Developmental regulation of AMPA-receptor properties in CA1 pyramidal neurons of rat hippocampus

Neuropharmacology 39 (2000) 931–942 www.elsevier.com/locate/neuropharm

Developmental regulation of AMPA-receptor properties in CA1 pyramidal neurons of rat hippocampus夽 Gerald Seifert a, Min Zhou 1,a, Dirk Dietrich a, Thekla B. Schumacher a, Andre Dybek b, Thomas Weiser c, Marion Wienrich c, Doris Wilhelm d, Christian Steinha¨user a,* a

Experimental Neurobiology, Neurosurgery, Bonn University, Sigmund-Freud-Str. 25, 53105 Bonn, Germany b Experimental Anesthesiology, Bonn University, Sigmund-Freud-Str. 25, 53105 Bonn, Germany c Department of CNS Research, Boehringer Ingelheim Pharma KG, 55216 Ingelheim, Germany d Janssen GmbH, Preclinical Research, Raiffeisenstr. 8, 41470 Neuss, Germany Accepted 19 August 1999

Abstract AMPA-receptor (AMPA-R) currents were recorded from CA1 pyramidal neurons in situ and after acute isolation from the hippocampus of 3- to 45-day-old rats. Membrane currents were analyzed by combining the patch clamp method with fast application techniques. The complete block of receptor currents by GYKI 53655 and the absence of modulation by Concanavalin A indicated that the cells exclusively expressed non-NMDA glutamate receptors of the AMPA subtype while functional kainate receptors could not be detected. The lowest sensitivity to kainate and NBQX was observed at postnatal day (p) 18. These changes might reflect a lower abundance of GluR1 at that developmental stage. A decrease of potentiation of receptor currents by cyclothiazide (CTZ), an acceleration of the recovery from CTZ potentiation and a faster and more complete desensitization of glutamate-evoked currents suggest an up-regulation of flop splice variants with increasing age. These functional data indicate that AMPA-R expression in CA1 pyramidal neurons varies during postnatal development which can be expected to influence the kinetics of synaptic transmission and the excitotoxic vulnerability as well.  2000 Elsevier Science Ltd. All rights reserved. Keywords: AMPA-receptors; Patch clamp; CA1 pyramidal neurons; Flip/flop splicing

1. Introduction Glutamate is the main excitatory neurotransmitter in the central nervous system and activates a large variety of ionotropic and metabotropic receptors (for review see Hollmann and Heinemann, 1994). It is thought that fast synaptic transmission is mainly mediated by ionotropic glutamate receptors (GluR) of the α-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid (AMPA) subtype (AMPA-R). Additionally, glutamate plays a crucial role as a neurotrophic factor that widely influences the regulation of neuronal circuitry and cytoarchitecture during 夽

The first two authors contributed equally to this work. * Corresponding author. Tel.: +49-228 2874669; fax: +49-228 2879121. E-mail address: [email protected] (C. Steinha¨user). 1 Present address: Division of Neurosurgery, Albany Medical College, Albany, NY 12208, USA.

development (McDonald and Johnston, 1990). AMPA-R activation was demonstrated to mediate neuroprotective actions (Bambrick et al., 1995) and to induce morphological changes in neurons (Mattson et al., 1988). Besides the physiological role of glutamate as an excitatory neurotransmitter, however, the excessive activation of GluRs leads to neurotoxicity and pathological changes in the nervous system (for review see Choi, 1992; Meldrum, 1994). Numerous studies have reported on the expression of AMPA-preferring GluR in hippocampal neurons. These functional studies have been complemented by evaluating receptor expression at the transcript level (Bochet et al., 1994; Geiger et al., 1995; Garaschuk et al., 1996) and by immunocytochemistry (Wenthold et al., 1996). At least four subunits, GluR1–4, constitute the AMPAR, and each of these subunits exist in different variants generated by alternative splicing (Hollmann and Heinemann, 1994). Recombinant receptor studies have demon-

0028-3908/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 2 1 2 - 9

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strated that functional properties of AMPA-Rs vary with subunit composition, alternative splicing, and RNA editing. The best-studied example of this type of regulation is the link between the relative abundance of the GluR2 subunit and Ca2+ permeability of the receptor pore. Furthermore, the relative affinities of some agonists and antagonists could be related to distinct subunit compositions (Stein et al., 1992; Nakanishi et al., 1990). Finally, the functional significance of the process of alternative splicing of AMPA-Rs could be unraveled with cyclothiazide (CTZ), a substance that distinguishes between the flip and flop versions of the receptors (Partin et al., 1994; Fleck et al., 1996). However, despite these important findings from expression studies, the composition of subunits that make up native AMPA-Rs in many neuronal cell types is still largely unknown. Early ligand binding studies demonstrated developmental changes in the expression of AMPA- and kainatereceptors in various brain areas (Insel et al., 1990; Miller et al., 1990). In the hippocampus, the number of binding sites showed an increase within the first 4 weeks of postnatal development. Subsequent in situ hybridization (Monyer et al., 1991; Pellegrini-Giampietro et al., 1992; Standley et al., 1995) and Northern blot analysis (Durand and Zukin, 1993) confirmed that the expression of AMPA-R subunits is altered during ontogenesis. Recently, single-cell reverse transcription–polymerase chain reaction (RT-PCR) experiments revealed that GluR1 and GluR2 were the main receptor subunits expressed in CA3 principal neurons (Geiger et al., 1995), while immunocytochemical analysis suggested that GluR1/2 and GluR2/3 were the dominant subtypes expressed in hippocampal CA1/CA2 neurons (Wenthold et al., 1996). The data on an age-dependent variation of AMPA-R expression in hippocampal neurons are still inconsistent and little evidence is available on accompanying changes in receptor functioning and pharmacology. To address this issue, we used the patchclamp technique to compare functional properties of AMPA-Rs expressed in CA1 pyramidal neurons during early postnatal development.

