Theta-Frequency Facilitation of AMPA Receptor-Mediated Synaptic Currents in the Principal Cells of the Medial Septum

John N. Armstrong and Brian A. MacVicar

Neuroscience Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
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DISCUSSION
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Armstrong, John N. and Brian A. MacVicar. Theta-Frequency Facilitation of AMPA Receptor-Mediated Synaptic Currents in the Principal Cells of the Medial Septum. J. Neurophysiol. 85: 1709-1718, 2001. Recent evidence suggests that Ca2+-permeable AMPA receptors display rapid, short-lasting current facilitation. In this study, we investigated the properties of AMPA receptor-mediated synaptic currents in medial septal neurons of the rat in an in vitro slice preparation. Immunocytochemistry with a selective antibody to the GluR2 subunit revealed that both choline acetyltransferase-containing and parvalbumin-containing neurons of the medial septum express no detectable GluR2 subunit immunoreactivity. We used whole cell voltage-clamp recordings to measure synaptically evoked AMPA receptor-mediated currents from medial septal neurons following stimulation of midline afferents. The GYKI 52466 (50 µM)- and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (20 µM)-sensitive AMPA receptor-mediated component of the synaptic response was isolated by blocking GABAA- and N-methyl-D-aspartate receptor-mediated currents with 30 µM bicuculline and 100 µM 2-amino-5-phosphonovaleric acid, respectively. In some cases, patched cells were filled with Lucifer yellow (0.1%) and imaged using 2-photon laser scanning microscopy. AMPA receptor-mediated currents that were observed in large medial septal neurons (20-30 µm) displayed rectification. These currents were sensitive to external application of philanthotoxin-343 (PhTx-343, 50 µM), a potent, high-affinity antagonist of Ca2+-permeable, GluR2-lacking AMPA receptors. Rectifying AMPA receptor-mediated currents also displayed a rapid increase in amplitude when evoked five times at low frequency such as 6 Hz. In contrast to currents observed in large medial septal neurons, AMPA-receptor mediated currents evoked in the remaining small (8-11 µm) neurons were nonrectifying and displayed rapid synaptic depression when stimulated five times at 6 Hz. The currents evoked in these cells were unaffected by external application of PhTx-343 and were therefore GluR2-containing AMPA receptors. The results of the present study demonstrate that the principal projection neurons of the medial septum contain PhTx-343-sensitive, GluR2-lacking AMPA receptors that display rapid current facilitation when stimulated at low frequencies.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

alpha -Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors mediate fast, excitatory glutamatergic synaptic transmission throughout the CNS. A single AMPA receptor is a heteromeric tetramer containing any of the four AMPA receptor subunits, GluR1-4 (Rosenmund et al. 1998). Of the four subunits the GluR2 subunit appears to be most abundant (Geiger et al. 1995). When the GluR2 subunit is present, the channel properties of the GluR2 subunit dominate the entire heteromeric receptor complex (Boulter et al. 1990; Nakanishi et al. 1990). Therefore several unusual features of AMPA receptor mediated synaptic transmission are observed when GluR2 subunits are not present. These unusual features include Ca2+ permeability (Burnashev et al. 1992; Hollmann et al. 1991; Hume et al. 1991), rectification (Hollmann et al. 1991; Rozov et al. 1998; Washburn et al. 1997) and sensitivity to block by external polyamines (Brackley et al. 1993; Herlitze et al. 1993; Washburn et al. 1997). Ca2+ impermeability is generated in the AMPA receptor through RNA editing of the GluR2 subunit (Hume et al. 1991; Seeburg 1996). Virtually all GluR2 subunits appear to be edited in the rat brain (Jonas and Burnashev 1995; Seeburg 1996). Therefore AMPA receptors lacking GluR2 subunits are the only native AMPA receptors that are Ca2+ permeable (Hollmann and Heinemann 1994; Washburn et al. 1997).

Rozov et al. (1998) recently showed that recombinant, calcium permeable AMPA receptors, display rapid, short lasting (<5 s) activity-dependent facilitation when stimulated at low frequencies (<50 Hz). Both the inward rectification and facilitation were the result of block of open AMPA receptor channels by intracellular polyamines (Rozov et al. 1998). The following study was designed to determine whether similar low-frequency facilitation could be synaptically activated at calcium permeable AMPA receptors in the medial septum.

Previous studies have suggested that some medial septal neurons possess GluR2-lacking, Ca2+-permeable AMPA receptors. First, AMPA receptor activation caused Ca2+ influx (Schneggenburger et al. 1993a,b; Waters and Allen 1998). Second, as few as 15% of these neurons contain detectable levels of GluR2/3 immunoreactivity (Page and Everitt 1995), whereas all medial septal neurons have been reported to contain GluR4 AMPA receptor subunits. Unfortunately the determination of the distribution of GluR2-lacking AMPA receptors has been hindered by the lack of a GluR2 antibody that did not show some high degree of cross-reactivity with GluR3.

