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Kainate receptors containing GluR5 subunits contribute to synaptic transmission and long-term potentiation.. Tse, Maric Tsz Lung. 2004

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Running head: K A I N A T E RECEPTORS IN T H E D E N T A T E G Y R U S  Kainate receptors containing GluR5 subunits contribute to synaptic transmission and long-term potentiation in the hippocampal dentate gyrus by Marie Tsz Lung Tse B.A., Concordia University, 2002  A THESIS S U B M I T T E D IN PARTIAL F U L F I L L M E N T OF T H E  REQUIREMENTS  FOR T H E D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Graduate Program in Neuroscience) We accept this thesis as conforming to the required standard:  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A August 2004 © Marie Tsz Lung Tse, 2004  JtJBCl THE UNIVERSITY OF BRITISH COLUMBIA  FACULTY OF GRADUATE STUDIES  Library Authorization  In presenting this thesis in partial fulfillment of the requirements for an a d v a n c e d d e g r e e at t h e University of British C o l u m b i a , I agree that the Library shall m a k e it freely available for reference a n d study. I further a g r e e that p e r m i s s i o n for e x t e n s i v e c o p y i n g of this thesis for scholarly p u r p o s e s m a y be g r a n t e d by t h e h e a d of m y d e p a r t m e n t or by his or her r e p r e s e n t a t i v e s . It is u n d e r s t o o d that copying or publication of this thesis for financial g a i n shall not be allowed without m y written p e r m i s s i o n .  0^1 N a m e of A u t h o r (please  print)  ID I  J^ociL  Date (dd/mm/yyyy)  Title of Thesis:  Degree:  Year:  Department of The University of British C o l u m b i a Vancouver, BC  Canada  p a g e 1 of 1  last updated:  20-M-04  Abstract  It has recently been shown that presynaptic kainate receptors can play a role in synaptic plasticity in the C A 3 region of the hippocampus (Bortolotto et al., 1999); however, a role for these receptors in synaptic transmission and synaptic plasticity in other regions of the brain has been difficult to establish. In the present experiments we show that kainate receptors can contribute significantly to synaptic transmission in the dentate gyrus (DG) of the hippocampus, and that furthermore, they appear to play a role in synaptic plasticity in this region. Kainate receptors in the D G appear to be located post-synaptically at excitatory synapses where they can influence synaptic plasticity by alleviating the M g  2 +  blockade of N M D A receptors. These results  demonstrate that kainate receptors in the D G play a vital post-synaptic role at excitatory synapses, and may contribute to learning and memory processes in this region.  ii  Contents Abstract  ii  Contents  iii  List of Tables  ...iv  List of Figures  v  Abbreviations  vi  Acknowledgements  vii  1. Introduction  1  1.1 Glutamate receptors as the basis of excitatory synaptic transmission  1  1.2 Subunit properties of a kainate receptor  2  1.3 Kainate receptors play a role in synaptic transmission in the hippocampus  4  2. Methods  6  3. Results  8  4. Discussion  15  5. Bibliography  19  iii  List of Tables Table 1. Selective pharmacological agents for kainate receptors.  iv  List of Figures Figure 1. Membrane topology of a kainate receptor subunit. Figure 2. Subunit composition of a kainate receptor. Figure 3. A schematic illustration of the medial and lateral perforant path projections. Figure 4. The GluR5 antagonist LY382884 attenuates synaptically evoked responses in the DG. Figure 5. N M D A and A M P A receptor antagonists failed to completely block evoked responses in the DG. Figure 6. Kainate receptor activation is required for the induction of LTP in the DG. Figure 7. LY382884 failed to block LTP induction in Mg -free A C S F . 2+  v  Abbreviations A C S F : Artificial cerebral spinal fluid A M P A : Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid C A 1 : Cornu ammonis 1 C A 3 : Cornu ammonis 3 DG: Dentate gyrus EC: Entorhinal cortex EPSC: Excitatory post-synaptic current EPSP: Excitatory post-synaptic potential GluR5: Glutamate receptor subunit 5 K A : Kainate LPP: Lateral perforant path mA: Milliamp ml: Milliliter MPP: Medial perforant path mV: Millivolt N M D A : N-methyl-D-aspartate u.M: Micromolar  vi  Acknowledgements  First and foremost, heartfelt appreciation goes to my supervisor Dr. Brian Christie, thank you for all the patient guidance and expert supervision throughout the two years, thank you for giving me opportunity