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INTRODUCTION |
There is good evidence to suggest that the amino acid glutamate or a related compound mediates excitatory synaptic transmission within the vertebrate olfactory bulb (Berkowicz et al. 1994
; Jacobson and Hamberger 1986
; Jacobson et al. 1986
; Trombley and Westbrook 1990
; Wellis and Kauer 1993
). Several classes of glutamate receptors have been identified in this brain area, including
-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate-gated ion channels (glutamate receptors), and previous reports have shown that fast synaptic excitation of principal neurons as well as inhibitory interneurons to a large extent is mediated by activation of these receptors (Berkowicz et al. 1994
; Jacobson and Hamberger 1986
; Jacobson et al. 1986
; Trombley and Westbrook 1990
; Wellis and Kauer 1993
).
In the CNS most neurons appear to express AMPA receptors that are also activated by the neurotoxin kainic acid (KA). These receptors rapidly desensitize when exposed to AMPA, give nondesensitizing currents when activated by KA, and have generally been considered to have a low Ca2+ permeability. In recent reports, however, it have been demonstrated that several types of neurons, including cortical inhibitory interneurons, express AMPA receptors that are highly Ca2+ permeable (Jonas et al. 1994
). Although the functional significance of this high divalent cation permeability is unknown, it is conceivable that, similarly to the situation in N-methyl-D-aspartate receptors, Ca2+ influx through these AMPA receptors may trigger intracellular Ca2+-dependent processes that ultimately control neuronal signal transmission. In the olfactory bulb, where neuronal interaction to a large extent occurs through reciprocal dendrodendritic synapses between principal neurons and inhibitory interneurons (Shepherd 1972
), the existence of Ca2+-permeable AMPA receptors most likely would have a particularly profound influence on synaptic performance. It was therefore of importance to examine in more detail the divalent cation permeability of native AMPA receptors expressed in neurons from the olfactory bulb, and in this report we show that interneurons, acutely dissociated or in primary culture, mainly express AMPA receptors with low Ca2+ permeability.
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METHODS |
Acutely dissociated neurons
Neurons from rat olfactory bulb were acutely prepared by enzymatic dissociation as described previously (Jacobson 1991
; Jacobson and Li 1992
).
Cell cultures
Primary cultures of mixed astroglial/neuronal cells were prepared from olfactory bulbs of newborn rats. According to a previously described method (Nilsson et al. 1993
), the cells were grown for 12-14 days on poly-L-lysine-coated glass coverslips in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum, 5% glucose, 2.5% insulin, 1% glutamine, 250 000 IU/l penicillin, and 0.5% streptomycin, all at pH 7.3. On day 6 the cultures were treated with 10
5 M cytosine-1-b-D-arabinofuranoside to decrease the nonneural cell proliferation. The medium was changed once the 1st wk and thereafter every other day. The majority of the cells (~60%) in these cultures were type 1 astrocytes, whereas ~30% were neurons [as evaluated with immunohistochemical techniques (glial fibrillary acidic protein and neuron-specific enolase/neurofilaments)].
Recordings
Experiments were carried out with the use of the whole cell configuration of the patch-clamp technique (Hamill et al. 1981
). Patch pipettes were fabricated from thick-walled borosilicate glass (code number GC150-10, Clarke Electromedical Instruments, Reading, UK). The diameters and the resistances of the tips were ~2.5 µm and 5-15 M
, respectively. The estimated series resistance was always <50 M
(Jacobson 1991
) and holding potentials were corrected for voltage errors due to series resistance. These corrections, however, were in most cases small (<10 mV) and had negligible effects on values for reversal potentials. Measured liquid junction potentials were <3 mV and corrections were not made for these errors. All experiments were performed at room temperature (18-22°C). The pipettes (reference and measuring electrodes) used for recording contained (in mM) 120 KF, 1 MgCl2, 1 CaCl2, 11 ethylene glycol-bis(
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid, and 10 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES) (acutely isolated neurons) or 140 CsCl, 1 MgCl2, 1 CaCl2, 11 ethylene glycol-bis(
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid, and 10 HEPES (cultured neurons), pH in both solutions adjusted to 7.2 with KOH.
External buffers used in the present study had the following ionic composition (in mM): 140 NaCl; 5 KCl; 1 or 10 MgCl2; 0 (nominally free Ca2+ solution), 1 (normal external medium), or 10 CaCl2; 10 HEPES; and 10 glucose; pH adjusted to 7.4 with NaOH: 60 CaCl2, 10 HEPES, and 10 glucose, osmolarity corrected by adding sucrose, pH adjusted to 7.4 with KOH: and 140 N-methylglucamine, 10 CaCl2, 10 HEPES, and 10 glucose, pH adjusted to 7.4 with HCl.