2. Methods Cell isolation was performed as previously described (Steinha¨user et al., 1990; Gu¨ndel et al., 1990) with few modifications. Briefly, female rats (postnatal days (p) 3– 45) were anesthetized (50% O2/50% CO2) and sacrificed by decapitation. Their brains were dissected, washed in oxygenated Ca2+-free bath solution at 6°C, placed onto the plastic stage of a tissue slicer, and cut in frontal orientation into 500-µm-thick slices. The slices were transferred to an O2-bubbled, Ca2+-free solution warmed up to 32–35°C (30–60 min), and incubated in papain (24 U/ml, supplemented with 0.24 mg/ml cysteine) contain-

ing solution (22°C). The digestion took 20, 30 and 50 min at p5, p18 and p45, respectively. After thorough washing, the slices were stored in oxygenated Ca2+-free solution for at least 60 min. For recording, the pyramidal layer of the CA1 subregion was carefully dissected to reduce contamination with other cell types, and the cells were dispersed by gentle trituration of tissues pieces with fire polished Pasteur pipettes (diameter 100–200 µm) under microscopic control (Telaval 3, Zeiss Jena, Germany). Therefore, most neurons in the suspension were shaped pyramidally and only those were selected in the present study (cf. Gu¨ndel et al., 1990). Cells were grouped as follows: p5 (p3–5), p18 (p9–18) and p45 (p26–45). Preparation of tissue slices for in situ recordings was similar as for isolated cells except that 400-µm slices were prepared with a vibratome (Ted Pella, Redding, USA) and transferred to a storage chamber (NaCl 124.0, KCl 3.0, CaCl2 1.6, MgSO4 1.8, NaH2PO4 1.25, NaHCO3 26.0, d-glucose 10.0 (mM); gassed with 95% O2/5% CO2). For recording, slices were placed in a submerged chamber on an inverted microscope and perfused with oxygen gassed standard bath solution (see below). Outside-out patches were obtained from CA1 pyramidal neurons and cells were identified by their location, electrophysiological properties and morphology as recovered by intracellular staining. Standard staining procedures were used to develop biocytin-filled cells in slices for later light microscopy (Horikawa and Armstrong, 1988). 2.1. Solutions, electrodes, and drugs The standard bath solution contained (in mM): 150 NaCl, 5 KCl, 2 MgSO4, 2 CaCl2, 10 glucose, 10 HEPES. In Ca2+-free solutions, CaCl2 was omitted and 1 mM sodium pyruvate was added. The pH of the external solution was adjusted to 7.4. Tetrodotoxin (TTX; 0.5 µM) was added to the solutions to block voltage-gated sodium currents. The standard pipette solution contained (in mM): 90 KCl, 30 KF, 0.5 CaCl2, 1 MgCl2, 5 BAPTA, 10 HEPES, 3 Na2-ATP, and 0.1 Na-GTP (pH 7.25). Outside-out patches were recorded with a pipette solution containing (in mM): 140 KCl, 2 MgCl2, 10 EGTA, 10 HEPES, 2 Na2-ATP, 0.1% biocytin (pH 7.3). Patch pipettes were pulled from borosilicate capillaries (Hilgenberg, Malsfeld, Germany) and subsequently fire polished. When filled with standard pipette solution, the pipette resistance was 1.5–3 M⍀ for isolated cells and 3–4 M⍀ for outside-out patches. CTZ was purchased from RBI (Natick, MA, USA), TTX from Alomone Labs (Jerusalem, Israel), and Na2ATP from Fluka (Switzerland). GYKI 53655 and NBQX were synthesized at the Department of Pharmaceutical Chemistry of Boehringer Ingelheim (Germany), and all the other salts and reagents were obtained from Sigma (St. Louis, MO, USA) or Merck (Darmstadt, Germany).