In the following study, we present evidence from immunocytochemistry using a GluR2-selective antibody that the principal projection neurons of the medial septum possess no detectable GluR2 immunoreactivity. AMPA receptor-mediated synaptic currents evoked in these neurons rectify are sensitive to blockade by philanthotoxin-343 (PhTx-343) and undergo rapid current facilitation when stimulated at 6 Hz.


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Protein isolation, SDS-PAGE, and Western blots

Total protein was isolated from rat brain tissue that was rapidly dissected and frozen on dry ice. Each 100 mg of tissue was then sonicated in 200 µl of 0.32 M sucrose and refrozen in 10-µl aliquots. Approximately 25 µg of protein was then diluted in sample buffer, electrophoretically separated on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) (Laemmli 1970), transferred to a Immobilon-P PVDF membrane (Millipore, Bedford, MA), and blotted with a rabbit anti-GluR2 polyclonal antibody (Chemicon, Temecula, CA) at 1:1,000 Tris-buffered saline. This antibody was raised against amino acid residues 827-842 from the C-terminus of GluR2 and was absorbed against a cross-contaminating 6-amino-acid GluR3 peptide to remove any cross reactivity to GluR3 (see Petralia et al. 1997). The immunoreactive signal was detected using a peroxidase-labeled goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) and an enhanced chemiluminesence kit (Amersham Pharmacia Biotech, Piscataway, NJ).

Immunocytochemistry

The immunocytochemistry protocol used in the present study was based on the protocol described by Sloviter and Nilaver (1987) as reported previously (Armstrong et al. 1996). Briefly, 10- to 30-day-old rats were anesthetized with pentobarbital sodium (100 mg/kg) and transcardially perfused with phosphate-buffered saline (PBS, pH 7.4) containing 0.1% sodium nitrite followed by 4% paraformaldehyde/0.4% glutaraldehyde. After 3 h additional glutaraldehyde-free fixation, the tissue was sectioned in the coronal or sagittal plane at 25 or 50 µm on a vibrating microtome (VT100, Leica Microsystems, Willowdale, ON). Collected sections were then washed in PBS and incubated in 1% hydrogen peroxide for 30 min. After rinsing several times in PBS, sections were incubated for 1 h in PBS containing 5% normal donkey serum. The sections were then transferred to a fresh solution of PBS containing 0.005% BSA and rabbit anti-GluR2 (1:3,000; Chemicon) and incubated overnight at room temperature. The next day, sections were rinsed several times in PBS and incubated for 1 h in PBS containing biotinylated donkey anti-rabbit IgG (1:1000, Jackson Immunoresearch Laboratories, West Grove, PA). Sections were then washed several times in PBS and incubated in streptavidin-peroxidase complex (1:1,000; ABC Elite; Vector Laboratories) for 1 h. Sections were washed again several times in PBS and reacted for 15 min in PBS containing 0.003 mgmL-1 glucose oxidase, 0.4 mgmL-1 ammonium chloride, 2 mgmL-1 beta (D)+glucose, and 0.01% diaminobenzidine-tetrachloride (DAB, Sigma, St. Louis, MO). Occasionally cobalt chloride was added to this solution to produce a black reaction product that was easier to visualize than the standard brown reaction product. Sequential black/brown DAB was carried out by first completing an entire reaction using the cobalt chloride enhanced DAB followed by an entire reaction using standard (brown) DAB. Finally, following 15 min in DAB solution, the sections were washed several times in PBS and mounted on gelatin-, chrom-alum-coated slides, air-dried overnight, dehydrated in a series of ethanols, cleared in xylene, and coverslipped with Permount (Sigma).

For co-localization studies, immunocytochemistry was first carried out for GluR2 as described above using a Cy5-conjugated donkey anti-rabbit secondary antibody (1:1,000, Jackson Immunoresearch Laboratories). The GluR2 labeled sections were then incubated overnight in PBS containing 0.005% BSA, 0.1% TritonX-100, mouse anti-parvalbumin (PARV; 1:20,000; Sigma) and goat anti-choline acetyltransferase (ChAT, 1:3,000; Chemicon). The following day the sections were rinsed several times in PBS and incubated overnight at 4°C in buffer containing Cy2-conjugated donkey anti-mouse IgG and Cy3-conjugated donkey anti-goat IgG (Jackson Immunoresearch Laboratories) at a dilution of 1:600 PBS containing 0.1% TritonX-100 and 0.005% BSA. Sections were then washed several times in PBS and mounted on gelatin-, chrom-alum coated slides, air-dried overnight, coverslipped with FluorSave (Calbiochem, La Jolla, CA) and imaged on a LSM510 Laser Scanning Axioplan 2 Microscope (Carl Zeiss Mikroskopie, Jena, Germany).

