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Functional Kainate-Selective Glutamate Receptors in Cultured Hippocampal Neurons (Excitatory Amino Acid Receptors/Hippocampus) JUAN LERMA*, ANA V

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Functional Kainate-Selective Glutamate Receptors in Cultured Hippocampal Neurons (Excitatory Amino Acid Receptors/Hippocampus) JUAN LERMA*, ANA V Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11688-11692, December 1993 Neurobiology Functional kainate-selective glutamate receptors in cultured hippocampal neurons (excitatory amino acid receptors/hippocampus) JUAN LERMA*, ANA V. PATERNAIN, JosE R. NARANJO, AND BRITT MELLSTR6M Departamento de Plasticidad Neural, Instituto Cajal, Consejo Superior de Investigaciones Cientfficas, Avenida Doctor Arce 37, 28002-Madrid, Spain Communicated by Michael V. L. Bennett, September 15, 1993 ABSTRACT Glutamate mediates fast synaptic transmis- experiments, the regional distribution of high-affinity sion at the majority of excitatory synapses throughout the [3H]kainate binding sites does not match the AMPA receptor central nervous system by interacting with different types of distribution but corresponds well to the brain areas with high receptor channels. Cloning of glutamate receptors has pro- susceptibility to the neurotoxic actions of kainate (e.g., vided evidence for the existence of several structurally related hippocampal CA3 field) (13). However, patch-clamp record- subunit families, each composed of several members. It has ings from adult hippocampal neurons have revealed that been proposed that KA1 and KA2 and GluR-5, GluR-6, and native glutamate receptors are similar to the AMPA-type GluR-7 families represent subunit classes of high-affinity kain- recombinant glutamate receptors expressed from cDNA ate receptors and that in vivo different kainate receptor sub- clones but have failed so far to detect receptor channels ofthe types might be constructed from these subunits in heteromeric kainate type (14, 15). The only apparently high-affinity kain- assembly. However, despite some indications from autoradio- ate receptor channels have been found in the peripheral graphic studies and binding data in brain membranes, no nervous system (16, 17), although they are also activated by functional pure kainate receptors have so far been detected in AMPA. brain cells. We have found that early after culturing, a high In the present study, carried out with cultured hippocampal percentage of rat hippocampal neurons express functional, cells, kainate was found to activate a fully desensitizing kainate-selective glutamate receptors. These kainate receptors inward current in a subset of neurons which could not be show pronounced desensitization with fast onset and very slow activated or cross-desensitized by AMPA. Functional prop- recovery and are also activated by quisqualate and domoate, erties of responses selectively activated by kainate match but not by a-amino-3-hydroxy-5-methylisoxazole-4-propion- those reported for GluR-6 receptors formed in exogenous ate. Our results provide evidence for the existence offunctional expression systems. glutamate receptors ofthe kainate type in nerve cells, which are likely to be native homomeric GluR-6 receptors. MATERIALS AND METHODS Glutamate receptors mediate transmission at the majority of Cells were mechanically dissociated from hippocampi of fast excitatory synapses in the vertebrate central nervous embryonic day 18 (E18) (unless otherwise stated) rat embryos system (1, 2). Different types of glutamate receptors coexist after treatment with trypsin (type I, Sigma) at 0.12 mg/ml for in the same synaptic contact, and they have been classified 15 min at 37°C and were seeded onto 35-mm Petri dishes from a pharmacological point of view as of the N-methyl-D- previously coated with poly(D-lysine) (5 mg/ml) and laminin aspartate (NMDA) type and of non-NMDA type (2, 3). The (4 gg/ml). Cells were incubated in Dulbecco's modified so-called non-NMDA receptors include a-amino-3-hydroxy- Eagle's medium supplemented with transferrin (0.1 mg/ml), 5-methylisoxazole-4-propionate (AMPA) receptors, which insulin (5 ,ug/ml), putrescine (100 ,uM), progesterone (20 nM), were initially characterized by rapid activation kinetics and SeO2 (30 nM), ovalbumin (0.1%), glucose (3.3 mM), sodium pyruvate (1 mM), glutamine (4 mM), and antibiotics in a fast desensitization, and kainate receptors, which were dif- humidified incubator at 37°C and 5% CO2. ferentiated from AMPA receptors in that they were nonde- Electrophysiological experiments were carried out from 4 sensitizing (e.g., ref. 1). However, molecular biology studies hr up to 17 days after plating. Membrane currents were have provided new insight into the functional and structural recorded in the whole-cell configuration of the patch-clamp diversity of NMDA and non-NMDA glutamate receptor technique (18) by using a List EPC-7 amplifier. Cells were channels (see ref. 4). These studies have indicated that all rapidly perfused by a fast perfusion system (19). Series glutamate receptor channels may be assembled from struc- resistance during whole-cell recording