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Mechanisms of bi-directional modulation of thalamocortical transmission in barrel cortex by presynaptic kainate receptors
Neuropharmacology 60 (2011) 832e841
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Mechanisms of bi-directional modulation of thalamocortical transmission in barrel cortex by presynaptic kainate receptors Jean-Sébastien Jouhanneau a,1, Simon M. Ball b, Elek Molnár b, John T.R. Isaac a, *, 2 a b
Developmental Synaptic Plasticity Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892-3701, USA MRC Centre for Synaptic Plasticity, School of Physiology and Pharmacology, University of Bristol, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 July 2010 Received in revised form 17 December 2010 Accepted 20 December 2010
Presynaptic kainate receptors play an important role in synaptic transmission and short-term plasticity to profoundly regulate network activity in many parts of the mammalian brain. In primary sensory neocortex, where short-term synaptic plasticity is important for receptive field structure and information processing, kainate receptors are highly expressed and regulate thalamocortical inputs, particularly during development. However, the mechanisms of the kainate receptor-dependent presynaptic regulation of thalamocortical transmission are unclear. We therefore investigated this issue using electrophysiology in neonatal thalamocortical slices of barrel cortex combined with pharmacology and biochemical analyses. We show that presynaptic kainate receptors can both facilitate or depress synaptic transmission depending on the extent of their activation. This bi-directional regulation is mediated in part by kainate receptors that directly influence thalamocortical axonal excitability, but also likely involves receptors acting at thalamocortical terminals to regulate transmitter release. The efficacy of kainate in regulating thalamocortical transmission is low compared to that reported for other inputs. Consistent with this low efficacy, our biochemical analyses indicate that the presynaptic kainate receptors regulating neonatal thalamocortical inputs likely lack the high kainate affinity GluK4 and 5 subunits. Thus thalamocortical transmission can be bi-directionally regulated by low affinity kainate receptors through two mechanisms. Such presynaptic regulation provides a potentially powerful mechanism to influence sensory processing during development of barrel cortex. Ó 2010 Published by Elsevier Ltd.
Keywords: Synaptic transmission Presynaptic mechanisms Kainate receptors Barrel cortex
1. Introduction Kainate-type glutamate receptors are widely expressed in the mammalian brain and play important roles in synaptic transmission
Abbreviations: LTP, long-term potentiation (LTP); EPSC, excitatory postsynaptic current; EPSP, excitatory postsynaptic potential; VB, ventrobasal complex of thalamus; DC, direct current; AMPAR, AMPA receptor; NMDAR, NMDA receptor; GluK1, kainate-type glutamate receptor subunit 1 (GluR5 in previous nomenclature); GluK2, kainate-type glutamate receptor subunit 2 (GluR6 in previous nomenclature); GluK3, kainate-type glutamate receptor subunit 3 (GluR7 in previous nomenclature); GluK4, kainate-type glutamate receptor subunit 4 (KA1 in previous nomenclature); GluK5, kainate-type glutamate receptor subunit 5 (KA2 in previous nomenclature). * Corresponding author. E-mail address: [email protected] (J.T.R. Isaac). 1 Present address: Department of Neuroscience, Max-Delbruck-Centrum for Molecular Medecine, Robert-Rossler-Strasse 10, D-13092 Berlin-Buch, Germany; Neurocure, Neuroscience Research Center, Universitaetsmedizin Charite, 10117 Berlin, Germany. 2 Eli Lilly and Company, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK. 0028-3908/$ e see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.neuropharm.2010.12.023
and neuronal excitability (Wisden and Seeburg, 1993; Bahn et al., 1994; Bettler and Mulle, 1995). Presynaptic kainate receptors are best characterized in hippocampus where they can up- or down-regulate both glutamatergic and GABAergic transmission (Kullmann, 2001; Lerma et al., 2001; Isaac et al., 2004), profoundly impacting circuit function. For example, kainate receptors regulate network activity in hippocampal CA3 and CA1 (Kullmann, 2001; Fisahn et al., 2004; Lauri et al., 2005) and contribute to epileptiform activity in hippocampus (Smolders et al., 2002). Presynaptic kainate receptors also play a role in long-term synaptic plasticity, being required for the induction of mossy fiber LTP (Bortolotto et al., 1999; Contractor et al., 2001; Lauri et al., 2001, 2003; Schmitz et al., 2003) and the expression of neonatal hippocampal CA1 LTP (Lauri et al., 2006). Kainate receptors are tetramers made up of combinations of GluK1-5 subunits (Bettler and Mulle, 1995; Collingridge et al., 2009). GluK1-3 subunits can form functional ion channels either as homomers or heteromers; however, GluK4 or 5 homomers are not functional and are not surface expressed in neurons (Gallyas et al., 2003; Ren et al., 2003; Isaac et al., 2004; Nasu-Nishimura
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et al., 2006). Kainate receptors can act both as ionotropic and metabotropic receptors (Lerma et al., 2001) and both modes of their signaling for presynaptic regulation by kainate receptors have been described (Frerking et al., 2001; Kidd et al., 2002; Lauri et al., 2003; Lerma, 2003; Pinheiro and Mulle, 2008). The mechanisms by which kainate receptors regulate presynaptic function are diverse (Kullmann, 2001; Isaac et al., 2004). Kainate receptors located at presynaptic terminals have been shown to up-regulate or downregulate glutamate or GABA release in a dose-dependent manner when activated by exogenous agonist and also by synapticallyreleased glutamate e.g. (Jiang et al., 2001; Lauri et al., 2001; Schmitz et al., 2001; Semyanov and Kullmann, 2001; Kidd et al., 2002). In addition to acting at presynaptic locations, kainate receptors have also regulate synaptic function by altering axonal excitability as shown in hippocampus for the mossy fibre input to CA3 (Kamiya and Ozawa, 2000; Schmitz et al., 2000) and for GABAergic interneuronal input to CA1 pyramidal neurons (Semyanov and Kullmann, 2001; Maingret et al., 2005). Importantly such axonal receptors have been shown to be physiologically activated by synaptically-released glutamate to regulate synaptic transmission (Schmitz et al., 2000; Semyanov and Kullmann, 2001; Contractor et al., 2003). Therefore, these studies show that presynaptic kainate receptors physiologically regulate neurotransmitter release through at least two distinct mechanisms and further that studying these receptors using exogenously applied agonist is a useful approach in understanding the mechanisms of their action. Kainate receptors are highly expressed and developmentally regulated in somatosensory pathways (Bettler and Mulle, 1995; Kerchner et al., 2001; Daw et al., 2007b). In layer 4 of neonatal barrel cortex presynaptic kainate receptors are found at thalamocortical inputs where they can be synaptically-activated during brief trains of activity to mediate a short-term depression (Kidd et al., 2002). Moreover, short-term plasticity plays an important role for in information processing in primary sensory neocortex (Abbott and Regehr, 2004). However, little is known about the properties or mechanisms of action of kainate receptors that regulate thalamocortical transmission. We therefore probed the mechanisms by which presynaptic kainate receptors can regulate thalamocortical transmission in neonatal barrel cortex. We show that kainate receptors can both up- and down-regulate neonatal thalamocortical transmission by at least two mechanisms. We find that presynaptic kainate receptors strongly influence axonal excitability; however, kainate receptors also act independently of this mechanism likely through direct regulation of glutamate release at terminals. In addition, we show that the efficacy of kainate in modulating thalamocortical transmission is low compared to other inputs in the brain, and consistent with this our biochemical analyses indicate that the presynaptic kainate receptors likely lack the high kainate affinity GluK4 and 5 subunits. This bi-directional regulation of thalamocortical transmission is predicted to be important in determining the dynamic properties of thalamocortical inputs hence influencing sensory processing in developing layer 4. 2. Materials and methods 2.1. Ethical approval All experiments were performed in accordance with the guidelines of the National Institutes of Health Animal Care and Use Committee guidelines. 2.2. Electrophysiology Thalamocortical slices (Agmon and Connors, 1991) were prepared from C57Bl/6 or 129SV/EV mouse pups aged between postnatal day (P) 3 and 7 (day of birth is P0), as previously described (Bannister et al., 2005; Daw et al., 2006). Animals were anaesthetised by inhalation of isofluorane and then decapitated with sharp scissors. The brain was rapidly removed and placed in ice-cold extracellular solution.
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Thalamocortical slices were then prepared as previously described (Daw et al., 2006). The data from slices prepared from C57Bl/6 mice are shown in Figs. 1e4. The 129SV/ EV mice were used for the histoblot experiments (Fig. 5). For electrophysiological recordings the extracellular solution contained: 119 mM NaCl, 2.5 mM KCl, 1 mM NaH2PO4, 26.2 mM NaHCO3, 11 mM glucose, 1.3 mM MgSO4, 2.5 mM CaCl2, saturated with 95% O2/5%CO2, pH 7.4. 50 mM picrotoxin and 50 mM D-AP5 were routinely included to block GABAA and NMDA receptors (NMDARs), respectively, in all experiments except those monitoring NMDAR-mediated EPSCs. To monitor NMDAR EPSCs, D-AP5 was omitted and 10 mM BAPTA was included in the intracellular solution. All recordings were performed at room temperature. Whole-cell patch-clamp recordings were made from visually-identified stellate cells in layer 4 barrel cortex (Bannister et al., 2005; Daw et al., 2006) using 4e7 MU electrodes (Fig. 1AeC). For whole-cell voltage-clamp recordings the intracellular solution was as follows: 135 mM CsMeSO4, 8 mM NaCl, 10 mM HEPES, 5 mM QX-314, 0.5 mM EGTA, 4 mM MgATP, 0.3 mM Na-GTP, pH 7.2, 285 mOsm. For current-clamp recordings the intracellular solution was as follows: 130 mM KMeSO4, 8.5 mM NaCl, 5 mM HEPES, 0.5 mM EGTA, 4 mM Mg-ATP, 0.5 mM Na-GTP, pH 7.2, 285 mOsm. The intracellular solution also contained 20 mM AlexaFluor488, which was used for visualizing the recorded neurons (Fig. 1B, C). Fluorescent signals were visualized using a cooled CCD camera (Hamamatsu Orca ER). Thalamocortical EPSCs were evoked by electrical stimulation of the VB thalamus (Agmon and Connors, 1991; Bannister et al., 2005; Daw et al., 2006) at a frequency of 0.2 Hz. Cells were voltage-clamped at 70 mV except when recording pharmacologically-isolated NMDAR-mediated EPSCs for which holding potential was þ30 mV. During most whole-cell recordings, fibre volley was also simultaneously recorded using a patch electrode filled with extracellular solution placed in the internal capsule (Fig. 1A) (Crair and Malenka, 1995). In other experiments the fibre volley in layer 4 was recorded; in these experiments an extracellular field potential elicited by stimulation in VB was first recorded and then 0 mM Ca2þ superfused onto the slice to isolate the fibre volley component. Signals were amplified by a Multiclamp 700B (Axon Instruments), filtered at 4 KHz, digitized at 10 KHz and analyzed using Signal software (CED). Input resistance, series resistance, DC and EPSC amplitude were displayed on-line. Series resistance was estimated as previously described (Daw et al., 2000) and if it varied by >20% during the recording, the cell was discarded. 2.3. Analysis For EPSCs recorded at 70 mV peak EPSC amplitude was measured, which is mediated predominately by AMPA receptors (Kidd and Isaac, 1999, 2001). The fibre volley amplitude was measured as the amplitude of the negative going deflection (Crair and Malenka, 1995). For statistical comparisons an averaged response amplitude over a 1.5 min period immediately before drug application was compared to the average over a 1.5 min period at the peak of the effect. Statistical analysis was performed using the Student’s t-test unless otherwise noted, paired or unpaired as appropriate. 2.4. Histoblot experiments Region-specific changes in distribution of GluK1/2 and GluK5 immunoreactivities were analyzed with previously characterized antibodies (Wenthold et al., 1994; Molnar et al., 1995) in 10 mm thick thalamocortical sections of P3, 5, 7 and adult brains of mice using an in situ blotting technique (‘histoblot’) (Tonnes et al., 1999; Gallyas et al., 2003; Jo et al., 2006). Nissl staining was performed on adjacent sections to confirm brain regions. Mean pixel density of histoblots was analyzed using previously established image quantification procedures (Kopniczky et al., 2005; Jo et al., 2006, n ¼ 4 (n ¼ one animal)).