Slice preparation

Slices containing rat basal forebrain were obtained from male and female neonatal Sprague-Dawley rat pups. All rat pups were housed with their dam and littermates according to the guidelines set by the Canadian Council on Animal Care. Successful whole cell recordings were made in slices obtained from 5- to 21-day-old rat pups; however, the majority of recordings were made in slices prepared from rat pups that were 9-10 days of age.

To obtain slices, rats were decapitated, their brains rapidly removed, and the basal forebrain containing the medial septum/diagonal band of Broca and surrounding structures blocked. This tissue block was rapidly fixed to a mounting tray and immersed in ice-cold (4°C) modified oxygenated (95% O2-5% CO2) artificial cerebrospinal fluid (ACSF) that contained (in mM) 205 sucrose, 2.0 KCl, 7.0 MgCl2, 0.5 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, and 11 D-glucose, pH 7.35. Coronal (300-400 µm) slices were then cut through the tissue block with a vibrating microtome (VT100, Leica Microsystems, Willowdale, ON) equipped with a sapphire blade (Delaware Diamond Knives, Wilmington, DE). Once cut, the slices were transferred to a storage chamber containing ACSF consisting of (in mM) 120 NaCl, 3.0 KCl, 1.4 MgSO4, 2 CaCl2, 1.5 KH2PO4, 26 NaHCO3, and 10 D-glucose, pH 7.35 (20°C).

Slices were then individually transferred from the storage chamber to a recording chamber on an upright microscope where they were submerged and anchored in rapidly flowing (1 ml/min), oxygenated ACSF (20°C). A bipolar stimulating electrode was placed in the ventral segment of the vertical limb of the diagonal band of Broca and afferent fibers were activated by a single, 50-V, 200-µs pulse every 15-30 s, or every 5 s for PhTx-343 experiments.

Whole cell recordings

Whole cell voltage-clamp recordings (Hamill et al. 1981) were obtained in the medial septum of the basal forebrain slice using the "blind-patch" technique (Blanton et al. 1989) with either an Axoclamp 2A or Axopatch 1D amplifier (Axon Instruments, Foster City, CA). All recordings were digitized at 5-10 kHz and filtered at 2 kHz. Patch electrodes were pulled from borosilicate thin-walled glass (1.5 mm OD; 150F-4, World Precision Instruments, Sarasota, FL) in three stages on a Flaming-Brown micropipette puller (model P-87; Sutter Instrument, Novato, CA). Patch electrodes had a resistance of 2-6 MOmega when filled with (in mM) 100 cesium methanesulfonate, 10 cesium-bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, 40 HEPES, and 5 QX-314, adjusted to pH 7.3 with cesium hydroxide. Unless indicated, the intracellular patch pipette solution contained 50 or 100 µM spermine.

Series resistance was monitored on-line using pClamp7.0 software (Axon Instruments) and also by delivery of a -5-mV voltage step before each evoked current. The average series resistance was 10.65 ± 1.07 MOmega . Data were uncorrected for this error. Data were not included from recordings in which the series resistance was >20 MOmega or changed by >20%.

Chemicals were obtained from the following suppliers: PhTx-343, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), bicuculline methchloride, and GYKI 52466, Research Biochemicals, Natick, MA; spermine, 2-amino-5-phosphonovaleric acid (APV), Sigma.

Lucifer yellow and 2-photon microscopy

To determine the morphology of cells displaying rectification and synaptic facilitation, patch pipettes were filled with 0.1% Lucifer yellow, and filled cells were imaged using a 2-photon laser-scanning microscope (Denk et al. 1990). Briefly, a Ti:Sapphire laser (710-1,000 nm, 76 MHz, 200 fs pulse width, Mira Model 900-F, Coherent Laser Group, Santa Clara, CA) that was excited by a 5-W solid-state diode-pumped, frequency-doubled, Nd:Vanadate laser (532 nm, Verdi, Coherent Laser Group) was directly coupled to a LSM510 Laser scanning Axioplan 2 microscope or Axioskop 2 fs mot (Carl Zeiss Mikroskopie). Lucifer yellow was excited at 890 nm and LSM510 software was used to reconstruct filled neurons in three dimensions and measure the diameter of each cell body.