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CTZ and NBQX were dissolved in dimethyl sulfoxide at 100 mM and 10 mM, respectively, before dilution in the bath solution.

lation was approximated by a double or triple exponential function. The degree of receptor desensitization and recovery from CTZ modulation were estimated by

2.2. Recording setup and fast application techniques

Desensitization⫽100% [(Ipeak⫺Iss)/(Ipeak)]

Membrane currents in isolated cells were obtained with the patch-clamp technique in the whole-cell mode. Current signals were amplified (EPC-9, HEKA elektronik, Lambrecht, Germany), filtered at 3 kHz, sampled at 10 kHz, and monitored with TIDA software (HEKA). Whole-cell capacitance (CM) was determined from the current responses elicited by 10 mV voltage steps depolarizing the cell from ⫺70 to ⫺60 mV (sampling rate 30 kHz, filter 13 kHz). The time constants of capacitance artifacts were determined by fitting a single exponential to the current decay. These parameters corresponded well to the EPC-9 readings obtained after electronic cancellation of the capacitance transients. The mean values of CM were 14.3±3.3 pF (n=46, p5), 19.2±6.6 pF (n=54, p18), and 22.0±8.7 pF (n=64, p45). CM and RS compensation (40–50%) were used to improve voltage clamp control. The external solutions could be rapidly exchanged by means of a pneumatically controlled application system. Using solutions of different ionic concentrations and open pipettes the 20–80% exchange time was 0.9–1.2 ms (Seifert et al., 1997). Glutamate evoked currents in outside-out patches (holding potential ⫺50 mV) were filtered at 3 kHz and sampled at 100 kHz (EPC7, List, Darmstadt, Germany; Digidata 1200 and pClamp 6.0, Axon Instruments, Foster City, USA). For application of a 100–120 ms pulse of l-glutamate to an excised patch, a two tube high speed solution exchange system driven by a pinch valve was used (Brett et al., 1986). The 20–80% solution exchange times ranged between 90 and 200 µs (open pipette). The 20–80% rise time of AMPA currents was 558±228 µs (n=12). Currents were discarded if their rise time was ⱖ1 ms. Electrophysiological measurements were performed at room temperature.

where Ipeak is the maximal receptor current, and Iss the steady-state current in the presence of the agonist, determined 300 ms or 20 s after the onset of drug application. Currents were rundown corrected by comparing test responses with the average of control amplitudes determined immediately before and after test application. All data are given as mean±SD. Significance differences were evaluated according to the two-sided Student’s ttest. The level of significance was set at p⬍0.05.

2.3. Data analysis Dose–response curves were fitted according to the Hill equation. Antagonist inhibition curves were fitted by the equation I⫽Imax{1/(1⫹([antagonist]/IC50)}

(1)

with Imax being the response at saturating agonist concentration, [antagonist] the concentration of the applied antagonist, and IC50 the concentration at half maximal inhibition of the agonist response. NBQX affinity was calculated according to the Cheng–Prussoff equation: Ki⫽IC50,NBQX/{1⫹([kainate]/EC50,kainate)}.

(2)

The recovery of the receptor responses from CTZ modu-

(3)

3. Results 3.1. No evidence for functional kainate receptors in CA1 pyramidal neurons The expression of the AMPA subtype of GluR in hippocampal neurons has been confirmed by both molecular-based studies and functional analysis. Two recent studies provided first evidence for the presence of functional kainate-preferring receptors in CA3 pyramidal cells in situ (Vignes and Collingridge, 1997; Castillo et al., 1997). To unmask a possible contribution of currents through kainate receptors in our acute preparation we used the 2,3-benzodiazepine GYKI 53655 (Partin and Mayer, 1996; Wilding and Huettner, 1995; Donevan et al., 1994), that successfully isolated kainate receptor currents by completely blocking AMPA-Rs in the same individual cell. During kainate application (0.5 mM), a high concentration of GYKI 53655 (0.1 mM) was coapplied with the agonist to ensure a complete block of AMPA-Rs. We observed a time-dependent but complete antagonism of the kainate-induced currents (Fig. 1A and C). Pre-incubation in GYKI 53655 completely suppressed the responses to kainate (n=6; Fig. 1B). Thus, the transient current observed without pre-incubation reflected the time course of onset of GYKI 53655 antagonism at AMPA-preferring receptors but not an activation of kainate-preferring receptors. Accordingly, after pre-incubation in Concanavalin A (ConA; 0.3 mg/ml; 3 min) plus GYKI 53655, a subsequent kainate application did not evoke any GYKI 53655 resistant currents (Fig. 1C; n=3). A recent report raised the questions whether GYKI 53655 at a concentration of 100 µM could also interfere with kainate-preferring receptors (Frerking et al., 1998; Bureau et al., 1999). Therefore, we reduced the GYKI 53655 concentration to 50 µM and activated receptor currents with 50 µM kainate. Subsequently, after washout of kainate, the cells were incubated in ConA contain-

