 150 M are higher than those reported for NMDA binding to either homomeric or heteromeric NMDA receptors (5 M–27 M; Moriyoshi et al., 1991; Laurie and Seeburg, 1994) or those that evoke currents in cultured OB neurons (29 M, saturating at about 300 M; Trombley and Westbrook, 1990). However, the EC50s for stimulated AGB labeling are similar to those reported for the visual sensory system using this same technique (Marc, 1999). In the OB of mammals, NMDA receptors are widely distributed in all cell classes (Moriyoshi et al., 1991). Electrophysiological experiments have demonstrated func-
tional NMDA receptors on mitral cells, granule cells and JG/PG cells (Berkowicz et al., 1994; Bardoni et al., 1996; Trombley and Westbrook, 1990; Aroniadou-Anderjaska et al., 1997, 1999). Histological techniques not only confirm NMDA receptor expression, but also provide the ability to distinguish NMDA receptor subtypes (NR1, NR2 [A–D] and NR3). The NR1 subunit and all four NR2 subtypes were identified within the OB using in situ hybridization (Watanabe et al., 1993). The NR1 subunit was located in all OB layers with the highest levels of expression in mitral cells and low to moderate levels of expression in the interneurons (Petralia et al., 1994a; Giustetto et al., 1997). Although these findings correspond well with our results showing: 1) low to moderate levels of NMDA receptormediated labeling in the interneurons and 2) mitral cells being more intensely labeled than the interneurons, one must be careful when comparing absolute AGB IR intensity, particularly between cell types. AGB labeling intensity is dependent upon cell volume, the number of iGluR channels present, the probability of a channel opening, and the permeability of AGB through these channels (Marc, 1999). Thus, the particular types of NMDA receptors expressed
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Fig. 6. Kainate receptors are not uniformly distributed in the OB. The left, center and right panels show AGB IR, an RGB composite of AGB or TH IR (red), glutamate IR (green) and GABA IR (blue) and a high magnification view of a portion of the RGB image, respectively. (A) No AGB labeling of neurons is noted in a section from the middle of a control OB, not exposed to KA. TH IR was mapped into the red channel of the control RGB images in place of AGB IR to illustrate TH-positive (TH⫹) cells (open arrowheads). TH⫹ cells have a thin red cytoplasmic ring, due to exclusion of TH from the nucleus. Anterior (B) and posterior (C) coronal sections of a KA- (250 M) stimulated preparation reveal the most intensely labeled neurons and dendritic neuropil were located in the ventral OB. RGB images in B and C, prepared as described in Fig. 1, reveal the presence of labeled (yellow) and unlabeled (green) mitral cells (M) and labeled (pink) and unlabeled (blue–purple) interneurons (arrowheads). KA stimulated preparations were tested in the presence of AGB (5 mM), APV (100 M), TTX (1 M) and sym 2206 (25 M). Dashed lines in B and C illustrate the dorsal–ventral division for cells in the GL/MCL. The GCL was similarly divided in half with a dorsal–ventral division (not shown). Asterisks in the AGB IR image in A denote debris. Scale bars⫽100 m low magnification in A and B; C: high magnification⫽25 m.
by a cell class would understandably contribute to the level of AGB labeling. However, because we noted similarities in the EC50s for NMDA-stimulated labeling of different cell
classes the NMDA receptors present cannot be distinguished based on NMDA sensitivity as reflected by levels of AGB permeation within each cell class.
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on either soma morphology or their location in the GCL, which determined their response profiles to excitatory synaptic input (Satou, 1990; Melendez-Ferro et al., 2001). Several types of interneurons are present in the mammalian glomerular layer based on their chemical composition and anatomical structure (Kosaka et al., 1997, 1998; Toida et al., 1998). Although we can distinguish two types of glomerular layer interneurons based on TH IR in control preparations (Edwards and Michel, 2002), loss of TH IR during NMDA stimulation prevents us from considering these two populations separately. Alternatively, differences in labeling between the interneurons could be due to variations in the ‘status’ of the cell. For example, differences in maturity levels of the interneurons may result in the differences in labeling, since interneurons are routinely replaced throughout adulthood (Byrd and Brunjes 1998, 2001). Membrane bi-stability as reported in mitral cells (Heyward et al., 2001) and recurrent inhibitory networks (Manor and Nadim, 2001) might also contribute to differential labeling. Kainate receptor expression
Fig. 7. Quantification of 250 m KA-stimulated neurons in dorsal and ventral regions of the OB. (A) Proportions (⫾S.D.) of labeled mitral, JG and granule cells located in the ventral OB were significantly greater than in the dorsal OB (Z test; ** P⬍0.01, * P⬍0.05). Numbers indicate the total number of neurons quantified for each cell class. While the dorsal OB contained more mitral cells and granule cells than the ventral OB the differences were not significant. Dorsal and ventral neurons are combined in control preparations, which show no differences between the two in AGB IR. (B) The average AGB intensity of ventrally located, KA-stimulated cells, was also significantly greater than those located dorsally. The data were normalized to account for differences in intensification by subtracting the average background AGB IR intensity from the overall average AGB IR intensity of each cell class from all three KA-stimulated preparations.