to my wonderful experience in graduate school, as well as expanding my knowledge of neuroscience and introduced me the tool of electrophysiology. Next, I would like to thank my family, especially my parents, Sylvia Lo and Man-Ho Tse, thank you for all your emotional support, always giving me encouragement when I needed, I would not have accomplished my study without your support. Thank you to my three committee members, Dr. Tim Murphy, Dr. Yu Tian Wang and Dr. Tony Philips, thank you for all your input on my experiment, and have inspired me to look at my project in new directions. Thank you to all the lab members and good friends in the department, always lending me an ear and toughing it out with me throughout these two years. A special thank you to Stephanie Lieblich, thank you for always keeping the lab neat and tidy, your dedication have greatly assisted my work at the lab; thank you to Heather Booth, all your assistant on my project, as well as the wonderful snowboarding days in the winter; thank you to Matt Hill and Orsha Magyar, thank you for the inspirational research conversations, as well as all the moral support outside of my study; thank you to Karl Wodtke's insightful suggestions to my experiments, and thank you for teaching me how to use power point. The present experiment was made possible by a grant from the Canadian Institute of Health Research, awarded to Dr. Brian Christie.  vii  1  1. INTRODUCTION 1.1 Glutamate receptor as the basis of excitatory synaptic transmission In the central nervous system, the fast excitatory effects of glutamate are mediated primarily by the N M D A (N-methyl-D-aspartate), A M P A (a-amino-3-5-methyl-4-isoxazole propionic acid), and K A (kainate) subtypes of ionotropic receptors (Dingledine et al., 1999). While the physiological roles of N M D A and A M P A receptors have been studied extensively, especially in the hippocampal formation, a lack of specific pharmacological agonists and antagonists for the K A receptor have hindered our understanding of how K A receptors contribute to neuronal signaling (Lerma, 1997; Wilding & Huettner, 1997). Recently, the development of specific receptor blockers for A M P A receptors and certain specific blockers for K A receptor subunits has provided progress to the understanding of the physiological contribution of K A receptors to excitatory synaptic transmission (for summary of glutamate receptor pharmacology, see Table 1).  Table 1. Selective pharmacological agents for glutamate receptors Drug  Functions  APV  Competitive antagonist of N M D A receptors  ATPA  Selective GluR5 agonist  CNQX  Competitive A M P A / K A receptor antagonist  Domoate  Kainate receptor agonist  Glutamate  Endogenous glutamate receptor ligand; agonist for N M D A , A M P A , kainate, and metabotropic glutamate receptor agonist  GYKI52466  Non-competitive A M P A receptor antagonist; less potent than GYKI53655  GYKI53655  Non-competitive A M P A receptor antagonist  Kainate  Kainate receptor agonist; also A M P A receptor agonist at high concentration  LY382884  Competitive GluR5 subunit antagonist  2  1.2 Subunit properties of kainate receptors Structurally, K A receptors are tetrameric protein complexes whose composition includes at least one of the five different subunits that are unique to K A receptors: GluR5, GluR6, GluR7, K A 1 , and K A 2 (also known as G l u , Glu 6, G l u , G l u i , and K5  K  K7  K  Glu 2 repectively; Hollmann and Heinemann, 1994). These subunits are not equal K  however, and only the GluR5-7 subunits have been shown to form functional homomeric channels by themselves; these subunits also show a high affinity of binding to kainate and glutamate and, receptors with GluR5-7 show low conductance to N a  +  (Sommer et al., 1992; Egebjerg and Heinemann, 1993; Schiffer et al., 1997). These subunits can also form distinct heteromeric receptors when they coassemble either with K A 1 or K A 2 subunits, or each other (Werner et al., 1991; Herb et al., 1992; Schiffer et al., 1997; Paternain et al., 2000). Unlike GluR5-7, K A 1 and K A 2 have a low binding affinity to kainate and glutamate, but exhibit high channel conductance to N a . A subunit editing site on GluR5 and GluR6, located at the channel pore of the +  receptor (Q621R) also determines the channel conductance of the receptor to C a . A 2+  schematic illustration of the membrane topology and subunit composition of a kainate receptor is shown on Figure 1 and 2 respectively.  