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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11688-11692, December 1993 Neurobiology Functional kainate-selective glutamate receptors in cultured hippocampal (excitatory receptors/) 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 throughout the [3H]kainate binding sites does not match the AMPA central by interacting with different types of distribution but corresponds well to the 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 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 (5 ,ug/ml), (100 ,uM), progesterone (20 nM), were initially characterized by rapid activation kinetics and SeO2 (30 nM), ovalbumin (0.1%), glucose (3.3 mM), pyruvate (1 mM), (4 mM), and 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- configuration of the patch-clamp diversity of NMDA and non-NMDA 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 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 (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 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 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 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. This B Kainate result suggests differences in channel structure between AMPA and native kainate-selective receptors. A B T= 18.4 ms 20 pA 165 mM Na' C 50 ms

1 30 500 ms pA 50 mM Ca /O Na FIG. 1. (A) Inward currents activated by glutamate receptor kainate agonists in hippocampal cells 2 days after dissociation. Responses to kainate (300 i&M), glutamate (300 ,uM), quisqualate (100 ,uM), NMDA [100 ;LM in the presence of 10 ,uM glycine (GLY)], and AMPA (500 10 jxM) were recorded from the same cell. The response to domoate (40 250 ms pA ,uM) is from a different cell (note the different calibration) but of similar characteristics. The duration of application is indi- FIG. 2. Rectification and low Ca2+ permeability of kainate- cated by the bar above each trace. Responses to domoate exhibited selective receptors in hippocampal cells. (A) Current-voltage rela- incomplete and slower desensitization [single exponential time con- tionship of kainate-induced peak current. Kainate (300 ,uM) was stant (r) of 160 ± 20 ms, n = 3)] and a very slow current decay after rapidly applied while holding the at different removal of the agonist (in this record r is 680 ms). Calibration bars voltages. Peak amplitudes are plotted versus holding potential. The are valid for all records except for domoate- and NMDA-induced solid line represents the fourth-order polynomial function fitted to the responses. (B) Onset ofdesensitization followed a single exponential data points. (B) Kainate (300 ,AM) was applied in the normal decay (solid line) with an average T of 19.3 ms. (C) Recovery from Na+-containing solution (upper record) and in a 0-Na+, high-Ca2+ desensitization was very slow. Successive pulses of kainate (600 ms, solution (lower record, 50 mM Ca2+; Na+ replaced by 115 mM 300 ,uM) were delivered after waiting for the indicated intervals (10, N-methyl-D-glucamine, buffered to pH 7.5 with HCl) at -80 mV. 20, and 30 s). At least 40 s were necessary to fully recover the initial There was no measurable inward current when Ca2+ was the only peak amplitude. charge carrier. Downloaded by guest on September 25, 2021 11690 Neurobiology: Lerma et al. Proc. Natl. Acad. Sci. USA 90 (1993) In about half of the cells studied (n = 169, 47%), kainate- absence of peak when both responses are present and sum- selective receptors appeared to coexist with receptors of the mate. In contrast to AMPA or NMDA receptors, desensiti- AMPA type. In 64 cells (18%), kainate-induced responses zation of kainate-selective receptor channels seemed to be were only ofthe type that kainate evokes in AMPA receptors complete, since any steady current remaining after the peak (i.e., nondesensitizing). Fifteen cells (4%) gave no responses response during a kainate pulse was cross-desensitized by a to AMPA or kainate. Even in cases where a transient current prepulse of AMPA (Fig. 3C). In contrast, preapplication of was not evident upon kainate perfusion, the presence of AMPA did not influence the kainate-induced peak current in kainate-selective receptors could be detected by predesen- cells with no AMPA response or when it was smaller than the sitizing AMPA receptors by applying a