Profound regulation of neonatal CA1 rat hippocampal GABAergic transmission by functionally distinct kainate receptor populations

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1 J Physiol (2005) pp Profound regulation of neonatal CA1 rat hippocampal GABAergic transmission by functionally distinct kainate receptor populations François Maingret 1,Sari E. Lauri 2,TomiTaira 2 and John T. R. Isaac 1,3 1 MRC Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, Bristol BS8 1TD, UK 2 Neuroscience Centre and Department of Biosciences, PO Box 65 (Viikinkaari1), University of Helsinki, Finland 3 NINDS, NIH, 35 Convent Drive, Bethesda, MD 20892, USA Neonatal hippocampus exhibits distinct patterns of network activity that are dependent on the interaction between inhibitory and excitatory transmission. Kainate receptors are ideally positioned to regulate this activity by virtue of their ability to regulate presynaptic function in GABAergic interneurones. Indeed, kainate receptors are highly expressed in neonatal hippocampal interneurones, yet the role and mechanisms by which they might regulate neonatal circuitry are unexplored. To address this we investigated the kainate receptor-dependent regulation of GABAergic transmission onto neonatal CA1 pyramidal neurones. Kainate receptor activation produced two distinct opposing effects, a very large increase in the frequency of spontaneous IPSCs, and a robust depression of evoked GABAergic transmission. The up-regulation of spontaneous transmission was due to activation of somatodendritic and axonal receptors while the depression of evoked transmission could be fully accounted for by a direct regulation of GABA release by kainate receptors located at the terminals. None of the effects of kainate receptor agonists were sensitive to GABA B receptor antagonists, nor was there any postsynaptic kainate receptor-dependent effects observed in CA1 pyramidal cells that could account for our findings. Our data demonstrate that kainate receptors profoundly regulate neonatal CA1 GABAergic circuitry by two distinct opposing mechanisms, and indicate that these two effects are mediated by functionally distinct populations of receptors. Thus kainate receptors are strategically located to play a critical role in shaping early hippocampal network activity and by virtue of this have a key role in hippocampal development. (Resubmitted 27 April 2005; accepted after revision 2 June 2005; first published online 9 June 2005) Corresponding author J. Isaac: NINDS, NIH, 35 Convent Drive, Bethesda, MD 20892, USA. isaacj@ninds.nih.gov Glutamate mediates the major part of the fast excitatory synaptic transmission in the brain and acts through three classes of ionotropic receptors: α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), N-methyl-d-aspartate (NMDA), and kainate receptors (Bettler & Mulle, 1995). Despite their widespread expression in the mammalian brain, little is known about the functions and physiological roles of kainate receptors in comparison with the more commonly studied AMPA and NMDA receptor subtypes (Lerma etal. 2001; Huettner, 2003). Kainate receptors are present at both postsynaptic and presynaptic locations (Kullmann, 2001; Lerma et al. 2001; Huettner, 2003; Lerma, 2003; Isaac et al. 2004). The presynaptic actions of kainate receptors have been of particular interest because they regulate both glutamatergic and GABAergic transmission, thus providing potential local circuit mechanisms for regulating network excitability. This has been best described in the CA1 region of hippocampus where presynaptic kainate receptors inhibit glutamatergic transmission (Chittajallu et al. 1996; Frerking et al. 2001) and can also negatively or positively regulate GABAergic transmission (Clarke et al. 1997; Rodriguez-Moreno et al. 1997, 2000; Frerking et al. 1999; Mulle et al. 2000; Cossart et al. 2001; Jiang et al. 2001; Kullmann, 2001; Semyanov & Kullmann, 2001; Isaac et al. 2004). Neonatal hippocampus expresses high levels of kainate receptor subunits which decline as development progresses (Bahn et al. 1994; Ritter et al. 2002). There is also prominent high frequency spontaneous network activity in neonatal hippocampus (Ben-Ari et al. 1989; Garaschuk. No claim to original US government works. DOI: /jphysiol

2 132 F. Maingret and others J Physiol et al. 1998; Palva et al. 2000; Lahtinen et al. 2002; Leinekugel et al. 2002). This requires appropriate glutamatergic and GABAergic synaptic activity (Khazipov et al. 1997; Garaschuk et al. 1998; Bolea et al. 1999; Palva et al. 2000; Lamsa et al. 2000), is critical to normal hippocampal development (Groc et al. 2002; Lauri et al. 2003), and is developmentally down-regulated such that it is no longer evident by the end of the second postnatal week (Ben-Ari et al. 1989; Garaschuk et al. 1998; Khazipov et al. 2004). This strong developmental correlation between kainate receptor expression and hippocampal spontaneous activity combined with the potential for modulation of glutamatergic and GABAergic circuits in hippocampus by kainate receptors suggests that they may play a critical role in regulating early network activity in the developing hippocampus. Here we investigate the role of presynaptic kainate receptors in regulating GABAergic transmission onto neonatal hippocampal CA1 pyramidal neurones. We show that neonatal CA1 GABAergic transmission is very sensitive to kainate receptor activation which produces two opposing effects: a very large increase in spontaneous IPSCs due to activation of somatodendritic and axonal receptors, and a strong depression of evoked IPSCs due to the presence of