3. Results 3.1. Kainate causes a dose-dependent depression or facilitation of thalamocortical transmission in layer 4 of developing barrel cortex Our previous work shows that a presynaptic GluK1-containing kainate receptor exists at thalamocortical inputs to neonatal layer 4 barrel cortex that can be synaptically-activated during brief trains of activity to cause a short-term depression (Kidd et al., 2002). To investigate the mechanisms for this presynaptic kainate receptordependent regulation, we studied the effects of bath application of kainate at different concentrations in thalamocortical slices prepared from mice aged postnatal day (P) 3e7 (Agmon and Connors, 1991; Crair and Malenka, 1995; Daw et al., 2006). In thalamocortical slices, barrels in layer 4 could be visualized under low power magnification with infra-red illumination (Fig. 1A, B). Wholecell recordings were made from visually-identified stellate cells, which make up w80% of neurons in layer 4 (Feldmeyer et al., 1999;
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Fig. 1. Dose-dependent depression or potentiation of neonatal thalamocortical synaptic transmission in layer 4 barrel cortex by bath-applied kainate. A, Left panel: low power infrared DIC image of a thalamocortical slice in the recording chamber (from P5 mouse). Right panel: schematic of thalamocortical slice (VB: ventrobasal nucleus of thalamus, RTN: thalamic reticular nucleus, IC: internal capsule, Str: striatum, WM: white matter, Hip: hippocampus), the location of the stimulating (stim), fiber volley recording (fibre volley) and whole-cell recording (WeC) electrodes are shown. Barrels are shown in red. B, Infra-red DIC image of barrels and superimposed fluorescence image of a stellate cell filled with Alexa 488. C, Higher magnification fluorescence image of the filled stellate cell (same cell as in B). D, Peak amplitude of the thalamocortical EPSC recorded in layer 4 stellate cells (filled circles; n ¼ 8) and fiber volley (fibre volley) recorded in the internal capsule (IC; open circles; n ¼ 10) in experiments in which 1 mM kainate was bath-applied. Lower graph is direct current (DC) recorded from the same cells. For this and subsequent graphs, each point is an average of 3 consecutive responses that are normalized to a five minute baseline immediately prior to drug application. E, EPSC amplitude (n ¼ 10), fibre volley (n ¼ 10) and DC (n ¼ 10) for experiments in which 10 mM kainate was applied. F, Example traces of EPSCs (left) and fibre volley responses (right) for experiment shown in G. G, EPSC amplitude and fibre volley (top) and DC (bottom) from an example experiment in which 3 mM kainate was bath-applied. H, Summary data for the 3 mM kainate experiments (EPSC: n ¼ 7; fibre volley: n ¼ 12; DC: n ¼ 7).
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Fig. 2. Dose response relationships of kainate for thalamocortical EPSC, fibre volley and DC. A, Dose response relationship for kainate on EPSC amplitude (small circles are from individual cells, large circles are mean values; 1 mM: n ¼ 8; 3 mM: n ¼ 7; 5 mM: n ¼ 2; 10 mM: n ¼ 10; 30 mM: n ¼ 5). B, Dose response for fibre volley recorded in IC (small open circles are from individual cells, large open circles are mean values; 1 mM kainate: n ¼ 10; 3 mM: n ¼ 12; 5 mM: n ¼ 2; 10 mM: n ¼ 10; 30 mM: n ¼ 4) and fibre volley recorded in layer 4 (small closed gray circles are from individual cells, large closed gray circles are mean values; n ¼ 7). C, Dose response relationship for DC (1 mM: n ¼ 8; 3 mM: n ¼ 7; 5 mM: n ¼ 2; 10 mM: n ¼ 10; 30 mM: n ¼ 5). Data for 1, 3 and 10 mM kainate are from the data sets shown in Fig. 1. D, Summary bar graph for the effect of bath application kainate on EPSC, fibre volley and DC amplitude.
Lefort et al., 2009), with patch electrodes containing the fluorescent dye AlexaFluor488 so that cell type and location could be readily determined (Daw et al., 2007a) (Fig. 1B, C). Thalamocortical EPSCs were elicited in layer 4 stellate cells by stimulation of thalamocortical axons in the ventrobasal (VB) thalamus (Fig. 1A) (Agmon and Connors, 1991; Crair and Malenka, 1995; Daw et al., 2006). In addition, the postsynaptic effect of agonist application was monitored by measuring the effects on the direct current (DC) during recordings. Previous studies using bath application of kainate have found that presynaptic kainate receptors containing GluK4 or GluK5 are activated with high efficacy by kainate. For example hippocampal mossy fiber-CA3 synapses are facilitated by 50 nM kainate through GluK2-containg receptors (Lauri et al., 2001; Schmitz et al., 2001; Contractor et al., 2003). However, presynaptic kainate receptors can also exhibit very low efficacy for agonist; indeed, a second very low efficacy presynaptic kainate receptor also exists at mossy fibreCA3 inputs composed of GluK2 and GluK3 subunits that is synaptically-activated, yet requires mM glutamate for activation (Pinheiro et al., 2007; Pinheiro and Mulle, 2008). We therefore investigated the effects of bath application of kainate on thalamocortical transmission using a range of concentrations. When we applied 1 mM kainate to thalamocortical slices there was no effect on the thalamocortical EPSC (Fig. 1D, 96.24 6.72, P ¼ 0.7622, n ¼ 8). However, at a concentration of 3 mM, kainate caused a reversible potentiation of the thalamocortical EPSC (Fig. 1FeH, 137.90 14.53, P ¼ 0.0337, n ¼ 8). At a concentration of 10 mM kainate caused a strong reversible depression of the EPSC (Fig. 1E, 27.43 7.34, P ¼ 0.0001, n ¼ 10). This depression was preceded by a transient facilitation, similar to what has previously been
observed at hippocampal mossy fibre-CA3 synapses (Schmitz et al., 2000) and is likely due to a slow rise in agonist concentration reaching the receptors. In addition, we found that 5 and 30 mM kainate depressed the EPSC (58.57 46.68, n ¼ 2; 20.35 7.82, P ¼ 0.0081, n ¼ 5; respectively). During the same experiments we also monitored the effects of bath-applied kainate on thalamocortical axonal excitability by recording the fiber volley using an extracellular electrode placed in the internal capsule (Crair and Malenka, 1995)(Fig. 1A). 1 mM kainate failed to have an effect on fibre volley (Fig. 1D, 99.10 7.35, P ¼ 0.2485, n ¼ 10), whereas 3 mM (Fig. 1FeH, 127.23 5.88, P ¼ 0.0074, n ¼ 12), 5 mM (137.03 4.47, n ¼ 2; Fig. 2B) and 10 mM (Fig. 1E, 125.58 4.66, P ¼ 0.0011, n ¼ 11) all caused a facilitation of fibre volley, and 30 mM caused a strong depression (35.77 8.23, P ¼ 0.1094, n ¼ 5; Fig. 2B). Although fibre volley recordings from the internal capsule provide a readout of thalamocortical axonal excitability (Crair and Malenka, 1995), it is possible that other axonal populations may contribute to the response. Therefore, we also tested whether kainate affects the fibre volley recorded in barrels in layer 4. To do this thalamocortical EPSPs were recorded extracellularly and then extracellular solution nominally containing zero Ca2þ was washed in to isolate the fibre volley, after which kainate was bath-applied. Using this approach we found that 10 mM kainate also causes a facilitation of the fibre volley in layer 4 (Fig. 2A, 129.19 8.95, P ¼ 0.0384, n ¼ 7), similar to that observed for fibre volley measured in the internal capsule (unpaired t-test P ¼ 0.0897). To determine whether bath application of kainate had any direct postsynaptic effect on stellate cells, we measured direct current (DC) during the whole-cell voltage-clamp recordings. Kainate at doses up to 10 mM caused only a small change in DC (on
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Fig. 3. Depression of thalamocortical synaptic transmission by 10 mM kainate is not blocked by the selective AMPAR antagonist GYKI53655, but is prevented by the non-selective AMPA and kainate receptor antagonist NBQX. A, Summary data for the effects of bath application of 10 mM kainate on thalamocortical NMDAR-mediated EPSC amplitude (recorded at a membrane potential of þ30 mV) in the presence of 25 mM GYKI53655 (n ¼ 9). Inset top: example traces taken at the times indicated. B, Summary data for effects of 10 mM kainate on fibre volley in the presence of GYKI53655 (n ¼ 6). C, Summary data for the effects of 10 mM kainate on NMDAR-mediated EPSC amplitude in the presence of 20 mM NBQX (n ¼ 5). Inset top: example traces taken at the times indicated. D, Summary data for effects of 10 mM kainate on fibre volley in the presence of NBQX (n ¼ 10).
