Brandel, Andrew

Differential Calcium Permeability and Current Mediation by TwoKainate Receptor Subtypes:

Implications for the Modulation of GABAergic Synaptic Transmission

INTRODUCTION

The glutamate system is very important to brain function, particularly for studies

examining long-term potentiation/depression. The system is also central in modulating inhibitory

transmission via the _-aminobutyric acid (GABA)ergic system. This study focuses on class of

glutamate receptors about which the least is known; the kainite receptor (KAR). Molecular

analysis has revealed five unique subunits for KARs; GluR5, GluR6, GluR7, KA1 and KA2.

GluR5 and GluR6 are both found in CA1 interneurons and mediate depressive effects on

inhibitory transmission. This action can be abolished by targeting GluR5 and GluR6

simultaneously, but not GluR5 by itself, leaving GluR6 as central to GABAergic depression via

KAR. Both subtypes can appear unedited (Q) or edited (R), naturally in the hippocampus. When

either unedited subtype is expressed, an inward rectifier current was observed, however when

either edited version was analyzed, the receptors mediated a linear current-voltage relationship. It

has also been shown that Ca2+-permeability is influenced by the Q/R editing site of GluR6. Both

forms of GluR6 are permeable, however GluR(Q) is significantly more efficient (3x) than the R

form.

In this study, we use patch-clamp recording and multi-photon confocal microscopy to

observe KAR-induced currents and Ca2+ signals, brought about by submicromolar ATPA, and

micromolar KA. We found ATPA induced a small, slowly rising, inwardly rectified, GluR5

current with a relatively small reversal potential. There was also no increase in Ca2+ levels during

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this condition. Micromolar KA, however, triggered large, GluR6 mediated current with a larger

reversal potential. In this condition, there was a substantial Ca2+ increase in the axon trunk and

the dendrites. Minor depolarization of interneurons brought about by KAR agonists facilitate

GABA release however, KAR agonists activate additional Ca2+ permeable GluR6 receptors, and

may over-depolarize interneurons, resulting in possible disinhibition.

We also build on prior studies related to KAR agonists and their depression of inhibitory

transmission, particularly in CA1 of the hippocampus. Recently, KAR agonist-induced

depression of fiber stimulation-evoked inhibitory postsynaptic currents (eIPSCs) has been a point

of interest due to its similarity to the convulsant and excitotoxic nature of kainate (KA). Studies

on the mechanisms of KA-induced depression of eIPSCs have resulted in some disagreement in

the field. Work has now shown that contrary to this disinhibition, KAR agonists facilitate GABA

release through spontaneous action potential (AP) firing in interneurons, or by increased

efficiency of low-release probability. It is possible that KAR agonists are triggering presynaptic

KARs to inhibit GABA release. Both presynaptic inhibition of GABA and postsynaptic

inhibition of GABA(A) currents, may contribute to the depressive effects of micromolar KAR

agonists.

In order to better understand GABAergic release as it is mediated by KARs, it is critical

that we understand the distribution and Ca2+-permeability of KAR channels.

METHODS

Preparation of slices

Brain slices were prepared as described previously (Jiang et al, 2001). Briefly, 14- to 20-day-old

(P14-P20) Sprague-Dawley rats were anaesthetized with pentobarbitone sodium (55 mg/kg) and

decapitated. Transverse brain slices of 300 µm were cut with a vibratome (TPI, St Louis, MO,

USA) in a cutting solution containing (mM): 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, 0.5 CaCl2, 10

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glucose, 26 NaHCO3 and 230 sucrose. Slices containing the hippocampus were incubated in the

slice solution gassed with 5 % CO2 and 95 % O2 for 1-7 hours and then transferred to a

recording chamber (1.5 ml) that was perfused with the slice solution gassed with 5 % CO2 and

95 % O2 at room temperature (23-24°C) for recording. The standard slice solution contained

(mM): 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgCl2, 2 CaCl2, 10 glucose and 26 NaHCO3 (pH

at 7.4 when gassed with 95 % O2 and 5 % CO2).