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ABSTRACT
INTRODUCTION
METHODS
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GluR2 immunoreactivity

Western blot analysis (n = 2) using a rabbit anti-GluR2 antibody revealed a single band of protein at an approximate molecular weight of 101 kDa as reported previously (108 kDa, Petralia et al. 1998; 102 kDA, Vissavajjhala et al. 1993) (see Fig. 1A). As shown in Fig. 1A, GluR2-immunoreactive protein was detected in most areas of the rat CNS but appeared to be most abundant in the hippocampus, striatum, and neocortex. Immunocytochemistry for GluR2 using the same polyclonal antibody (n = 2; Fig. 1, B-H) revealed dense immunoreactive neuronal somata and dendrites throughout most areas of the CNS. Figure 1B shows an example of the distribution of GluR2 immunoreactivity that was observed in 50-µm parasagittal sections through the rat brain. Figure 1, C-F, shows examples of the GluR2 immunoreactivity that was observed in 25-µm coronal sections through the hippocampus and neocortex. Despite the widespread abundance of GluR2 immunoreactivity, no detectable GluR2-immunoreactive neurons were observed in the midline area of the medial septum (Fig. 1, G and H). However, GluR2-immunoreactive neurons were found in the lateral zone of the medial septum.



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Fig. 1. Characterization of GluR2 immunoreactivity throughout the CNS of the rat. A: Western blot detection of GluR2-immunoreactive protein isolated from several different areas of the CNS. GluR2-immunoreactive protein was found in all areas of the CNS but was relatively more abundant in the hippocampus (Hip), striatum (CPu), and neocortex (Ctx). B: GluR2 immunoreactivity in a parasagittal section (50 µm) through the rat brain. Note the abundant GluR2 immunoreactivity throughout the hippocampus and striatum. C: GluR2 immunoreactivity in a coronal section (25 µm) through the hippocampus. D: GluR2-immunoreactive neurons throughout the CA1 pyramidal cell layer of the hippocampus. Note the dendritic labeling. E and F: low-power (×5, E) and high-power (×40, F) photomicrographs of GluR2-immunoreactive neurons throughout the neocortex of the rat. G and H: low- and higher-power photomicrographs depicting the relative paucity of GluR2 immunoreactivity through the level of the medial septum (MS). Note the labeled neurons throughout most areas of the rat brain, including the lateral septum (LS), yet very few GluR2-immunoreactive neurons were detected in the midline zone of the medial septum or the vertical (VDB) and horizontal limbs (HDB) of the diagonal band of broca. WB, whole brain; Cb, cerebellum; CPu, caudate putamen; Thal, thalamus; Brst, brain stem; Olf, olfactory bulbs. Scale bars = 4 mm (B), 625 µm (C and E), 78.125 µm (D and F), 2.50 mm (G), and 1.25 mm (H).

Specifically, co-localization studies (n = 2) using triple immunofluorescence and confocal microscopy revealed that the ChAT containing and PARV containing neurons of the medial septum had no detectable co-localization of the GluR2 AMPA receptor subunit immunoreactivity (Fig. 2).



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Fig. 2. The principal neurons of the MS are not immunoreactive for the AMPA receptor subunit GluR2. A: a low-power (×1.25) photomicrograph of parvalbumin (PARV, black) and anti-choline acetyltransferase (ChAT, brown) immunoreactivity throughout a coronal section of the rat brain at the level of the MS. B: a higher-power (×2.5) photomicrograph showing the distribution of PARV- and ChAT-immunoreactive neurons throughout the basal forebrain of the rat. Note that both types of neurons were found throughout the midline region of the medial septum/diagonal band complex, an area that showed little GluR2 immunoreactivity (see Fig. 1H). C: high-power (×40) photomicrograph of PARV- and ChAT-immunoreactive neurons in the basal forebrain of the rat. D-F: confocal micrographs of PARV (green)- and ChAT (red)-labeled neurons in the MS. D: a low-power (×20) confocal image showing that PARV and ChAT immunocytochemistry resulted in the labeling of 2 separate populations of MS neurons. E and F: higher-power (×63 and ×100) confocal images of PARV- and ChAT-immunoreactive neurons in the MS. Note that both neurons were relatively large with very similar morphological features. G-N: labeling of PARV (green)-, ChAT (red)-, and GluR2 (purple)-immunoreactive neurons in the MS of the rat. Note that none of the PARV- and ChAT-labeled neurons were positive for GluR2. However, a small unidentified population of neurons in the medial septum expressed the GluR2 AMPA receptor subunit. Scale bars = 2.50 mm (A), 1.25 mm (B), 78.125 µm (C), and 50 µm (D-N).