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ing solution (1 mg/ml) supplemented with 50 µM GYKI 53655 (for 5 min, n=7). The control kainate current density was 58.1±18.9 pA/pF. Co-application of kainate and GYKI 53655 (50 µM each) never produced transient inward currents at ⫺70 mV. A small sustained current was observed in three cells (p45; 2.7±0.7 pA/pF, i.e. 4.6% of the control) while in the remaining cells (n=2, p18; n=2, p45) GYKI 53655 completely blocked the responses. Subsequent ConA treatment neither enhanced amplitudes nor changed the kinetics of the kainate responses. Finally, we tested the responses to low concentration of kainate (10 µM) before and after preincubation in ConA in the absence of GYKI 53655. The control current density was 9.5±3.7 pA/pF (n=7) and remained unchanged after ConA application (9.2±3.6 pA/pF; Fig. 1D). The kainate EC50 values observed here (Table 1) were about one order of magnitude higher than those observed at high-affinity kainate receptors (EC50苲20 µM, Lerma et al., 1993; Wilding and Huettner, 1997). At lower kainate concentrations (10 or 50 µM), the responses were also characterized by a significant steady-state component (cf. Figs. 1D and 2A), but not by the rapid desensitization that is typically observed in cells expressing kainate-preferring receptors. Collectively, our findings lend strong support to the notion that in our preparation, the glutamate and kainate responses were due to activation of AMPA-Rs rather than kainate receptors. Therefore, in the following these receptors will be referred to as AMPA-Rs. 3.2. AMPA-R current density and kainate affinity To obtain a measure independent of changes in surface area, current densities were calculated from the steady-state kainate responses (0.5 mM kainate, ⫺70 mV) and the corresponding CM. Current densities did not change during development, amounting to 89±40 pA/pF (n=33), 121±99 pA/pF (n=28), and 113±65 pA/pF (n=56) at p5, p18 and p45, respectively. Fig. 1. CA1 pyramidal neurons express AMPA-Rs. (A) After a control application of kainate (500 µM), co-application of GYKI 53655 (100 µM) and kainate (500 µM) resulted in a residual, transient current response. The time constant of current decay was 76 ms. (B) The antagonism of GYKI 53655 was reversible after washout. The residual response elicited by kainate plus GYKI 53655 completely disappeared when the cell was preincubated in GYKI 53655 (100 µM, 1 min). (C) The block of the residual response was reversible after washout. Preincubation in ConA (0.3 mg/ml, 3 min) together with GYKI 53655 failed to uncover any response of putative kainate-preferring receptors during a subsequent agonist application. All recordings were obtained from the same p5 pyramidal neuron (V=-70 mV). (D) The upper panel gives a microphotograph of an isolated CA1 pyramidal neuron at p11 (scale bar: 50 µm). After a control application of 10 µM kainate, the cell was incubated in 1 mg/ml ConA for 5 min and then kainate was co-applied together with ConA (V=-70 mV). The response remained unchanged after ConA treatment.

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Table 1 Developmental changes in agonist and antagonist potenciesa

Kainate EC50 (µM) Hill coefficient nH NBQX IC50 (nM) GYKI 53655 IC50 (µM)

p5

p18

p45

226±39 (4) 1.33±0.25 191±90 (6) 5.9±0.5 (4)¶

320±91 (4)* 1.23±0.15 366±102 (6)† 3.9±1.2 (4)

180±53 (5) 0.97±0.16 203±40 (5) 3.5±1.0 (5)

a

Values in parentheses give the cell numbers. The significance of the difference between p18 and p45 is indicated by * (kainate EC50), between p18 and all other ages by † (NBQX IC50), and between p5 and all other ages by ¶ (GYKI 53655 IC50).

rents obtained at different concentrations (V=⫺70 mV) were normalized to control responses elicited by 1 mM kainate in the same cell. The EC50 ranged between 180±53 µM (n=5; p45) and 320±91 µM (n=4; p18) (Table 1) and were statistically different. 3.3. Competitive antagonism of kainate responses by NBQX

Fig. 2. The affinity of kainate to AMPA-Rs changed during development. Steady-state kainate currents were used to construct doseresponse curves (V=-70 mV). (A) Current traces obtained from the same individual p10 cell. (B) Test current amplitudes at different concentrations were normalized to the corresponding response evoked by 1 mM kainate in the same cell. Dose-response curves are demonstrated for p18 (triangles) and p45 cells (dots). Smooth lines give the best fit according to the Hill equation (I=Imax{1/[1+(EC50/[kainate])nH]}). Each data point represents the mean of pooled data from four cells at p18 (EC50=320 µM, Hill coefficient nH=1.23) and five cells at p45 (EC50=180 µM, nH=0.97), bars give SD. The p5 curve was located between these two curves (not shown). Note the left shift in the doseresponse curves at p45. The change of kainate affinity between p18 and p45 curves was statistically significant.