Our observation of a wide range of labeling of granule cells and JG cells is suggestive of differential NMDA receptor expression within the interneuron populations. Glial cells, misidentified as granule cells or glomerular interneurons, may contribute to this wide range of labeling; however, fish glia do not express NMDA receptors (Clasen et al., 1995). Heterogeneous labeling could indicate that our groupings of interneurons in the zebrafish OB actually comprise several subpopulations. In mammals, three types of granule cells have been described based on the projection pattern of their gemmules into the external plexiform layer (Mori et al., 1983) and differences in Toluidine Blue staining of the soma (Struble and Walters, 1982). In fish, two types of granule cells were distinguished based
In mammals, KA receptor subtypes (KA1–2; GluR5–7) appear to be heterogeneously expressed by mitral cells, PG cells and granule cells (Egebjerg et al., 1991; Herb et al., 1992; Petralia et al., 1994b; Montague and Greer, 1999). The functional significance of this heterogeneous distribution remains to be established. Fish express a KA receptor in the OB that is highly homologous to the mammalian GluR6 (Ziegra et al., 1992; Wo and Oswald, 1994) and distributed in the granule cell and glomerular layers (Maler and Monaghan, 1991). Consistent with the observation that KA receptors are expressed in the fish OB, application of an AMPA/KA receptor antagonist to the OB decreased activity of neurons not thought to be mitral cells (Cinelli and Salzberg, 1990). However, the specific cell types expressing KA receptors in the glomerular layer of the fish OB were not known until recently when we demonstrated that KA stimulates labeling of virtually all zebrafish OB neurons and implicated AMPA/KA receptors in odor-stimulated signaling in the four groups of neurons we identified (Edwards and Michel, 2002). When the OB was stimulated with KA in the absence of the AMPA receptor blocker virtually all neurons in both the dorsal and ventral regions were robustly labeled (Edwards and Michel, 2002). In the present study we differentiated KA receptor activation from AMPA receptor activation. In contrast to NMDA-stimulated labeling where all neurons were stimulated, even the highest concentration of KA tested stimulated labeling of only a portion of each neuron group. The variable labeling observed in the presence of the AMPA receptor antagonist sym 2206 suggests that some neurons in each group either express fewer KA receptors, different KA receptor subtypes or no KA receptors. The simplest explanation for the reduction in KA-stimulated labeling noted in the presence of the AMPA receptor antagonist, is expression patterns predominated by AMPA receptors in unlabeled cells and KA receptors in labeled cells. Direct examination of the expression patterns with immunocyto-
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chemical or in situ hybridization techniques is required to resolve this issue. Alternatively, sym 2206 has been shown to block all KA-stimulated labeling at higher concentrations (100 M) and approximately 40% of membrane currents mediated by recombinant KA2/GluR6 receptors (Pelletier et al., 1996; Bleakman et al., 2002). Therefore, we cannot exclude the possibilities that some KA receptor subtypes may be blocked by 25 M sym 2206, or alternatively, are impermeable to AGB. To our knowledge our study provides the first evidence for regional differences in KA receptor expression in the OB. Kainate receptor activation labeled significantly more neurons in the ventral OB. A portion of all neuron types was labeled suggesting that the regional difference in KAstimulated labeling was not due to heterogeneous stimulation of cell types. The fish OB is divided in two regions by the projection patterns of the mitral cell axons into the medial and lateral olfactory tracts (Satou, 1990). Whether the observed dorsal–ventral KA receptor distributions we observed map in any way to this anatomical division remains to be determined. Collectively, our findings indicate differential expression of KA receptors in the OB, and suggest that KA receptor activation may affect bulbar signaling differently than AMPA receptor activation.
CONCLUSION In conclusion, the current report reveals that cells expressing NMDA receptors are distributed throughout the OB, but that cells expressing KA receptors are preferentially distributed ventrally. This suggests that the NMDA receptor is an important component at most glutamatergic synapses, but differences in KA receptor expression may uniquely regulate specific synaptic functions in particular regions of the OB. Acknowledgements—We thank Dr. Larry J. Stensaas for his critical review of this manuscript. Supported by National Institutes of Health grants 2RO1-DC-01418 and 5PO1-NS-07938.
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J. G. Edwards and W. C. Michel / Neuroscience 122 (2003) 1037–1047 neuronal networks with recurrent inhibitory connectivity. J Neurosci 21:9460 –9470. Marc RE (1999) Mapping glutamatergic drive in the vertebrate retina with a channel-permeant organic cation. J Comp Neurol 407:47– 64. Melendez-Ferro M, Perez-Costas E, Rodriguez-Munoz R, GomezLopez MP, Anadon R, Rodicio MC (2001) GABA immunoreactivity in the olfactory bulbs of the adult sea lamprey Petromyzon marinus L. Brain Res 893:253–260. Montague AA, Greer CA (1999) Differential distribution of ionotropic glutamate receptor subunits in the rat olfactory bulb. J Comp Neurol 405:233–246. Mori K, Kishi K, Ojima H (1983) Distribution of dendrites of mitral, displaced mitral, tufted, and granule cells in the rabbit olfactory bulb. J Comp Neurol 219:339 –355. Moriyoshi K, Masu M, Ishii T, Shigemoto R, Mizuno N, Nakanishi S (1991) Molecular cloning and characterization of the rat NMDA receptor. Nature 354:31–37. Nakanishi S, Masu M, Bessho Y, Nakajima Y, Hayashi Y, Shigemoto R (1994) Molecular diversity of glutamate receptors and their physiological functions. Experientia Supplements 71:71–80. O’Dell T, Christensen BN (1986) N-methyl-D-aspartate receptors coexist with kainate and quisqualate receptors on single isolated catfish horizontal cells. Brain Res 381:359 –362. Pelletier JC, Hesson DP, Jones KA, Costa AM (1996) Substituted 1,2-dihydrophthalazines: potent, selective, and noncompetitive inhibitors of the AMPA receptor. J Med Chem 39:343–346. Petralia RS, Wang YX, Wenthold RJ (1994a) The NMDA receptor subunits NR2A and NR2B show histological and ultrastructural localization patterns similar to those of NR1. J Neurosci 14:6102– 6120. Petralia RS, Yokotani N, Wenthold RJ (1994b) Light and electron microscope distribution of the NMDA receptor subunit NMDAR1 in
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the rat nervous system using a selective anti-peptide antibody. J Neurosci 14:667–696. Satou M (1990) Synaptic organization, local neuronal circuitry, and functional segregation of the teleost olfactory bulb. Prog Neurobiol 34:115–142. Smeraski CA, Dunwiddie TV, Diao LH, Finger TE (1999) NMDA and non-NMDA receptors mediate responses in the primary gustatory nucleus in goldfish. Chem Senses 24:37–46. Struble RG, Walters CP (1982) Light microscope differentiation of two populations of rat olfactory bulb granule cells. Brain Res 236:237– 251. Toida K, Kosaka K, Heizmann CW, Kosaka T (1998) Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb: III. Structural features of calbindin D28K-immunoreactive neurons. J Comp Neurol 392:179 –198. Trombley PQ, Westbrook GL (1990) Excitatory synaptic transmission in cultures of rat olfactory bulb. J Neurophysiol 64:598 –606. Van Epps HA, Yim CM, Hurley JB, Brockerhoff SE (2001) Investigations of photoreceptor synaptic transmission and light adaptation in the zebrafish visual mutant nrc. Invest Ophthalmol Vis Sci 42:868 – 874. Watanabe M, Inoue Y, Sakimura K, Mishina M (1993) Distinct distributions of five N-methyl-D-aspartate receptor channel subunit mRNAs in the forebrain. J Comp Neurol 338:377–390. Wo ZG, Oswald RE (1994) Transmembrane topology of two kainate receptor subunits revealed by N-glycosylation. Proc Natl Acad Sci USA 91:7154 –7158. Zar JH (1984) Biostatistical analysis. Englewood Cliffs, New Jersey: Prentice-Hall, Inc. Ziegra CJ, Willard JM, Oswald RE (1992) Biochemical characterization of kainate receptors from goldfish brain. Mol Pharmacol 42: 203–209. Ziff EB (1999) Recent excitement in the ionotropic glutamate receptor field. Ann NY Acad Sci 868:465–473.