Electrophysiology

The recording chamber was placed on the stage of an Olympus BX51 upright microscope

(Olympus Optical Co., NY, USA) equipped with differential inference contrast (DIC) optics, and

cells were visualized with a 63x water immersion lens. Two electrical manipulators (TS Products

Co., Arleta, CA, USA) were mounted on the stage in opposing positions and moved along a

plane 18° to the horizontal. Patch electrodes with a resistance of 4-7 MΩ were pulled from KG-

33 glass capillaries (i.d. 1.0 mm, o.d. 1.5 mm, Garner Glass Co., Claremont, CA, USA) using a

P-97 electrode puller (Sutter Instrument Co., Novato, CA, USA). Interneurons whose somata

were located in the stratum radiatum of the CA1 area were patched in the whole-cell voltage-

clamp configuration (Hamill et al, 1981) and recorded at a holding potential of -60 mV using an

Axopatch 200B amplifier (Axon Instruments, Inc., Burlingame, CA, USA). The pipette solution

contained (mM): 120 K-gluconate, 10 KCl, 1 MgCl2, 10 HEPES, 0.1 EGTA, 0.025 CaCl2, 1

ATP, 0.2 GTP, and 4 glucose (pH adjusted to 7.2 with KOH). A seal resistance < 5 GΩ was

rejected, and experiments with a holding current > -200 pA were rejected. We started

experiments after access resistance remained relative constant (5 - 10 min), and recordings with

changes in series resistance >10% of control were rejected.

Calcium imaging

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A customized 2-photon laser scanning Olympus BX61WI microscope with a 60x objective lens

was used to detect Ca2+ signals. A Mai/TaiTM laser (the solid-state laser company, Mountain

View, CA) tuned to 810 nm was used for excitation. Red and green fluorescence were detected

simultaneously by two photomultiplier tubes. Image acquisition was controlled by Olympus

software Fluoview FV300 (Olympus America INC, Melville, NY). In the transfluorescence

pathway, a 565 nm dichroic mirror was used to separate green and red fluorescence. HQ525/50

and HQ605/50 filters were placed in the “green” and “red” pathways, respectively to eliminate

transmitted or reflected excitation light (Chroma Technology Corp., Rockingham, VT, U.S.A.).

Neurons were loaded with dyes through the patch pipette for 20 min before image acquisition.

Alexa Fluo-4 was used to outline the neuronal structures, and Fluo-4 to detect Ca2+ signals. To

measure Ca2+ dynamics, the fluorescence was collected by scanning at 4 Hz in a surface

scanning mode (XYT). Fluorescence was averaged from the specified structures to obtain F (t).

Baseline fluorescence (F0) was the average of four images during control and ∆F/F was

calculated as (∆F/F)(t) = (F(t)-F0)/F0.

Local application of the KAR agonists

In the presence of 1 µM TTX, 50 µM SYM 2206 and 50 µM APV, whole-cell KAR currents

were induced by pressure application of ATPA (1 _M) or KA (3 µM) through a pipette

positioning near the patched interneuron. 3-5 psi pressure for 0.5 - 2 s duration was applied to the

puff pipette through a picospritzer III (Parker Hannifin Corporation General Valve Operation,

Fairfield, NJ, USA).

Immunocytochemistry for GluR5/6/7 proteins

Four 25- to 35-day-old rats were anesthetized with 100 mg/kg sodium pentobarbital injected

intraperitoneally. Each rat was transcardially perfused with: (1) 20 ml normal saline containing

1000 units/ml heparin, (2) 50 ml 3.75% Acrolein and 2% paraforaldehyde in 0.1 M phosphate

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buffer (PB; pH=7.4), and (3) 200 ml 2% paraforaldehyde in PB. The brains were removed and

cut into 5-6 mm blocks which were immersed in the last fixative for 1 hour. Sections (450 _m)

containing the hippocampal formation were cut with a vibratome and collected in PB. Sections

were pretreated with 1% sodium borohydride and then in 10% normal serum of the species of the

secondary antibody (goat) in buffer for 1 hour at 4°C as described previously (Huntley et al,