high concentration of kainate-selective component. These observations indicate AMPA and then rapidly switching to a kainate-containing that the steady current that followed the initial peak during solution. In such experiments, a clear peak current did the kainate application is due exclusively to the activation of appear at the beginning of kainate application (Fig. 3A, AMPA receptors and that AMPA does not desensitize kain- arrow), indicating the presence of channels of the kainate- ate-selective receptors. selective type. The transient current was followed by a Although most experiments were done using cells disso- progressively developing response ascribable to recovery of ciated from hippocampi obtained from E18 embryos, tran- previously desensitized AMPA receptors and their subse- sient responses induced by kainate were also found in cells quent activation by kainate. Transient responses to kainate acutely dissociated from hippocampi from E17, E19, and E20 were also evidenced by rapidly applying kainate while con- embryos and postnatal day 0 (P0) and P2 neonates. Cells tinuously perfusing a high concentration of AMPA (500 ,uM) expressing a pure population of kainate-selective receptors (data not shown), indicating that AMPA is not a competitive were more frequently found in young cultures. Twenty-three inhibitor at kainate-selective receptors. This transient cur- percent of the cells studied 2 days after plating (n = 105) rent could not be demonstrated in 18% of the cells (e.g., Fig. responded exclusively with a transient response to kainate, 3B), perhaps because ofthe lack ofkainate-selective channels whereas only 8% of the cells were found with this charac- It also 3B teristic after 4 days in vitro (n = 12). Conversely, kainate in these cells. is apparent from Fig. that kainate induced both peak and steady currents in 44% of the cells 2 responses at AMPA receptors have a slower rise. Different days after plating, while 67% of the cells showed this mixed rise time to peak and steady currents should account for the response when studied 2 days later (i.e., 4 days in vitro). Fig. 4A shows currents evoked by various concentrations A of kainate in a hippocampal with an apparently pure kainate AMPA kainate population of kainate-selective receptors. Concentrations of kainate lower than 1 AM did not induce any noticeable current. Half-maximal activation was achieved with about 20 ,uM (EC50 = 22 AM; 90% confidence interval, 10.6-33.4 ,uM; n = 10) (Fig. 4B). A similar analysis in cells showing exclusively nondesensitizing responses to kainate (i.e., ex- I""~~~4 / IPA pressing only receptors of the AMPA type) revealed a lower 400 ms sensitivity to kainate (EC50 = 240 ,uM; 90%o confidence interval, 90-386 ,uM; n = 4). Hill coefficients were in both B cases close to unity (1.1 and 0.9, respectively). The different kainate AMPA kainate sensitivity to kainate of either response is also illustrated in a cell expressing both receptors in Fig. 4C. A low concen- tration of kainate elicited a totally desensitizing response whereas, in the same cell, a high concentration ofkainate not only increased the amplitude of the peak response but also 40 evoked a substantial steady current. pA Kainate-selective receptors showed different pharmaco- logical properties than AMPA receptors. The known com- 400 ms petitive antagonist of AMPA receptors CNQX (10 ,uM) (24) C slightly attenuated the peak current induced by kainate in kainate AMPA kainate cells where no AMPA responses were detected or where this component was minor (22 + 4.9% inhibition, 300 ,uM kainate, n = 4) (Fig. 5A). In contrast, CNQX (10 ,uM) largely blocked the induction of nondesensitizing currents by kainate in cells not expressing kainate-selective receptors (85 ± 2.6% of = 20 inhibition with 300 uM kainate, n 4) (Fig. SB). In cells pA showing both responses, the introduction of 10 uM CNQX 400 ms greatly reduced the steady component of the response (82 ± FIG. 3. Kainate-selective and AMPA receptors coexist in the 2.3% inhibition, 300 ,uM kainate, n = 4), unmasking the same cell. (A) Unmasking of kainate-induced transient responses transient current (Fig. SC). Thus, CNQX is a less potent (arrows) by previous desensitization of AMPA receptors. The rapid blocker of kainate-selective receptors than of AMPA recep- switch from an AMPA-containing