(Accepted 1 July 2003)
PHARMACOLOGICAL CHARACTERIZATION OF IONOTROPIC GLUTAMATE RECEPTORS IN THE ZEBRAFISH OLFACTORY BULB J. G. EDWARDS AND W. C. MICHEL*
are heterogeneously expressed at olfactory sensory neuron (OSN)-mitral/tufted cell, OSN-periglomerular (PG) cell, mitral cell–PG cell and mitral cell– granule cell synapses (Berkowicz et al., 1994; Bardoni et al., 1996; Isaacson, 2001). The NMDA receptors are more uniformly distributed across these cell types than are the AMPA/KA receptors (Petralia et al., 1994b; Montague and Greer, 1999). Differential AMPA/KA receptor distribution has been proposed as one mechanism mediating different roles for glutamate within the mammalian OB (Montague and Greer, 1999). Each iGluR subtype has unique properties including activation/deactivation kinetics, ion permeability, voltage-dependence and kinase regulation. Variations in the subunit composition of each of the three iGluR subtypes further contribute to unique cellular responses elicited by glutamate (Nakanishi et al., 1994). Since iGluR activation has been shown to contribute to other functions such as mediating long-term changes in gene expression, modulating second messenger signaling pathways and differentially affecting long-term potentiation/depression (reviewed by Ziff, 1999) it is of critical importance to correlate the response of different subtypes of iGluRs with their distribution and level of expression. In contrast to the numerous studies in the mammalian OB, much less is known about iGluR function in the fish OB. Several earlier studies hinted to the significance of iGluRs in the fish OB. All three iGluRs were found present in the glomerular and internal cell layers (Flynn et al., 1997; Maler and Monaghan, 1991). Antagonists to either AMPA/KA or NMDA receptors altered olfactory nerve-stimulated activity in the OB (Cinelli and Salzberg, 1990). We recently established the presence of functional AMPA/KA receptors and NMDA receptors in zebrafish OB neurons and demonstrated that odor-stimulated labeling of OB neurons could be completely blocked by a mixture of NMDA and AMPA/KA receptor antagonists (Edwards and Michel, 2002). Application of either NMDA or KA stimulated labeling of most bulbar neurons. However, direct activation and neuronal labeling by either of these iGluR agonists was not isolated from potential indirect activation resulting from glutamate release by agonist-activated neurons. Therefore, in the current study we examined the distribution of iGluR subtypes in greater detail by testing the ability of glutamate agonists, NMDA or KA, to stimulate activitydependent labeling in the presence of either an AMPA/KA receptor antagonist (6-cyano-7-nitroquinoxaline-2,3-dione; CNQX) or an NMDA receptor antagonist (D-2-amino-5phosphono-valeric acid; APV) and an AMPA receptor antagonist (sym 2206), respectively.
University of Utah School of Medicine, Department of Physiology, 410 Chipeta Way, Room 155, Salt Lake City, UT 84108-1297, USA
Abstract—The distribution of N-methyl-D-aspartate- (NMDA) and kainic acid- (KA) sensitive ionotropic glutamate receptors (iGluR) in the zebrafish olfactory bulb was assessed using an activity-dependent labeling method. Olfactory bulbs were incubated with an ion channel permeant probe, agmatine (AGB), and iGluR agonists in vitro, and the labeled neurons containing AGB were visualized immunocytochemically. Preparations exposed to 250 M KA in the presence of a NMDA receptor antagonist (D-2-amino-5-phosphono-valeric acid) and an ␣-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist (sym 2206), revealed KA receptor-mediated labeling of approximately 60 –70% of mitral cells, juxtaglomerular cells, tyrosine hydroxylase-positive cells and granule cells. A higher proportion of ventral olfactory bulb neurons were KA-sensitive. Application of 333 M NMDA in the presence of an AMPA/KA receptor antagonist (6-cyano-7-nitroquinoxaline-2,3-dione) resulted in NMDA receptor-mediated labeling of almost all neurons. The concentrations eliciting 50% of the maximal response (effective concentration: EC50s) for NMDA-stimulated labeling of different cell types were not significantly different and ranged from 148 M to 162 M. These results suggest that while NMDA receptors with similar binding affinities are widely distributed in the neurons of the zebrafish olfactory bulb, KA receptors are heterogeneously expressed among these cells and may serve unique roles in different regions of the olfactory bulb. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: agmatine, NMDA, kainate, sym 2206, activitydependent labeling.