1993). Sections were then incubated in the identical solution in addition of the primary antibody

GluR5/6/7 (1:500, mouse IgM monoclonal; BD Biosciences Inc, USA) for 72 hr at 4°C. Sections

were incubated with the appropriate biotinylated secondary antibody (goat anti-mouse IgM,

1:500, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for overnight at

4°C. Then sections were incubated with the appropriate ABC reagent (1:500, Jackson

ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Immunoperoxidase labeling was

visualized by incubating sections in 0.05M Tris-HCl buffer (pH=7.6) containing 3,3‘-

diaminobenzidine (DAB, 0.025%) and hydrogen peroxide (0.003%). After reaction with DAB,

sections were postfixed for 1 hr in 1% osmium tetroxide. Sections were then dehydrated in

ethanol followed by immersion in acetone, infiltrated and embedded in Embed (EMS, Fort

Washington, PA, USA). Blocks were polymerized at 60°C for 48 hr. Thick sections (0.50 _m)

were cut and stained with toluidine blue O and viewed with the light microscope to ensure proper

orientation and preservation. Poorly preserved tissue was discarded. Ultrathin sections were cut

at 60-70_m, collected on 200-mesh copper grids and counterstained with uranyl acetate and lead

citrate (Chan et al, 1990). The sections were examined and photographed on a Hitachi 7000

electron microscope. Control sections were incubated in solutions from which the primary

antisera were replaced by 1 % nonimmune mouse serum, whereas the rest of the protocol

remained the same as described above. Two blocks were cut from each rat brain, and four grids

were obtained from each block. Each grid had 20 fields.

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Data analysis

Recording signals were filtered through an 8-pole Bessel low-pass filter with a 2 kHz cut-off

frequency and sampled by a PCLAMP 8.1 program (Axon Instruments Inc.) with an interval of

50 µs or 200 µs. Data were further processed with Origin 5.1 (Microcal Software, Inc,

Northampton, MA, USA) and CorelDraw 9.0 (Corel Co. Ontario, Canada) programs. Statistical

data are presented as mean ± SEM if not otherwise indicated.

RESULTS

Different Currents Induced by ATPA and KA

To test KAR currents in interneurons, we performed whole-cell voltage-clamp recordings in

CA1 stratum radiatum. A solution of 1_M ATPA or 3_M KA was puffed using an auxiliary

pipette. Due to surrounding solutions, exact concentrations may be slightly off. APV, a NMDA

receptor antagonist, Tetrodotoxin (TTX), a voltage-dependant Na+ channel blocker, and SYM

2206, an AMPA receptor blocker, were perfused 5 minutes prior to puff, in order to isolate KAR

function. A 5mV hyperpolarized step was used to monitor input resistance. ATPA induced a

slow rising inward current in over 93% of interneurons. The mean amplitude of these currents at

-80mV was -30.5 ± 7.4 pA. It can be concluded that this current was a GluR5-current, because at

this concentration, ATPA is selective for that subtype of KAR. In half the cells recorded 3 _M

KA induced both a slowly rising GluR5 component, and an additional large and fast current.

ATPA below 1 _M never induced such a large and fast current, implicating a non-GluR5

component in the KA-mediated current. (Fig 1a) Previous research by other groups suggests that

both GluR5 and GluR6 subunits are expressed in CA1 interneurons (Bureau et al., 1999;

Paternain et al., 2000; Mule et al., 2000). It is also been found that KA1, KA2 and GluR7 do not

form functional homoeric channels (Werner et al 1991; Herb et al., 1992; Kamboj et al., 1992;

Bettler et al., 1992; Lomeli et al., 1992) and furthermore GluR7 RNA is not found in CA1 at all

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(Bettler et al., 1990; Egebjerg et al., 1991; Lomeli et al., 1992; Bureau et al. 1999). Based on

these findings, it can be reasonably assumed that the KA-induced non-GluR5 current is most

likely a result of GluR6. Both currents were blocked with application of _M DNQX in the

perfusion solution (Fig 1B), proving them to be KAR-mediated.