average < 15 pA; Fig. 1D: 1 mM kainate, 11.82 4.12 pA, P ¼ 0.0168,n ¼ 8, E: 10 mM kainate, 11.22 3.04 pA, P ¼ 0.0022, n ¼ 10, G, H: 3 mM kainate, 2.08 2.06 pA, P ¼ 0.1601, n ¼ 7). In separate current-clamp experiments we determined whether this small change in DC was sufficient to cause spiking in stellate cells and found that 10 mM kainate did not cause any consistent depolarization in stellate cells, rather a small but significant hyperpolarization was observed (Supplementary Fig. 1, 3.72 1.08 mV, P ¼ 0.0184, n ¼ 5). Taken together our analysis of the effects of kainate on thalamocortical transmission demonstrates that kainate facilitates synaptic transmission at 3 mM, but depresses at 5 mM and higher doses (Fig. 2A). In contrast, the thalamocortical fibre volley is facilitated by 3, 5 and 10 mM kainate, only depressing at 30 mM (Fig. 2B). Finally, at doses of up to 10 mM, kainate does not cause any large change in DC (Fig. 2C) and does not induce spiking in stellate cells. These findings indicate that kainate bi-directionally affects thalamocortical synaptic transmission via a presynaptic mechanism, consistent with our previous study (Kidd et al., 2002). Further, there is a dissociation in the dose response between effects on the thalamocortical EPSC and fibre volley. This demonstrates that, although kainate affects thalamocortical axonal excitability, this mechanism cannot account for all the effects on the thalamocortical EPSC suggesting that kainate also directly affects the properties of presynaptic terminals. Our
analysis also shows that the efficacy of kainate in regulating transmission is in the low micromolar range. As highlighted in Supplementary Fig. 2A, B this efficacy is approximately 100 fold lower compared to the high efficacy kainate receptor at hippocampal mossy fibres (Lauri et al., 2001; Schmitz et al., 2001; Contractor et al., 2003). 3.2. Kainate affects thalamocortical transmission through actions at kainate receptors Although kainate is selective for kainate receptors over AMPA receptors (AMPARs), it has significant activity at AMPARs at doses >1 mM (Clarke et al., 1997; Mulle et al., 2000). Therefore, since kainate only affects thalamocortical transmission at doses of 3 mM and greater, it is important to investigate whether action at AMPARs contributes to the effects we observe. To address this, we investigated whether the effects of 10 mM kainate were altered in the presence of the selective AMPAR antagonist GYKI53655 (25 mM) (Wilding and Huettner, 1995; Kidd and Isaac, 1999). To monitor thalamocortical synaptic transmission under these conditions of AMPAR blockade, we recorded NMDAR-mediated EPSCs at a membrane potential of þ30 mV. We found that 10 mM kainate produced a transient facilitation followed by a depression of the NMDAR-mediated EPSC (Fig. 3A; 44.10 13.43, P ¼ 0.0357, n ¼ 9) and also facilitated fibre volley (Fig. 3B; 118.42 6.06, P ¼ 0.0195,
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Fig. 4. Kainate regulates thalamocortical axonal excitability. A, Summary data for the effects of 10 mM kainate on antidromic spike probability evoked by electrical stimulation of thalamocortical axons recorded in VB neurons (voltage-clamp at 70 mV) in the presence of 25 mM GYKI53655 (n ¼ 5). Inset top: traces from an example experiment taken at the times indicated. Inset right: schematic of recording/stimulating configuration. B, Summary bar graph of effects of 10 mM kainate on antidromic spike probability. C, Summary data for effects of 10 mM kainate on DC in VB neurons in GYKI53655. D, Summary bar graph of effects of 10 mM kainate on DC.
n ¼ 6) in the presence of GYKI53655, similar to the effects observed on fibre volley and AMPAR-mediated EPSCs in the absence of GYKI53655. This finding demonstrates that AMPARs do not substantially contribute to the effects of kainate at doses of 10 mM or less. We also tested whether NBQX, at a dose (20 mM) that blocks both AMPA and kainate receptors, prevents the effects of kainate. NBQX blocked both the depression of the NMDAR EPSC and the facilitation of fibre volley by 10 mM kainate (Fig. 3C 84.57 9.12, P ¼ 0.4012, n ¼ 5, D 98.47 6.67, P ¼ 0.8603, n ¼ 10, respectively). Some run down of the NMDA EPSC was observed in these experiments; however, this was not statistically significant and likely reflects the decrease in NMDAR function that can be observed in neurons during whole-cell recording. Taken together, these analyses show that the effects of 10 mM kainate on EPSC and fibre volley are not mediated by AMPARs, but rather are due to activation of kainate receptors. In addition, the similar effects of kainate on AMPA and NMDA receptor-mediated EPSCs provide further strong evidence for the presynaptic locus of kainate receptors regulating thalamocortical transmission.
3.3. Kainate receptors present on thalamocortical axons regulate axonal excitability The increase in fibre volley caused by 3, 5 and 10 mM kainate suggests that kainate receptors can regulate thalamocortical axonal excitability, as has been reported for hippocampal mossy fibers (Kamiya and Ozawa, 2000; Schmitz et al., 2000) and CA1 interneuron axons (Semyanov and Kullmann, 2001). However, an alternative possibility is that kainate acts on somatodendritic kainate receptors present on presynaptic neurons in VB causing excitation that indirectly increases the excitability of thalamocortical axons. Therefore, to test whether kainate receptors present on thalamocortical axons regulate axonal excitability we made wholecell voltage-clamp recordings from VB neurons and monitored antidromic action potentials evoked by a stimulating electrode placed in the internal capsule (Fig. 4A right inset). This approach allows isolation of effects of bath-applied kainate on axonal receptors because somatic voltage-clamp prevents any depolarization due to activation of somatodendritic receptors from
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Fig. 5. Kainate receptors in neonatal layer 4 barrel cortex contain GluK2/3, but lack GluK5 subunits. A, Pseudocolour images of immunoreactivity levels of histoblots from 10 mm thick thalamocortical sections for the antibody indicated (top) and at the different ages as indicated (left). Extreme left hand panels are Nissl stains of adjacent thalamocortical sections (red box indicates approximate position of barrel cortex). Scale bar is 1 mm and applies to all images. B, Quantitation of GluK5 immunoreactivity for layer 4 barrel cortex (n ¼ 4). C, Quantitation of GluK2/3 immunoreactivity for layer 4 barrel cortex (n ¼ 4).
influencing antidromic action potential generation (Semyanov and Kullmann, 2001). We used a stimulus intensity that evoked action potentials in approximately 50% of trials and then monitored the effect of kainate on antidromic action potential probability in the presence of GYKI53655. Under these conditions we found that 10 mM kainate caused a reversible increase in action potential probability (Fig. 4A, B, 0.78 0.14, P ¼ 0.04, n ¼ 5) providing strong evidence for the presence of kainate receptors on thalamocortical axons that can facilitate action potential generation. In the same experiments we observed an increase in DC in VB neurons with kainate application (Fig. 4C, D, 38.32 10.78 pA, P ¼ 0.1935, n ¼ 5); however, the DC increase was variable between cells. Thus while it is likely that kainate receptors are present on somatodendritic membranes of VB neurons, our data strongly indicates that kainate receptors located on thalamocortical axons regulate axonal excitability. 3.4. Kainate receptors containing GluK2/3, but lacking the high kainate affinity GluK5 subunit, are expressed in neonatal layer 4 barrel cortex The low micromolar doses of kainate needed to produce effects on transmission indicate that the kainate receptors regulating the thalamocortical input have low affinity for kainate. Native kainate receptors are typically heteromers made up from combinations of
GluK1-5 and heteromeric subunit composition affects agonist efficacy, kinetics and signaling (Lerma et al., 2001; Pinheiro and Mulle, 2008). The presence of GluK4 or 5 yields kainate receptors with high affinity for kainate, producing nM efficacy for kainate in regulating synaptic transmission e.g., (Contractor et al., 2003). Therefore, one possibility to explain the low efficacy of kainate in regulating neonatal thalamocortical transmission is that the kainate receptors lack GluK4 and 5. We investigated this issue using histoblot analysis (Tonnes et al., 1999; Jo et al., 2006) in which protein expression levels in different brain regions are analyzed by performing immunolabelling of nitrocellulose membranes onto which brain sections have been blotted. Since a number of brain sections can be blotted onto the same membrane, protein levels can be compared for multiple brain regions within and between brains. We applied this analysis to thalamocortical brain slices prepared from P3, P5 and P7 animals, the age range used for our electrophysiology experiments, as well as slices from P14 and adults (Fig. 5A). Histoblots were probed for GluK5 and GluK2/3 (note that no good GluK1, GluK2, GluK3 or GluK4 specific antibodies exist). In layer 4 barrel cortex we found very low levels of GluK5 expression in the first postnatal week, approximately six-fold lower than GluK5 levels in adult, with GluK5 levels increasing dramatically by P14 (Fig. 5A, B). Previous studies have shown that mRNA for GluK4, the other high affinity subunit, is never observed in neocortex at any developmental stage