Glutamate acting through metabotropic and ionotropic receptors serves as the primary excitatory neurotransmitter in the brain. Three ionotropic glutamate receptor (iGluR) subtypes, the N-methyl-D-aspartate (NMDA), ␣-amino-3hydroxyl-5-methylisoxazole-4-propionic acid (AMPA) and kainic acid (KA) receptors, mediate glutamate-induced fast synaptic currents. In the olfactory bulb (OB), these iGluRs *Corresponding author. Tel: ⫹1-801-585-5420; fax: ⫹1-801-5813476. E-mail address: [email protected] (W. C. Michel). Abbreviations: ACSF, artificial cerebrospinal fluid; AGB, agmatine; AMPA, ␣-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid; APV, D-2-amino-5-phosphono-valeric acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt; EC50, concentration eliciting 50% of the maximal response; GCL, granule cell layer; GL/MCL, glomerular layer/ mitral cell layer; IR, immunoreactivity; iGluRs, ionotropic glutamate receptors; JG, juxtaglomerular; KA, kainic acid; NMDA, N-methyl-D-aspartate; OB, olfactory bulb; OSN, olfactory sensory neuron; PG, periglomerular; TH, tyrosine hydroxylase; TTX, tetrodotoxin.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00544-X
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The current study advances understanding of the glutamatergic system in the teleost OB by characterizing the distribution of functional NMDA- and KA-stimulated neurons with an activity-dependent labeling technique. The labeling procedure takes advantage of the ability of agmatine (AGB) to permeate active iGluRs, while not permeating other cation channels, such as voltage activated Na⫹ and Ca2⫹ channels, or Na⫹-coupled transporters (Marc, 1999; Edwards and Michel, 2002). Using pharmacological techniques to isolate AGB labeling mediated by specific iGluR agonists, we report on the widespread cellular distribution of NMDA receptors and the more regional expression of KA receptors within the zebrafish OB. The variation in expression of iGluRs may have important functional implications with regards to odor-stimulated activity in the OB.
EXPERIMENTAL PROCEDURES Animal handling Zebrafish (Danio rerio; Hamilton Buchanan), obtained from a commercial supplier (Scientific Hatcheries, Huntington Beach, CA, USA), were housed in recirculating aquaria (40 – 80 L at 28 °C) with fluorescent lighting (12 h-light/dark cycle) and fed (Tetramin: Tetra Werke, Melle, Germany) daily. The University of Utah Institutional Animal Care and Use Committee approved all experimental procedures. Every effort was made to minimize the numbers of animals used and to ensure there was a minimal amount of suffering.
Activity-dependent labeling The OBs and telencephalons were dissected from decapitated zebrafish at room temperature in artificial cerebrospinal fluid (ACSF; see Solutions for composition). To begin an experiment OBs were transferred into 28.5 °C ACSF for 5 min. Then, OBs were incubated with the appropriate iGluR agonists and antagonists in ACSF, together with tetrodotoxin (TTX; 1 M) and 5 mM AGB for 10 min. To isolate NMDA receptor-mediated labeling, NMDA (12 M–3 mM) and CNQX (AMPA/KA receptor antagonist; 50 M) were employed. To isolate kainate (KA) receptor-mediated labeling, KA (2–250 M), APV (NMDA receptor antagonist; 100 M) and sym 2206 (AMPA receptor antagonist; 0.1–100 M) were employed. Finally, the OBs were rinsed (5 min) in ACSF to remove exogenous AGB, fixed (see Solutions for composition) and stored at 4 °C overnight. Control bulbs were exposed to 5 mM AGB in ACSF without iGluR agonists/antagonists. When used, GluR antagonists were included in the ACSF bathing the OBs during all three phases of the experiment.
Immunohistochemistry The fixed tissue was dehydrated, embedded in Eponate plastic, sectioned and processed for immunohistochemistry (Edwards and Michel, 2002). To minimize inherent variation in immunohistochemical procedures, OBs used in iGluR agonist concentration response experiments were processed as a group (Marc, 1999). Individual plastic-embedded OBs were first horizontally thick-sectioned (130 m) and a single section from the middle of each OB was mounted with up to six sections from other OBs onto a new plastic block. This block of grouped OB tissues was horizontally thin-sectioned (50 –100 nm) and each composite section containing cross-sections through all OBs was placed into a separate well of Teflon-coated spot slides (Erie Scientific, NH, USA). To reconstruct the location of stimulated neurons, KA-exposed prepara-
tions were coronally sectioned at 0.5 m and sections were collected at 12 m steps throughout the entire OB. Rabbit polyclonal anti-AGB, anti-GABA and anti-glutamate (1:100; Signature Immunologics, Salt Lake City, UT, USA) were applied to consecutive sections and visualized by silver intensification after treatment with a goat anti-rabbit nanogold secondary antibody (Amersham, Buckinghamshire, England). Anti-tyrosine hydroxylase (TH; 1:100; Chemicon, Temecula, CA, USA) was visualized with biotinylated goat anti-rabbit secondary antibody and streptavidinconjugated nanogold (1 nm) to improve the signal to noise ratio.