kainate receptors at terminals that directly inhibit GABA release. These findings indicate that there are two functionally distinct populations of kainate receptors that can differentially regulate GABAergic transmission in neonatal CA1. Thus kainate receptors are ideally situated in the neonate to play a critical role in regulating early hippocampal network activity. Methods Electrophysiology Hippocampal slices (400 µm thick) were prepared from 5- to 6-day-old Wistar rat pups (day 0 is the day of birth) using standard techniques and in accordance with United Kingdom Home Office regulations (Animals (Scientific Procedures) Act 1986). Briefly animals were deeply anaesthetized using sodium pentobarbital (60 mg kg 1 i.p.), decapitated and then transverse hippocampal slices were prepared as described (Daw et al. 2000). The extracellular solution was as follows (mm): 119 NaCl, 2.5 KCl, 1.0 NaH 2 PO 4, 26.2 NaHCO 3, 2.5 CaCl 2, 1.3 MgSO 4,11glucose, saturated with 95% O 2 5% CO 2.Inthe majority of experiments room temperature (22 25 C) was used, but for the mipsc experiments the bath temperature was C. Whole-cell patch-clamp recordings from visually identified CA1 pyramidal neurones were made using electrodes (3 5 M ) filled with the following intracellular solution (mm): 134 CsMeSO 4,4 NaCl, 10 Hepes, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP, ph 7.2, 285 mosmol l 1. Monosynaptic pharmacologically isolated IPSCs recorded at a holding potential of 50 mv were evoked using local extracellular stimulation of axons in stratum radiatum at a frequency of 0.1 Hz in the presence of d( )-2-amino-5-phosphonopentanoic acid (d-ap5) (50 µm) to block NMDA receptors and 1,2,3,4- tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulf onamide (NBQX) (2 µm) or GYKI53655 (25 µm) to block AMPARs. For recordings from interneurones, CA1 stratum radiatum interneurones were visually identified based on their soma shape and localization. Perforated patch-clamp recordings were made as previously described (Lamsa & Taira, 2003) with electrodes (12 14 M ) tip-filled with a solution containing (in mm): 135 potassium gluconate, 10 Hepes, 5 EGTA, 4 Mg-ATP, 0.5 Na-GTP, 2 KCl, 2 Ca(OH) 2, 290 mosmol l 1, ph 7.2, and then backfilled with the same solution containing gramicidin (100 µgml 1 )oramphotericin-b (500 µgml 1 ). It was necessary to use the perforated patch-clamp configuration since antidromic action current success rate was not stable under whole-cell recording conditions. To evoke antidromic action currents, a bipolar stimulation electrode was placed in stratum oriens close to the site of recording (Semyanov & Kullmann, 2001). The stimulation intensity was adjusted to yield an approximately 50% success rate for evoking action currents at a baseline frequency of 0.1 Hz, in the presence of NBQX (2 µm), pictrotoxin (100 µm), CGP55845A (1 µm) and d-ap5 (50 µm). The interneurones were voltage clamped at 70 mv throughout the experiment. Under these conditions changes in threshold for antidromically evoked action currents produced by kainate receptor agonists are due to the regulation of axon excitability by kainate receptors (Semyanov & Kullmann, 2001). Analysis Data were recorded using an Axopatch 200B amplifier, filtered at 5 khz and acquired at 10 khz on computer. Series resistance and input resistance were measured on-line during recordings as previously described (Daw et al. 2000). Cells were rejected if series resistance changed by > 20% during recordings. For evoked events the LTP program was used for data acquisition (Anderson &Collingridge, 2001). For spontaneous and miniature events data were acquired using Axoscope software (Axon Instruments, Union City, CA, USA) and analysed off line using MiniAnalysis software (Synaptosoft Inc., Decatur, GA, USA). Spontaneous events were detected by setting the threshold value for detection at three times the root mean square of background noise, followed by visual confirmation of appropriate detection (Fitzjohn et al. 2001). The success rate of action currents was calculated by dividing the number of successes by the total number

3 J Physiol Kainate receptors on neonatal CA1 interneurones 133 of trials for a given epoch (35 50 trials for each epoch). Pooled data are presented as means ± s.e.m. Statistical analysis was performed using Student s t test, and P < 0.05 was considered significant. All drugs were obtained from Tocris except LY382884, which was kindly supplied by Eli Lilly and Co. Results Monosynaptic IPSCs evoked by local electrical stimulation of GABAergic axons in stratum radiatum were recorded from CA1 pyramidal neurones voltage-clamped at a holding potential of 50 mv in hippocampal slices prepared from 5- to 6-day-old rats. The evoked IPSCs (eipscs) were pharmacologically isolated in the presence of 50 µmd-ap5 and 2 µm NBQX. Under these conditions the evoked currents were outward going and were fully and reversibly blocked by the GABA A receptor antagonist bicuculline (10 µm; Fig. 1A C). across the kainate receptor subunits (Clarke et al. 1997; Mulle et al. 2000). Kainate, when bath applied at 1 µm in the presence of 2 µm NBQX and 1 µm CGP55845A, also caused a robust depression of eipsc amplitude in neonatal CA1 pyramidal neurones (amplitude = 37 ± 4% of baseline, P < 0.001; n = 9; Fig. 2A C), similar to that observed with 1 µm ATPA. Thus in neonatal hippocampus, both ATPA and kainate depress evoked GABAergic transmission onto CA1 pyramidal cells in a mechanism independent of GABA B receptors. No change in the input resistance of the CA1 pyramidal neurones was observed either during ATPA (1 µm) or kainate (1 