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(Werner et al., 1991; Herb et al., 1992; Bahn et al., 1994); therefore, the additional lack of GluK5 strongly suggests that there is a lack of expression of subunits with high kainate binding affinity in layer 4 barrel cortex during the first postnatal week. There was considerable GluK2/3 immunoreactivity detected in layer 4 in the first postnatal week, which by P7 was comparable to adult levels (Fig. 5A, C). A similar profile for GluK5 and GluK2/3 was observed in inner layers of barrel cortex and in VB thalamus (Fig. 5A). Thus, the histoblot analysis shows that kainate receptor subunits exist in layer 4 barrel cortex during the first postnatal week; however, kainate receptors likely lack the high kainate affinity GluK4 and 5 subunits providing the basis for the low efficacy of kainate in regulating thalamocortical transmission in layer 4 that we observe. 4. Discussion Presynaptic receptors regulating neurotransmitter release represent an important mechanism for determining the dynamic properties of synapses in many areas of the mammalian brain. Prominent amongst such receptors are presynaptic kainate receptors that regulate short-term plasticity at glutamatergic and GABAergic synapses in many brain areas including hippocampus and neocortex (Kullmann, 2001; Isaac et al., 2004; Pinheiro and Mulle, 2008). Here we now show that presynaptic kainate receptors can both up- and down-regulate thalamocortical transmission in developing layer 4 barrel cortex through at least two mechanisms. In one mechanism they increase or decrease axonal excitability, and in a second regulate transmission independently of axonal excitability, likely due to direct action at thalamocortical terminals. The kainate receptors mediating these effects exhibit low efficacy for kainate, lacking the GluK4 and GluK5 subunits that confer high affinity for kainate. These findings taken together with previous work showing that synaptic activation of kainate receptors at this input mediates a short-term depression (Kidd et al., 2002) demonstrate that kainate receptors provide a mechanism for the bi-directional regulation of thalamocortical transmission in developing layer 4 barrel cortex. We find that bath application of kainate affects both the thalamocortical synaptic response and the fibre volley. This is similar to hippocampal mossy fiber-CA3 transmission; however, the dosee response relationship for kainate regulating thalamocortical responses is shifted to the right, demonstrating w100 fold lower efficacy of kainate compared to that reported by a number of groups for mossy fibers (Kamiya and Ozawa, 2000; Schmitz et al., 2000; Lauri et al., 2001; Schmitz et al., 2001b; Contractor et al., 2003). The low efficacy of kainate in regulating thalamocortical transmission can be explained by a lack of expression of the high kainate binding affinity GluK4 and 5 subunits in early postnatal layer 4 barrel cortex. High efficacy of kainate correlates with a presence of these high kainate binding affinity subunits in the receptor complex (Werner et al.,1991; Herb et al., 1992). Consistent with a major role for GluK5 in the high efficacy of kainate, the nM effects of kainate at mossy fibres are absent in GluK5 KO mice (Contractor et al., 2003; Ruiz et al., 2005). Moreover, recent work shows that the GluK5 subunit mediates the metabotropic effects of kainate receptors and this subunit is selectively activated by nM kainate (Ruiz et al., 2005) (but see (Fernandes et al., 2009)). For the ionotropic actions of kainate receptors, the evidence indicates that there is typically a mM efficacy of kainate or glutamate that is not strongly influenced by the presence of GluK4 or 5 subunits (Lerma et al., 2001). Native ionotropic kainate receptors with similar kainate efficacies (1e10 mM) to that observed in the present study, have been reported in a number of preparations (Patneau et al., 1994; Sahara et al., 1997; Wilding and Huettner,
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1997; Pemberton et al., 1998). Notably, a recent study shows that a low affinity GluK2 and GluK3-containing kainate receptor is also present presynaptically at mossy fibre-CA3 inputs, is activated by mM concentrations of glutamate and is activated by synapticallyreleased glutamate (Pinheiro et al., 2007). This study, therefore, indicates that low affinity presynaptic kainate receptors can be activated by endogenous glutamate release to physiologically regulate synaptic transmission. Application of kainate produces a modulation of thalamocortical fibre volley that is due to the presence of axonal receptors on thalamocortical afferents. Such direct regulation of axonal excitability by kainate receptors has been shown previously to occur in axons of local circuit interneurons in CA1 hippocampus (Semyanov and Kullmann, 2001; Maingret et al., 2005) and in mossy fibre axons in CA3 hippocampus (Kamiya and Ozawa, 2000; Schmitz et al., 2000). Importantly these previous studies show that synapticallyreleased glutamate can activate the axonal receptors pointing to a physiological role in regulating synaptic transmission. Our new data represent the first evidence for such a mechanism occurring in neocortex and on an axonal population that projects significant distances across many neocortical layers. Although in the present study we do not address how such axonal receptors are physiologically activated, by analogy to mossy fibres one possibility is that glutamatergic activity in deep layers 5 and 6 of neocortex could activate the axonal receptors. This would provide a novel mechanism by which local neocortical activity within the column could modulate its ascending input. Our previous study of presynaptic kainate receptors in barrel cortex (Kidd et al., 2002) showed that the GluK1 selective agonist ATPA depressed thalamocortical transmission and that a GluK1 antagonist, LY382884, blocks short-term depression elicited during brief trains of synaptic activity. Those findings are consistent with the present results; however, our new findings now show that pharmacological activation of kainate receptors using the broader spectrum agonist, kainate, reveals that kainate receptors facilitate thalamocortical transmission when activated with low doses of kainate in addition to the depression observed at high doses. The facilitation of axonal excitability observed with 3 mM kainate could be the mechanisms that entirely accounts for the facilitation of the thalamocortical EPSC at this dose. At higher doses of kainate (5 and 10 mM) the fibre volley is still facilitated suggesting that this mechanism remains in play at these higher concentrations. However the EPSC is depressed with 5 and 10 mM kainate indicting that a second mechanism mediating depression overrides the increase in axonal excitability. This latter mechanism is most likely due to kainate receptors acting at thalamocortical terminals because previous work shows that short-term depression induced by brief high frequency trains of activity at this input is blocked by a kainate receptor antagonist (Kidd et al., 2002). It is unlikely that postsynaptic mechanisms contributes to the depression induced by kainate because there is only a very small change in DC on application of 10 mM kainate even though this concentration produces a robust depression in transmission. In addition, both the NMDA and AMPA recepor-mediated EPSCs are similarly depressed by 10 mM kainate suggesting a common presynaptic mechanism for this action. However, it is also possible that for bath-applied kainate indirect mechanisms may contribute, for example involving activation of presynaptic GABAB, CB1 or mGlu receptors (Kullmann, 2001; Lourenco et al., 2010). Our findings emphasize the central role played by kainate receptors in regulating presynaptic function. Much of the previous work has focused on presynaptic kainate receptors in hippocampal circuits (Kullmann, 2001; Isaac et al., 2004; Pinheiro and Mulle, 2008); however, the physiological roles of such presynaptic kainate receptor mechanisms are unclear. The present work now characterizes the regulation of transmission by kainate receptors in primary
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sensory neocortex where synaptic dynamics are well established to be critical for circuit function (Abbott and Regehr, 2004). The presence of at least two mechanisms for the bi-directional kainate receptor-dependent regulation of developing thalamocortical transmission thus provides a potentially powerful combination for the regulation of synaptic dynamics at this input that likely has important impact on information processing in layer 4. Author contributions J-S.J. performed all the electrophysiology experiments, analyzed the data and designed the electrophysiology experiments. S.M.B. performed the histoblot experiments, analyzed the data and designed the histoblot experiments. E.M. provided guidance for S.M.B., helped with histoblot data analysis and provided intellectual input on the project. J.T.R.I. provided guidance for J-S.J., wrote the manuscript, and was responsible for overall project design and conception. Acknowledgements We are very grateful to Dr. Christophe Mulle for advice on the study and comments on the manuscript. This work supported by the MRC (G0601509, E.M.), Wellcome Trust (J.T.R.I., E.M.) and NINDS Intramural Program (J.T.R.I.). SMB was an MRC funded PhD student. Appendix. Supplementary data Supplementary data associated with this article can be found in the on-line version, at doi:10.1016/j.neuropharm.2010.12.023. References Abbott, L.F., Regehr, W.G., 2004. Synaptic computation. Nature 431, 796e803. Agmon, A., Connors, B.W., 1991. Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro. Neuroscience 41, 365e379. Bahn, S., Volk, B., Wisden, W., 1994. Kainate receptor gene expression in the developing rat brain. J. Neurosci. 14, 5525e5547. Bannister, N.J., Benke, T.A., Mellor, J., Scott, H., Gurdal, E., Crabtree, J.W., Isaac, J.T., 2005. Developmental changes in AMPA and kainate receptor-mediated quantal transmission at thalamocortical synapses in the barrel cortex. J. Neurosci. 25, 5259e5271. Bettler, B., Mulle, C., 1995. Review: neurotransmitter receptors. II. AMPA and kainate receptors. Neuropharmacology 34, 123e139. Bortolotto, Z.A., Clarke, V.R., Delany, C.M., Parry, M.C., Smolders, I., Vignes, M., Ho, K.H., Miu, P., Brinton, B.T., Fantaske, R., Ogden, A., Gates, M., Ornstein, P.L., Lodge, D., Bleakman, D., Collingridge, G.L., 1999. Kainate receptors are involved in synaptic plasticity. Nature 402, 297e301. Clarke, V.R., Ballyk, B.A., Hoo, K.H., Mandelzys, A., Pellizzari, A., Bath, C.P., Thomas, J., Sharpe, E.F., Davies, C.H., Ornstein, P.L., Schoepp, D.D., Kamboj, R.K., Collingridge, G.L., Lodge, D., Bleakman, D., 1997. A hippocampal GluR5 kainate receptor regulating inhibitory synaptic transmission. Nature 389, 599e603. Collingridge, G.L., Olsen, R.W., Peters, J., Spedding, M., 2009. A nomenclature for ligand-gated ion channels. Neuropharmacology 56, 2e5. Contractor, A., Swanson, G., Heinemann, S.F., 2001. Kainate receptors are involved in short- and long-term plasticity at mossy fiber synapses in the hippocampus. Neuron 29, 209e216. Contractor, A., Sailer, A.W., Darstein, M., Maron, C., Xu, J., Swanson, G.T., Heinemann, S.F., 2003. Loss of kainate receptor-mediated heterosynaptic facilitation of mossy-fiber synapses in KA2/ mice. J. Neurosci. 23, 422e429. Crair, M.C., Malenka, R.C., 1995. A critical period for long-term potentiation at thalamocortical synapses. Nature 375, 325e328. Daw, M.I., Chittajallu, R., Bortolotto, Z.A., Dev, K.K., Duprat, F., Henley, J.M., Collingridge, G.L., Isaac, J.T., 2000. PDZ proteins interacting with C-terminal GluR2/3 are involved in a PKC-dependent regulation of AMPA receptors at hippocampal synapses. Neuron 28, 873e886. Daw, M.I., Bannister, N.V., Isaac, J.T., 2006. Rapid, activity-dependent plasticity in timing precision in neonatal barrel cortex. J. Neurosci. 26, 4178e4187. Daw, M.I., Ashby, M.C., Isaac, J.T., 2007a. Coordinated developmental recruitment of latent fast spiking interneurons in layer IV barrel cortex. Nat. Neurosci. 10, 453e461. Daw, M.I., Scott, H.L., Isaac, J.T., 2007b. Developmental synaptic plasticity at the thalamocortical input to barrel cortex: mechanisms and roles. Mol. Cell. Neurosci. 34, 493e502.