Image analysis Eight-bit digital images of stained material were captured using a Zeiss Axioplan 2 microscope fitted with bright field illumination and a CCD video camera connected to a Pentium-based personal computer using Axiovision software (Zeiss Inc., Germany). The images included a horizontal section of OBs exposed to NMDA and an anterior and posterior coronal section from KA-stimulated preparations (n⫽3 fish per condition). Cell types were identified based on size, location and pattern of glutamate, GABA and TH immunoreactivity (IR) from images (RGB images; Adobe Photoshop 5.5, San Jose, CA, USA) created from consecutive, registered sections using PCI software (Richmond Hill, Ontario, Canada). The mitral cells were found in the glomerular layer/mitral cell layer (GL/MCL) and characterized by large glutamate-positive and GABA-negative somas. Three smaller but similarly sized, GABA-positive and glutamate-positive interneuron types could be distinguished on the basis of TH IR and location. In the GL/MCL, the TH⫹ neurons contained high levels of cytoplasmic TH while the juxtaglomerular (JG) cells did not. The third interneuron type, the granule cells, was also TH-negative but found in the granule cell layer (GCL). Density-scaled images were then inverted, assigned quantifiable pixel-intensity values (0⫽white, 255⫽black) and cell types were independently masked using Image-Pro Plus 4.0 (Media Cybernetics Inc., Silver Spring, MD, USA). Each set of NMDA dose response data was normalized to correct for small variations in immunostaining frequently observed across material processed on different days. Normalization involved determining the average AGB pixel intensity value of the most weakly labeled group of cells, the granule cells exposed to 12 M NMDA. This value was subtracted from the average AGB IR levels for all cell types in all preparations from a given set of grouped OB tissue, so that granule cells exposed to 12 M NMDA were equal to zero. To avoid pseudoreplication effects from the large number of cells sampled in each preparation, statistical comparisons are based on the average AGB IR from all neurons of a given class in each preparation. The NMDA concentration eliciting 50% of the maximal response (EC50) for NMDA receptorstimulated labeling was determined from three separate dose response experiments and then averaged. Average NMDA receptor affinities were compared across cell types using an ANOVA (Tukey’s post hoc test). Cells were considered significantly stimulated by KA if their average AGB IR intensities were greater than three S.D.s above the background intensity. The average background intensity was determined for each image from three areas of interest at different locations in the GL/MCL that did not contain labeled cell bodies. A Z-test (Zar, 1984) was employed to determine significant (P⬍0.05) differences between the proportions of labeled cells in KA stimulated versus control preparations, and to compare between KA-stimulated cells located in the different regions of the OB. The average AGB IR intensities of KA-stimulated cells were compared for significance using an ANOVA (Tukey’s post hoc test).
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Fig. 1. (Caption overleaf).
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Solutions ACSF was composed of the following (in mM): NaCl, 131; NaHCO3, 20; KCl, 2; KH2PO4, 1.25; MgSO4, 2; CaCl2, 2.5; glucose, 10, adjusted to pH 7.4 after equilibration for at least 1 h with 95% O2/5% CO2. PB contained (in mM): NaH2PO4, 64; Na2HPO4, 15; CaCl2, 24; glucose, 167, with a pH of 7.4. Fixative contained 2.5% glutaraldehyde, 1% paraformaldehyde, 3% sucrose and 0.01% CaCl2 in 0.1 M PB, adjusted to pH 7.4. NMDA, NMDA receptor antagonist APV, kainate and AGB were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). The AMPA/KA receptor antagonist CNQX was purchased from Sigma-RBI Corporation (Natick, MA, USA). The AMPA receptor antagonist sym 2206 was purchased from Tocris (Ballwin, MO, USA). Sym 2206 and CNQX were dissolved in DMSO and ddH2O to 50 mM and 21 mM then diluted serially in ACSF to final working concentrations of 0.1–100 M and 50 M, respectively, on the day of use. APV was dissolved in 0.5 M NaOH to 10 mM, diluted in ACSF to a final working concentration of 100 M and adjusted to pH 7.4 on the day of use. NMDA and KA were dissolved in ACSF containing 5 mM AGB to a concentration of 3 mM and 250 M, respectively.
RESULTS NMDA receptor distribution and sensitivity Using AGB labeling as a measure of NMDA receptor activation we previously suggested that NMDA receptors were expressed by virtually all neurons within the OB (Edwards and Michel, 2002). In the current study we confirmed the expression of NMDA receptors in the interneuron populations and determined the EC50s for NMDA receptor-stimulated labeling of mitral cells, granule cells and pooled TH⫹ and JG cells. To isolate NMDA-stimulated labeling, experiments were conducted in the presence of TTX to block neuronal firing and the AMPA/KA receptor antagonist CNQX (50 M). NMDA stimulated the labeling of bulbar neurons in dose-dependent fashion (Fig. 1). At higher doses of NMDA (111 M–3 mM), virtually all neurons were labeled. However, NMDA stimulation significantly reduced TH IR making discrimination of JG and TH⫹ cells impossible. Consequently, data for interneurons located in the glomerular layer were pooled for all NMDA concentrations. Quantification of AGB IR revealed the dose dependent increase in NMDA-stimulated labeling of mitral cells, granule cells and JG/TH⫹ cells from 12 M to 333 M (Fig. 2). Neuronal labeling decreased at 1 mM and 3 mM NMDA relative to the peak labeling observed at 333 M (see Figs. 1 and 2), suggesting potential excitotoxic effects of NMDA stimulation at these higher concentrations. NMDA binding affinities were estimated using the EC50 as measured from raw data with 333 M taken as the maximum response. The EC50s for NMDA-stimulated labeling of mitral cells, granule cells and JG/TH⫹ cells were 161.6⫾12.6 M,
Fig. 2. Neurons in the zebrafish OB have similar EC50s for NMDA stimulated labeling. The data plotted represent the average AGB IR from three separate concentration response experiments (⫾S.E.M.). For each concentration response series, the average AGB pixel intensity was determined as the average AGB IR of all neurons from a given cell class in a representative section. Average AGB intensities (⫾S.E.M.) for mitral cells (■), JG cells/TH-positive cells (Œ) and granule cells (F) are graphed and were used to measure EC50s.