Synaptically evoked currents

Whole cell voltage clamp recordings were obtained from medial septal neurons in coronal brain slices to investigate the properties of AMPA receptor-mediated synaptic transmission (n = 79 cells). An initial survey with extracellular field recordings indicated that placing the stimulating electrode close to the midline ventral to the medial septum produced the largest synaptic response (data not shown).

Whole cell recordings revealed that three distinct postsynaptic currents could be reliable evoked in all medial septal neurons (Fig. 3). An inhibitory, bicuculline-sensitive (30 µM) GABAA receptor-mediated current was evoked in all medial septal neurons (Fig. 3, A and B). These neurons also displayed an excitatory APV-sensitive (100 µM) N-methyl-D-aspartate (NMDA) receptor-mediated current (Fig. 3, C and D). The remaining bicuculline- and APV-insensitive currents were completely blocked by NBQX (20 µM; Fig. 3, E and F).



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Fig. 3. Whole cell voltage-clamp recordings revealed three distinct currents evoked by stimulation of midline afferents. Currents were measured from -80 to +80 mV in 20-mV steps. Data presented in this figure were generated from a single cell. A: an example of the complex currents evoked by stimulation of midline afferents. B: the I-V relationship of the inhibitory, bicuculline-sensitive (30 µM), GABAA receptor-mediated component of the synaptic current (mean ± SE). This relationship was determined by subtracting the currents observed in bicuculline from the currents observed in control conditions. C: an example of the current evoked in the presence of 30 µM bicuculline. D: the I-V relationship of the excitatory, APV-sensitive (100 µM), NMDA receptor-mediated component of the synaptic current (mean ± SE). This relationship was determined by measuring the amplitude of the evoked current 30 ms following stimulation at the various potentials. E: an example of the current evoked in the presence of 30 µM bicuculline and 100 µM APV. F: the high-affinity AMPA receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (20 µM), successfully blocked the bicuculline- and APV-resistant current.

When the polyamine spermine (50 or 100 µM) was included in the patch pipette, synaptically evoked NBQX-sensitive currents could be classified as either rectifying or nonrectifying (Fig. 4) based on a best-fit polynomial (1st vs. 2nd order) regression analysis of the current voltage relationship. First, 30% (8/28) of the patched cells displayed synaptically evoked currents that were best fit with a first-order polynomial regression analysis. Thus there was a linear or nonrectifying relationship between peak current and holding potential from a range of -80 to +80 mV (Fig. 4, A and B). The currents evoked in these cells were unaffected by external application of PhTx-343 (97.0 ± 8.0% of control), a potent, high-affinity antagonist of Ca2+-permeable, GluR2-lacking AMPA receptors (2/2; Fig. 4C). In contrast, NBQX-sensitive currents evoked in the remaining (70%, 20/28) neurons were best fit by a second-order polynomial regression analysis and therefore displayed "inward" rectification of the relationship between peak current and holding potential from a range of -80 to +80 mV (Fig. 4, D and E). The currents evoked in these cells were blocked (19.85 ± 1.45% of control) by external application of PhTx-343 (50 µm), demonstrating that rectifying, NBQX-sensitive currents were mediated by AMPA receptors that contain very few or no GluR2 AMPA receptor subunits (3/3; Fig. 4F). The inward rectification observed in the present study was similar to the rectification that has been described in previous reports of AMPA receptor-mediated currents in cells that lack GluR2 mRNA (Washburn et al. 1997).



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Fig. 4. AMPA receptor-mediated currents evoked in the majority of MS neurons displayed rectification and were blocked by external application of the potent high-affinity Ca2+-permeable AMPA receptor antagonist philanthotoxin-343 (PhTx-343). A: an example of a nonrectifying NBQX-sensitive current that was evoked at different holding potentials from -80 to +80 mV (20-mV steps) in the presence of 30 µM bicuculline and 100 µM APV (average traces, n = 4-5). B: these currents were best fit by a first order polynomial regression analysis and exhibited nonrectifying current voltage relationship (all data points are shown for this cell). C: nonrectifying NBQX-sensitive currents were not affected by 50 µM PhTx-343. D: an example of a rectifying NBQX-sensitive current that was evoked at different holding potentials from -80 to +80 mV (20-mV steps) in the presence of 30 µM bicuculline and 100 µM APV (average traces, n = 2-5). E: these currents were best fit by a second-order polynomial regression analysis and exhibited a rectifying current voltage relationship. F: rectifying NBQX-sensitive currents were 80% blocked by 50 µM PhTx-343.