Recombinant receptor studies have shown that the presence of specific subunits in the receptor complexes confers on them a distinct agonist affinity. Hence, changes in agonist affinity during development could be indicative of variations in receptor subunit composition. Fig. 2 gives dose–response curves for kainate together with a representative current family. Steady-state cur-

Until now NBQX has been demonstrated to be the most potent AMPA-R antagonist. It inhibits AMPA-R responses through a competitive mechanism (Donevan and Rogawski, 1993; Parsons et al., 1994). Recombinant receptor studies have indicated that the potency of NBQX depends on receptor subunit combination and it was suggested that this antagonist can be used to detect variations in subunit expression (Stein et al., 1992). To test for age-associated changes in the NBQX effect, dose–inhibition curves were determined with kainate (0.5 mM) as a receptor agonist (Fig. 3). Data were taken at the end of the responses and normalized to corresponding control currents evoked by 0.5 mM kainate in the same cell. As for kainate affinities, NBQX potencies did not continuously change with increasing age (Table 1). In the absence of the agonist, NBQX occupied half the receptors at concentrations of 59 nM (p5), 143 nM (p18), and 54 nM (p45), estimated according to the Cheng–Prussoff equation (Eq. (2)). 3.4. Noncompetitive antagonism of kainate responses by GYKI 53655 As mentioned above, GYKI 53655 has been demonstrated to act as a highly selective antagonist at both native and recombinant AMPA-Rs. The block produced by this 2,3-benzodiazepine occurred in a noncompetitive fashion, was voltage independent, and failed to show use-dependence, indicating an allosteric blocking mechanism (Donevan and Rogawski, 1993). To reveal ontogenetic changes in AMPA-R modulation by GYKI 53655 in CA1 neurons, different concentrations of the antagonist (0.1–100 µM) were co-applied with 0.5 mM kainate (V=⫺70 mV; Fig. 4). The inhibition curves were constructed taking the data at the end of the current

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Fig. 3. Competitive antagonism of kainate responses by NBQX. A series of NBQX concentrations were co-applied each with 500 µM kainate to construct NBQX inhibitory curves. The drug-resistant current amplitudes were normalized to corresponding responses produced by 500 µM kainate in the same cell. (A) A representative current family obtained from one individual p4 pyramidal neuron. (B) NBQX inhibitory curves for p18 (dots) and p45 cells (triangles). The p45 curve was located between these two curves (not shown). Each data point represents mean and SD of pooled data from six cells at p18 and six cells at p5. Data were fitted by Eq. (1) (smooth lines). No continuous change in NBQX potencies was observed with increasing age. There was, however, a significant left shift of the p18 curve (IC50=366 nM) compared with p45 (IC50=203 nM) and p5 (IC50=191 nM).

traces. We observed an increase in antagonist affinity with proceeding maturation (Table 1). The onset of GYKI 53655 block of kainate responses was accelerated with increasing concentration of the antagonist. Assuming a bimolecular receptor binding of GYKI 53655, a rate constant for concentration-dependent binding (kon) was calculated (Donevan and Rogawski, 1993). The monoexponential time constant of onset of block was determined at various GYKI 53655 concentrations and different ages. The reciprocal of the time constants was plotted against the GYKI 53655 concentration and the apparent rate constant of binding was determined from the slope of the best straight line fit (Fig. 4C). Thus, the rate constants, kon, of GYKI 53655 binding amounted to 9.7×104 M⫺1 s⫺1, 2.5×105 M⫺1 s⫺1, and 2.3×105 M⫺1 s⫺1 at p5, p18 and p45, respectively. Table 1 demonstrates that neurons at p5 displayed the lowest GYKI 53655 potency and these cells also showed the slowest onset kinetics of GYKI block.

Fig. 4. Noncompetitive antagonism of kainate responses by GYKI 53655. GYKI 53655 at various concentrations was co-applied with 500 µM kainate to obtain GYKI 53655 inhibition curves. Data were taken at the end of the current traces and were normalized to control responses evoked by 500 µM kainate in the same cell. (A) Representative current traces obtained from one individual p35 pyramidal neuron. (B) GYKI 53655 inhibitory curves analyzed at p5 (circles) and p45 (triangles) were fitted by Eq. (1) (smooth lines). Each data point represents mean and SD of four cells at p5 and five cells at p45. The p5 curves differed significantly with the other age groups. The p18 curve was located between the two curves (not shown), indicating an increase in GYKI 53655 potencies with maturation. (C) Kainate (500 µM) was simultaneously applied with GYKI 53655 (0.1-100 µM) and the time course of the onset of block was determined by single exponential fits. The onset rates were calculated as the reciprocal of the time constants and plotted against the GYKI 53655 concentration. Each data point represents mean and SD of four to six different cells. The dotted lines indicate the best straight line fits.

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3.5. Age-dependent modulation of AMPA-Rs by cyclothiazide Research on recombinant receptors has found that CTZ can modulate AMPA-Rs in a splicing-dependent manner, producing pronounced attenuation of desensitization at flip versus flop variants (Partin et al., 1994). We used this drug to reveal developmental variations in flip/flop expression in CA1 neurons. After control application of 100 µM kainate, the cells were preincubated in CTZ-containing solution (ⱖ20 s) and were subsequently exposed to the agonist again. In all cells, we observed a potentiation of kainate responses by CTZ. However, the potentiation was clearly dependent on the stage of postnatal development (Fig. 5). Maximal potentiation was produced at p5 (873±288%, n=6) and p18 (814±65%, n=6), while at p45, CTZ potentiation was significantly less (375±54%, n=5). Besides its distinct modulatory effect on the AMPAR splice variants, the affinity of CTZ to flip and flop forms differed significantly. The slow dissociation of CTZ from the flip versions caused a marked delay in the recovery from CTZ modulation (Partin et al., 1994; Fleck et al., 1996). We analyzed the recovery of glutamate responses from CTZ potentiation for further infor-