µm) application (ATPA data: baseline = 761 ± 51 M, Kainate receptor agonists depress evoked IPSCs in neonatal CA1 pyramidal neurones in a mechanism independent of AMPA or GABA B receptors To test whether kainate receptor activation can regulate GABAergic transmission in neonatal rat hippocampus we bath applied the GluR5 selective kainate receptor agonist (RS)-2-amino-3-(3-hydroxy)-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) (Clarke et al. 1997; Cossart et al. 1998; Rodriguez-Moreno et al. 2000). ATPA (1 µm) produced a rapid and reversible depression of eipsc amplitude (eipsc amplitude = 35 ± 4% of baseline, P < 0.001; n = 7; Fig. 1D F), similar to that reported previously for older animals (Clarke et al. 1997; Rodriguez-Moreno et al. 2000). There is considerable debate about the mechanism by which kainate receptor agonists cause the depression of eipscs (Kullmann, 2001; Lerma et al. 2001). One mechanism that has been proposed is that the intense excitation of GABAergic interneurones by somatodendritic kainate receptors causes the release of GABA that then activates inhibitory GABA B receptors present at GABAergic terminals onto CA1 pyramidal cells (Frerking et al. 1999). To test whether this mechanism is involved, ATPA (1µm) was applied in the presence of the potent and selective GABA B receptor antagonist CGP55845A (1 µm; Davies et al. 1993). Under these conditions ATPA produced a robust reversible depression of eipsc amplitude (amplitude = 37 ± 5% of baseline, P < 0.001; n = 8; Fig. 2D) that was indistinguishable from the depression produced in the absence of GABA B receptor blockade (P = 0.85). We also investigated the effects of kainate, an agonist that shows less selectivity between AMPA and kainate receptors than ATPA, but broader spectrum activity Figure 1. Evoked GABA A receptor-mediated IPSCs recorded in neonatal hippocampal CA1 pyramidal neurones are depressed by the kainate receptor agonist ATPA A, averaged eipscs (recorded at a holding potential of 50 mv) from an example experiment before, during and after application of 10 µm bicuculline (for this and subsequent figures: IPSCs shown are averages of 15 consecutive traces, stimulus artefacts were digitally removed, time of stimulation indicated by arrow). B, eipsc amplitude versus time for the same experiment as in A (for this and subsequent figures: each point is the amplitude averaged from 4 consecutives traces). C, pooled data showing the reversible block of eipsc amplitude by bicuculline (n numbers shown above each bar; P < 0.001). D, averaged eipscs from an example experiment showing the reversible effects of application of ATPA (1 µm). E, eipsc amplitude versus time for the same experiment as in D. F, pooled data showing eipsc amplitude versus time for all experiments in which ATPA (1 µm) was applied (n = 7). For this figure all recordings were in the presence of 50 µm D-AP5 and 2 µm NBQX.

4 134 F. Maingret and others J Physiol ATPA = 756 ± 55 M, P = 0.93, n = 8; kainate data: baseline = 757 ± 44 M, kainate = 752 ± 48 M, P = 0.95, n = 9), indicating that a postsynaptic change in membrane properties does not contribute to the depression in eipscs observed on application of these agonists. We routinely used 2 µm NBQX to selectively block AMPA receptors in our experiments. NBQX at low micromolar concentrations has been reported to show good selectivity for AMPA receptors over the kainate receptors regulating GABAergic transmission in hippocampus (Bureau et al. 1999; Mulle et al. 2000). To test this in our experiments, we compared the depression produced by ATPA application in the presence of 2 µm NBQX with that in the presence of the more selective AMPA receptor antagonist GYKI53655 (Paternain et al. 1995; Wilding & Huettner, 1995). When ATPA (1 µm) was bath applied in the presence of GYKI53655 (25 µm) and CGP55845A (1 µm), a reversible depression of eipsc amplitude occurred (amplitude = 29 ± 4% of baseline, P < 0.001; n = 6; Fig. 2D), that was very similar to that observed in the presence of 2 µm NBQX (P = 0.25). Like ATPA, the effect of 1 µm kainate was of similar magnitude when GYKI53655 (25 µm), rather than NBQX, was used to block AMPA receptors (amplitude = 32 ± 2% of baseline, P < 0.001; n = 4; P = 0.67 versus 1 µm kainate in the presence of 2 µm NBQX; Fig. 2D). Therefore in neonatal hippocampus, 2 µm NBQX shows as good selectivity as GYKI53655 between AMPA receptors and the kainate receptors regulating eipscs. Moreover, this pharmacology demonstrates no role for AMPA receptors in the depression of eipscs induced by ATPA or kainate. We also tested whether a higher dose of kainate produced additional depression of the eipsc. When 30 µm kainate was bath applied, no additional depression in eipsc amplitude was observed (amplitude = 35 ± 3% of baseline, P < 0.05; n = 3; P = 0.87 versus 1 µm kainate; Fig. 2D). Figure 2. The depression of evoked IPSCs by kainate receptor agonists is due to activation of kainate receptors and independent of GABA B receptors A, averaged eipscs from an example experiment before, during and after application of 1 µm kainate in the presence of 50 µm D-AP5, 2 µm NBQX and 1 µm CGP55845A. B, eipsc amplitude versus time for the same experiment as in A. C, pooled data showing eipsc amplitude versus time for all experiments in which kainate (1 µm) was applied in the presence of 50 µm D-AP5, 2 µm NBQX and 1 µm CGP55845A (n = 9). D, summary bar graph showing pooled data for the effects of ATPA or kainate under various conditions ( P < 0.05; P < 0.001; n numbers shown above each bar). E, pooled data showing eipsc amplitude versus time for all experiments in which