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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Mechanisms of bi-directional modulation of thalamocortical transmission in barrel cortex by presynaptic kainate receptors Jean-Sébastien Jouhanneau a,1, Simon M. Ball b, Elek Molnár b, John T.R. Isaac a, *, 2 a b
Developmental Synaptic Plasticity Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892-3701, USA MRC Centre for Synaptic Plasticity, School of Physiology and Pharmacology, University of Bristol, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 July 2010 Received in revised form 17 December 2010 Accepted 20 December 2010
Presynaptic kainate receptors play an important role in synaptic transmission and short-term plasticity to profoundly regulate network activity in many parts of the mammalian brain. In primary sensory neocortex, where short-term synaptic plasticity is important for receptive field structure and information processing, kainate receptors are highly expressed and regulate thalamocortical inputs, particularly during development. However, the mechanisms of the kainate receptor-dependent presynaptic regulation of thalamocortical transmission are unclear. We therefore investigated this issue using electrophysiology in neonatal thalamocortical slices of barrel cortex combined with pharmacology and biochemical analyses. We show that presynaptic kainate receptors can both facilitate or depress synaptic transmission depending on the extent of their activation. This bi-directional regulation is mediated in part by kainate receptors that directly influence thalamocortical axonal excitability, but also likely involves receptors acting at thalamocortical terminals to regulate transmitter release. The efficacy of kainate in regulating thalamocortical transmission is low compared to that reported for other inputs. Consistent with this low efficacy, our biochemical analyses indicate that the presynaptic kainate receptors regulating neonatal thalamocortical inputs likely lack the high kainate affinity GluK4 and 5 subunits. Thus thalamocortical transmission can be bi-directionally regulated by low affinity kainate receptors through two mechanisms. Such presynaptic regulation provides a potentially powerful mechanism to influence sensory processing during development of barrel cortex. Ó 2010 Published by Elsevier Ltd.
Keywords: Synaptic transmission Presynaptic mechanisms Kainate receptors Barrel cortex
1. Introduction Kainate-type glutamate receptors are widely expressed in the mammalian brain and play important roles in synaptic transmission
Abbreviations: LTP, long-term potentiation (LTP); EPSC, excitatory postsynaptic current; EPSP, excitatory postsynaptic potential; VB, ventrobasal complex of thalamus; DC, direct current; AMPAR, AMPA receptor; NMDAR, NMDA receptor; GluK1, kainate-type glutamate receptor subunit 1 (GluR5 in previous nomenclature); GluK2, kainate-type glutamate receptor subunit 2 (GluR6 in previous nomenclature); GluK3, kainate-type glutamate receptor subunit 3 (GluR7 in previous nomenclature); GluK4, kainate-type glutamate receptor subunit 4 (KA1 in previous nomenclature); GluK5, kainate-type glutamate receptor subunit 5 (KA2 in previous nomenclature). * Corresponding author. E-mail address: [email protected] (J.T.R. Isaac). 1 Present address: Department of Neuroscience, Max-Delbruck-Centrum for Molecular Medecine, Robert-Rossler-Strasse 10, D-13092 Berlin-Buch, Germany; Neurocure, Neuroscience Research Center, Universitaetsmedizin Charite, 10117 Berlin, Germany. 2 Eli Lilly and Company, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK. 0028-3908/$ e see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.neuropharm.2010.12.023
and neuronal excitability (Wisden and Seeburg, 1993; Bahn et al., 1994; Bettler and Mulle, 1995). Presynaptic kainate receptors are best characterized in hippocampus where they can up- or down-regulate both glutamatergic and GABAergic transmission (Kullmann, 2001; Lerma et al., 2001; Isaac et al., 2004), profoundly impacting circuit function. For example, kainate receptors regulate network activity in hippocampal CA3 and CA1 (Kullmann, 2001; Fisahn et al., 2004; Lauri et al., 2005) and contribute to epileptiform activity in hippocampus (Smolders et al., 2002). Presynaptic kainate receptors also play a role in long-term synaptic plasticity, being required for the induction of mossy fiber LTP (Bortolotto et al., 1999; Contractor et al., 2001; Lauri et al., 2001, 2003; Schmitz et al., 2003) and the expression of neonatal hippocampal CA1 LTP (Lauri et al., 2006). Kainate receptors are tetramers made up of combinations of GluK1-5 subunits (Bettler and Mulle, 1995; Collingridge et al., 2009). GluK1-3 subunits can form functional ion channels either as homomers or heteromers; however, GluK4 or 5 homomers are not functional and are not surface expressed in neurons (Gallyas et al., 2003; Ren et al., 2003; Isaac et al., 2004; Nasu-Nishimura
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et al., 2006). Kainate receptors can act both as ionotropic and metabotropic receptors (Lerma et al., 2001) and both modes of their signaling for presynaptic regulation by kainate receptors have been described (Frerking et al., 2001; Kidd et al., 2002; Lauri et al., 2003; Lerma, 2003; Pinheiro and Mulle, 2008). The mechanisms by which kainate receptors regulate presynaptic function are diverse (Kullmann, 2001; Isaac et al., 2004). Kainate receptors located at presynaptic terminals have been shown to up-regulate or downregulate glutamate or GABA release in a dose-dependent manner when activated by exogenous agonist and also by synapticallyreleased glutamate e.g. (Jiang et al., 2001; Lauri et al., 2001; Schmitz et al., 2001; Semyanov and Kullmann, 2001; Kidd et al., 2002). In addition to acting at presynaptic locations, kainate receptors have also regulate synaptic function by altering axonal excitability as shown in hippocampus for the mossy fibre input to CA3 (Kamiya and Ozawa, 2000; Schmitz et al., 2000) and for GABAergic interneuronal input to CA1 pyramidal neurons (Semyanov and Kullmann, 2001; Maingret et al., 2005). Importantly such axonal receptors have been shown to be physiologically activated by synaptically-released glutamate to regulate synaptic transmission (Schmitz et al., 2000; Semyanov and Kullmann, 2001; Contractor et al., 2003). Therefore, these studies show that presynaptic kainate receptors physiologically regulate neurotransmitter release through at least two distinct mechanisms and further that studying these receptors using exogenously applied agonist is a useful approach in understanding the mechanisms of their action. Kainate receptors are highly expressed and developmentally regulated in somatosensory pathways (Bettler and Mulle, 1995; Kerchner et al., 2001; Daw et al., 2007b). In layer 4 of neonatal barrel cortex presynaptic kainate receptors are found at thalamocortical inputs where they can be synaptically-activated during brief trains of activity to mediate a short-term depression (Kidd et al., 2002). Moreover, short-term plasticity plays an important role for in information processing in primary sensory neocortex (Abbott and Regehr, 2004). However, little is known about the properties or mechanisms of action of kainate receptors that regulate thalamocortical transmission. We therefore probed the mechanisms by which presynaptic kainate receptors can regulate thalamocortical transmission in neonatal barrel cortex. We show that kainate receptors can both up- and down-regulate neonatal thalamocortical transmission by at least two mechanisms. We find that presynaptic kainate receptors strongly influence axonal excitability; however, kainate receptors also act independently of this mechanism likely through direct regulation of glutamate release at terminals. In addition, we show that the efficacy of kainate in modulating thalamocortical transmission is low compared to other inputs in the brain, and consistent with this our biochemical analyses indicate that the presynaptic kainate receptors likely lack the high kainate affinity GluK4 and 5 subunits. This bi-directional regulation of thalamocortical transmission is predicted to be important in determining the dynamic properties of thalamocortical inputs hence influencing sensory processing in developing layer 4. 2. Materials and methods 2.1. Ethical approval All experiments were performed in accordance with the guidelines of the National Institutes of Health Animal Care and Use Committee guidelines. 2.2. Electrophysiology Thalamocortical slices (Agmon and Connors, 1991) were prepared from C57Bl/6 or 129SV/EV mouse pups aged between postnatal day (P) 3 and 7 (day of birth is P0), as previously described (Bannister et al., 2005; Daw et al., 2006). Animals were anaesthetised by inhalation of isofluorane and then decapitated with sharp scissors. The brain was rapidly removed and placed in ice-cold extracellular solution.
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Thalamocortical slices were then prepared as previously described (Daw et al., 2006). The data from slices prepared from C57Bl/6 mice are shown in Figs. 1e4. The 129SV/ EV mice were used for the histoblot experiments (Fig. 5). For electrophysiological recordings the extracellular solution contained: 119 mM NaCl, 2.5 mM KCl, 1 mM NaH2PO4, 26.2 mM NaHCO3, 11 mM glucose, 1.3 mM MgSO4, 2.5 mM CaCl2, saturated with 95% O2/5%CO2, pH 7.4. 50 mM picrotoxin and 50 mM D-AP5 were routinely included to block GABAA and NMDA receptors (NMDARs), respectively, in all experiments except those monitoring NMDAR-mediated EPSCs. To monitor NMDAR EPSCs, D-AP5 was omitted and 10 mM BAPTA was included in the intracellular solution. All recordings were performed at room temperature. Whole-cell patch-clamp recordings were made from visually-identified stellate cells in layer 4 barrel cortex (Bannister et al., 2005; Daw et al., 2006) using 4e7 MU electrodes (Fig. 1AeC). For whole-cell voltage-clamp recordings the intracellular solution was as follows: 135 mM CsMeSO4, 8 mM NaCl, 10 mM HEPES, 5 mM QX-314, 0.5 mM EGTA, 4 mM MgATP, 0.3 mM Na-GTP, pH 7.2, 285 mOsm. For current-clamp recordings the intracellular solution was as follows: 130 mM KMeSO4, 8.5 mM NaCl, 5 mM HEPES, 0.5 mM EGTA, 4 mM Mg-ATP, 0.5 mM Na-GTP, pH 7.2, 285 mOsm. The intracellular solution also contained 20 mM AlexaFluor488, which was used for visualizing the recorded neurons (Fig. 1B, C). Fluorescent signals were visualized using a cooled CCD camera (Hamamatsu Orca ER). Thalamocortical EPSCs were evoked by electrical stimulation of the VB thalamus (Agmon and Connors, 1991; Bannister et al., 2005; Daw et al., 2006) at a frequency of 0.2 Hz. Cells were voltage-clamped at 70 mV except when recording pharmacologically-isolated NMDAR-mediated EPSCs for which holding potential was þ30 mV. During most whole-cell recordings, fibre volley was also simultaneously recorded using a patch electrode filled with extracellular solution placed in the internal capsule (Fig. 1A) (Crair and Malenka, 1995). In other experiments the fibre volley in layer 4 was recorded; in these experiments an extracellular field potential elicited by stimulation in VB was first recorded and then 0 mM Ca2þ superfused onto the slice to isolate the fibre volley component. Signals were amplified by a Multiclamp 700B (Axon Instruments), filtered at 4 KHz, digitized at 10 KHz and analyzed using Signal software (CED). Input resistance, series resistance, DC and EPSC amplitude were displayed on-line. Series resistance was estimated as previously described (Daw et al., 2000) and if it varied by >20% during the recording, the cell was discarded. 2.3. Analysis For EPSCs recorded at 70 mV peak EPSC amplitude was measured, which is mediated predominately by AMPA receptors (Kidd and Isaac, 1999, 2001). The fibre volley amplitude was measured as the amplitude of the negative going deflection (Crair and Malenka, 1995). For statistical comparisons an averaged response amplitude over a 1.5 min period immediately before drug application was compared to the average over a 1.5 min period at the peak of the effect. Statistical analysis was performed using the Student’s t-test unless otherwise noted, paired or unpaired as appropriate. 2.4. Histoblot experiments Region-specific changes in distribution of GluK1/2 and GluK5 immunoreactivities were analyzed with previously characterized antibodies (Wenthold et al., 1994; Molnar et al., 1995) in 10 mm thick thalamocortical sections of P3, 5, 7 and adult brains of mice using an in situ blotting technique (‘histoblot’) (Tonnes et al., 1999; Gallyas et al., 2003; Jo et al., 2006). Nissl staining was performed on adjacent sections to confirm brain regions. Mean pixel density of histoblots was analyzed using previously established image quantification procedures (Kopniczky et al., 2005; Jo et al., 2006, n ¼ 4 (n ¼ one animal)).