162.1⫾16.2 M and 148⫾24 M, respectively (⫾ 1 S.D., n⫽3 concentration response series each), and not significantly different from one another (P⬎0.05). At 333 M, NMDA stimulated a range of labeling intensities that varied across the cell types. The peak of AGB intensity was highest in mitral cells but the range of labeling intensities was broader for the granule cells and JG cells (Fig. 3). Kainate receptor distribution Our previous study had demonstrated that at high concentrations, KA stimulates labeling of virtually all bulbar neurons (Edwards and Michel, 2002). This earlier study did not, however, attempt to discriminate between KA-stimulated labeling via KA receptors versus AMPA receptors. In the current study, KA receptor activation was isolated from AMPA receptor activation using the AMPA receptor antagonist, sym 2206. An appropriate dose of sym 2206 was established by testing concentrations of KA (100 M) and AMPA (50 M) known to elicit near saturating responses (Hollmann et al., 1989; Marc, 1999; Edwards and Michel, 2002; data not shown) in the presence of several sym 2206 doses. All AMPA- (n⫽4) and KA- (n⫽2) stimulated labeling was blocked by 100 M sym 2206 (Fig. 4). In the presence of 25 M sym 2206, KA-stimulated labeling persisted (n⫽2) but AMPA-stimulated labeling was completely blocked in one of two preparations with only a few, lightlylabeled mitral cells observed in the other (see Fig. 4).
Fig. 1. NMDA stimulates agmatine (AGB) labeling of bulbar neurons in a dose-dependent fashion. (A) OB incubated with AGB (5 mM), CNQX (50 M), TTX (1 M) and increasing concentrations of NMDA (12 M–3 mM). (B) High magnification images of enclosed areas in A illustrate labeled cells. The corresponding RGB image of registered and inverted AGB (red), glutamate (green) and GABA (blue) IR was used to identify cell types. Mitral cells (M) ranged in color from heavily labeled yellow cells to unlabeled green cells. Granule cells are present in the GCL and JG and TH⫹ cells are present in the GL/MCL. Interneurons are either pink (heavily labeled; arrow) or purple (lightly labeled; arrowhead). Note differential labeling within the granule cell population in B (333 M and 3 mM). Lines in A (3 mM) denote GCL, GL/MCL and nerve layer (NL) boundaries. A representative control image is shown in Fig. 6A. Abbreviations: C, caudal; L, lateral; M, medial; R, rostral. Scale bars⫽100 m (A); B⫽25 m.
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to visualize KA-stimulated labeling, while assuring AMPA receptor blockade, we used 25 M sym 2206 for subsequent experiments. Kainate also stimulated labeling of OB neurons in a dose-dependent fashion (Fig. 5). The lowest concentration of KA that stimulated labeling was 4 M; maximal labeling was noted at 125–250 M KA. In contrast to NMDAstimulated preparations, many cells in each cell class in KA-stimulated preparations were unlabeled, even at high KA concentrations. In addition, TH IR persisted during stimulation with higher KA concentrations allowing us to examine TH⫹ and JG cell responses separately. Perhaps of greater interest was the finding that KA-mediated labeling was not distributed uniformly in the OB. The majority of labeled cells and labeled dendritic neuropil were present in the ventral region throughout the OB (Fig. 6). To explore the spatial distribution of KA-stimulated neurons we serial sectioned each entire OB and analyzed labeling on one anterior coronal section and one posterior coronal section from each preparation (n⫽3). To examine labeling in the dorsal and ventral aspects of the bulb, the bulb was divided in half at the point of maximum height. There was a significantly (P⬍0.01) higher percentage of neurons labeled in the ventral and dorsal regions of KAstimulated preparations than in control preparations (Fig. 7A). Similar distributions of KA-stimulated neurons in both these regions were noted in each of the planes of section; therefore, the data from both anterior and posterior sections were pooled. The proportion of KA-stimulated cells ranged from approximately 75–95% in the ventral OB versus 45–58% in the dorsal OB. The proportion of labeled TH⫹ cells was greater in the ventral OB but not significantly so. The average AGB intensity of mitral cells and JG cells that were considered KA-stimulated was also significantly (P⬍0.05) greater for those in the ventral versus the dorsal OB (Fig. 7B). Granule cells and TH⫹ cells showed a similar but non-significant trend in average AGB intensity.
DISCUSSION
Fig. 3. Histograms of AGB intensity reveal the range of NMDA-stimulated labeling in the different cell populations. Data are plotted for all cells analyzed in the 333 M NMDA-stimulated preparations (n⫽3), previously summarized in Fig. 2. (A) Mitral cells (n⫽87 cells) were the most intensely labeled neuron population but had the narrowest distribution. The maximum pixel intensity value for mitral cells was 240 (255⫽black) and therefore were not saturated. (B) Granule cell (n⫽670 cells) labeling intensity was more widely distributed showing a peak at approximately 150 and a left shoulder indicating a wide range of weakly stimulated cells. (C) Pooled TH⫹/JG cells (n⫽137 cells) also had a wide distribution of labeling intensities. The AGB intensities for each preparation exposed to 333 M NMDA were normalized by either adding or subtracting a constant offset such that the overall average intensities of granule cells exposed to 12 M NMDA were equal.
Lower sym 2206 concentrations (5 M, 1 M or 0.1 M) failed to block AMPA-mediated labeling (n⫽2). Therefore,
Although iGluRs have been studied in the fish locomotor regions (Ali et al., 2000), retina (O’Dell and Christensen, 1986; Ayoub et al., 1998; Maguire et al., 1998; Connaughton and Nelson, 2000; Van Epps et al., 2001), spinal cord (Brodin and Grillner, 1985) and vagal lobe (Smeraski et al., 1999), little is known about their cellular distribution or role in the OB. The current report describes the functional labeling of neurons in the zebrafish OB following NMDA receptor activation and demonstrates that stimulated neurons are distributed throughout the OB. On the other hand, neurons labeled via KA receptor activation were most abundant in the ventral OB. Our observation that NMDA receptor but not KA receptor activation reduced TH IR is a further indication that each receptor mediates unique cellular functions. NMDA receptor expression Several earlier studies hinted to the significance of NMDA receptors in the fish OB. The NR1 subunit of the NMDA
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Fig. 4. The AMPA receptor antagonist, sym 2206, was used to isolate kainate (KA) receptor-mediated labeling of bulbar neurons. (A, B) Sym 2206 at 100 M blocked both KA- (100 M) and AMPA- (50 M) stimulated AGB labeling. (C, D) At 25 M, sym 2206 continued to block AMPA-mediated labeling but not KA-mediated labeling. (E, F) Low concentrations of sym 2206 (5 M) failed to block either KA- or AMPA-mediated labeling. All sections are oriented coronally. Abbreviations: D, dorsal; L, lateral; M, medial; V, ventral. Scale bar⫽100 m.