ATPA (1 µm) was applied in the presence of 2 µm NBQX, 1 µm CGP55845A and 10 µm LY (n = 9). F, summary data comparing the effects of ATPA and kainate (1 and 30 µm) inthe presence of 20 µm NBQX, or 2 µm NBQX and 10 µm LY ( P < 0.01; n numbers shown above each bar). All these experiments are in the presence of 50 µm D-AP5 and 1 µm CGP55845A. ATPA and kainate depress evoked IPSCs via a kainate receptor-dependent mechanism To determine whether ATPA and kainate depress evoked GABAergic transmission onto CA1 pyramidal cells through the activation of kainate receptors, we investigated the effects of two kainate receptor antagonists: NBQX at a dose (20 µm) that is not selective between AMPA and kainate receptors, and LY (10 µm) which shows good selectivity for GluR5-containing kainate receptors over AMPA receptors (Bortolotto et al. 1999; Lauri et al. 2001). When ATPA (1 µm) was bath applied in the presence of 20 µm NBQX, no depression of eipsc amplitude was observed (amplitude = 93 ± 6% of baseline, n = 5, P = 0.52; Fig. 2F). The effects of 1 µm ATPA were also completely blocked by 10 µm LY (amplitude = 102 ± 7% of baseline, n = 9, P = 0.97; Fig. 2E and F). LY when applied on its own, however, had no effect on eipsc amplitude indicating that there is no tonic kainate receptor-dependent regulation of evoked GABAergic transmission under our recording conditions (Fig. 2E and F). LY (10 µm) also blocked the effects of1 µm kainate (amplitude = 87 ± 4% of baseline, n = 6, P = 0.42; Fig. 2F), although it did not block the depression of eipsc amplitude induced by 30 µm kainate (amplitude = 39 ± 7% of baseline, n = 3,

5 J Physiol Kainate receptors on neonatal CA1 interneurones 135 P < 0.01; P = 0.61 versus 30 µm kainate in the absence of LY382884; Fig. 2F). These data demonstrate that the depression of GABAergic transmission onto neonatal CA1 pyramidal neurones induced by ATPA and kainate (1 µm) isdue to the actions of these agonists on kainate receptors. The lack of effect of the GluR5-selective antagonist, LY382884, on the depression induced by 30 µm kainate could be due to a displacement of the antagonist by this high dose of agonist, due to kainate acting at non-glur5 kainate receptor subunits, or by activity of this concentration of kainate at AMPA receptors. However these experiments were performed in the presence of 2 µm NBQX, a concentration twice that previously shown to completely block the effects of 30 µm kainate on AMPA receptors in CA1 interneurones (Mulle et al. 2000), and which in the present experiments fully blocks AMPA receptor-mediated EPSCs. Therefore effects mediated by AMPA receptor activation seem unlikely. Furthermore, LY at a concentration of 10 µm has been shown to effectively block kainate receptor-mediated currents evoked by 30 µm kainate (Bortolotto et al. 1999), also arguing against a simple displacement of the antagonist by this dose of kainate. Thus our data indicate that 30 µm kainate depresses eipscs by activity at non-glur5 kainate receptor subunits. This suggests that kainate receptor agonists can depress evoked GABAergic transmission by actions mediated by either GluR5 or other non-glur5 kainate receptor subunits. Whether this represents activity at distinct populations of kainate receptors, or independent activation of the same heteromeric receptor population by agonist binding to different subunits, is unknown. (389 ± 164-fold change), wash = ± Hz, P < for kainate versus baseline, n = 8), in addition to a depression in the eipsc which was simultaneously monitored (Fig. 3A and C). ATPA and kainate cause kainate receptor-dependent increases in spontaneous IPSC frequency Previous studies of hippocampal slices from adolescent and adult animals have shown that kainate receptor agonists cause an increase in the frequency of spontaneous action potential-dependent IPSCs (sipscs, e.g. Cossart et al. 1998; Frerking et al. 1998; Bureau et al. 1999; Mulle et al. 2000; Rodriguez-Moreno et al. 2000). To investigate whether this also occurs in neonatal hippocampus we recorded sipscs in CA1 pyramidal cells and monitored the effects of agonist application. Application of either 1 µm ATPA (Fig. 3A, B and F) or 1µm kainate (Fig. 3C, D and F) in the presence of NBQX (2 µm) and CGP55845A (1 µm) produced a very large increase in sipsc frequency (sipsc frequency for 1 µm ATPA: baseline = ± Hz, ATPA = 3.89 ± 0.49 Hz (339 ± 137-fold change), wash = ± Hz, P < for ATPA versus baseline, n = 11; for 1 µm kainate: baseline = ± Hz, kainate = 3.16 ± 0.78 Hz Figure 3. Bath application of ATPA or kainate causes a kainate receptor-dependent increase of sipsc frequency in CA1 pyramidal cells A, example experiment showing the effect of ATPA (1 µm) application on eipsc amplitude and sipsc frequency recorded in the same cell (bin width = 40 s). B, example traces from this experiment showing sipscs recorded before, during and after the application of 1 µm ATPA. C, example experiment showing the effect of kainate (1 µm) application on eipsc amplitude and sipsc frequency recorded in the same cell (bin width = 40 s). D, example traces from this experiment showing sipscs recorded before, during and after the application of 1 µm kainate. E, example traces from an experiment showing sipscs recorded before, during and after the application of 1 µm ATPA (left panel) or 1 µm kainate (right panel) in the presence of 10 µm LY F, summary data comparing the effects of ATPA (1 µm) and kainate (1 and 30 µm) onsipsc frequency in the absence and presence of 10 µm LY ( P < 0.001; n numbers shown above each bar). All experiments in the presence of 50 µm D-AP5, 2 µm NBQX and 1 µm CGP55845A.