Cell size

We examined the morphology of neurons to determine whether there was a correlation between synaptic rectification and cell size. Whole cell voltage-clamp recordings were made to characterize AMPA receptor-mediated currents, and medial septal neurons were subsequently filled and imaged using 2-photon laser scanning microscopy. The neurons that displayed current rectification were significantly larger in diameter [25.4 ± 1.9 (SE) µm; n = 5] than the neurons that displayed nonrectification [10.3 ± 1.2; n = 3; F(1,6) = 31.98, P < 0.01; see Fig. 5]. Previous reports have demonstrated that medial septal ChAT- and PARV-containing neurons are larger that the non-PARV-containing GABAergic neurons (see Jakab and Leranth 1995). These results are consistent with the conclusion that the GluR2-lacking AMPA synaptic responses are found in the cholinergic- and PARV-containing GABAergic projection neurons.



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Fig. 5. Two-photon imaging revealed that neurons that expressed rectifying, PhTx-343-sensitive AMPA receptor-mediated currents were large (20-30 µm) while neurons expressing nonrectifying PhTx-343-insensitive currents were small (8-12 µm). A: a 2-photon laser scanning image of a filled medial septal neuron that exhibited a rectifying current-voltage relationship. This image is a 3-dimensional reconstruction of the neuron from a stack of images that was obtained in 2.21-µm increments over 57 µm in the z plane. B: an image of the same large cell shown in A through the mid soma level where cell diameter measurements were taken. C: an image of a small cell that exhibited a nonrectifying current-voltage relationship. This image was taken through a single z plane (2.21 µm) through the mid soma level where the cell diameter measurements were taken. D: a plot of the mean evoked current amplitude (±SE) as function of the different holding potentials for the cell depicted in A and B. E: a plot of the mean evoked current amplitude (±SE) as function of different holding potential for the small cell depicted in C. F: the mean (±SE) soma diameter of neurons that displayed a rectifying current-voltage relationship was significantly larger than the diameter of MS neurons that displayed a nonrectifying current voltage relationship.

Facilitation

Next we investigated the response of AMPA receptor-mediated synaptic currents to repetitive stimulation. First, the relationship between the amplitude of the peak synaptic current and membrane potential was obtained to determine if the AMPA receptor-mediated synaptic response rectified as described in the preceding text. The isolated AMPA receptor-mediated synaptic currents were then evoked five times at rates ranging from 1 to 20 Hz (n = 26). All of the rectifying AMPA receptor-mediated currents showed amplitude facilitation during repetitive synaptic activation. Both the amount of facilitation and the frequency that produced the maximum facilitation varied somewhat for each cell. However, the largest relative increases were observed when currents were evoked at frequencies of 6-10 Hz (25 ± 10% at 1 Hz vs. 62 ± 14% at 6 Hz and 55 ± 24% at 10 Hz; currents were normalized to the first in each series of 5 evoked currents; n = 16 for facilitation). Figure 6, A and C, shows an example of facilitation at 6 Hz in a cell that displayed a rectifying AMPA receptor-mediated synaptic response.



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Fig. 6. Facilitation of rectifying AMPA receptor-mediated currents during 6 Hz stimulation. A and C: illustration of an example of the type of facilitation observed at 6 Hz in a cell that displayed a rectifying AMPA receptor-mediated synaptic response (mean ± SE). The facilitation of synaptic currents in 1 cell is shown in a plot in C. Currents (in both C and D) were normalized to the 1st synaptic response evoked by the 1st pulse in the 1st 6-Hz train. B and D: an example is shown of the type of synaptic depression observed at 6 Hz in another cell that displayed a nonrectifying AMPA receptor-mediated synaptic response (mean ± SE). E and F: both facilitating, rectifying (E) and depressing, nonrectifying (F) synaptic responses were blocked by GYKI 52466 (50 µM), the potent AMPA-receptor antagonist. The time courses of the GKI 52466 effects are plotted, and examples of synaptic currents before and after GYKI 52466 are shown in the insets. G: an example of a facilitating synaptic current is shown before and after GYKI 52466. There were little or no residual facilitating synaptic currents that were GYKI 52466 resistant.

In contrast to these facilitating currents, AMPA receptor-mediated currents evoked in neurons that displayed nonrectifying I-V curves depressed when stimulated at frequencies ranging from 1 to 20 Hz (n = 10). Figure 6, B and D, shows an example of the type of synaptic depression observed at 6 Hz in a neuron that contained a nonrectifying AMPA receptor-mediated response.

Next we examined the sensitivity of both types of synaptic responses to the potent AMPA receptor antagonist, GYKI 52466 (Donevan and Rogawski 1993; Paternain et al. 1995; Zorumski et al. 1993). GYKI 52466 (50 µM) blocked both the rectifying, facilitating synaptic currents (Fig. 6E; 93.6 ± 4.6% depression, n = 5) and the linear, depressing synaptic currents (Fig. 6F; 95.4 ± 1.2% depression, n = 5). The synaptic currents were also evoked at 6 Hz before and after GYKI 51466 to ensure that the facilitating currents were also AMPA-receptor mediated (Fig. 6G).