3  Figure 1. Membrane topology of a kainate receptor subunit. Each kainate receptor subunit contains three membrane spanning domains ( M l , M 3 , and M4), and a p-loop (M2), which constitutes the pore of the channel. Ligand binding sites are at the extracellular space next to the M l and M3 domains (adapted from Dingledine et al., 1999).  Ligand binding domain  Figure 2. Subunit composition of a kainate receptor. Each kainate receptor is composed of four subunits. The diagram illustrates the site of the p-loop  forming the  channel pore, and the ligand binding sites at the extracellular space. A Q/R editing site is present close to the channel pore (Q/R621, represented by white circle); unedited receptors contain a high channel conductance to C a , and display a linear gating 2+  property to monovalent cations (adopted from Dingledine et al., 1999).  4 1.3. Kainate receptors play a role in synaptic transmission in the hippocampus Radioactive in situ hybridization has shown that both hippocampal pyramidal neurons and granule cells express the GluR5-7 subunits, with the GluR6 subunit being most prevalent (Wisden and Seeburg, 1993; Bahn et al., 1994; Bureau et al., 1999; Paternain et al., 2000; Kohama and Urbanski, 1997), when compared to the GIuR5 and GluR7 subunits (Petralia et al., 1994; Porter et al., 1997; Bureau et al., 1999; Bailey et al., 2001). In the hippocampal C A 3 subfield, it has become apparent that kainate receptors can play an important neurophysiological role at the mossy fiber synapse. Presynaptic kainate receptors appear to act to depress both mossy fiber and associational-commissural inputs (Vignes et al., 1998; Contractor et al., 2000; Karniya and Ozawa, 2000; Frerking et al., 2001; Ji and Staubli, 2002), while post-synaptic K A receptors appear to contribute to mossy fiber evoked EPSCs (Castillo et al., 1997; Vignes and Collingridge, 1997; Mulle et al., 1998), and frequency-dependent facilitation (Schmitz et al., 2001; Contractor et al., 2001; Lauri et al., 2001) and act also at excitatory synapses onto interneurons (Cossart et al., 1998; Frerking et al., 1998). It has been reported that K A receptors in the C A 3 can modulate the release of transmitter in the mossy-fiber-CA3 synapse. Schmitz et al. (2000) reported that incubation of low dose of kainate (200nM) enhanced presynaptic fiber volley, as well as increased cell excitability. When kainate is added to the synapse at high dose (10u.M), on the other hand, presynaptic fiber volley amplitude became suppressed. Post-synaptic K A receptors in the C A 3 synapse can be activated in a frequency-dependent manner. Post-synaptic EPSC was seen to be enhanced after repetitive stimulation of the afferent fiber (at 25 or 100Hz). The frequency-dependent nature of the K A receptors can also be seen in kainate-dependent LTP at the mossy fiber synapse, which was also subjective to block by the selective GluR5 antagonist,  5 LY382884 (Bortolotto et al., 1999). In the CA1 region, K A receptors also modulate both excitatory and inhibitory synaptic transmission (Chittajallu et al., 1996; Clark et al., 1997; Rodriguez-Moreno et al. 1997; Frerking et al., 1998 ). Chittajallu and colleagues (1996) provided first evidence that K A receptors play a role on transmitter release in the CA1 region of the hippocampus. It has been observed that bath perfusion of kainic acid produced a dose-dependent reduction of glutamate current in the CA1 pyramidal neurons. This effect was inhibited when slices were added with an A M P A / K A receptor blocker, C N Q X . Fluorescent imaging also revealed that bath application of kainic acid induces presynaptic C a  2 +  signals (Kamiya & Ozawa, 1998), and paired-pulse facilitation was  also enhanced when slices were exposed to kainate, further suggesting the existence of presynaptic K A receptors. Although it is evident that K A receptors function in an ionotropic manner, pharmacological studies also reported that K A receptors can exhibit metabotropic actions. The application of metabotropic enzyme inhibitors for protein kinase C and phospholipase C can suppress the release of G A B A from the CA1 interneurons (Rodriguez-Moreno et al., 1997), indicating that K A receptors are capable of initiating G-protein coupled cascades for cell signaling. Although the expression of K A receptors in the dentate gyrus was reported previously, their physiological roles in the dentate gyrus have not yet been established. Despite the intense concentration of K A receptors in this region of the hippocampus, Lerma et al. (1997) reported that all responses were blocked by the selective A M P