3. Results 3.1. Kainate causes a dose-dependent depression or facilitation of thalamocortical transmission in layer 4 of developing barrel cortex Our previous work shows that a presynaptic GluK1-containing kainate receptor exists at thalamocortical inputs to neonatal layer 4 barrel cortex that can be synaptically-activated during brief trains of activity to cause a short-term depression (Kidd et al., 2002). To investigate the mechanisms for this presynaptic kainate receptordependent regulation, we studied the effects of bath application of kainate at different concentrations in thalamocortical slices prepared from mice aged postnatal day (P) 3e7 (Agmon and Connors, 1991; Crair and Malenka, 1995; Daw et al., 2006). In thalamocortical slices, barrels in layer 4 could be visualized under low power magnification with infra-red illumination (Fig. 1A, B). Wholecell recordings were made from visually-identified stellate cells, which make up w80% of neurons in layer 4 (Feldmeyer et al., 1999;
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Fig. 1. Dose-dependent depression or potentiation of neonatal thalamocortical synaptic transmission in layer 4 barrel cortex by bath-applied kainate. A, Left panel: low power infrared DIC image of a thalamocortical slice in the recording chamber (from P5 mouse). Right panel: schematic of thalamocortical slice (VB: ventrobasal nucleus of thalamus, RTN: thalamic reticular nucleus, IC: internal capsule, Str: striatum, WM: white matter, Hip: hippocampus), the location of the stimulating (stim), fiber volley recording (fibre volley) and whole-cell recording (WeC) electrodes are shown. Barrels are shown in red. B, Infra-red DIC image of barrels and superimposed fluorescence image of a stellate cell filled with Alexa 488. C, Higher magnification fluorescence image of the filled stellate cell (same cell as in B). D, Peak amplitude of the thalamocortical EPSC recorded in layer 4 stellate cells (filled circles; n ¼ 8) and fiber volley (fibre volley) recorded in the internal capsule (IC; open circles; n ¼ 10) in experiments in which 1 mM kainate was bath-applied. Lower graph is direct current (DC) recorded from the same cells. For this and subsequent graphs, each point is an average of 3 consecutive responses that are normalized to a five minute baseline immediately prior to drug application. E, EPSC amplitude (n ¼ 10), fibre volley (n ¼ 10) and DC (n ¼ 10) for experiments in which 10 mM kainate was applied. F, Example traces of EPSCs (left) and fibre volley responses (right) for experiment shown in G. G, EPSC amplitude and fibre volley (top) and DC (bottom) from an example experiment in which 3 mM kainate was bath-applied. H, Summary data for the 3 mM kainate experiments (EPSC: n ¼ 7; fibre volley: n ¼ 12; DC: n ¼ 7).
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Fig. 2. Dose response relationships of kainate for thalamocortical EPSC, fibre volley and DC. A, Dose response relationship for kainate on EPSC amplitude (small circles are from individual cells, large circles are mean values; 1 mM: n ¼ 8; 3 mM: n ¼ 7; 5 mM: n ¼ 2; 10 mM: n ¼ 10; 30 mM: n ¼ 5). B, Dose response for fibre volley recorded in IC (small open circles are from individual cells, large open circles are mean values; 1 mM kainate: n ¼ 10; 3 mM: n ¼ 12; 5 mM: n ¼ 2; 10 mM: n ¼ 10; 30 mM: n ¼ 4) and fibre volley recorded in layer 4 (small closed gray circles are from individual cells, large closed gray circles are mean values; n ¼ 7). C, Dose response relationship for DC (1 mM: n ¼ 8; 3 mM: n ¼ 7; 5 mM: n ¼ 2; 10 mM: n ¼ 10; 30 mM: n ¼ 5). Data for 1, 3 and 10 mM kainate are from the data sets shown in Fig. 1. D, Summary bar graph for the effect of bath application kainate on EPSC, fibre volley and DC amplitude.
Lefort et al., 2009), with patch electrodes containing the fluorescent dye AlexaFluor488 so that cell type and location could be readily determined (Daw et al., 2007a) (Fig. 1B, C). Thalamocortical EPSCs were elicited in layer 4 stellate cells by stimulation of thalamocortical axons in the ventrobasal (VB) thalamus (Fig. 1A) (Agmon and Connors, 1991; Crair and Malenka, 1995; Daw et al., 2006). In addition, the postsynaptic effect of agonist application was monitored by measuring the effects on the direct current (DC) during recordings. Previous studies using bath application of kainate have found that presynaptic kainate receptors containing GluK4 or GluK5 are activated with high efficacy by kainate. For example hippocampal mossy fiber-CA3 synapses are facilitated by 50 nM kainate through GluK2-containg receptors (Lauri et al., 2001; Schmitz et al., 2001; Contractor et al., 2003). However, presynaptic kainate receptors can also exhibit very low efficacy for agonist; indeed, a second very low efficacy presynaptic kainate receptor also exists at mossy fibreCA3 inputs composed of GluK2 and GluK3 subunits that is synaptically-activated, yet requires mM glutamate for activation (Pinheiro et al., 2007; Pinheiro and Mulle, 2008). We therefore investigated the effects of bath application of kainate on thalamocortical transmission using a range of concentrations. When we applied 1 mM kainate to thalamocortical slices there was no effect on the thalamocortical EPSC (Fig. 1D, 96.24 6.72, P ¼ 0.7622, n ¼ 8). However, at a concentration of 3 mM, kainate caused a reversible potentiation of the thalamocortical EPSC (Fig. 1FeH, 137.90 14.53, P ¼ 0.0337, n ¼ 8). At a concentration of 10 mM kainate caused a strong reversible depression of the EPSC (Fig. 1E, 27.43 7.34, P ¼ 0.0001, n ¼ 10). This depression was preceded by a transient facilitation, similar to what has previously been
observed at hippocampal mossy fibre-CA3 synapses (Schmitz et al., 2000) and is likely due to a slow rise in agonist concentration reaching the receptors. In addition, we found that 5 and 30 mM kainate depressed the EPSC (58.57 46.68, n ¼ 2; 20.35 7.82, P ¼ 0.0081, n ¼ 5; respectively). During the same experiments we also monitored the effects of bath-applied kainate on thalamocortical axonal excitability by recording the fiber volley using an extracellular electrode placed in the internal capsule (Crair and Malenka, 1995)(Fig. 1A). 1 mM kainate failed to have an effect on fibre volley (Fig. 1D, 99.10 7.35, P ¼ 0.2485, n ¼ 10), whereas 3 mM (Fig. 1FeH, 127.23 5.88, P ¼ 0.0074, n ¼ 12), 5 mM (137.03 4.47, n ¼ 2; Fig. 2B) and 10 mM (Fig. 1E, 125.58 4.66, P ¼ 0.0011, n ¼ 11) all caused a facilitation of fibre volley, and 30 mM caused a strong depression (35.77 8.23, P ¼ 0.1094, n ¼ 5; Fig. 2B). Although fibre volley recordings from the internal capsule provide a readout of thalamocortical axonal excitability (Crair and Malenka, 1995), it is possible that other axonal populations may contribute to the response. Therefore, we also tested whether kainate affects the fibre volley recorded in barrels in layer 4. To do this thalamocortical EPSPs were recorded extracellularly and then extracellular solution nominally containing zero Ca2þ was washed in to isolate the fibre volley, after which kainate was bath-applied. Using this approach we found that 10 mM kainate also causes a facilitation of the fibre volley in layer 4 (Fig. 2A, 129.19 8.95, P ¼ 0.0384, n ¼ 7), similar to that observed for fibre volley measured in the internal capsule (unpaired t-test P ¼ 0.0897). To determine whether bath application of kainate had any direct postsynaptic effect on stellate cells, we measured direct current (DC) during the whole-cell voltage-clamp recordings. Kainate at doses up to 10 mM caused only a small change in DC (on
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Fig. 3. Depression of thalamocortical synaptic transmission by 10 mM kainate is not blocked by the selective AMPAR antagonist GYKI53655, but is prevented by the non-selective AMPA and kainate receptor antagonist NBQX. A, Summary data for the effects of bath application of 10 mM kainate on thalamocortical NMDAR-mediated EPSC amplitude (recorded at a membrane potential of þ30 mV) in the presence of 25 mM GYKI53655 (n ¼ 9). Inset top: example traces taken at the times indicated. B, Summary data for effects of 10 mM kainate on fibre volley in the presence of GYKI53655 (n ¼ 6). C, Summary data for the effects of 10 mM kainate on NMDAR-mediated EPSC amplitude in the presence of 20 mM NBQX (n ¼ 5). Inset top: example traces taken at the times indicated. D, Summary data for effects of 10 mM kainate on fibre volley in the presence of NBQX (n ¼ 10).