Finally, to determine the relationship between rectification and the change in current amplitude when stimulated at 6 Hz, we quantified the degree of rectification by dividing the sum of the peak current amplitude evoked at +60, +40, and +20 mV by the sum of the peak current amplitude evoked at -60, -40, and -20 mV. Using this measure of rectification, cells that displayed AMPA receptor-mediated currents with little current amplitude at membrane potentials >0 mV (rectification) had a small rectification ratio. There was a statistically significant negative correlation between rectification ratio and the change in current amplitude at 6 Hz (Fig. 7A; Pearson r = -0.507, P < 0.05). This significant correlation came about because the rectification ratio of the currents that displayed facilitation were significantly smaller (0.38 ± 0.05) than the rectification ratio of the currents that displayed synaptic depression [0.77 ± 0.09; Fig. 7B; F(1,15) = 15.821, P < 0.05].



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Fig. 7. Relationship between rectification and synaptic changes at 6 Hz. A: the degree of rectification was quantified by dividing the sum of the peak current amplitude evoked at +60, +40, and +20 mV by the sum of the peak current amplitude evoked at -60, -40, and -20 mV. Using this measure of rectification, cells that displayed AMPA receptor-mediated currents with little current amplitude at membrane potentials 0 mV (rectification) had a small rectification ratio. This was plotted against the normalized change in synaptic current amplitude recorded during 6-Hz stimulation in the same cell at -70-mV holding potential. There was a significant negative correlation between rectification ratio and the change in synaptic current amplitude. B: the rectification ratio of the currents that displayed facilitation were significantly smaller (0.38 ± 0.05;  in A) than the rectification ratio of the currents that displayed synaptic depression (0.77 ± 0.09;  in A).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we have demonstrated that the AMPA receptor-mediated responses recorded in the principal projection neurons of the medial septum display rapid facilitation following theta-like stimulation of midline afferents. These synaptic currents were mediated by AMPA receptors because they were blocked by either PhTx-343 or GYKI 52466. Our triple immunocytochemistry indicated that the large principal medial septal projection neurons containing either ChAT or PARV were not GluR2 immunoreactive. We suggest that this form of rapid, reversible AMPA receptor-mediated synaptic plasticity may provide medial septal neurons with a unique postsynaptic mechanism for controlling synaptic gain because synaptic potentials can be quickly enhanced in an activity-dependent manner when stimulated repetitively at theta rates.

Rozov et al. (1998) first showed that facilitation at calcium permeable AMPA receptors depends on intracellular polyamines and is the result of use-dependent relief of polyamine block during consecutive AMPA receptor channel openings. Rozov et al. (1998) initially used recombinant, unedited, Ca2+-permeable, homomeric GluR2 AMPA receptors to demonstrate that Ca2+-permeable AMPA receptors facilitate when repetitively activated at low frequencies. In a subsequent study, Rozov and Burnashev (1999) demonstrated that facilitation of AMPA receptors occurs at synapses between neocortical pyramidal cells and neighboring interneurons. This facilitation reduced the rate of paired-pulse depression and was postulated to play an important role in the functioning of the neocortex during low-frequency activity. The results of the study presented here shows that GluR2-lacking AMPA receptors activated by synaptic stimulation in the medial septum also display a similar form of rapid current facilitation.

The results of the present study indicate that this facilitation occurs in the principal projection neurons of the medial septum at AMPA receptor-mediated synapses that lack the GluR2 receptor subunit. Very little GluR2-specific immunoreactivity was observed around the midline regions of the medial septum where the large medial septal PARV- and ChAT-containing neurons are localized (see Jakab and Leranth 1995). Also, triple immunofluorescent labeling of ChAT, PARV, and GluR2 revealed no colocalization of GluR2 immunoreactivity with either ChAT or PARV. Finally, rectification of PhTx-343-sensitive AMPA receptor-mediated currents was observed in neurons that were significantly larger than medial septal neurons that displayed PhTx-343-insensitive, nonrectifying currents (see Fig. 5). ChAT- and PARV-positive medial septal neurons have been reported to be larger in size then the GABAergic type III neurons that are found in the lateral aspects of the medial septum (see Jakab and Leranth 1995). Furthermore both types of synaptic responses were mediated by AMPA receptor activation because the potent AMPA antagonist GYKI 52466 blocked them.