937

mation on developmental changes in alternative splicing. The decay of current in the presence of glutamate was recorded immediately after removal of CTZ. Analysis of responses to a longer application (⬎30 s) of glutamate was not possible in most of the experiments because the neurons were frequently damaged by the large-amplitude currents recorded after treatment with CTZ (see also Fleck et al., 1996). The decay of responses to glutamate was fit by the sum of two or three exponentials. In cells where either the amplitude of one of the components was near zero or the time constants of two components were nearly equal, a double exponential was used (14 out of 20 cells). Several findings indicated an up-regulation of flop variants during maturation. First, the recovery from CTZ potentiation, determined 20 s after glutamate application was much more complete at p45 compared with p5 and p18. Second, a predominant fast time constant (i.e. ⬎50% of the decay characterized by tf⬍1 s) was observed at the more mature stage. Third, a predominant slow time constant (i.e. ⬎50% of the decay characterized by ts⬎10 s), was found at p5 and p18 (Table 2, Fig. 6). These data agreed well with the findings on kainate potentiation by CTZ (Fig. 5). 3.6. Outside-out patch analysis confirms agedependent changes in receptor kinetics To further substantiate age-dependent changes in flip/flop splicing and to exclude putative artifacts due to the isolation procedure, receptor current kinetics were compared in CA1 neurons in situ. The cells were filled with biocytin (Fig. 7A) and subsequently glutamate (1 mM) was rapidly applied to an outside-out patch isolated from the same cell (holding potential ⫺50 mV). The current decay could be fitted well by a double exponential (Fig. 7B). The time constants of desensitization, tfast and tslow, did not vary with age, but their relative amplitudes ms, differed significantly (p18: tfast=7.4±2.1 46.1±16.2%; tslow=28.3±5.2 ms, 53.9±16.2%; n=7, and p45: tfast=7.6±1.5 ms, 69.3±15.5%; tslow=27.2±3.5 ms, 30.7±15.5%; n=5) (Fig. 7C). In addition to the faster kinetics, receptor desensitization was more complete in the older cells. The steady-state currents 100 ms after the onset of glutamate application reached 7.9±3.3% (p18) and 3.6±2.7% (p45) of the corresponding maximum currents with the differences being statistically significant.

4. Discussion Fig. 5. Potentiation of kainate-activated receptor currents by CTZ. (A) The potentiation of kainate (100 µM) currents by CTZ (100 µM) was age-dependent. At p11, preincubation in CTZ resulted in a 820% increase in current amplitudes, whereas in the p31 cell, the potentiation amounted to only 360% (V=-70 mV). (B) Histogram illustrating the CTZ potentiation (100 µM) of kainate currents (100 µM) as a function of postnatal age. The decrease in potentiation at p45 was significant (*) compared with CTZ modulation at p5 and p18.

4.1. Lack of functional kainate-preferring receptors in CA1 pyramidal neurons In situ hybridization has demonstrated kainate-preferring receptors in the hippocampus (Bettler et al., 1990; Herb et al., 1992; Wisden and Seeburg, 1993) and cul-

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Table 2 Changes in recovery of glutamate responses from CTZ potentiationa

Recovery (%) 20 s after CTZ removal tf (s) ti (s) ts (s)

p5

p18

p45

55±10 (6)

56±16 (8)

83±17*(6)

0.4±0.3 (5) 25.6±12.4% 3.0±1.2 (3) 41.7±26.0% 14.1±3.8 (6) 57.8±27.5%

0.5±0.1 (5) 31.4±18.7% 3.3±2.2 (4) 23.4±17.2% 23.7±13.4 (8) 68.7±16.8%

0.7±0.2 (6) 55.6±25.5%† 3.9±1.5 (4) 15.6±12.0% 12.3±2.8 (6) 30.9±15.2%¶

a Values in parentheses give the cell numbers. The significance of the difference between p45 and all other ages is indicated by *, between p5 and p45 by †, and between p18 and p45 by ¶. The fast time constant (tf ) was defined as being ⬍1 s, the intermediate time constant (ti) between 1 and 10 s, and the slow time constant (ts) ⬎10 s.