Fig. 1. ATPA- and KA-induced currents in CA1 interneurons. (A) Left trace, puff of 1 _M ATPA induced a slowly rising inward current(GluR5 current). Right trace, puff of 3 _M KA induced a slowly rising GluR5 component (1) and an additional, fast rising component (2).Dotted line indicated the GluR5 component. The number at the left of the trace indicates holding potential in mV. (B) ATPA- and KA-inducedcurrents were blocked by 10 _M DNQX. Data are representative chosen from 10 experiments. (C) Replacement of extracellular NaCl withNMDG-Cl abolished the ATPA-induced current (left panel), but not the KA-induced current (right panel).

To test Ca2+ components of the currents, extracellular NaCL was replaced by NMDG-CL in 5

cells (Fig 1C), in order to isolate Ca2+ activity. The GluR5 current was destroyed, however, the

GluR6 component was left intact in 50% of neurons. We then examined the reversal potential for

the GluR5 and GluR5 currents by holding cells at varying membrane potentials (Fig 2.). The

ATPA-induced GluR5 currents reversed around 0mV and inwardly rectified. However, KA-

induced GluR5 currents in nearly 62.5% of cells reversed somewhere between +50 and +60 mV

(Fig 2B). In the remaining cells of this condition, GluR6 currents were linear reversed around

+30 and +40 mV (Fig 2C). The mean amplitude of both forms of currents was plotted (Fig 2D)

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and the components of the KA current are well defined. The GluR5 current is small, displays

inward rectification and a reversal potential of +1.4 ± 0.9 mV. The KA-induced additional

current is large, displays inward rectification, but has a reversal potential of +57.4 ± 3.8 mV. The

linear current induced by KA is also large, displays a linear I-V curve, and maintains a reversal

potential of +31.0 ± 4.7 mV. Given this reversal potential, it would seem that the GluR6 channel

is Ca2+-permeable.

Fig. 2. Current-voltage relationship (I-V curve) for ATPA- and KA-induced currents. (A) Whole-cell voltage-clamp recordings showingATPA-induced currents with different holding potentials. The number at the left of each trace indicates holding potentials in mV. The currentreversed around 0 mV. (B) KA-induced inward rectifier currents with different holding potentials in a representative interneuron. The currentreversed between +50 and +60 mV. (C) KA-induced inward linear currents in a representative interneuron. The current reversed between +30and +40 mV. (D) The I-V curves for ATPA- (open circle) and KA-induced inward rectifier (closed circle) and linear (open square) currents.

KA induced minor increases in Ca2+ in the soma of interneurons. We used multi-photon confocal

microscopy combined with patch-clamp techniques to observe Ca2+-permeability of both the

GluR5 and GluR5 channels. By using both techniques concurrently, we were able to compare

calcium signaling with electrophysiology in real time. Contained in the pipette solution for the

whole-cell recording was Fluo4 (100 _M), which is used to monitor Ca2+ signals, and Alex

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Fluo-594 (100 _M), which indicates the outline of the interneuron. ATPA (0.5 _M, 7.5 _l of 0.1

mM ATPA) or KA (3 _M, 4.5 _l of 1mM) was applied to the bath in the presence of APV, SYM

2206, and TTX. Once the soma was identified, we found basal Ca2+ states in the soma to be

higher than the more peripheral regions, attributable to loading bias. In a representative cell, 0.5

_M ATPA did not induce a rise in Ca2+ in the soma (Fig 3A), while a small GluR5 current was

still induced. Bath application of KA did not induce a GluR6 component as was described from

puff results, likely due to a lack of synchronization. Pooled data from 4 KA cells from 10

experiments displayed Ca2+ signals during ATPA application (Fig 3E) and were not significantly

different from control data. Ca2+ signals during KA application were significantly different from

control, however. (Con, P<0.01, paired t-test).