Standard chemicals and enzymes for tissue dissociation were obtained from Sigma Chemical (St. Louis, MO).
Drugs were dissolved in appropriate external bathing medium and were applied by local microperfusion with the use of a parallel arrangement of fused silica capillaries or pressure injection (Jacobson 1991
; Jacobson and Li 1992
). The composition of the external medium was then controlled by the flow pipe system (acutely isolated cells) or full bath application (cultured neurons). Because of complex binding of kainic acid (KA) to Ca2+, the effective concentration of KA in 60 mM Ca2+ solution was adjusted according to Gu and Huang (1991)
.
Signals were recorded with a List L/M EPC-7 patch-clamp amplifier and were digitized and stored on videotape (20 kHz, PCM 2 A-D VCR adaptor, Medical Systems, New York, NY). For spectral analysis of whole cell currents, the signal from the video adaptor was low-pass filtered with an eight-pole Butterworth filter (1 kHz) and digitized at 2 kHz. Records were divided into 0.5-s blocks before calculation of the spectral density and the mean power spectrum was calculated by averaging all power spectra obtained from these blocks (>20). To obtain the power spectrum due to application of agonist, the spectrum obtained before application of the agonist was subtracted from that obtained during its application. The resulting power spectra were then fitted by a single or double Lorentzian function with the use of a least-squares Levenberg-Marquardt algorithm with proportional weighting (FigP software, Biosoft, Cambridge, UK). The quality of the fitted curve was judged from t ratios of fitted parameters, plot of residuals, and parameter correlations. Background noise was in most cells
1 order of magnitude lower than drug-induced noise. For acutely isolated cells apparent single-channel conductances were estimated according to
=
2/[(E
Er)Im(1
Po)] where
2 is the current variance, Im is the mean current, E is the holding potential, Er is the reversal potential, and Po is the probability of channels being open, which was estimated from the dose-response relationship.
2 was obtained from fitted power spectra. For cultured neurons, the expression above was rewritten to
2 =
(E
Er)(Im
Im2/n) and a fit was made directly to experimental data where Im and
2 were calculated from digitized records (0.25-s blocks) and n then provides an estimate of the number of channels.
Current-voltage (I-V) relationships were obtained either from current responses to KA at different holding potentials or from voltage ramps (
90 or
70 mV to 50 or 90 mV, duration 3 or 6 s, pClamp software, Axon Instruments). To obtain the I-V relationship due to application of agonist, the ramp obtained before application of the agonist was subtracted from that obtained during its application.
Data are presented as means ± SE. Differences between data sets were assessed with the use of either analysis of variance followed by a least significant difference test or, when appropriate, a t-test.
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RESULTS |
Dissociated cells
Similar to our previous studies, all recordings were made from cells that had cell bodies <20 µm diam and two or more processes of 10-100 µm (Jacobson 1991
; Jacobson and Li 1992
). As we have reported previously, application of KA to these cells generates a nondesensitizing current that is associated with an increase in current noise (Jacobson 1991
; Jacobson and Li 1992
) (Fig. 1). The dose-response relationship of this current was adequately fitted by the logistic equation with a EC50 value of 137 ± 30 (SE) µM and a Hill coefficient of 1.00 ± 0.15 (n = 4), and the amplitude of the current was attenuated by coapplication of kynurenate (250-500 µM) or 6-cyano-7-nitroquinoxaline-2,3-dione (10 µM) (data not shown).

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| FIG. 1.
Typical example of the effect of external Ca2+ ion concentration on current responses to kainic acid (KA) at different holding potentials and the corresponding current-voltage (I-V) curves. Left: whole cell currents produced by application of KA (100 µM, indicated by bars) to an acutely isolated interneuron held under voltage clamp ( 70 and +30 mV) in external bathing solutions containing different Ca2+ ion concentrations. Right: corresponding I-V relationship generated by a voltage ramp where the holding potential was changed from 70 to 40 mV during a 3-s period. The ramp response obtained before application of KA was subtracted from that obtained during exposure to KA and the signal was low-pass filtered at 500 Hz.