was 8-20 Mfl. A turally homologous subunits that can be grouped into families 100-pA response would produce a voltage error of0.8-2 mV. according to sequence characteristics. On the basis of ligand As series resistance compensation (60-70%) did not improve affinity, the GluR-A to -D (GluR-1 to -4) subunits are con- notably the response amplitude or speed, it was not routinely stituents of AMPA receptor channels (5-8), whereas the used. Currents were filtered at 1 kHz and acquired at a GluR-5, GluR-6, GluR-7, and KA subunits form recombinant sampling rate of 1-2 kHz into a personal computer for receptors with high affinity for kainate (9-11). However, analysis and display purposes. The external solution was 165 kainate gates AMPA receptors and AMPA can activate mM NaCl/2.5 mM KCl/0.5 mM CaCl2/20 mM glucose/10 receptors formed by particular combinations of the high- mM Hepes, pH 7.5. Mg2+ was omitted to prevent block of affinity kainate receptor subunits (4). Thus, the functional NMDA receptor channels. In high-Ca2+ solutions, Ca2+ distinction between AMPA and kainate receptors is unclear substituted for Na+. To the re- and the existence of pure kainate or AMPA receptors in the keep osmolarity constant, brain has been questioned (10, 12). In autoradiographic Abbreviations: NMDA, N-methyl-D-aspartate; AMPA, a-amino-3- hydroxy-5-methylisoxazole-4-propionate; CNQX, 6-cyano-7- The publication costs of this article were defrayed in part by page charge nitroquinoxaline-2,3-dione; En, embryonic day n; Pn, postnatal day payment. This article must therefore be hereby marked "advertisement" n. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. Downloaded by guest on September 25, 2021 11688 Neurobiology: Lerma et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11689 maining Na+ was replaced by N-methyl-D-glucamine. Pi- potent neurotoxin domoic acid (40 AM) induced a partially pettes were filled with 130 mM CsCH3SO3/20 mM CsCl/0.5 desensitizing response, the deactivation of which was very mM CaCl2/2 mM MgCl2/10 mM EGTA/10 mM Hepes, pH slow upon domoate washout (single exponential decay time 7.2. constant of 715 ± 98 ms, n = 4). Responses to domoate were NMDA and kainate were purchased from Sigma. Quis- also cross-desensitized by the previous application of kain- qualate, AMPA and 6-cyano-7-nitroquinoxaline-2,3-dione ate. In some cells, perfusion of NMDA plus glycine evoked (CNQX) were from Tocris Neuramin (Bristol, U.K.). Do- small inward currents which, as in the case illustrated in the moic acid was generously provided by A. Novelli (University figure, commonly consisted of a few 50-pS channel openings of Oviedo, Spain). NS-102 was a gift from T. Drejer (Neu- which could be observed as single steps. The onset of rosearch, Glostrup, Denmark). desensitization of kainate-activated currents in most cases followed a single exponential decay with a time constant of RESULTS 19.3 ± 0.9 ms (300 ,uM kainate, n = 42) (Fig. 1B). In some In 108 of 356 cells studied (30%), AMPA (500 i&M) failed to cells the decaying phase was better fitted by the sum of two induce any response. In these cells a fast desensitizing inward exponentials, the fast component being predominant (time current was observed upon rapid kainate perfusion (Fig. 1A), constants of 17.1 ± 1.5 and 146 ± 17.2 ms, with relative and glutamate (300 ,uM) and quisqualate (100 ,uM) induced amplitudes of 58% and 42%, respectively, n = 8). Recovery responses with similar shapes. Responses induced by gluta- from the desensitized state was very slow (Fig. 1C). Full mate and quisqualate were cross-desensitized by the previ- recovery of kainate-induced peak current took about 40 sec, ous application of kainate, and kainate induced no response indicating that kainate-selective receptors recover from de- after glutamate or quisqualate perfusion (data not shown), sensitization considerably more slowly than NMDA or indicating that all agonists were able to activate and totally AMPA receptors. desensitize the same population ofreceptors. In contrast, the The shape of the peak current-voltage relationship re- vealed strong inward rectification (Fig. 2A), a phenomenon A observed in some homo- and heteromeric assemblies of Kainate Domoate AMPA receptors as well as in recombinant glutamate recep- 20 tors insensitive to AMPA (4, 10, 20). Since inward rectifica- IpA tion and Ca2+ permeability are two phenomena linked to the 400 Ms presence of a glutamine in the Q/R site of the second transmembrane domain of non-NMDA glutamate receptor subunits (21-23), we measured peak currents induced by fast Glutamate Quisqualate application ofkainate when Ca2+ was the only charge carrier. *I" AeP okh If these channels were equally permeable to Ca2+ and Cs+, a substantial inward current should be expected at -80 mV NMDA + GLY AMPA in a high-Ca2+ (50 mM) solution, since the reversal potential for the induced current would be about -10 mV. No inward |¶1~IPA 20 current was detected under these circumstances in the cells 400 ms tested (n = 6) (Fig. 2B), suggesting that these kainate- selective receptors have a very low Ca2+ permeability.
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