Fig. 3. Ca2+ signals in the soma induced by ATPA and KA.(A) Upper images, Fluo-4 fluorescence levels in the soma of arepresentative cell before (Con) and after application of 0.5_M ATPA (ATPA). Bottom images, Alexa Fluo-594fluorescence signals showing the morphology of the soma.Scale bar, 5 _m. (B) The time course of Ca2+ signals (Ca2+,_F/F) and whole-cell current (IKAR) for the experiment in A.(C) Upper images, Fluo-4 fluorescence levels in the soma ofthe same cell in A (upper image) before (Con) and afterapplication of 3 _M KA (KA). (D) The time course of Ca2+signals (Ca2+) and whole-cell current (IKAR) for theexperiment in C. KA induced a slight increase in Ca2+ levelsin the soma. (E) Pooled data for ATPA- (ATPA) and KA(KA)-induced increase in Ca2+ levels in the soma. n = 4 cells.

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Ca2+ Signals in the Axon Trunk and Dendrites

Next, CA2+ signals in the axonal trunk were compared to whole-cell electrophysiology with two-

photon confocal microscopy. 0.5 _M ATPA did not induce any Ca2+ rise (Fig 4A) in the axon

trunk, but a GluR5 current was induced. This would seem to indicate that the GluR5 channel is

not Ca2+-permeable or at a minimum does not utilize Ca2+. 3 _M KA on the other hand did

induce a Ca2+ increase in the axon trunk (Fig 4C). The Ca2+ rise was not observed in the early

part of the current, suggesting that the first component is mediated by a Ca2+-impermeable

channel. The Ca2+ influx lasted longer than the inward current and recovered ~5-10 minutes

following current recovery to basal state. Pooled data showed that increase in Ca2+ in the axonal

trunk following KA application was significantly larger than that in the soma (0.25 ± 0.04

P<0.01, t-test).

Fig. 4. Ca2+ signals in the axonal trunk induced by ATPAand KA. (A) Left images, Fluo-4 fluorescence levels in theaxon trunk of a representative cell before (Con) and afterapplication of 0.5 mM ATPA (ATPA). Scale bar, 2 mm. Rightimages, Alexa Fluo-594 fluorescence signals. Bottom picture,projection of the Alexa Fluo-594 fluorescence showing the cellstructure including the selected axon trunk (squared area). (B)The time course of Ca2+ signals (Ca2+) and whole-cellcurrent (IKAR) for the experiment in A. (C) Fluo-4fluorescence levels in the axon trunk before (Con) and afterapplication of 3 mM KA (KA). (D) The time course of Ca2+signals (Ca2+) and whole-cell current (IKAR) for theexperiment in C. (E) Pooled data for ATPA- (ATPA) and KA(KA)-induced increase in Ca2+ levels in the axonal trunk. n =4 cells.

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In a representative cell (Fig 5A), 0.5 _M ATPA did not increase Ca2+ levels in the dendrites

although a small GluR5 current was observed. 3 _M KA induced both a large two-component

current and a Ca2+ increase in the dendrites and spine. The increase of Ca2+ in the dendrites and

spines due to KA was delayed and significantly greater than baseline (P = 0.82, paired t-test).

Fig. 5. Ca2+ signals in the dendrite induced by ATPA and KA. (A) Upper images, Fluo-4 fluorescence levels in the dendrite of arepresentative cell before (Con) and after application of 0.5 _M ATPA (ATPA). Scale bar, 2 mm. Bottom images, Alexa Fluo-594 fluorescencesignals showing the morphology of dendrite. (B) The time course of Ca2+ signals (Ca2+) and whole-cell current (IKAR) for the experiment inA. (C) Fluo-4 fluorescence levels in the dendrite before (Con) and after application of 3 _M KA (KA). Ca2+ increased in both the dendrite (d)and spine (s) during KA application. (D) The time course of Ca2+ signals (Ca2+) and whole-cell current (IKAR) for the experiment in C. (E)Pooled data for ATPA- (ATPA) and KA (KA)-induced increase in Ca2+ levels in the dendrites. n = 4 cells.