6 136 F. Maingret and others J Physiol The effect of ATPA (1 µm) on sipsc frequency was fully antagonized by 10 µm LY (baseline = ± Hz, ATPA + LY = ± Hz (1.13 ± 0.03-fold change), wash = ± Hz, P = 0.96 for ATPA + LY versus baseline, n = 5; Fig. 3E and F). However, LY only partially blocked the effects of 1 µm kainate (baseline = ± Hz, 1 µm kainate + LY = 1.77 ± 0.15 Hz (109 ± 25-fold change), wash = ± Hz, P < for 1 µm kainate + LY versus baseline, n = 4; Fig. 3E and F), despite this treatment producing the same magnitude increase in sipsc frequency in the absence of antagonist as 1 µm ATPA (P = 0.82 ATPA versus 1 µm kainate). We also tested the effects of 30 µm kainate on sipsc frequency. Kainate at this dose produced a similar frequency increase to the other two agonist treatments (baseline = ± Hz, 30 µm kainate = 4.60 ± 0.45 Hz (324 ± 99-fold change), wash = ± Hz, P < for 30 µm kainate versus baseline, n = 3; Fig. 3F), but this was unaffected by coapplication of LY (baseline = ± Hz, 30 µm kainate + LY = 3.60 ± 0.56 Hz (330 ± 25-fold change), wash = ± Hz, P < for 30 µm kainate + LY versus baseline, n = 3; P = 0.24 for 30 µm kainate versus 30 µm kainate + LY382884; Fig. 3F). These findings demonstrate that sipsc frequency in neonatal CA1 hippocampus is extremely sensitive to kainate receptor activation. The differential blockade of the increase in sipsc by LY depending upon agonist and dose used, indicate that activation of different kainate receptor subunits can produce similar effects on sipsc frequency. Figure 4. Kainate receptor activation produces an inward current and regulates axonal excitability in CA1 interneurones A, DC measured during a perforated patch-clamp recording from a stratum radiatum interneurone showing the responses to kainate receptor agonists (1 µm) and the effects of the antagonist LY (10 µm). B, summary data from experiments like A ( P < 0.05, P < 0.01, P < 0.001, n numbers indicated above the bars). C, single experiment showing the effects of kainate receptor activation on the success rate of antidromically evoked action currents. D, action currents from the experiment in C, taken at the time points indicated. E, summary data for the effects of ATPA (1 µm), kainate (1 µm) and LY (10 µm) on action current success rate (normalized to baseline success rate; P < 0.005, P < 0.05, n numbers shown above each bar). Kainate receptor activation produces an inward current and regulates axonal excitability in CA1 interneurones To investigate the mechanisms by which kainate receptor agonists regulate GABAergic transmission onto CA1 pyramidal neurones, we made patch-clamp recordings from CA1 stratum radiatum interneurones. We investigated the effects of kainate receptor agonist application on somatodendritic kainate receptors by monitoring the inward current produced by agonists in the presence of NBQX (2 µm), pictrotoxin (100 µm), CGP55845A (1 µm) and d-ap5 (50 µm). Both ATPA (1 µm) and kainate (1 µm) application produced inward currents in CA1 interneurones (Fig. 4A and B). The effect of ATPA (1 µm) was completely blocked by LY (10 µm); however, an inward current remained when 1 µm kainate was applied in the presence of the antagonist (Fig. 4A and B). These findings are consistent with activation of interneuronal somatodendritic kainate receptors contributing to the increase in sipsc frequency in CA1 pyramidal neurones observed with application of these antagonists. In further support of this, the inward current and increase in sipsc frequency show a very similar pharmacology in that the effects of ATPA on both these parameters are fully blocked by LY382884, while the kainate-evoked increase in sipsc frequency and the inward current both only show a partial sensitivity to LY This suggests that the same populations of receptors are involved in mediating both the increase in spontaneous transmission and the inward current. In addition to a somatodendritic kainate receptordependent mechanism regulating sipsc frequency,

7 J Physiol Kainate receptors on neonatal CA1 interneurones 137 there is evidence that axonal excitability in CA1 interneurones is also regulated by kainate receptors (Semyanov & Kullmann, 2001). Therefore this axonal mechanism could also contribute to the change in spontaneous GABAergic transmission we observe with kainate receptor agonist application. To investigate this we studied the effects of kainate receptor agonist application on axon excitability by monitoring antidromically evoked action currents in the same cells in which direct current (DC) data were collected. Under our conditions of voltage clamp and blockade of AMPA, NMDA, GABA A and GABA B receptors, the effects of kainate receptor agonists on antidromically evoked action currents can be attributed to axonal kainate receptors (Semyanov & Kullmann, 2001). Action currents were evoked with a 50% baseline success rate and application of either kainate (1 µm) or ATPA (1µm) caused a reversible increase in success rate (success rate ratio (drug/baseline), ATPA: 1.48 ± 0.17, P = 0.004, n = 6; kainate: 2.15 ± 0.41, P = 0.015, n = 5; Fig. 4C E). The effects of ATPA were fully blocked by LY (10 µm); however, the effects of kainate showed little sensitivity to this antagonist (ATPA + LY382884: 0.86 ± 0.09, P = 0.93, n = 3; kainate + LY382884: 1.98 ± 0.73, P = 0.025, n = 3; Fig. 4C E). These data demonstrate that in addition to somatodendritic depolarization contributing to the kainate receptor-dependent increase in sipsc frequency, kainate receptor activation also increases the excitability of axons. Both these mechanisms therefore are likely to contribute to the high sensitivity of spontaneous GABAergic transmission to kainate receptor agonists in the neonate. Kainate receptor agonists cause a decrease in miniature IPSC frequency but not amplitude Much controversy surrounds the mechanism by which kainate receptor agonists cause the depression in eipscs observed in CA1 pyramidal neurones (Frerking et al. 1999; Rodriguez-Moreno etal. 2000; Mulleetal. 