receptor has been cloned from the electric fish (Bottai et al., 1997) and antibodies for it distinguished a few unidentified cells containing NMDA receptors in the platyfish OB (Flynn et al., 1997). NMDA receptors were also identified in the glomerular layer and internal cell layer of fish using autoradiography (Maler and Monaghan, 1991), suggesting
their expression by one or more cell types in the glomerular layer and by granule cells. Using optical imaging, NMDA receptor function in the OB was ascertained when the NMDA receptor antagonist APV increased olfactory nervestimulated activity (Cinelli and Salzberg, 1990). This was suggested to be a direct effect on mitral cells, although an
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Fig. 5. Kainate stimulates a dose-dependent increase in OB neuron labeling. The OBs incubated with AGB (5 mM), APV (100 M), TTX (1 M), sym 2206 (25 M) and (A) 4 M, (B) 67 M or (C) 250 M KA revealed a dose-dependent increase in neuronal labeling. Corresponding high magnification images of the enclosed areas, illustrate the labeling within some mitral cells (M; arrows). All sections are oriented transversely. Scale bars⫽100 m for low magnification; 25 m for high magnification images. Asterisks in the AGB IR images in A illustrate debris.
indirect effect mediated by inhibitory neurons is possible. We more recently demonstrated that NMDA receptor activation was required during odor stimulation of the OB and that all the neuron types of both the glomerular layer and internal cell layer of the zebrafish OB expressed NMDA receptors (Edwards and Michel, 2002). The current study confirms that NMDA receptors are widely expressed in fish OB neurons, including all interneurons. Based on affinity (EC50), all cell types seemingly express similar NMDA receptors. The EC50 values we report of approximately 150 M are higher than those reported for NMDA binding to either homomeric or heteromeric NMDA receptors (5 M–27 M; Moriyoshi et al., 1991; Laurie and Seeburg, 1994) or those that evoke currents in cultured OB neurons (29 M, saturating at about 300 M; Trombley and Westbrook, 1990). However, the EC50s for stimulated AGB labeling are similar to those reported for the visual sensory system using this same technique (Marc, 1999). In the OB of mammals, NMDA receptors are widely distributed in all cell classes (Moriyoshi et al., 1991). Electrophysiological experiments have demonstrated func-
tional NMDA receptors on mitral cells, granule cells and JG/PG cells (Berkowicz et al., 1994; Bardoni et al., 1996; Trombley and Westbrook, 1990; Aroniadou-Anderjaska et al., 1997, 1999). Histological techniques not only confirm NMDA receptor expression, but also provide the ability to distinguish NMDA receptor subtypes (NR1, NR2 [A–D] and NR3). The NR1 subunit and all four NR2 subtypes were identified within the OB using in situ hybridization (Watanabe et al., 1993). The NR1 subunit was located in all OB layers with the highest levels of expression in mitral cells and low to moderate levels of expression in the interneurons (Petralia et al., 1994a; Giustetto et al., 1997). Although these findings correspond well with our results showing: 1) low to moderate levels of NMDA receptormediated labeling in the interneurons and 2) mitral cells being more intensely labeled than the interneurons, one must be careful when comparing absolute AGB IR intensity, particularly between cell types. AGB labeling intensity is dependent upon cell volume, the number of iGluR channels present, the probability of a channel opening, and the permeability of AGB through these channels (Marc, 1999). Thus, the particular types of NMDA receptors expressed
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Fig. 6. Kainate receptors are not uniformly distributed in the OB. The left, center and right panels show AGB IR, an RGB composite of AGB or TH IR (red), glutamate IR (green) and GABA IR (blue) and a high magnification view of a portion of the RGB image, respectively. (A) No AGB labeling of neurons is noted in a section from the middle of a control OB, not exposed to KA. TH IR was mapped into the red channel of the control RGB images in place of AGB IR to illustrate TH-positive (TH⫹) cells (open arrowheads). TH⫹ cells have a thin red cytoplasmic ring, due to exclusion of TH from the nucleus. Anterior (B) and posterior (C) coronal sections of a KA- (250 M) stimulated preparation reveal the most intensely labeled neurons and dendritic neuropil were located in the ventral OB. RGB images in B and C, prepared as described in Fig. 1, reveal the presence of labeled (yellow) and unlabeled (green) mitral cells (M) and labeled (pink) and unlabeled (blue–purple) interneurons (arrowheads). KA stimulated preparations were tested in the presence of AGB (5 mM), APV (100 M), TTX (1 M) and sym 2206 (25 M). Dashed lines in B and C illustrate the dorsal–ventral division for cells in the GL/MCL. The GCL was similarly divided in half with a dorsal–ventral division (not shown). Asterisks in the AGB IR image in A denote debris. Scale bars⫽100 m low magnification in A and B; C: high magnification⫽25 m.
by a cell class would understandably contribute to the level of AGB labeling. However, because we noted similarities in the EC50s for NMDA-stimulated labeling of different cell
classes the NMDA receptors present cannot be distinguished based on NMDA sensitivity as reflected by levels of AGB permeation within each cell class.