No Ca2+ Permeable KARs in the Terminals of Interneurons

Research by another group suggests that there is a subtype of KARs that plays an inhibitory role

on GABA release vis-à-vis a G-protein coupled pathway (Rodriguez-Moreno, Lerma 1998;

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Rodriguez-Moreno et al., 2000). We tested whether the KA-induced Ca2+ rise would occur at the

axonal terminals. Axon positions were located by tracing down the axon in the distal direction

from the soma (Fig 6). Neither 0.5 _M ATPA nor 4 _M KA induced any Ca2+ increase at the

axon terminals, however somatic recordings displayed KAR-mediated currents, as described

earlier. Pooled data displayed neither agonist increased Ca2+ signaling in axon terminals. These

results suggest that there are no Ca2+ permeable KAR channels in axonal terminals of CA1

interneurons. Projections of all the regions of the interneuron during KA application showed

increased Ca2+ limited to the axonal trunk and dendrites, suggesting restricted distribution to

these two regions.

Fig. 6. Ca2+ signals in the terminals induced by ATPA andKA. (A) Upper images, Fluo-4 fluorescence levels in the terminalsof a representative cell before (Con) and after application of 0.5_M ATPA (ATPA). Scale bar, 2 _m. Bottom images, Alexa Fluo-594 fluorescence signals (red) showing the morphology of theterminals. (B) The time course of Ca2+ signals (Ca2+) and whole-cell current (IKAR) for the experiment in A. (C) Fluo-4fluorescence levels in the terminals before (Con) and afterapplication of 3 _M KA (KA). (D) The time course of Ca2+signals (Ca2+) and whole-cell current (IKAR) for the experimentin C. (E) Pooled data for ATPA- (ATPA) and KA (KA)-inducedincrease in Ca2+ levels in the terminals. n = 5 cells. (F) Projectionof Fluo-4 (left panel) fluorescence levels and Alexa Fluo-594morphology (right panel) of the soma (s), dendrite (d), axon (a),and terminals (t).

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Immunocytochemistry for KAR Proteins in Presynaptic Terminals

Ca2+ imaging revealed no Ca2+ permeable KAR channels in the axonal terminals. In order to test

for Ca2+-impermeable KARs in terminals, GluR5/6/7 protein antibodies were used to measure

immunreactivity by light and electron microscopy (EM). The antibody staining at the light level

indicated immunoreactivity in the soma and major processes of interneurons in the statum

radiatum (Fig 7) and pyramidal layer. Symmetric synapses were defined by a point of close

opposition between a terminal and a dendrite, whereby the membranes are precisely parallel and

the postsynaptic membrane displays a fine postsynaptic density. Asymmetric synapses were

qualified by heavy postsynaptic density. Immunoreactivity in presynaptic terminals was

observed in 9.7% of cells, from 152 fields. Immunoreactivity in postsynaptic spines, however,

was found in a quarter of asymmetric synapses, which turned out to be putative glutamatergic

synapses. The percentage of GABAergic synapses that have KAR receptors in their presynaptic

terminals is therefore significantly lower than putative glutamatergic synapses who have KAR in

their postsynaptic spines. This is supported by a limited number of GABAergic synapses which

contain presynaptic GluR5/6/7 proteins.

Fig. 7. Immunoassay for GluR5/6/7 proteins in CA1interneurons. (A) Immunoreactivity in the CA1 area revealed by alight microscope. The somata and major processes (dendrites oraxons) of interneurons in the stratum rediatum (St. rad) andpyramidal neurons in the pyramidal layer (Pyr) were stained byGluR5/6/7 antibody. Scale bar, 50 _m. (B) A control EM pictureshowing symmetric (arrow) and asymmetric (arrow head)synapses. (C) An EM picture showing immunoreactivity in apresynaptic terminal of a symmetric synapse (arrow). (D) An EMpicture showing immunoreactivity in a postsynaptic spine of anasymmetric synapse (arrow head). Scale bars in B, C, and D, 0.1_m.