2000; Kullmann, 2001; Semyanov & Kullmann, 2001; Kang et al. 2004). There is evidence that the intense excitation of CA1 interneurones by kainate receptor agonists resulting in the high frequency of spontaneous events can account for the depression in evoked transmission through two parallel mechanisms, one involving GABA B receptor-mediated presynaptic inhibition, and the other a postsynaptic decrease in input resistance (Frerking et al. 1999). In addition it has recently been reported that kainate receptors postsynaptically located on CA1 pyramidal neurones can also contribute to postsynaptic changes in membrane properties that lead to a depression in eipscs (Kang et al. 2004). Moreover, other studies have provided evidence for further mechanisms, involving a kainate receptor-dependent increase in axon excitability (Semyanov & Kullmann, 2001) and a direct regulation of Ca 2+ -dependent but not Ca 2+ -independent release at terminals (Jiang et al. 2001). However, there is also evidence that the effects of kainate receptor activation on sipsc frequency can be dissociated from those on eipsc amplitude (Rodriguez-Moreno et al. 2000). Thus it is unclear what the mechanism(s) is for the kainate receptor-dependent depression in evoked GABAergic transmission onto CA1 pyramidal neurones. One way to determine if a presynaptic receptor regulates transmitter release by a direct action at the terminal is to investigate the effects of agonist application on action potential-independent miniature events recorded in the presence of tetrodotoxin (TTX; MacDermott et al. 2001). Achange in the frequency of miniature events indicates a direct regulation of release by a receptor located at the terminal, while a change in the amplitude and/or kinetics of the events points to a postsynaptic mechanism of regulation. Kainate receptor agonists have been reported to reduce the frequency of miniature IPSCs (mipscs) in CA1 pyramidal neurones from adolescent animals with little effect on amplitude (Rodriguez-Moreno et al. 1997; Cossart et al. 1998; Rodriguez-Moreno & Lerma, 1998); however, in other studies little or no effect on mipsc frequency was observed (Frerking et al. 1998, 1999; Bureau et al. 1999; Jiang et al. 2001). The reasons for the differences between these studies are not clear, and the mechanism for the kainate receptor-dependent depression of evoked inhibitory transmission is still controversial. To determine whether there is a direct regulation of GABAergic transmission in neonatal hippocampus by kainate receptors located at terminals, we investigated the effects of ATPA on mipscs recorded from CA1 pyramidal neurones in the presence of 0.5 µm TTX, 50 µm d-ap5, 2 µm NBQX, and 1 µm CGP55845A. At room temperature the frequency of mipscs was prohibitively low; therefore we recorded at a temperature of C, which enhanced basal mipsc frequency. In separate experiments we confirmed that ATPA (1 µm) produced a robust depression of eipsc amplitude at this temperature (eipsc amplitude = 38 ± 2% baseline, P < 0.001, n = 5; not illustrated). Application of 1 µm ATPA produced a robust depression of mipsc frequency (baseline = 0.57 ± 0.06 Hz, ATPA = 0.19 ± 0.02 Hz (35 ± 5% of baseline), P < 0.001, n = 7; Fig. 5A, B and E), but had no effect on mipsc kinetics or amplitude (baseline = 20.7 ± 2.2 pa, ATPA = 19.0 ± 2.2 pa, P = 0.60, n = 7; Fig. 5C, D and E). 1 µm kainate also caused a similar large decrease in mipsc frequency (baseline = 0.60 ± 0.06 Hz, kainate = 0.19 ± 0.04 Hz (31 ± 5% of baseline), P < 0.001, n = 5; Fig. 5E) with no change in amplitude (baseline = 20.3 ± 1.6 pa, kainate = 19.1 ± 1.7 pa, P = 0.64, n = 5; Fig. 5E). The effects of kainate on mipscs frequency were

8 138 F. Maingret and others J Physiol blocked by 10 µm LY (baseline = 0.59 ± 0.08 Hz, kainate + LY = 0.45 ± 0.06 Hz (77 ± 2% of baseline), P = 0.23, n = 4; Fig. 5E) with no change in amplitude (baseline = 20.0 ± 1.8 pa, kainate + LY = 18.7 ± 1.6 pa, P = 0.62, n = 4; Fig. 5E). Taken together with the effects of ATPA, this indicates that the regulation of mipsc frequency is mediated via kainate receptors containing the GluR5 subunit. The decrease in mipsc frequency combined with a lack of effect on mipsc amplitude or kinetics strongly indicates that GABAergic synapses in neonatal CA1 are directly regulated by a presynaptic kainate receptor located at the terminal which acts to decrease the probability of GABA release. This decrease in mipsc frequency is sufficient to account for all of the depression in evoked transmission. Therefore this presynaptic mechanism can fully explain the depression of evoked transmission. Discussion Kainate receptors are highly expressed in neonatal hippocampus (Bahn et al. 1994; Ritter et al. 2002) and ideally located in the circuit to modulate network activity by influencing both excitatory and inhibitory transmission (Kullmann, 2001; Lerma et al. 2001; Huettner, 2003; Lerma, 2003; Isaac et al. 2004). We show that neonatal CA1 GABAergic transmission is profoundly regulated by kainate receptors through two opposing mechanisms: (1) kainate receptors that directly inhibit the probability of release at GABAergic terminals and, (2) a functionally distinct population of somatodendritic and axonal kainate receptors that very strongly up-regulate action potential-dependent spontaneous GABAergic transmission. None of the effects of kainate receptor activation are dependent on GABA B receptor activation, and no postsynaptic effects on CA1 pyramidal neurones were observed that could account for the actions of the kainate receptor agonists. Thus in neonatal CA1, kainate receptors located on GABAergic interneurones strongly regulate GABAergic transmission by two distinct opposing mechanisms. Figure 5. Kainate receptor agonists cause a reduction in the frequency but not amplitude of mipscs in neonatal CA1 pyramidal neurones A, example