average < 15 pA; Fig. 1D: 1 mM kainate, 11.82 4.12 pA, P ¼ 0.0168,n ¼ 8, E: 10 mM kainate, 11.22 3.04 pA, P ¼ 0.0022, n ¼ 10, G, H: 3 mM kainate, 2.08 2.06 pA, P ¼ 0.1601, n ¼ 7). In separate current-clamp experiments we determined whether this small change in DC was sufficient to cause spiking in stellate cells and found that 10 mM kainate did not cause any consistent depolarization in stellate cells, rather a small but significant hyperpolarization was observed (Supplementary Fig. 1, 3.72 1.08 mV, P ¼ 0.0184, n ¼ 5). Taken together our analysis of the effects of kainate on thalamocortical transmission demonstrates that kainate facilitates synaptic transmission at 3 mM, but depresses at 5 mM and higher doses (Fig. 2A). In contrast, the thalamocortical fibre volley is facilitated by 3, 5 and 10 mM kainate, only depressing at 30 mM (Fig. 2B). Finally, at doses of up to 10 mM, kainate does not cause any large change in DC (Fig. 2C) and does not induce spiking in stellate cells. These findings indicate that kainate bi-directionally affects thalamocortical synaptic transmission via a presynaptic mechanism, consistent with our previous study (Kidd et al., 2002). Further, there is a dissociation in the dose response between effects on the thalamocortical EPSC and fibre volley. This demonstrates that, although kainate affects thalamocortical axonal excitability, this mechanism cannot account for all the effects on the thalamocortical EPSC suggesting that kainate also directly affects the properties of presynaptic terminals. Our
analysis also shows that the efficacy of kainate in regulating transmission is in the low micromolar range. As highlighted in Supplementary Fig. 2A, B this efficacy is approximately 100 fold lower compared to the high efficacy kainate receptor at hippocampal mossy fibres (Lauri et al., 2001; Schmitz et al., 2001; Contractor et al., 2003). 3.2. Kainate affects thalamocortical transmission through actions at kainate receptors Although kainate is selective for kainate receptors over AMPA receptors (AMPARs), it has significant activity at AMPARs at doses >1 mM (Clarke et al., 1997; Mulle et al., 2000). Therefore, since kainate only affects thalamocortical transmission at doses of 3 mM and greater, it is important to investigate whether action at AMPARs contributes to the effects we observe. To address this, we investigated whether the effects of 10 mM kainate were altered in the presence of the selective AMPAR antagonist GYKI53655 (25 mM) (Wilding and Huettner, 1995; Kidd and Isaac, 1999). To monitor thalamocortical synaptic transmission under these conditions of AMPAR blockade, we recorded NMDAR-mediated EPSCs at a membrane potential of þ30 mV. We found that 10 mM kainate produced a transient facilitation followed by a depression of the NMDAR-mediated EPSC (Fig. 3A; 44.10 13.43, P ¼ 0.0357, n ¼ 9) and also facilitated fibre volley (Fig. 3B; 118.42 6.06, P ¼ 0.0195,
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Fig. 4. Kainate regulates thalamocortical axonal excitability. A, Summary data for the effects of 10 mM kainate on antidromic spike probability evoked by electrical stimulation of thalamocortical axons recorded in VB neurons (voltage-clamp at 70 mV) in the presence of 25 mM GYKI53655 (n ¼ 5). Inset top: traces from an example experiment taken at the times indicated. Inset right: schematic of recording/stimulating configuration. B, Summary bar graph of effects of 10 mM kainate on antidromic spike probability. C, Summary data for effects of 10 mM kainate on DC in VB neurons in GYKI53655. D, Summary bar graph of effects of 10 mM kainate on DC.
n ¼ 6) in the presence of GYKI53655, similar to the effects observed on fibre volley and AMPAR-mediated EPSCs in the absence of GYKI53655. This finding demonstrates that AMPARs do not substantially contribute to the effects of kainate at doses of 10 mM or less. We also tested whether NBQX, at a dose (20 mM) that blocks both AMPA and kainate receptors, prevents the effects of kainate. NBQX blocked both the depression of the NMDAR EPSC and the facilitation of fibre volley by 10 mM kainate (Fig. 3C 84.57 9.12, P ¼ 0.4012, n ¼ 5, D 98.47 6.67, P ¼ 0.8603, n ¼ 10, respectively). Some run down of the NMDA EPSC was observed in these experiments; however, this was not statistically significant and likely reflects the decrease in NMDAR function that can be observed in neurons during whole-cell recording. Taken together, these analyses show that the effects of 10 mM kainate on EPSC and fibre volley are not mediated by AMPARs, but rather are due to activation of kainate receptors. In addition, the similar effects of kainate on AMPA and NMDA receptor-mediated EPSCs provide further strong evidence for the presynaptic locus of kainate receptors regulating thalamocortical transmission.
3.3. Kainate receptors present on thalamocortical axons regulate axonal excitability The increase in fibre volley caused by 3, 5 and 10 mM kainate suggests that kainate receptors can regulate thalamocortical axonal excitability, as has been reported for hippocampal mossy fibers (Kamiya and Ozawa, 2000; Schmitz et al., 2000) and CA1 interneuron axons (Semyanov and Kullmann, 2001). However, an alternative possibility is that kainate acts on somatodendritic kainate receptors present on presynaptic neurons in VB causing excitation that indirectly increases the excitability of thalamocortical axons. Therefore, to test whether kainate receptors present on thalamocortical axons regulate axonal excitability we made wholecell voltage-clamp recordings from VB neurons and monitored antidromic action potentials evoked by a stimulating electrode placed in the internal capsule (Fig. 4A right inset). This approach allows isolation of effects of bath-applied kainate on axonal receptors because somatic voltage-clamp prevents any depolarization due to activation of somatodendritic receptors from
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Fig. 5. Kainate receptors in neonatal layer 4 barrel cortex contain GluK2/3, but lack GluK5 subunits. A, Pseudocolour images of immunoreactivity levels of histoblots from 10 mm thick thalamocortical sections for the antibody indicated (top) and at the different ages as indicated (left). Extreme left hand panels are Nissl stains of adjacent thalamocortical sections (red box indicates approximate position of barrel cortex). Scale bar is 1 mm and applies to all images. B, Quantitation of GluK5 immunoreactivity for layer 4 barrel cortex (n ¼ 4). C, Quantitation of GluK2/3 immunoreactivity for layer 4 barrel cortex (n ¼ 4).
influencing antidromic action potential generation (Semyanov and Kullmann, 2001). We used a stimulus intensity that evoked action potentials in approximately 50% of trials and then monitored the effect of kainate on antidromic action potential probability in the presence of GYKI53655. Under these conditions we found that 10 mM kainate caused a reversible increase in action potential probability (Fig. 4A, B, 0.78 0.14, P ¼ 0.04, n ¼ 5) providing strong evidence for the presence of kainate receptors on thalamocortical axons that can facilitate action potential generation. In the same experiments we observed an increase in DC in VB neurons with kainate application (Fig. 4C, D, 38.32 10.78 pA, P ¼ 0.1935, n ¼ 5); however, the DC increase was variable between cells. Thus while it is likely that kainate receptors are present on somatodendritic membranes of VB neurons, our data strongly indicates that kainate receptors located on thalamocortical axons regulate axonal excitability. 3.4. Kainate receptors containing GluK2/3, but lacking the high kainate affinity GluK5 subunit, are expressed in neonatal layer 4 barrel cortex The low micromolar doses of kainate needed to produce effects on transmission indicate that the kainate receptors regulating the thalamocortical input have low affinity for kainate. Native kainate receptors are typically heteromers made up from combinations of
GluK1-5 and heteromeric subunit composition affects agonist efficacy, kinetics and signaling (Lerma et al., 2001; Pinheiro and Mulle, 2008). The presence of GluK4 or 5 yields kainate receptors with high affinity for kainate, producing nM efficacy for kainate in regulating synaptic transmission e.g., (Contractor et al., 2003). Therefore, one possibility to explain the low efficacy of kainate in regulating neonatal thalamocortical transmission is that the kainate receptors lack GluK4 and 5. We investigated this issue using histoblot analysis (Tonnes et al., 1999; Jo et al., 2006) in which protein expression levels in different brain regions are analyzed by performing immunolabelling of nitrocellulose membranes onto which brain sections have been blotted. Since a number of brain sections can be blotted onto the same membrane, protein levels can be compared for multiple brain regions within and between brains. We applied this analysis to thalamocortical brain slices prepared from P3, P5 and P7 animals, the age range used for our electrophysiology experiments, as well as slices from P14 and adults (Fig. 5A). Histoblots were probed for GluK5 and GluK2/3 (note that no good GluK1, GluK2, GluK3 or GluK4 specific antibodies exist). In layer 4 barrel cortex we found very low levels of GluK5 expression in the first postnatal week, approximately six-fold lower than GluK5 levels in adult, with GluK5 levels increasing dramatically by P14 (Fig. 5A, B). Previous studies have shown that mRNA for GluK4, the other high affinity subunit, is never observed in neocortex at any developmental stage