A antagonist GYKI53655 (150 u M , administered in the presence of N M D A and G A B A antagonists). Later, Behr and colleagues (2002), showed that the activation of K A receptors at presynaptic terminals in the D G can reduce G A B A release in slices obtained from kindled animals, and to a lesser extent in tissue obtained from control animals. This may indicate that the role of kainate receptors in this region is dependent  6 upon their activation by high frequency activity like that exhibited during kindling, and that their role in the D G may differ from the role they play in other hippocampal subregions. To test this hypothesis, we used the selective GluR5 antagonist, LY382884, to examine the role of K A receptors in synaptic transmission and plasticity in the D G LY382884 has been used by previous experiments, and have shown to be selective on K A receptor channels containing a GluR5 subunit (Bortolotto et al., 1999).  2. M E T H O D S Slice preparation. Male Spraque-Dawley rats (2 to 4 weeks old), obtained from the U B C animal colony, were used in the present experiment (UBC Animal Care Protocol AO 1-0088). A l l surgical and handling procedures were in accordance with the guidelines of the Canadian Council of Animal Care and the Animal Care Committee of the University of British Columbia. Hippocampal slices were cut as described previously (Christie et al., 2000; Froc et al., 2003) and individual slices were transferred to the recording chamber as needed. Experiments were performed at room temperature in A C S F composed of (in mM): 125 N a C l ; 2.5 K C I ; 1.25 N a H P 0 ; 28.0 2  4  N a H C 0 ; 2.0 C a C l ; 1.0 M g C l ; 25.0 Dextrose. Bicuculline ( l u M ) was routinely 3  2  2  added to the bathing medium during recordings to block GABA (y-amino-butyric acid) A  inhibitory synaptic activity. To isolate or block specific receptor subtypes, the following compounds were used: LY382884 (GluR5 antagonist, 10 u M , Lilly, IA); GYKI53655 ( A M P A antagonist, 5 - 5 0 uM, Sigma); D - A P V ( N M D A antagonist, 200 u M , Sigma) and C N Q X ( A M P A / K A antagonist, 20 uM). Electrophysiological responses were obtained using 1-3 MQ. recording electrodes filled with A C S F and a Dagan BVC-700 amplifier. Responses from the dentate gyrus were elicited with a sharpened tungsten electrode using biphasic current pulses (120 us, 10-400 uA) and a digital stimulus isolation unit (Getting Instruments, C A ) as described  7 previously. A n Olympus BX50wi microscope (lOx objective) was used to visually position both the recording and stimulating electrodes for each experiment. Electrodes were positioned in the middle third of the molecular layer for M P P (Medial Perforant Path) recordings, and in the outer third (adjacent to the fissure, distal from the granule cell layer) for L P P (Lateral Perforant Path) recordings (Figure 3). A l l evoked responses were continuously elicited at 15s intervals, except during the application of the conditioning stimulation. A stable 15 min baseline period was required before any of the receptor antagonists were utilized in an experiment. For LTP experiments, four bursts of 50 pulses at 100Hz (30s between bursts) were used as the conditioning stimuli. Following any manipulation, single pulse stimulation was resumed at 15s intervals for a minimum of 30 min. A l l data were acquired at 5-10 kHz, and the initial slope of the negative going waveform was used to assess changes in synaptic efficacy (Christie et al., 2000; Froc et al., 2003). Paired-pulse stimuli (50ms interpulse interval; 6-8 stimulations) were applied in all experiments. The normalized difference between the slopes of the two responses was calculated and is presented as a percentage change. In all figures, each data point represents the mean ± S E M of one minute of data (i.e., average of 4 responses).  8  MPP  |_pp  Figure 3. A schematic illustration of the medial and lateral perforant path projections. The medial perforant path (MPP) is positioned at the middle third of the dentate molecular layer, whereas the lateral perforant path (LPP) is located at the outer third of the molecular layer. Both of the perforant paths originate from the pyramidal cells from the entorhinal cortex (EC).  