Our conclusion that neurons within the medial septum contain Ca2+-permeable, GluR2-lacking AMPA receptors is consistent with previous reports where fura-2 measurements (Schneggenburger et al. 1993a,b) or voltage clamping (Waters and Allen 1998) demonstrated substantial AMPA receptor-mediated Ca2+ influx in these neurons. Previous studies of the distribution of AMPA receptor subunits GluR1-4 within the basal forebrain of the rat demonstrated that GluR2/3 immunoreactivity was present within 15% of the cholinergic neurons (Page and Everitt 1995). Until recently the rigorous determination of the distribution of GluR2-lacking AMPA receptors in the septum was hindered by the lack of a GluR2-specific antibody that did not cross-react with GluR3. In the present study, we used a GluR2-selective antibody that did not cross-react with GluR3 to demonstrate the lack of detectable GluR2 immunoreactivity in the large neurons of the medial septum. The main AMPA receptor subunit expressed in medial septal neurons is reported to be GluR4 (Page and Everitt 1995).

Several other regions of the brain also possess neurons that express GluR2-lacking AMPA receptors. For example, electrophysiological data indicate that a subpopulation of GABAergic interneurons in both the hippocampus and neocortex possess Ca2+-permeable, inwardly rectifying AMPA receptors (Jonas and Burnashev 1995; McBain et al. 1999). Generally, it is believed that most principal neurons possess AMPA receptors with low Ca2+ permeability while some interneurons possess AMPA receptors with high Ca2+ permeability (Seeburg 1996). However, Ca2+-permeable AMPA receptors have also been found in the principal projection neurons of the cochlear nucleus (Otis et al. 1995), the medial nucleus of the trapezoid body (Geiger et al. 1995), and projection neurons in the dorsal horn of the spinal cord (Kyrozis et al. 1995).

Rozov et al. (1998) previously demonstrated that facilitation of currents through a recombinant AMPA receptor that contains unedited GluR2 AMPA receptor subunits depend on intracellular polyamines and occurs entirely postsynaptically. Our study suggests that a similar mechanism contributes to facilitation at the native GluR2-lacking synapse in medial septum. In the present study, facilitation was only observed in PhTx-343-sensitive AMPA receptors that displayed rectification. The responses were clearly mediated by AMPA receptors because the facilitating synaptic currents were blocked by GYKI 52466, the potent AMPA-receptor antagonist.

AMPA receptor-mediated, low-frequency facilitation may play an important role in the normal functioning of the septohippocampal circuit. ChAT- and PARV-containing neurons account for all of the medial septal neurons that project to the hippocampus and are therefore considered the "principal" neurons of the medial septum (Jakab and Leranth 1995). These neurons are critical for the generation of hippocampal theta activity (Bland 1986; Smythe et al. 1992) and hippocampal-dependent learning and memory (Eichenbaum et al. 1990; Packard and McGaugh 1992; Sutherland and Rodriguez 1989; Whishaw and Maaswinkel 1998; Whishaw and Tomie 1997; Whishaw et al. 1995). Medial septal ChAT- and PARV-containing neurons are part of a large ascending midline pathway that generates hippocampal theta activity (Bland 1986). This pathway originates in the rostral pons and ascends through the medial hypothalamus to the medial septum/vertical limb of the diagonal band of Broca (Kocsis and Vertes 1994; Oddie et al. 1994; Smythe et al. 1991). A portion of the ascending midline input to the medial septum arises from glutamatergic neurons in the supramammillary area of the hypothalamus (Leranth and Kiss 1996).

The remaining medial septal neurons that are not ChAT or PARV immunoreactive appear to be small GABAergic interneurons that occupy the lateral zones of the MS (Jakab and Leranth 1995). These neurons do not project to the hippocampus (Freund 1989). They are the neurons in present study that contained nonrectifying, GluR2-containing AMPA receptors, and these neurons displayed synaptic depression when stimulated at 6 Hz.

In conclusion, our study suggests that differential localization of GluR2 AMPA receptor subunit within the principal and nonprincipal cells of the medial septum may result in the potent enhancement of glutamatergic synaptic inputs to the septohippocampal pathway at low frequencies.


    ACKNOWLEDGMENTS

The authors thank D. Feighan, M. Johns, and K. Whalen for technical assistance and C. Armstrong, S. Mulligan, and B. Kuzmiski for reading an earlier version of the manuscript. C. Armstrong assisted in the preparation of the photomicrographs. B. A. MacVicar is an Alberta Heritage Foundation for Medical Research and Medical Research Council (MRC) Senior Scientist.

This work was supported by the MRC of Canada.


    FOOTNOTES

Address for reprint requests: B. A. MacVicar, Dept. of Physiology and Biophysics, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada (E-mail: macvicar{at}ucalgary.ca).

Received 6 October 2000; accepted in final form 22 December 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society