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on either soma morphology or their location in the GCL, which determined their response profiles to excitatory synaptic input (Satou, 1990; Melendez-Ferro et al., 2001). Several types of interneurons are present in the mammalian glomerular layer based on their chemical composition and anatomical structure (Kosaka et al., 1997, 1998; Toida et al., 1998). Although we can distinguish two types of glomerular layer interneurons based on TH IR in control preparations (Edwards and Michel, 2002), loss of TH IR during NMDA stimulation prevents us from considering these two populations separately. Alternatively, differences in labeling between the interneurons could be due to variations in the ‘status’ of the cell. For example, differences in maturity levels of the interneurons may result in the differences in labeling, since interneurons are routinely replaced throughout adulthood (Byrd and Brunjes 1998, 2001). Membrane bi-stability as reported in mitral cells (Heyward et al., 2001) and recurrent inhibitory networks (Manor and Nadim, 2001) might also contribute to differential labeling. Kainate receptor expression
Fig. 7. Quantification of 250 m KA-stimulated neurons in dorsal and ventral regions of the OB. (A) Proportions (⫾S.D.) of labeled mitral, JG and granule cells located in the ventral OB were significantly greater than in the dorsal OB (Z test; ** P⬍0.01, * P⬍0.05). Numbers indicate the total number of neurons quantified for each cell class. While the dorsal OB contained more mitral cells and granule cells than the ventral OB the differences were not significant. Dorsal and ventral neurons are combined in control preparations, which show no differences between the two in AGB IR. (B) The average AGB intensity of ventrally located, KA-stimulated cells, was also significantly greater than those located dorsally. The data were normalized to account for differences in intensification by subtracting the average background AGB IR intensity from the overall average AGB IR intensity of each cell class from all three KA-stimulated preparations.
Our observation of a wide range of labeling of granule cells and JG cells is suggestive of differential NMDA receptor expression within the interneuron populations. Glial cells, misidentified as granule cells or glomerular interneurons, may contribute to this wide range of labeling; however, fish glia do not express NMDA receptors (Clasen et al., 1995). Heterogeneous labeling could indicate that our groupings of interneurons in the zebrafish OB actually comprise several subpopulations. In mammals, three types of granule cells have been described based on the projection pattern of their gemmules into the external plexiform layer (Mori et al., 1983) and differences in Toluidine Blue staining of the soma (Struble and Walters, 1982). In fish, two types of granule cells were distinguished based
In mammals, KA receptor subtypes (KA1–2; GluR5–7) appear to be heterogeneously expressed by mitral cells, PG cells and granule cells (Egebjerg et al., 1991; Herb et al., 1992; Petralia et al., 1994b; Montague and Greer, 1999). The functional significance of this heterogeneous distribution remains to be established. Fish express a KA receptor in the OB that is highly homologous to the mammalian GluR6 (Ziegra et al., 1992; Wo and Oswald, 1994) and distributed in the granule cell and glomerular layers (Maler and Monaghan, 1991). Consistent with the observation that KA receptors are expressed in the fish OB, application of an AMPA/KA receptor antagonist to the OB decreased activity of neurons not thought to be mitral cells (Cinelli and Salzberg, 1990). However, the specific cell types expressing KA receptors in the glomerular layer of the fish OB were not known until recently when we demonstrated that KA stimulates labeling of virtually all zebrafish OB neurons and implicated AMPA/KA receptors in odor-stimulated signaling in the four groups of neurons we identified (Edwards and Michel, 2002). When the OB was stimulated with KA in the absence of the AMPA receptor blocker virtually all neurons in both the dorsal and ventral regions were robustly labeled (Edwards and Michel, 2002). In the present study we differentiated KA receptor activation from AMPA receptor activation. In contrast to NMDA-stimulated labeling where all neurons were stimulated, even the highest concentration of KA tested stimulated labeling of only a portion of each neuron group. The variable labeling observed in the presence of the AMPA receptor antagonist sym 2206 suggests that some neurons in each group either express fewer KA receptors, different KA receptor subtypes or no KA receptors. The simplest explanation for the reduction in KA-stimulated labeling noted in the presence of the AMPA receptor antagonist, is expression patterns predominated by AMPA receptors in unlabeled cells and KA receptors in labeled cells. Direct examination of the expression patterns with immunocyto-
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chemical or in situ hybridization techniques is required to resolve this issue. Alternatively, sym 2206 has been shown to block all KA-stimulated labeling at higher concentrations (100 M) and approximately 40% of membrane currents mediated by recombinant KA2/GluR6 receptors (Pelletier et al., 1996; Bleakman et al., 2002). Therefore, we cannot exclude the possibilities that some KA receptor subtypes may be blocked by 25 M sym 2206, or alternatively, are impermeable to AGB. To our knowledge our study provides the first evidence for regional differences in KA receptor expression in the OB. Kainate receptor activation labeled significantly more neurons in the ventral OB. A portion of all neuron types was labeled suggesting that the regional difference in KAstimulated labeling was not due to heterogeneous stimulation of cell types. The fish OB is divided in two regions by the projection patterns of the mitral cell axons into the medial and lateral olfactory tracts (Satou, 1990). Whether the observed dorsal–ventral KA receptor distributions we observed map in any way to this anatomical division remains to be determined. Collectively, our findings indicate differential expression of KA receptors in the OB, and suggest that KA receptor activation may affect bulbar signaling differently than AMPA receptor activation.
CONCLUSION In conclusion, the current report reveals that cells expressing NMDA receptors are distributed throughout the OB, but that cells expressing KA receptors are preferentially distributed ventrally. This suggests that the NMDA receptor is an important component at most glutamatergic synapses, but differences in KA receptor expression may uniquely regulate specific synaptic functions in particular regions of the OB. Acknowledgements—We thank Dr. Larry J. Stensaas for his critical review of this manuscript. Supported by National Institutes of Health grants 2RO1-DC-01418 and 5PO1-NS-07938.
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(Accepted 1 July 2003)