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(Werner et al., 1991; Herb et al., 1992; Bahn et al., 1994); therefore, the additional lack of GluK5 strongly suggests that there is a lack of expression of subunits with high kainate binding affinity in layer 4 barrel cortex during the first postnatal week. There was considerable GluK2/3 immunoreactivity detected in layer 4 in the first postnatal week, which by P7 was comparable to adult levels (Fig. 5A, C). A similar profile for GluK5 and GluK2/3 was observed in inner layers of barrel cortex and in VB thalamus (Fig. 5A). Thus, the histoblot analysis shows that kainate receptor subunits exist in layer 4 barrel cortex during the first postnatal week; however, kainate receptors likely lack the high kainate affinity GluK4 and 5 subunits providing the basis for the low efficacy of kainate in regulating thalamocortical transmission in layer 4 that we observe. 4. Discussion Presynaptic receptors regulating neurotransmitter release represent an important mechanism for determining the dynamic properties of synapses in many areas of the mammalian brain. Prominent amongst such receptors are presynaptic kainate receptors that regulate short-term plasticity at glutamatergic and GABAergic synapses in many brain areas including hippocampus and neocortex (Kullmann, 2001; Isaac et al., 2004; Pinheiro and Mulle, 2008). Here we now show that presynaptic kainate receptors can both up- and down-regulate thalamocortical transmission in developing layer 4 barrel cortex through at least two mechanisms. In one mechanism they increase or decrease axonal excitability, and in a second regulate transmission independently of axonal excitability, likely due to direct action at thalamocortical terminals. The kainate receptors mediating these effects exhibit low efficacy for kainate, lacking the GluK4 and GluK5 subunits that confer high affinity for kainate. These findings taken together with previous work showing that synaptic activation of kainate receptors at this input mediates a short-term depression (Kidd et al., 2002) demonstrate that kainate receptors provide a mechanism for the bi-directional regulation of thalamocortical transmission in developing layer 4 barrel cortex. We find that bath application of kainate affects both the thalamocortical synaptic response and the fibre volley. This is similar to hippocampal mossy fiber-CA3 transmission; however, the dosee response relationship for kainate regulating thalamocortical responses is shifted to the right, demonstrating w100 fold lower efficacy of kainate compared to that reported by a number of groups for mossy fibers (Kamiya and Ozawa, 2000; Schmitz et al., 2000; Lauri et al., 2001; Schmitz et al., 2001b; Contractor et al., 2003). The low efficacy of kainate in regulating thalamocortical transmission can be explained by a lack of expression of the high kainate binding affinity GluK4 and 5 subunits in early postnatal layer 4 barrel cortex. High efficacy of kainate correlates with a presence of these high kainate binding affinity subunits in the receptor complex (Werner et al.,1991; Herb et al., 1992). Consistent with a major role for GluK5 in the high efficacy of kainate, the nM effects of kainate at mossy fibres are absent in GluK5 KO mice (Contractor et al., 2003; Ruiz et al., 2005). Moreover, recent work shows that the GluK5 subunit mediates the metabotropic effects of kainate receptors and this subunit is selectively activated by nM kainate (Ruiz et al., 2005) (but see (Fernandes et al., 2009)). For the ionotropic actions of kainate receptors, the evidence indicates that there is typically a mM efficacy of kainate or glutamate that is not strongly influenced by the presence of GluK4 or 5 subunits (Lerma et al., 2001). Native ionotropic kainate receptors with similar kainate efficacies (1e10 mM) to that observed in the present study, have been reported in a number of preparations (Patneau et al., 1994; Sahara et al., 1997; Wilding and Huettner,
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1997; Pemberton et al., 1998). Notably, a recent study shows that a low affinity GluK2 and GluK3-containing kainate receptor is also present presynaptically at mossy fibre-CA3 inputs, is activated by mM concentrations of glutamate and is activated by synapticallyreleased glutamate (Pinheiro et al., 2007). This study, therefore, indicates that low affinity presynaptic kainate receptors can be activated by endogenous glutamate release to physiologically regulate synaptic transmission. Application of kainate produces a modulation of thalamocortical fibre volley that is due to the presence of axonal receptors on thalamocortical afferents. Such direct regulation of axonal excitability by kainate receptors has been shown previously to occur in axons of local circuit interneurons in CA1 hippocampus (Semyanov and Kullmann, 2001; Maingret et al., 2005) and in mossy fibre axons in CA3 hippocampus (Kamiya and Ozawa, 2000; Schmitz et al., 2000). Importantly these previous studies show that synapticallyreleased glutamate can activate the axonal receptors pointing to a physiological role in regulating synaptic transmission. Our new data represent the first evidence for such a mechanism occurring in neocortex and on an axonal population that projects significant distances across many neocortical layers. Although in the present study we do not address how such axonal receptors are physiologically activated, by analogy to mossy fibres one possibility is that glutamatergic activity in deep layers 5 and 6 of neocortex could activate the axonal receptors. This would provide a novel mechanism by which local neocortical activity within the column could modulate its ascending input. Our previous study of presynaptic kainate receptors in barrel cortex (Kidd et al., 2002) showed that the GluK1 selective agonist ATPA depressed thalamocortical transmission and that a GluK1 antagonist, LY382884, blocks short-term depression elicited during brief trains of synaptic activity. Those findings are consistent with the present results; however, our new findings now show that pharmacological activation of kainate receptors using the broader spectrum agonist, kainate, reveals that kainate receptors facilitate thalamocortical transmission when activated with low doses of kainate in addition to the depression observed at high doses. The facilitation of axonal excitability observed with 3 mM kainate could be the mechanisms that entirely accounts for the facilitation of the thalamocortical EPSC at this dose. At higher doses of kainate (5 and 10 mM) the fibre volley is still facilitated suggesting that this mechanism remains in play at these higher concentrations. However the EPSC is depressed with 5 and 10 mM kainate indicting that a second mechanism mediating depression overrides the increase in axonal excitability. This latter mechanism is most likely due to kainate receptors acting at thalamocortical terminals because previous work shows that short-term depression induced by brief high frequency trains of activity at this input is blocked by a kainate receptor antagonist (Kidd et al., 2002). It is unlikely that postsynaptic mechanisms contributes to the depression induced by kainate because there is only a very small change in DC on application of 10 mM kainate even though this concentration produces a robust depression in transmission. In addition, both the NMDA and AMPA recepor-mediated EPSCs are similarly depressed by 10 mM kainate suggesting a common presynaptic mechanism for this action. However, it is also possible that for bath-applied kainate indirect mechanisms may contribute, for example involving activation of presynaptic GABAB, CB1 or mGlu receptors (Kullmann, 2001; Lourenco et al., 2010). Our findings emphasize the central role played by kainate receptors in regulating presynaptic function. Much of the previous work has focused on presynaptic kainate receptors in hippocampal circuits (Kullmann, 2001; Isaac et al., 2004; Pinheiro and Mulle, 2008); however, the physiological roles of such presynaptic kainate receptor mechanisms are unclear. The present work now characterizes the regulation of transmission by kainate receptors in primary
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sensory neocortex where synaptic dynamics are well established to be critical for circuit function (Abbott and Regehr, 2004). The presence of at least two mechanisms for the bi-directional kainate receptor-dependent regulation of developing thalamocortical transmission thus provides a potentially powerful combination for the regulation of synaptic dynamics at this input that likely has important impact on information processing in layer 4. Author contributions J-S.J. performed all the electrophysiology experiments, analyzed the data and designed the electrophysiology experiments. S.M.B. performed the histoblot experiments, analyzed the data and designed the histoblot experiments. E.M. provided guidance for S.M.B., helped with histoblot data analysis and provided intellectual input on the project. J.T.R.I. provided guidance for J-S.J., wrote the manuscript, and was responsible for overall project design and conception. Acknowledgements We are very grateful to Dr. Christophe Mulle for advice on the study and comments on the manuscript. This work supported by the MRC (G0601509, E.M.), Wellcome Trust (J.T.R.I., E.M.) and NINDS Intramural Program (J.T.R.I.). SMB was an MRC funded PhD student. Appendix. Supplementary data Supplementary data associated with this article can be found in the on-line version, at doi:10.1016/j.neuropharm.2010.12.023. References Abbott, L.F., Regehr, W.G., 2004. Synaptic computation. Nature 431, 796e803. Agmon, A., Connors, B.W., 1991. Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro. Neuroscience 41, 365e379. Bahn, S., Volk, B., Wisden, W., 1994. Kainate receptor gene expression in the developing rat brain. J. Neurosci. 14, 5525e5547. Bannister, N.J., Benke, T.A., Mellor, J., Scott, H., Gurdal, E., Crabtree, J.W., Isaac, J.T., 2005. Developmental changes in AMPA and kainate receptor-mediated quantal transmission at thalamocortical synapses in the barrel cortex. J. Neurosci. 25, 5259e5271. Bettler, B., Mulle, C., 1995. Review: neurotransmitter receptors. II. AMPA and kainate receptors. Neuropharmacology 34, 123e139. Bortolotto, Z.A., Clarke, V.R., Delany, C.M., Parry, M.C., Smolders, I., Vignes, M., Ho, K.H., Miu, P., Brinton, B.T., Fantaske, R., Ogden, A., Gates, M., Ornstein, P.L., Lodge, D., Bleakman, D., Collingridge, G.L., 1999. Kainate receptors are involved in synaptic plasticity. Nature 402, 297e301. Clarke, V.R., Ballyk, B.A., Hoo, K.H., Mandelzys, A., Pellizzari, A., Bath, C.P., Thomas, J., Sharpe, E.F., Davies, C.H., Ornstein, P.L., Schoepp, D.D., Kamboj, R.K., Collingridge, G.L., Lodge, D., Bleakman, D., 1997. A hippocampal GluR5 kainate receptor regulating inhibitory synaptic transmission. Nature 389, 599e603. Collingridge, G.L., Olsen, R.W., Peters, J., Spedding, M., 2009. A nomenclature for ligand-gated ion channels. Neuropharmacology 56, 2e5. Contractor, A., Swanson, G., Heinemann, S.F., 2001. Kainate receptors are involved in short- and long-term plasticity at mossy fiber synapses in the hippocampus. Neuron 29, 209e216. Contractor, A., Sailer, A.W., Darstein, M., Maron, C., Xu, J., Swanson, G.T., Heinemann, S.F., 2003. Loss of kainate receptor-mediated heterosynaptic facilitation of mossy-fiber synapses in KA2/ mice. J. Neurosci. 23, 422e429. Crair, M.C., Malenka, R.C., 1995. A critical period for long-term potentiation at thalamocortical synapses. Nature 375, 325e328. Daw, M.I., Chittajallu, R., Bortolotto, Z.A., Dev, K.K., Duprat, F., Henley, J.M., Collingridge, G.L., Isaac, J.T., 2000. PDZ proteins interacting with C-terminal GluR2/3 are involved in a PKC-dependent regulation of AMPA receptors at hippocampal synapses. Neuron 28, 873e886. Daw, M.I., Bannister, N.V., Isaac, J.T., 2006. Rapid, activity-dependent plasticity in timing precision in neonatal barrel cortex. J. Neurosci. 26, 4178e4187. Daw, M.I., Ashby, M.C., Isaac, J.T., 2007a. Coordinated developmental recruitment of latent fast spiking interneurons in layer IV barrel cortex. Nat. Neurosci. 10, 453e461. Daw, M.I., Scott, H.L., Isaac, J.T., 2007b. Developmental synaptic plasticity at the thalamocortical input to barrel cortex: mechanisms and roles. Mol. Cell. Neurosci. 34, 493e502.
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