traces showing mipscs from a single experiment before and during 1 µm ATPA application (all data in A D from the same cell). B, mipsc frequency plotted versus time for this experiment. C, mipsc amplitude plotted versus time for an example 180-s period during baseline (left) and in the presence of 1 µm ATPA (right). D, frequency histograms of mipsc amplitude for the same 180-s periods (as in C) during baseline (left; N = 124 events) and in the presence of 1 µm ATPA (right; N = 56 events). Lower insets show mipsc averages constructed from all events in the histogram for each data set; upper right inset shows the two mipsc averages superimposed (control: black trace, ATPA: grey trace, scale bar 30 ms). E, summary data for the effect of 1 µm ATPA, 1 µm kainate and 1 µm kainate plus 10 µm LY on mipsc frequency (left; P < 0.001) and mipsc amplitude (right; n numbers indicated above bars). All these experiments were in the presence of 50 µm D-AP5, 2 µm NBQX, 1 µm CGP55845A and 0.5 µm TTX. Mechanism of the kainate receptor-dependent depression of evoked GABAergic transmission There is controversy surrounding the mechanisms by which kainate receptor agonists depress evoked GABAergic transmission onto CA1 pyramidal neurones (Frerking et al. 1999; Mulle et al. 2000; Rodriguez-Moreno et al. 2000; Kullmann, 2001; Semyanov & Kullmann, 2001; Kang et al. 2004). Evidence has been presented supporting roles for a direct inhibition of GABA release from terminals (Rodriguez-Moreno et al. 1997, 2000; Rodriguez-Moreno & Lerma, 1998), a modulation of axonal excitability (Semyanov & Kullmann, 2001; Kang et al. 2004), an indirect GABA B receptor-mediated effect (Frerking et al. 1999), and for postsynaptic mechanisms (Frerking et al. 1999; Kang et al. 2004). In the present study we show

9 J Physiol Kainate receptors on neonatal CA1 interneurones 139 that kainate receptor activation produces a depression of eipscs with no change in postsynaptic input resistance, and that kainate or ATPA causes a robust depression of mipsc frequency with no effect on mipsc amplitude or kinetics. The decrease in mipsc frequency is sufficient to account for all the depression of evoked transmission. Moreover, application of kainate receptor agonists does not suppress axonal excitability, but rather increases it. Finally the effects of kainate receptor agonist application on eipscs are insensitive to GABA B receptor blockade allowing us to exclude GABA B receptor activation as a mechanism. Taken together all these findings strongly argue for a direct kainate receptor-mediated inhibition of GABA release at GABAergic terminals as the mechanism for the depression of eipscs by kainate receptor agonists. Previous studies on slices from adolescent animals have also shown that kainate receptor agonists decrease mipsc frequency, although some change in mipsc amplitude was also noted (Rodriguez-Moreno et al. 1997; Cossart et al. 1998; Rodriguez-Moreno & Lerma, 1998). However, in other studies evidence against such a mechanism has also been provided (Frerking et al. 1998, 1999; Bureau et al. 1999; Jiang et al. 2001). The reduction in mipsc frequency we observe in the neonatal hippocampus is larger and much more robust than that reported in older animals (Rodriguez-Moreno et al. 1997; Cossart et al. 1998; Rodriguez-Moreno & Lerma, 1998), which suggests that this mechanism is developmentally down-regulated, perhaps explaining some of the controversy in the literature. There is evidence that the kainate receptors in CA1 interneurones regulating evoked IPSCs and mipscs operate via a mechanism involving a pertussis-sensitive G-protein and PKC (Rodriguez-Moreno & Lerma, 1998) as has also been described for kainate receptors regulating glutamate release in CA1 (Frerking et al. 2001). The precise details of how ionotropic kainate receptors can couple to this transduction pathway are unclear, but it is quite possible that this mechanism may also occur in the neonate and underlie the inhibition of GABAergic transmission we observe in the present study. Mechanism of the kainate receptor-dependent increase in spontaneous GABAergic transmission CA1 interneurones are intensely excited by activation of somatodendritic kainate receptors, and this is the primary mechanism proposed in previous studies for the increase in sipsc frequency commonly observed with agonist application (e.g. Cossart et al. 1998; Frerking et al. 1998, 1999; Bureau et al. 1999; Mulle et al. 2000; Rodriguez-Moreno et al. 2000). However an additional mechanism also exists which is regulation of axonal excitability by kainate receptors (Semyanov & Kullmann, 2001). Our data show that both these mechanisms are likely to contribute to the very large increase in sipsc frequency we observe with kainate receptor agonist application. The effect we see is very much greater than those previously reported (Cossart et al. 1998; Frerking et al. 1998; Bureau et al. 1999; Mulle et al. 2000), which were from studies on older animals (a 300-fold increase in the neonate compared with a 6-fold increase in older animals). This primarily appears to be due to the much lower baseline frequency of sipscs in the neonate, since the absolute sipsc frequency achieved with agonist application in the neonate is not greater than that produced by agonist stimulation in older animals. Our pharmacological analyses indicate that the increase of sipsc frequency can be produced by activation of different kainate receptor subunits. This is evident when the pharmacology of the selective GluR5 agonist ATPA and the broader spectrum agonist kainate (Clarke et al. 1997) are compared. The effects of ATPA on sipsc frequency, DC and axonal excitability are fully blocked by the GluR5-selective antagonist LY as expected; however, the effects of 1 µm kainate on these parameters show much less sensitivity to this antagonist. Moreover, the effects of 30 µm kainate are completely insensitive to LY This indicates that