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The current response to KA had a near linear/slightly outwardly recifying I-V relationship with an average reversal potential at +7 mV in normal external medium (1 mM CaCl2 and 1 MgCl2, electrodes containing KF solution, Fig. 1, Table 1). Increasing the external Ca2+ ion concentration to 10 mM (in the presence of 1 mM MgCl2) markedly suppressed the amplitude (to 59% of the KA-evoked current in normal external medium at a holding potential of
70 mV), whereas omission of Ca2+ ions (nominally Ca2+-free solution) enhanced the amplitude (to 130% of the amplitude observed in normal external medium at a holding potential of
70 mV) of the current (Table 1, Fig. 1). These effects were not associated with any marked shift in reversal potentials (Table 1, Fig. 1). Because Mg2+ is the other major divalent cation present in vivo, it was of interest to investigate the effects of this ion on KA-evoked currents. Mg2+ ions (10 mM) were, however, less efficient than Ca2+ ions in causing a reduction of KA-evoked inward currents (P < 0.001 compared with the effects of Ca2+, Fig. 2, Table 1). In fact, the depressant effect of Mg2+ was marginally significant (i.e., P = 0.05). Raising the Mg2+ concentration to 10 mM (electrodes containing KF solution) gave no significant shift in the I-V relationship (Table 1).
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TABLE 1.
Effects of external divalent cation concentration on reversal potentials and current amplitudes acutely isolated interneurons
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| FIG. 2.
Example of the current response to KA obtained in high external Mg2+ ion concentration. Whole cell currents were produced by application of KA (100 µM, indicated by bars) to an acutely isolated interneuron held under voltage clamp in external bathing solutions containing low (1 mM) and high (10 mM) concentrations of Mg2+ ions. The concentration of Na+ and Ca2+ ions was kept constant at 140 and 1 mM, respectively. Holding potential: 70 mV.
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To observe a clear shift in the reversal potential for the evaluation of the Ca2+ permeability, we increased the external concentration of Ca2+ to 60 mM (0 mM Mg2+, electrodes containing KF solution), a procedure that not only further depressed the amplitude of the KA-evoked inward current but also caused a marked leftward shift in the reversal potential (Table 1, Fig. 3).

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| FIG. 3.
Typical example of the effect of high (60 mM) external Ca2+ concentration on the I-V relationship. Whole cell currents were produced by application of KA (100 µM). The voltage ramp changed the holding potential from 70 to 40 mV during a 3-s period. The ramp response obtained before application of KA was subtracted from that obtained during exposure to KA and the signal was low-pass filtered at 500 Hz.
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As we have reported elsewhere, analysis of the increase in current variance produced by KA gives power spectra that in most cases are best fitted by the sum of two Lorentzian functions (Jacobson 1991
; Jacobson and Li 1992
) (Fig. 4, Table 2). Changes in the external Ca2+ concentration had no significant effect on corner frequencies or the relative contribution to the total variance of the two components. The increase in total current variance produced by KA was, however, attenuated by increased external Ca2+ ion concentration, and there was also a reduction of the apparent single-channel conductance estimated from the fitted power spectra at 10 mM (Table 2). The poor variance signal obtained in external medium containing higher divalent cation concentrations prevented accurate measurements from being made in 60 mM Ca2+.

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| FIG. 4.
Example of the effect of external Ca2+ ion concentration on power spectra obtained from spectral analysis of the increase in current variance produced by administration of KA to acutely isolated interneurons. The spectra were best fitted by the sum of 2 Lorentzian functions ( ). Corner frequencies were 14 and 212 Hz (1 mM) and 10 and 175 Hz (10 mM). The corresponding apparent single-channel conductances estimated from the spectra were 5.1 and 3.4 pS.
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TABLE 2.
Effects of external Ca2+ ion concentration on parameters obtained by spectral analysis acutely isolated interneurons
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Cells in primary culture
All recordings were made from small-diameter neurons (<20 µm) with a bipolar appearance, and a majority of these cells produced inward nondesenzitizing currents when exposed to KA at a holding potential of
70 mV. Similar to acutely isolated neurons, the current responses to KA showed nearly linear/outwardly rectifying I-V relationships with a positive average reversal potential in normal external medium (containing 1 mM CaCl2 and 1 MgCl2, electrodes containing CsCl solution, Fig. 5, Table 3). Substituting N-methylglucamine for Na+ ions and increasing the Ca2+ ion concentration to 10 mM gave a leftward shift in the average reversal potential (range
40 to
68 mV, electrodes containing CsCl solution) and caused a marked reduction in the amplitude of the KA-evoked inward current (Fig. 5, Table 3). Of the 14 cells tested in N-methylglucamine buffer, 3 gave I-V relationships in response to KA that showed no clear inward current.

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| FIG. 5.
Typical examples of I-V relationships of KA (100 µM)-evoked currents obtained from cultured neurons in low Ca2+ (normal external medium) and Na-free high Ca2+ (N-methylglucamine was substituted for Na+). Data obtained from different cells. Holding potential: 70 mV.