3. R E S U L T S  In our initial experiments, we placed our electrodes to selectively stimulate and record from the medial aspects of the D G as shown in Figure 4A. These electrode placements routinely resulted in paired-pulse depression when two stimuli were administered at a short interpulse interval (50 ms). To determine whether the selective GluR5 antagonist itself had any effects on synaptic transmission, A C S F containing LY382884 (10 u.M) was used following the establishment of stable baseline responses in normal ACSF. To our surprise, A C S F containing the selective GluR5 antagonist reduced the field EPSP by M P P stimulation by 38.1 ± 1.8% (n=5; Figure 4). Paired-pulse depression in these slices remained unchanged (Baseline: 19.97 ± 4.10%, LY382884: 16.44 ± 8.34% of paired-pulse depression, p>.05), even after the response had stabilized with LY382884 present in the bath, indicating that LY382884 was likely acting at post-synaptic receptors. The more lateral aspects of the perforant path in the D G represent a physically and pharmacologically separate input independent of the M P P (Hjorth-Simonsen,  9  A  -15  -10  -5  B Recording  Stimulation  Recording  0  5  10  15  20  25  Stimulation  30  Time (minutes)  Di  Dii  -15  -10  -5  0  5  10  15  20  25  30  Time (minutes)  Figure 4. The G l u R 5 antagonist LY382884 attenuates synaptically evoked responses in the D G . Schematic diagram showing the position of electrodes used when stimulating and recording from the medial aspects of the D G (A) versus those used when recording from the terminal regions of the granule cell dendrites for lateral perforant path synapses (B). Graph of medial (C) and lateral (D) perforant path evoked responses in the dentate gyrus prior to and following inclusion of the competitive GluR5 antagonist LY382884 (lOuM) in the ACSF. (Cii, Dii) Representative traces taken just prior to (1), and 25 minutes following (2), the application of the drug. Scale bars: 0.25mv; 50 ms.  10 1972). With electrodes positioned as shown in Figure 4B, we reliably obtained paired-pulse facilitation with stimuli administered at 50 ms interpulse intervals, indicating that we were activating LPP fibers (McNaughton and Barnes, 1978; Christie and Abraham, 1992; Froc et al., 2003). In these experiments, the addition of LY382884 produced a 29 ± 6.2% reduction in the LPP evoked EPSP (Figure ID). The difference between the M P P and LPP in the degree of response suppression by LY382884 was not significantly different (p>0.05). Paired-pulse facilitation in the LPP was not affected by LY382884, again indicating a post-synaptic locus for any GluR5 mediated contribution to synaptic transmission in this pathway (Baseline: 15.67 ± 8.31%, LY382884: 22.58 ± 6.35% of paired-pulse facilitation, p>.05). In a second series of experiments we attempted to reveal any putative kainate mediated component of synaptic transmission in the M P P and L P P by blocking A M P A receptors with G Y K I 53655 (5 or 50 uM) and N M D A receptors with D - A P V (200 uM). As in the previous experiments, inhibition was blocked in these experiments. When a concentration of 5 u.M was used, G Y K I 53655 reduced M P P elicited EPSPs by 62.8 + 1.6%) (n=6), and LPP elicited responses by 86.1 + 3.7% (n=6). Attempts to wash-out the block were generally ineffective, with responses only recovering approximately 50%o of the amount they were blocked over a 45 minute period (data not shown). Despite the fact that this would indicate that this concentration of G Y K I 53655 provides an effective block of all A M P A receptors in a slow perfusion system like that used here, we also performed these experiments using a higher concentration of GYKI53655 (50 u.M). At this concentration, M P P responses were reduced 90.1% ± 3.1% (n = 7), while LPP responses were reduced 83.3 ± 6.0% (n = 6; Figure 5). Thus, in neither case were evoked responses in the D G completely blocked, despite being exposed to 50 L I M GYKI53655 for prolonged periods (up to 45 minutes). The residual responses could be antagonized with the non-specific K A / A M P A antagonist C N Q X ,  11 indicating that these were indeed glutamate-mediated responses (Figure 6). Together, the preceding experiments indicate that GluR5 subunits play a functional role at both M P P and L P P synapses in an area known to be involved in learning and memory processes. This led us to speculate that kainate receptors might play an active role in LTP in this region. To control for the fact that LY382884 reduces the size of EPSPs in the DG, we used EPSPs of a similar magnitude in control slices when performing these experiments (LY382884 = 0.09 + 0.03 mV; Con. = 0.13 ± 0.08 mV; t )=0.719; p=0.5). Under the LY382884 conditions, evoked responses in the M P P (5  exhibited a significant degree of STP (7.01 ± 1.17%, p < 0.05), although this