solution (200 AM) to a kainate (300 tors. Conversely, the newly developed glutamate antagonist ,uM) solution generates a transient inward current at the beginning, NS-102 (5-nitro-6,7,8,9-tetrahydrobenzo[g]indole-2,3-dione- followed by a slowly developing response due to the recovery of 3-oxime) (25) was able to block reversibly the peak current AMPA receptors from the desensitized state. (B) The same protocol (Fig. 5 A and C) but not the steady response induced by as in A revealed the absence of kainate-selective receptors in a subset kainate (Fig. S B and C). In cells where no peak response was of hippocampal cells. Note that the slower rise of kainate responses NS-102 affected the nondesen- at AMPA receptors accounts for the absence of an isolated peak in detected, (3 ,uM) minimally A Left. (C) In cells with small AMPA responses, the application of sitizing response induced by kainate at AMPA receptors (10 = AMPA (500 uM) before kainate (300 ,uM) cross-desensitized com- ± 2.6% of inhibition, n 6) (Fig. 5B). Because of the rapid pletely the steady response to kainate (arrow) but did not affect the desensitization of kainate-selective receptors, full dose- initial peak. inhibition curves were not attempted (i.e., IC50 values would Downloaded by guest on September 25, 2021 Neurobiology: Lerma et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11691 A A Kainate rl% ~ CNQX NS-102 1 Mp 3 sIM 1_q_JM kainate kainate kainate B._,...... --;b .-...... ffi *l 4

10 10 IpA pA B 0.5 s 400 ms 400 0 CA, B CNQX NS-102 300 C+ kainate CD 200 = ED Ce 0 100 3 CD

0 1 10 100 1000 Kainate WM) C 400 ms 10 pM 300pM CN(QX NS-1 02 jP~~~~~-- C kainate kainiatO,.,.rkainate 1 0 50 I5A t...... pA 0.5 S 250 ms 400 ms D FIG. 4. Dose-response relations for kainate in hippocampal kainate AMPA kainate AMPA neurons expressing kainate-selective or AMPA-type receptors. (A) ftr" Desensitizing currents activated by different concentrations of kain- ate in a cell with a pure population ofkainate-selective receptors. (B) Dose-response curves for kainate obtained from cells showing only transient responses (e) and from cells with nondesensitizing re- sponses to kainate (o). The amplitude values obtained in different cells were normalized (at 10 ,uM and 100 ,uM for peak amplitude and 50 steady responses, respectively) and pooled for computer fitting. pA Points are the mean ± SEM. The smooth curves are the least-square fits of I = Iml/[l + (EC5o/K)']. Ix, the maximum current 400 ms response, EC50, the agonist concentration at half-maximal current, and n, the Hill coefficient, were fit as free parameters. The estimated FIG. 5. Different pharmacological properties for kainate- EC50 values for kainate acting at each type ofreceptor are indicated. selective and AMPA receptors. (A-C) The effect of CNQX (10 PsM) (C) In cells expressing both kainate-selective and AMPA receptors, and NS-102 (3 pM) on the response to kainate (300 pM) in cells kainate activated the transient current exclusively at low concen- expressing only kainate-selective receptors (A), only AMPA recep- trations, whereas at higher concentrations both responses were tors (B), or both kainate-selective and AMPA receptors (C). (D) The apparent. effect of the drug aniracetam (5 mM) on kainate-selective and AMPA receptors. Aniracetam did not modify transient or steady be overestimated), but these results indicate different phar- currents induced by kainate, but it greatly potentiated responses to macological properties for kainate-selective and AMPA re- AMPA (200 uM). ceptors and provide an example for pharmacological sepa- does not activate these responses. In addition, the kainate- ration of kainate-induced currents at AMPA- and kainate- selective receptor has a much higher affinity for kainate than selective receptors. In addition, while AMPA-induced has the AMPA receptor. The fast desensitization of kainate- transient responses were enhanced by aniracetam (290 t 32% selective receptors may to overestimation of the value ofcontrol peak with 5 mM, n = 7), this nootropic drug did not for half-maximal activation in dose-response curves. How- affect peak amplitude or desensitization of transient re- ever, the EC5o value for kainate was 10-fold lower when sponses induced by kainate (Fig. SD), indicating that kainate- calculated from data obtained in cells with