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TABLE 3.
Effects of external Ca2+ ion concentration on reversal potentials and current amplitudes cultured neurons
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The values of the parameters obtained from an analysis of the increase in current variance produced by application of KA to cultured neurons were not markedly different from corresponding values for acutely isolated neurons. Corner frequencies were estimated to 19 ± 6 (SE) Hz and 109 ± 44 (SE) Hz (from 4 cells), respectively, and the apparent single-channel conductance was 4.1 ± 1.1 (SE) pS (from 4 cells).
Determination of Ca2+ permeability
For estimation of the Ca2+ permeability, the Goldman-Hodgkin-Katz constant field theory (Goldman 1943
; Hodgkin and Katz 1949
; Jan and Jan 1976
; Lewis 1979
; Lewis and Stevens 1979
) and the Eyring rate [symmetrical 2-barrier, 1-site (2B1S)] model (Eyring et al. 1949
; Lewis and Stevens 1979
) were used. Ion activities derived from Shatkay (1968)
and from the formula by Pitzer and Kim (1974)
were used in all calculations.
For acutely dissociated neurons, our average reversal potential data yielded an apparent pCa2+/pK+ permeability ratio of 0.18 when fitted by the constant field equation (assuming equal permeability to K+ and Na+ ions), whereas the Eyring 2B1S model gave a pCa2+/pK+ permeability ratio of 0.06. The constant field model, however, was unable to satisfactorily account for both reversal potential and current amplitudes. In contrast, our data yielded current amplitudes (in % of the amplitude obtained at
70 mV in normal external medium, 1 mM Ca2+ and 1 mM Mg2+) of 115, 59, and 8% in nominally Ca2+-free solution and 10 mM and 60 mM Ca2+ buffer, respectively, when fitted by the 2B1S model.
For the 2B1S model to reproduce obtained experimental data, it was necessary to include a surface charge (0.005 charges per A2) and to set the fraction of the total electrical potential drop from the outside to the binding site, i.e., the electrical distance at 0.24 (Gu and Huang 1991
; Lewis and Stevens 1979
). Although these values are somewhat arbitrary chosen, inclusion of a surface charge was necessary to satisfactorily reproduce current amplitudes at negative membrane potentials, whereas an electrical distance of <0.5 had to be included to get an outward rectification. The weak effect of Mg2+ (10 mM) on current amplitude was entirely explained by screening of the surface charge (predicted current amplitude 85% of amplitude in normal external medium, ions assumed impermeant). The energy barrier heights (Gb) and well depths (Gw) used were (in RT units) Gb(Na+) = Gb(K+) = 10, Gb(Ca2+) = 12.75, Gw(Na+) = Gw(K+) =
3.5, and Gw(Ca2+) =
5.85. As before, equal permeability to K+and Na+ was assumed.
When our average data for cultured neurons were fitted, the 2B1S model gave a pCa2+/pK+ permeability ratio of 0.14, a value not markedly different from the corresponding value for acutely isolated neurons. The Gb and Gw values used were then (in RT units) Gb(Na+) = Gb(K+) = 10.1, Gb(Ca2+) = 12.1, Gw(Na+) = Gw(K+) =
3.05, and Gw(Ca2+) =
7.94. The corresponding value obtained from the constant field equation was 0.40.
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DISCUSSION |
In the present study recordings were made from small neurons, which, as has been reported previously (Jacobson 1991
; Jacobson and Li 1992
; Trombley and Westbrook 1990
), most likely correlate with inhibitory granule/periglomerular cells, the most numerous cell types in the olfactory bulb (Shepherd 1972
). A majority of these cells expresses glutamate receptors, and to activate the AMPA type of glutamate receptors and to circumvent problems due to desensitization we used KA as the agonist. Although we cannot rule out the possibility that we also activated KA receptors, currents flowing through these receptors must have been small, because 1) it was not possible to detect transient KA-activated currents even after administration of 6-cyano-7-nitroquinoxaline-2,3-dione or after desensitization of AMPA receptors (Jacobson and Li 1992
) and 2) I-V relationships displayed an outward, not an inward, rectification, which would have been expected if substantial currents were flowing through KA receptors (Lerma et al. 1993
).