different kainate receptor subunits can contribute to, and independently fully drive, the increase in spontaneous GABAergic transmission. Furthermore, there is a dissociation of the effects of 1 µm kainate on eipsc amplitude and sipsc frequency by LY The effects of this dose of kainate on eipsc amplitude are fully blocked by 10 µm LY382884, yet under these conditions 1 µm kainate still causes a large increase in sipsc frequency. A block of the effects of 1 µm kainate by LY on mipsc frequency is consistent with this, indicating that GluR5-containing kainate receptors regulate GABA release at terminals, while the changes in spontaneous action potential-dependent transmission can be driven by GluR5-containing or GluR5-lacking receptors with a similar efficacy. This finding adds further weight to the idea that two functional populations of kainate receptors differentially regulate sipscs and evoked GABAergic transmission. A recent study, using selective agonists and antagonists and knock-out mice, indicates that GluR5-containing receptors are required for the regulation of GABA release at terminals (Christensen et al. 2004). Although in this study the authors conclude that there is no role for GluR5-containing receptors in driving the increase in sipsc frequency, their findings are consistent with ours since they show that the GluR5-selective agonist ATPA causes a robust increase in sipsc frequency. Moreover, in that study an extensive analysis of the pharmacology of the kainate induced increase in sipsc frequency was not performed. The increase in spontaneous GABAergic transmission is in direct opposition to the effects of kainate receptor activation on evoked and miniature IPSCs, suggesting the existence of two functionally distinct

10 140 F. Maingret and others J Physiol populations of kainate receptors that can differentially regulate neonatal GABAergic circuitry: a pro-gabaergic population that provides somatodendritic depolarization and increases axonal excitability, and an anti-gabaergic population at terminals inhibiting transmitter release. It is not clear if the population that regulates axonal excitability is spatially distinct from those at the terminals; however, pharmacologically they are distinct. The receptors regulating axonal excitability are less sensitive to ATPA than to kainate and the increase in axonal excitability produced by kainate is insensitive to LY However, those regulating mipsc frequency are very sensitive to these compounds. This suggests that two populations of axonal receptors exist with different subunit compositions: a GluR5-lacking population that can increase axonal excitability and a GluR5-containing population that reduce GABA release probability, although the GluR5-containing receptors may also influence axonal excitability. Previous studies suggest that activation of kainate receptors on CA1 interneurones with low doses of agonists can under certain conditions produce an increase in GABAergic transmission onto CA1 pyramidal cells (Jiang et al. 2001) or CA1 interneurones (Cossart et al. 2001). In other studies submicromolar doses of kainate have also been shown to elicit a robust increase in sipsc frequency in CA1 pyramidal cells in the absence of a depression in evoked or miniature GABAergic transmission (Cossart et al. 1998; Frerking et al. 1998; Rodriguez-Moreno et al. 2000; Mulle et al. 2000; Cossart et al. 2001). Although we have not investigated this issue in the present paper, it is likely that submicromolar doses of kainate will activate neonatal CA1 interneurones due to the very high sensitivity to kainate receptor agonists we observe at this developmental stage. In future studies it would be of interest to determine if such low doses of agonists can facilitate GABAergic transmission under certain conditions and investigate the effects of activation of kainate receptors by endogenous glutamate release. Regulation of GABAergic circuitry in neonatal CA1 by kainate receptors GABA A receptors are typically thought of as mediating inhibitory events, since in adolescent and adult hippocampus their activation produces a hyperpolarization of membrane potential combined with membrane shunting. However, in neonatal hippocampus, the chloride reversal potential is such that GABA A receptor activation typically produces a depolarization of membrane potential and can generate action potential firing, independent of glutamate receptor activation (Ben-Ari et al. 1989, 1997). As GABA receptors still have the capability to produce shunting inhibition at this developmental stage, their net effect during intense activity of the network can be either inhibitory or excitatory (Gao et al. 1998; Lamsa et al. 2000). Nevertheless the kainate receptor-dependent regulation of GABAergic transmission we describe in the neonate is likely to have a very different role from that proposed in previous studies on older animals. The existence of pro-gabaergic kainate receptors located both somatodendritically and axonally in CA1 interneurones, and an anti-gabaergic population of kainate receptors present at GABAergic terminals onto CA1 pyramidal neurones suggests that different patterns of activity are likely to activate these two populations. The pro-gabaergic population may sense large scale activity in CA1, for example as a result of synchronous activation of a large number of inputs from CA3. In contrast, the anti-gabaergic kainate receptors located at terminals may sense more local spill-over of glutamate from neighbouring glutamatergic afferents, and provide a localized suppression of GABA release in regions of the dendritic tree where glutamatergic synapses are highly active. Thus, in conclusion, kainate receptors in neonatal CA1 play distinct roles in regulating GABAergic transmission. They can differentially regulate GABAergic transmission, probably in response to different types of activity. 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