The Journal of Neuroscience, August 1993, 13(E): 34983509 Hippocampal Neurons Exhibit Cyclothiazide-sensitive Rapidly Desensitizing Responses to Kainate Doris K. Patneau,’ Ladislav Vyklicky, Jr.,* and Mark L. Mayer’ ‘Laboratory of Cellular and Molecular Neurophysiology, NICHD, NIH, Bethesda, Maryland 20892 and *Institute of Physiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, The Czech Republic In whole-cell recordings from mammalian CNS neurons, Glutamate receptors in the rat CNS are known to be formed AMPA-preferring glutamate receptors exhibit strong desen- from several gene families (Sommer and Seeburg, 1992). Ex- sitization in response to AMPA, glutamate, and quisqualate, periments on receptors generatedusing in vitro expression sys- but not to kainate or domoate. Such desensitization is re- tems provide evidence for AMPA-preferring subtypes assem- duced by lectins, by the nootropic drug aniracetam, and by bled from combinations of glutamate receptor subunits GluRl- diazoxide. None of these compounds strongly modulate re- GluR4 (Boulter et al., 1990) also referred to asGluR-A through sponses to kainate and domoate, consistent with the ap- GluR-D (Keinanen et al., 1990) and kainate-preferring subtypes parent lack of desensitization to these agonists. We now assembledfrom GluRS-GluR7 (Bettler et al., 1990, 1992; Ege- report experiments on hippocampal neurons in which re- bjerg et al., 1991) most likely in combination with IL41 and sponses to kainate were strongly potentiated by cyclothia- KA2 (Werner et al., 1991; Herb et al., 1992). At AMPA-pre- zide, a benzothiadiazine diuretic and antihypertensive drug ferring glutamate receptors, kainate acts with low potency and structurally related to diazoxide. Cyclothiazide increased the produces nondesensitizing responseswhile AMPA and gluta- maximum response to a saturating concentration of kainate mate produce rapidly and strongly desensitizing responses by approximately 300% and produced a shift to the left in (Sommer et al., 1990). In contrast, kainate is a potent agonist the kainate dose-response curve. Because cyclothiazide at kainate-preferring glutamate receptors and producesstrongly was considerably more effective than aniracetam in reducing desensitizing responseswhile AMPA is inactive or of very low desensitization evoked by glutamate, we tested the possi- potency (Egebjerg et al., 1991; Herb et al., 1992). Numerous bility that potentiation of responses to kainate was due to studieshave reported nondesensitizingresponses to kainate and block of a previously undetected component of desensiti- domoate and rapidly desensitizing responsesto AMPA, gluta- zation in the response to kainate itself. In outside-out patch- mate, and quisqualatein hippocampal neurons, consistent with es responses to rapid perfusion of 3 mu kainate showed the expressionof AMPA-preferring glutamate receptors (e.g., 34% desensitization, the onset of which developed with a Kiskin et al., 1986; Trussell et al., 1988; Mayer and Vyklicky, time constant of 2.2 msec. Desensitization of responses to 1989; Patneau and Mayer, 1990, 1991; Jonas and Sakmann, kainate was abolished by 100 I.IM cyclothiazide, as was the 1992). Compared to their action on hippocampal neurons, kai- much stronger desensitization evoked by glutamate and nate and domoate are more potent agonistsat dorsal root gan- AMPA. Cyclothiazide also slowed the rate of deactivation of glion (DRG) neurons and produce strongly desensitizing re- responses to kainate recorded after return to agonist-free sponses,consistent with the expression of kainate-preferring solution. Current-voltage plots for control responses to kai- receptorsin sensoryneurons (Huettner, 1990; Wong et al., 1992). nate exhibited outward rectification that was associated with The different functional responseof hippocampal neurons to a reduction in the amount of desensitization on depolariza- AMPA and kainate raisesthe question as to why someagonists tion. Both effects were absent in the presence of cyclothia- produce desensitization at AMPA-preferring receptors while zide, suggesting that rectification of responses to kainate others apparently do not. Previously we characterized the ac- was due to the voltage dependence of desensitization. The tions of a series of willardiines on hippocampal neurons and complete block of desensitization produced by cyclothia- proposed that agonist-dependentdifferences in relative affinity zide provides a powerful new tool for analysis of allosteric for the active and desensitizedstates of AMPA-preferring gluta- regulatory mechanisms at AMPA-preferring glutamate re- mate receptors could account for variation in the degree of ceptors. desensitization evoked by different agonists (Patneau et al., [Keywords: kainate, AMPA, desensitization, hippocam- 1992a). According to this hypothesis, kainate binds to the de- pus, excitatory amino acids, cyclothiazide, glutamate re- sensitizedstate(s) of AMPA-preferring glutamate receptorswith ceptor, allosteric regulation, concentration jumpj much lower affinity than doesAMPA, glutamate, or quisqualate (Patneau and Mayer, 1991; Mayer et al., 1992). Our hypothesis raised two issues.First, kainate might indeed produce desen- Received Nov. 25, 1992; revised Feb. 3, 1993; accepted Feb. 24, 1993. sitization at AMPA-preferring glutamate receptors,but lesspro- We thank Christine Winters for preparation of cell cultures, Drs. C. E. Jahr and G. L. Westbrook for advice on constructing the piezoelectric element perfusion found than occurs with AMPA or glutamate, and too rapid to system, Dr. M. J. Benveniste for reading the manuscript, and Drs. K. Yamada measurewith whole-cell recording. Second, substancesthat re- and S. Rothman for sharing results prior to publication. duce desensitization at AMPA receptors should potentiate re- Correspondence should be addressed to Dr. M. L. Mayer, Building 49, Room 5A78, National Institutes of Health, Bethesda, MD 20892. sponsesto kainate. The lectins and drugs that have been tested Copyright 0 1993 Society for Neuroscience 0270-6474/93/133496-14$05.00/O previously for activity at AMPA-preferring glutamate receptors The Journal of Neuroscience, August 1993, f3(8) 3497 have relatively weak effects on desensitization. Although ani- aniracetam, from Hoffman-La Roche (Nutley, NJ). Cyclothiazide and racetam (5 mM) produces nearly lo-fold potentiation of equi- aniracetam were dissolved in dimethyl sulfoxide (DMSO) at 20 and 500 mM, respectively, before dilution with extracellular solution, In several librium responses to glutamate, substantial (84%) desensitiza- experiments the control solution also contained 0.5% DMSO (the con- tion still occurs in the presence of 5 mM aniracetam (Vyklicky centration present in extracellular recording solution containing 100 PM et al., 199 1). The relief of desensitization at AMPA-preferring cyclothiazide). Salts, biochemicals, and excitatory amino acids were glutamate receptors by the lectins concanavalin A and WGA is purchased from Aldrich, Diagnostic Chemicals, Molecular Probes, Sig- even less than produced by aniracetam (Mayer and Vyklicky, ma, and Tocris Neuramin. Analysis. In many cases agonist-evoked currents exhibited significant 1989; Vyklicky et al., 199 l), and suggests that these substances rundown, particularly in outside-out patches; this was time dependent fail to affect responses to kainate because their effect on desen- rather than activity dependent, and apparently unaffected by cyclothia- sitization is weak. zide, suggesting that rundown is not due to a cyclothiazide-sensitive, The recent discovery that diazoxide and some structurally absorbing desensitized state. The rundown was much slower in whole- cell recording, in nucleated macropatches, and in nystatin vesicles. related derivatives are considerably more efficacious than ani- Whenever possible control responses recorded before and after an ex- racetam in reducing desensitization at AMPA-preferring gluta- perimental manipulation were used to correct for rundown. In com- mate receptors (Yamada and Rothman, 199 1, 1992; Yamada, parisons between agonists (e.g., kainate and glutamate) applications were 1992) offered the possibility to reexamine whether desensiti- alternated and several sequential responses averaged. Because the wash- zation is evoked by kainate. We report here experiments that out of cyclothiazide was so slow, analysis of the degree of potentiation by cyclothiazide was performed by comparing control responses im- show that kainate produces substantial, but very rapid, desen- mediately preceding the application of cyclothiazide with the first few sitization at AMPA-preferring glutamate receptors in hippo- potentiated responses. Decay time constants and rates of onset of de- campal neurons, and that such desensitization is completely sensitization were fit with one or the sum of two exponentials using blocked by cyclothiazide, a benzothiadiazine related in structure PCLAMP (Axon Instruments). Dose+response curves were fit to the equa- to diazoxide (Yamada, 1992). tion I = I,,, x (l/(1 + (EC,,/[ligand])‘)), (1) Materials and Methods where I-, is the response at a saturating concentration of ligand; EC,,, Cell culture and recording techniques. Newborn SpragucDawley rats the concentration of ligand producing a half-maximal response; [ligand], were killed by decapitation, and the hippocampi dissected. Neurons the concentration of ligand, and n, the Hill coefficient. Data are given were plated on a confluent glial cell feeder layer as previously described