Similar to other reports, we found a marked reduction of KA-evoked current amplitudes when the the Ca2+ concentration was increased from 1 to 10 mM, suggesting that interactions between ions are important in the ion permeation process (Gu and Huang 1991
). Consistent with this view, spectral analysis of the increase in current variance produced by KA in 10 mM Ca2+ indicated a reduced apparent single-channel conductance compared with normal solution and gave no indication of significantly altered gating characteristics. Although KA is known to produce openings with multiple conductance levels, the conductance estimated from current variance will give a weighted average of all these levels (Colquhoun and Hawkes 1977
; Cull-Candy and Usowicz 1987
). The decrease in the apparent single-channel conductance also shows that the effect of Ca2+ was just not simply due to the decrease in Po, because this would have caused an increase of the apparent conductance {according to
=
2/[(E
Er)Im(1
Po)]}, not the opposite. It has also previously been shown that an increase in the external concentration of Ca2+ does not affect the EC50 value for KA (Gu and Huang 1991
).
The estimation of the Ca2+ permeability was based on two models: the Goldman-Hodgkin-Katz constant field theory (Goldman 1943
; Hodgkin and Katz 1949
; Jan and Jan 1976
; Lewis 1979
; Lewis and Stevens 1979
) and the Eyring rate 2B1S model (Eyring et al. 1949
; Lewis and Stevens 1979
).
With the use of the constant field equation, the pCa2+/pK+ permeability ratio was estimated to 0.18 for freshly isolated interneurons and 0.40 for cultured interneurons, values that are close to those described for low-divalent-cation-permeable AMPA receptors expressed in other CNS neurons (Gu and Huang 1991
; Iino et al. 1990
, 1991
, 1994
; Jonas et al. 1994
). Although the constant field equation assumes independent ion fluxes, interactions can be accounted for through screening of surface charges, and previous authors have considered the existence of such charges in attempts to explain blocking actions of divalent cations and deviations from predictions made by the constant field equation (Ascher and Nowak 1988
; Iino et al. 1990
; Randle et al. 1988
). Despite the inclusion of a surface charge, however, the constant field equation was unable to satisfactorily account for both reversal potential and current amplitudes and we therefore fitted our data by the use of the simple Eyring 2B1S rate model. In this model, the ion channel is viewed as an aqeous pore with a binding site inside, which ions bind to on their way through the channel, thereby occluding other ions from the pore. Ions that bind to the site with higher affinity (i.e., Ca2+) pass through more slowly and block the ion species with lower affinities for the site (i.e., Na+ and K+). The permeability ratio pCa2+/pK+, defined as exp[
Gb(Ca2+)]/exp[
Gb(K+)], was 0.06 for acutely dissociated interneurons and 0.14 for cultured interneurons. Although in general the values of the parameters of rate models are not uniquely determined, inclusion of the rectification properties and estimates of single-channel conductances severely restricts the parameter space and it can be estimated that to reproduce all experimental data the pCa2+/pK+ permeability ratio has to be <0.18. The finding that the simple 2B1S model reproduces experimental data reasonably well is in close agreement with the results from Gu and Huang (1991)
and gives further support for the view that ion competion and surface charges are important determinants for ion permeation through glutamate receptors. Because ion competition in the permeation process cannot be ignored, values for the permeability ratio determined by the constant field equation would therefore be overestimated.
In conclusion, our results suggest that a large population of the interneurons in the rat olfactory bulb mainly expresses AMPA receptors with low permeability to Ca2+ and that the low permeability partly results from a strong interaction with the ion channel pore. Whether this interaction, which is sufficiently strong at physiological concentrations of Ca2+ to influence ion permeation properties, has any functional implication still remains to be elucidated. A low Ca2+ permeability of the neuronal AMPA receptors in the olfactory bulb is supported by studies in which in situ hybridization is used (Hollman and Heinemann 1994), which show a large abundance of the mRNA encoding glutamate receptor 2, the subunit that in its edited form gives low-Ca2+-permeable AMPA receptors. However, it is not known to what extent editing occurs and it also has not been established whether the glutamate receptor 2-mRNA fraction is fully translated to functional receptor/ion channel complexes.
Because none of the acutely isolated interneurons tested in buffers containing 60 mM Ca2+ (total 7) gave any sign of high divalent cation permeability, it means that, at P < 0.05, <23% of the test population would have been highly permeable. The corresponding figure for cultured interneurons is then 20%, and if all tested neurons are considered to belong to the same population, the amount would be 16%.
In view of our results and given the fact that interneurons in the olfactory bulb also respond to N-methyl-D-aspartate (Jacobson and Li 1992
; Trombley and Westbrook 1990
), it appears likely that N-methyl-D-aspartate receptors and voltage-dependent Ca2+ channels provide for a major route of synaptic, including dendrodendritic synaptic Ca2+ ion influx. However, we cannot rule out the possibility that, because of the minimal influence from cell isolation/recording conditions that conclude a loss of activity from distal dendritic processes, receptors located on distant processes have different ion permeability properties.