ture studies demonstrated a co-expression of functional kainate-preferring receptors with AMPA-Rs by embryonic (Lerma et al., 1993; Paternain et al., 1995) and postnatal (Wilding and Huettner, 1997) hippocampal neurons. In contrast, evidence for the expression of functional kainate receptors in the CNS in vivo was lacking until a short time ago. Recently, however, it has been shown that in CA3 neurons synaptic stimulation and exogenous kainate activated kainate-preferring receptors (Vignes and Collingridge, 1997; Castillo et al., 1997; Mulle et al., 1998). In the CA1 region, postsynaptic kainate receptors have been detected in interneurons (Cossart et al., 1998; Frerking et al., 1998) but not in pyramidal neurons (Castillo et al., 1997; Lerma et al., 1997; Frerking et al., 1998; but see Bureau et al., 1999). We also failed to detect these receptors in the acutely isolated CA1 pyramidal cells since (i) low concentrations of kainate never elicited transient responses, (ii) the kainate responses were sensitive to GYKI 53655, and (iii) the kainate-evoked currents were not modulated by ConA. 4.2. Variation of pharmacological properties of AMPA-Rs during development AMPA-Rs are thought to possess a pentameric or tetrameric stoichiometry. Thus, cells expressing two or more distinct subunits probably will produce multiple receptor complexes with different properties. Single-cell PCR (Geiger et al., 1995; Garaschuk et al., 1996) and immunoprecipitation (Wenthold et al., 1996) studies showed that a number of AMPA-R subunits and receptor complexes usually coexist in each individual neuron. Recently, Fleck et al. (1996) compared data from recombinant receptors with AMPA-Rs expressed in cultured hippocampal neurons. They came to the conclusion that in native cells, the complex kinetics of glutamate current recovery from CTZ modulation reflected the heterogeneity in subunit composition of AMPA-R channels. Assuming that this is a sensitive assay to distinguish

between receptor complexes differing in splice variants and subunits, our data from acutely isolated neurons confirmed such heterogeneity. In the present experiments, two or three time constants of recovery from CTZ potentiation were found, and their relative amplitudes were under developmental regulation (see below). Recently, it was shown that AMPA-Rs of CA1/CA2 pyramidal neurons mainly comprise heteromeric GluR1/GluR2 and GluR2/GluR3 receptor complexes, with another 8% of the receptors being assembled from homomeric GluR1 (Wenthold et al., 1996). These results were obtained from rats at p30–p40, making them directly comparable to our functional data. We compared AMPA-R functioning in CA1 pyramidal neurons between p3 and p45. Our results closely matched wholecell data on kainate potency reported by Pidoplichko et al. (1996) (EC50=271 µM, p20), while a higher value was obtained from outside-out patches (EC50=474 µM, Jonas and Sakmann, 1992). Notably, we failed to find a progressive change of kainate affinity with proceeding maturation. Recombinant receptor studies have demonstrated that the kainate affinities of homomeric GluR1, heteromeric GluR1/GluR3 and GluR1/GluR2 are remarkably higher than of those receptors composed of GluR2/GluR3 (Hollmann et al., 1989; Nakanishi et al., 1990; Stein et al., 1992). It is noteworthy that the present experiments revealed a parallel change in the affinities of both an agonist and a competitive antagonist at AMPA-Rs which is difficult to explain assuming a simple drug–receptor interaction. Interestingly, Stein et al. (1992) suggested that AMPA-R agonists and antagonists bind to different receptor substructures and that the binding sites can vary between different subunit combinations. Altogether, in comparing recombinant receptor findings with the present functional analysis, we suggest that multiple AMPA-R complexes are co-expressed in the CA1 principal neurons, with the proportion of GluR1 containing heteromers probably being transiently decreased at p18. The GYKI 53655 potencies found here were some-

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Fig. 6. Recovery of glutamate responses from CTZ potentiation. Recordings were obtained from cells at postnatal ages as indicated. After preincubation in CTZ (100 µM, 25 s), l-glutamate (1 mM) was applied at V=-70 mV. In the cells obtained at p5 and p11, current decay was fitted by a double exponential. Twenty seconds after glutamate application, the receptor currents still reached 48% (p5) and 44% (p11) of the initial amplitude. In contrast, in the p31 cell, a three exponential fit was necessary for approximation, with recovery being almost complete (85%) at the end of the recording.

what lower than those reported for cultured hippocampal (IC50=1.5 µM, Donevan et al., 1994), forebrain (IC50=1.2 µM, Waters and Allen, 1998), and cortical neurons (IC50=0.8 µM, Wilding and Huettner, 1995). Although the action of 2,3 benzodiazepines is assumed to occur in a noncompetetive fashion by an allosteric mechanism (Donevan and Rogawski, 1993), other studies indicated an influence of these substances on the agonist binding site (Parsons et al., 1994; Rammes et al., 1996). These reports demonstrated a right shift of kainate dose– response curves by GYKI 52466 and a change of onset and offset kinetics of AMPA-evoked currents by GYKI 53655. Thus, in our study, by using a relatively high

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Fig. 7. Outside-out patches obtained from hippocampal slices. (A) Microphotograph of a biocytin-filled CA1 pyramidal neuron at p45. Before a patch was excised the cell was held for 2 min in the whole cell configuration to allow diffusion of biocytin into the cell. (B) Receptor currents were evoked by rapid application of a 100 ms l-glutamate (1 mM, -50 mV) pulse. Patches were isolated from cells at p45 (same cell as in (A)) and p15. Currents were scaled to peak amplitude (absolute amplitudes were 323 pA, p15 and 249 pA, p45). Note the faster and almost complete desensitization of the older cell. Bi-exponential fits (dashed lines) revealed that the faster decay was due to differences in the relative amplitudes of fast (tf) and slow (ts) time constants. The traces represent averages of ten sweeps. (C) Mean values and SD of fast and slow time constants are given together with the corresponding relative amplitudes (open circles, p18, n=7; filled squares, p45, n=5). While tf and ts were virtually identical in both age groups, the corresponding relative amplitudes differed significantly (marked by *).