desensitizing selective receptors lack the allosteric site for aniracetam responses than when estimated from cells showing nonde- present in AMPA receptors (23). Aniracetam did not influ- sensitizing responses. Glutamate receptors with a similar ence steady responses induced by kainate at AMPA recep- affinity and with desensitizing properties when activated by tors, in agreement with previous reports (e.g., ref. 26). kainate have been observed in dorsal root ganglion cells in culture (17). However, unlike dorsal root ganglion cell re- DISCUSSION ceptors, the high-affinity kainate receptors we have found in hippocampal cells (i) are not activated by AMPA, (ii) are In this study we have demonstrated the existence in hippo- totally desensitized in the presence ofligand, (iii) exhibit fast campal neurons of glutamate receptors that are selectively and monoexponential desensitizing kinetics, (iv) are less activated by kainate. Kainate-evoked responses undergo sensitive to CNQX than AMPA receptors, and (v) may rapid and total desensitization, the recovery from which is coexist with AMPA receptors. Consequently, our results rather slow. Other glutamate receptor agonists such as glu- demonstrate the existence of functional receptors of the tamate, quisqualate, or domoate are also able to gate kainate- kainate type (i.e., selectively activated by kainate), not selective receptors. AMPA, even at high concentrations, previously known to be present in native membranes. Downloaded by guest on September 25, 2021 11692 Neurobiology: Lerma et al. Proc. Natl. Acad. Sci. USA 90 (1993) It is important to determine the molecular identity of Dr. A. Novelli for the generous gift of domoic acid, Dr. J. Drejer kainate-selective receptor channels in native membranes. (from Neurosearch) for a sample ofNS-102, and Dr. B. Sakmann for The pharmacological profile and the fast and the total desen- providing us with in-press material. Aniracetam was kindly provided by Productos Roche (Madrid). This work was supported in part by sitization of kainate-selective receptors are consistent with grants to J.L. from Direccion General de Investigaci6n Cientifica y properties observed in GluR-6 homomeric assemblies ex- Tecnica and Fondo de Investigaciones Sanitarias de la Seguridad pressed in mammalian cells (10, 27). GluR-6 subunits form Social, to J.R.N. from Comunidad Aut6noma de Madrid, and to homomeric channels which are the only receptor channels B.M. from Glaxo S.A. known to be gated by kainate but not by AMPA (10, 27-29), and, as in the present report, AMPA does not cross- 1. Mayer, M. L. & Westbrook, G. L. (1987) Prog. Neurobiol. desensitize homomeric GluR-6 receptors (28). Furthermore, (Oxford) 28, 197-276. recent data from the expression ofGluR-6 subunits show that 2. Collingridge, G. L. & Lester, R. A. (1989) Pharmacol. Rev. 40, editing of transmembrane region 1 may affect divalent 143-210. permeability (27). While the Q/R site in transmembrane 3. Watkins, J. C. & Evans, R. H. (1983) Annu. Rev. Pharmacol. Toxicol. 21, 165-204. region 2 determines the rectification properties (10, 20, 27), 4. Sommer, B. & Seeburg, P. H. (1992) Trends Pharmacol. Sci. in the same way as for the AMPA receptors, the Ca2+ 13, 291-2%. permeability ofhomomeric GluR-6 channels is changed in the 5. Boulter, J., Hoilmann, M., O'Shea-Greenfield, A., Hartley, opposite way. Expression of the GluR-6(Q) variant edited in M., Deneris, E., Maron, C. & Heinemann, S. (1990) Science transmembrane region 1 generates channels with very low 249, 1033-1037. Ca2+ permeability but with prominent inward rectification 6. Keinanen, K., Wisden, W., Sommer, B., Werner, P., Herb, A., (27). As illustrated in Fig. 2, we found cells expressing Verdoorn, T. A., Sakmann, B. & Seeburg, P. H. (1990) Science kainate-selective receptors with these same properties. Al- 249, 556-560. though we do not discard the existence of cells expressing 7. Nakanishi, N., Shneider, N. A. & Axel, R. (1990) Neuron 5, 569-581. Ca2+-permeable, kainate-selective receptors showing in- 8. Sakimura, K., Morita, T., Kushiya, E. & Mishina, M. (1992) wardly or outwardly rectifying current-voltage relationships, Neuron 8, 267-274. these results strongly suggest that the kainate-selective glu- 9. Bettler, B., Boulter, J., Hermans-Borgmeyer, I., O'Shea- tamate receptors described in this report correspond to Greenfield, A., Deneris, E. S., Moll, C., Borgmeyer, U., assemblies of GluR-6 subunits. In keeping with this sugges- Hoilmann, M. & Heinemann, S. (1990) Neuron 5, 583-595. tion, we have found that about 20%o ofthe cells in our cultures 10. Herb, A., Burnashev, N., Werner, P., Sakmann, B., Wisden, express the GluR-6 , as determined by in situ hybrid- W. & Seeburg, P. H. (1992) Neuron 8, 775-785. ization. In addition, reverse transcriptase-PCR analysis us- 11. Sommer, B., Burnashev, N., Verdoorn, T. O., Keinanen, K., ing RNA harvested from single cells (see ref. 30) exhibiting Sakmann, B. & Seeburg, P. H. (1992) EMBO J. 11, 1651-1656. 