DISCUSSION

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In this study we investigated unique currents mediated by two subtypes of Kainate

Receptors, in the hippocampus. The ATPA activated subtype mediated a small, slowly rising

current that displayed inward rectification. Since it is known that ATPA is a selective agonist for

the GluR5 receptor subunit (Clarke et al., 1997; Cossart et al., 1998; Paternain et al., 2000), it

can be assumed that the subtype of KAR likely contains the GluR5 subunit. The reversal

potential we observed, as well as a Na+ replacement study and Ca2+ imaging experiments

suggest that this KAR subtype is Ca2+-impermeable. The inward rectification of the ATPA-

current further suggests that the GluR5 subunit is in the unedited form (Q) at the Q/R site. While

other groups have shown a Ca2+-permeable GluR5(Q) in dorsal root ganglion (DRG) neurons

(Lee et al., 2001), our work shows a different function in interneurons in CA1.

Micromolar KA activated the other subtype. This current was large and fast, and either

displayed inward rectification or a linear I-V curve. Results from reversal potential, Na+

replacement, and Ca2+ imaging revealed a Ca2+-permeable nature. Knowing that interneurons

hippocampus express predominantly GluR5 and GluR6 subunits (Burea et al., 1999; Parternain

et al., 2000; Mule et al., 2000), this subtype most likely contains the GluR6 subunit. Previously,

it was shown that between 80 and 90 percent of GluR6 receptors in the brain of animals at this

age of development are in the edited form (R) (Bernard, Khrestchatisky 1994; Cha et al., 1993;

Paschen, Djuricic 1994, 1995; Bernard et al., 1999). Based on the reversal potential observed

(+57.4 ± 3.8 mV in 62.5% of cells) it seems to be the GluR6(Q) form is present in these CA1

interneurons. The other version of this current we observed displayed an inward linear I-V curve

and a reversal potential +31.0 ± 4.7 mV. These results suggest that there are also GluR6 (R)

forms of this channel in this region, thought not at the proportion thought to dominate the CNS

expression of GluR6.

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Distribution of the KA-activated, Ca2+-permeable subtype of KARs was mimicked by

KA-induced CA2+ signaling. Another group previously showed that GluR6(R) is also permeable

to Ca2+ although significantly (3x) less than GluR6(Q) (Egebjerg, Heinemann 1993). KA

induced Ca2+ signals restricted to the axonal trunk and dendrites, suggested a distribution bias for

those regions of this subtype. KA induction of small Ca2+ levels in the soma, on the other hand,

suggest a relatively low distribution there for this subtype. No Ca2+ signals were detected in the

terminals, however. This result supports the notion of no increase for eIPSCs during application

of KAR agonists, because there would be a Ca2+ increase at the presynaptic terminals if this was

the case. However, these results are not in agreement with a postulate of KAR agonist-activated

Ca2+-permeable GluR6 in the terminals to attenuate GABA release. EM indicated a mere 9.7% of

symmetric synapses have GluR5/6/7 immunoreactivity in the presynaptic terminals, suggesting a

limited role for KARs in KA-induced disinhibition at the terminals. This is affirmed by a limited

number of GABAergic synapses that have KAR in their presynaptic terminals. By the same

token, these experiments cannot exclude the theory of KARs in the axonal trunk and other areas

mediating a G-protein-coupled cascade, and therein activating a diffusible intracellular

messenger that would further down inhibit GABA release.

The slight depolarization of interneurons following Ca2+-impermeable GluR5 receptor

activation, may facilitate AP-driven GABA release, due to increased width of APs which would

increase open time for synaptic voltage-dependent Ca2+ channels to activate (Kang et al, 2004).

Since the GluR6-mediated current is faster, larger and has a greater positive reversal potential

than the GluR5 current, this depolarization by activation of GluR6 receptors would be inherently

stronger. The results of the GluR6 experiments, namely their distribution in the axonal trunk and

dendrites, are in agreement with previously reported large depolarizing effects of KAR agonists

at these areas (Frerking et al., 1998; Semyanov, Kullmann 2001; Kang et al., 2004).

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Furthermore, micromolar concentrations of KAR agonists seems to activate additional GluR6

channels may over-depolarize interneurons and contribute to depression of inhibitory

transmission, commonly associated with KA application. It is also possible that KA-triggered

CA2+ influx may signal additional intracellular Ca2+-dependent signaling pathways that lead to

inhibition of GABA release.

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