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kainate concentration (500 µM) to overcome age-dependent differences in kainate affinity, the somewhat higher IC50 for GYKI 53655 could indicate such an allosteric interference of the antagonist with the AMPA-R binding site. However, data from cultured cortical neurons apparently do not support this idea (Wilding and Huettner, 1995). A lower GYKI 53655 sensitivity was also observed in recombinat AMPA-Rs and it was speculated that as yet unknown modulatory proteins could be responsible for such differences (Partin and Mayer, 1996). The rate constants for the onset of block by GYKI 53655 found in the isolated CA1 pyramidal cells fit well the data from cultured hippocampal neurons (Donevan et al., 1994). 4.3. Switch in splice variant expression during maturation Alternative splicing of AMPA-R subunits (Sommer et al., 1990) controls the extent of receptor desensitization in a subunit-dependent manner, with the flop version of GluR3 and GluR4 producing very fast current desensitization (Mosbacher et al., 1994). In contrast, in heteromeric subunit combinations GluR2 flip produces slow receptor desensitization kinetics (Geiger et al., 1995; Mosbacher et al., 1994; Partin et al., 1994). Previous in situ hybridization studies have indicated that these splice variants display distinct expression profiles in the developing brain. Prenatal neurons mainly express flip forms of GluR1–4 that persist throughout life. The flop forms do not significantly appear until early postnatal stages, and then are co-expressed with flip forms in many cells (Sommer et al., 1990; Monyer et al., 1991). The selective AMPA-R modulator, CTZ, offers the possibility of functionally distinguishing between flip/flop variants. It has been shown that receptors carrying the flip forms exhibit a greater sensitivity to CTZ than those receptors assembled from flop (Partin et al., 1994). Furthermore, the kinetics of recovery from CTZ potentiation differed considerably for both splice variants (Partin et al., 1994; Fleck et al., 1996). In our study, a significant decrease in potentiation of the neuronal kainate responses was found between p18 and p45. Additional evidence reflecting a relative up-regulation of flop versions was the age-related speed-up of the recovery of glutamate responses from CTZ modulation (Table 2, Fig. 6). Furthermore, the relative amplitude of tf, probably indicating receptor complexes largely assembled from flop version subunits, also increased with maturation (Table 2). A complex kinetics of recovery from CTZ potentiation was a typical property of all the cells analyzed (Fig. 6). However, the portion of cells which had to be approximated by a three exponential fit (30%) was obviously lower than in cultured hippocampal neurons (67%, Fleck et al., 1996). Comparing the

recovery kinetics of glutamate responses after CTZ modulation of native and recombinant AMPA-Rs, previous work suggested a large variability in subunit and splice variant expression in the native cells (Fleck et al., 1996). Our data demonstrate that such a diverse expression of AMPA-Rs is not only observed in culture but in situ as well. The gating properties of the AMPA-R channels also critically depend on alternative splicing. Outside-out patches were isolated from CA1 pyramidal neurons in situ to compare glutamate evoked current kinetics at different ages. We found a significantly faster and more complete desensitization of receptor currents in the older cells. This data fits well with the results obtained from the acutely suspended cells and makes it unlikely that the latter were affected by the isolation procedure. All these functional changes strongly supported the assumption that early in postnatal life CA1 pyramidal neurons mainly express flip splice variants while the flop portion increases with continued maturation. Interestingly, an opposite chronological order of flip and flop variant expression of AMPA-Rs was found in CA1 astrocytes (Seifert et al., 1997). In the hippocampus, quisqualate- and AMPAmediated neurotoxicity peaks early in postnatal development (for review see McDonald and Johnston, 1990), which is compatible with a dominant expression of the flip forms of neuronal AMPA-Rs throughout the brain before p8 (Monyer et al., 1991). Our functional findings revealed a slightly delayed switch in splice variants. However, the data fit well into the general changes as discussed in the reports mentioned above, since they also predict a higher vulnerability for very early postnatal CA1 pyramidal neurons and point to a developmental change of glutamate efficacy.

Acknowledgements We gratefully acknowledge the excellent technical assistance of I. Krahner and thank Dr. M. Brenner (Boehringer Ingelheim KG) who provided NBQX and GYKI 53655. We also thank Dr. B.W. Urban for helpful discussion. This research was supported by Bundesministerium fu¨r Forschung und Technologie, Fonds der Chemischen Industrie, and Deutsche Forschungsgemeinschaft (SFB 400, Graduiertenkolleg 246).

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