12. Lambolez, B., Curutchet, P., Stinnakre, J., Bregestovski, P., prominent transient responses to kainate resulted in an Rossier, J. & Prado de Carvalho, L. (1990) Nature (London) intense hybridization signal with a GluR-6 probe (unpub- 347, 26. lished work). 13. Young, A. B. & Fagg, G. H. (1990) Trends Pharmacol. Sci. 11, An important feature of kainate-selective receptors was 126-133. that they were found in young cultures. Hippocampal cells 14. Jonas, P. & Sakmann, B. (1992) J. Physiol. (London) 455, dissociated from E17, E18, E19, and E20 embryos and P0 and 143-171. P2 pups showed desensitizing kainate responses upon rapid 15. McBain, C. & Dingledine, R. (1993) J. Physiol. (London) 462, application of kainate when studied 4 hr after plating. These 373-392. responses were found in neurons to 3-4 16. Agrawal, S. G. & Evans, R. H. (1986) Br. J. Pharmacol. 87, consistently up days 345-355. in culture. Neurons kept for longer periods showed promi- 17. Huettner, J. E. (1990) Neuron 5, 255-266. nent transient responses to AMPA and large steady re- 18. Hamill, 0. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, sponses to kainate, which probably explained the failure to S. J. (1981) Pflugers Arch. 391, 85-100. detect smaller currents of kainate-selective receptors. Brief 19. Lerma, J. (1992) Neuron 8, 343-352. responses to kainate were observed also after 6-7 days in 20. Egebjerg, J. & Heinemann, S. F. (1993) Proc. Natl. Acad. Sci. culture, but exclusively after predesensitizing with a high USA 90, 755-759. AMPA concentration (e.g., Fig. 3A). Another possibility is 21. Verdoorn, T. A., Burnashev, N., Monyer, H., Seeburg, P. H. that these channels might be located in particular zones of & Sakmann, B. (1991) Science 252, 1715-1718. dendrites or nerve more difficult to 22. Burnashev, N., Monyer, H., Seeburg, P. H. & Sakmann, B. terminals, becoming (1992) Neuron 8, 189-198. detect by patch-clamp techniques as the processes elongate. 23. Hume, R. J., Dingledine, R. & Heinemann, S. (1991) Science In fact, the membrane capacitance of those cells where 253, 1028-1031. transient kainate response was the only response or it was 24. Honore, T., Davies, S. N., Drejer, J., Fletcher, E. J., Jacob- predominant was low (8.4 ± 0.5 pF, n = 132), confirming the sen, P., Lodge, D. & Nielsen, F. E. (1988) Science 241, small appearance (about 840 p,m2, if one assumes 1 puF/cm2 701-703. as specific membrane capacitance) of these cells. 25. Johansen, T. H., Drejer, J., Watjen, F. & Nielsen, E. 0. (1993) In summary, our results provide evidence for the existence Eur. J. Pharmacol. 246,195-204. in hippocampal neurons of a functional glutamate receptor 26. Ito, I., Tanabe, S., Kohda, A. & Sugiyama, H. (1990) J. that has high affinity for kainate and that is not sensitive to Physiol. (London) 424, 533-543. 27. Kholer, M., Burnashev, N., Sakmann, B. & Seeburg, T. H. AMPA. Our data strongly suggest that homomeric GluR-6(Q) (1993) Neuron 10, 491-500. assemblies form functional kainate-selective receptors in 28. Egebjerg, J., Bettler, B., Hermans-Borgmeyer, I. & Heine- neuronal membranes. The functional significance of this mann, S. (1991) Nature (London) 351, 745-748. glutamate receptor is not known. 29. Morita, T., Sakimura, K., Kushiya, E., Yamazaki, M., Me- guro, H., Araki, K., Abe, T., Mori, K. J. & Mishina, M. (1992) We thank Dr. M. V. L. Bennett for helpful discussions, Drs. W. Mol. Brain Res. 14, 143-146. Bufio, R. Martfn del Rio and M. Casado for critical reading of the 30. Lambolez, B., Audinat, E., Bochet, P., Crepel, F. & Rossier, manuscript, Drs. J. Chowen and C. Blakeley for English corrections, J. (1992) Neuron 9, 247-258. Downloaded by guest on September 25, 2021