was significantly smaller than that normally observed in control slices (p<0.05). Moreover, LTP was completely abolished in the M P P in the presence of LY382884 (-5.84 ± 2.49%, p > 0.05%), as measured 25-30 minutes post-conditioning (Figure 7). This is in sharp contrast to the robust STP (51.10 ± 8.80%, t  (4)  = 7.42, p < 0.05) and LTP (39.27 ±  5.58%, t( ) = 10.72, p < 0.05) we obtained in control slices. Similarly, when 4  conditioning stimuli were administered to equivalent sized EPSPs in the LPP, we only induced LTP in normal A C S F (44.16 ± 11.72%) and not in the presence of LY382884 (6.69 ± 7.68%, t ) = 1.43, p > 0.05). Interestingly, STP (50.72 ± 8.34%, t (4  (4)  = 5.25, p <  0.05) was not affected by the addition of LY382884 in this path and was similar to that obtained in control slices (61.41 ± 11.87%; Figure 7). These experiments indicate that kainate receptors play a role in LTP induction in both the medial and lateral perforant path inputs to the dentate gyrus; however, the nature of their role remained unclear.  12  Ai  [1] MPP (Baseline)  [2] M P P (5uM G Y K I )  [3] MPP (Baseline)  [4] M P P (50uM G Y K I )  [5] LPP (Baseline)  [6] LPP ( 5 M M G Y K I )  25  g  APV+GYKI53655  Ml [3]  U  *S  -25  a  f  -50  Ui Q_  ..75  » A P V + G Y K I 5 3 6 5 5 (5uM)  [4]  A P V + G Y K I 5 3 6 5 5 (50uM)  CL UJ  -100 -20  -10  0  10  20  30  40  Time (minutes)  Bi  Bii  25  1/ APV + GYKI5365  | 5 ]  [7i  £ ~ o 55 QUi CL UJ  •  %  2 5  [7] LPP (Baseline)  [8] L P P ( 5 0 u M G Y K I )  "5° -75  • APV+GYKI53655 (5uM)  -100 -20  A P V + G Y K I 5 3 6 5 5 (50uM) -10  0  10  20  30  40  Time (minutes)  Figure 5. NMDA and  AMPA receptor antagonists failed to completely block  evoked responses in the DG. The time course for the blockade of both medial (Ai) and lateral (Bi) evoked responses is shown both prior to and following the wash-in of A C S F containing A P V (200 uM) and GYKI53655 (5 or 50uM). Representative traces for the medial (Aii) and the lateral (Bii) perforant path evoked responses are illustrated on the right. Note the residual response left in all instances.  13-  A P V + GYKI53655  -15  5  15  25  35  45  35  45  Time (minutes) B  APV + GYKI53655  -15  5  15  25  Time (minutes) Figure 6. Evoked responses in the medial (A) and lateral (B) perforant paths were challenged b y an AMPA/ kainate receptor antagonist (CNQX, 20uM). C N Q X abolished all evoked response followed by the N M D A and A M P A receptor antagonists, suggesting residual responses obtained in the presence of A P V and GYKI53655 is kainate receptor-mediated.  14  Medial Perforant Path  A 75  w  1  50  a 25  a,  en  -25 -15  o  15  30  T i m e (minutes)  B  Lateral Perforant Path  lOO T  15  O  30  T i m e (minutes)  Figure 7. Kainate receptor activation is required for the induction of LTP in the DG. Evoked responses in the medial (A) and lateral (B.) perforant path fail to exhibit LTP when the GluR5 antagonist, LY382884, is present during the application of the conditioning stimulation (open circles). LTP is readily induced with these same conditioning stimuli in both pathways in normal  A C S F  (filled circles).  15 One role kainate receptors may play in the D G is to provide either a source of depolarization or a conformational change that results in the alleviation of the N M D A receptor blockade by M g . To determine whether this is in fact the case, we attempted 2 +  to induce LTP in the presence of LY382884 in a Mg -free solution. As is shown in 2+  Figure 8, application of conditioning stimuli in this situation induced a significant degree of LTP in the medial path (33.2 + 2.4%; t )=2.77, p<0.05). Thus, removal of the (4  Mg  2 +  blockade of the N M D A receptor circumvented the requirement for kainate  receptor activation that we observed in normal ACSF. Similar results were also obtained for recordings where LTP was induced in recordings using lateral perforant path stimulation sites (32.1 + 5.3; t =6.02, p<0.05). (5)  4. DISCUSSION Our finding that LY382884 resulted in a substantial decrease in the size of evoked EPSPs in both the medial and lateral perforant path inputs to the dentate gyrus molecular layer comes as quite a surprise given that most immunohistochemical work does not indicate that a high concentration of GluR5 receptors are localized in this region (Paternain et al., 2000). Never-the-less, the receptors that are located in this region appear to play a role in synaptic transmission in the DG. Furthermore, the paired-pulse data would indicate that these receptors are located at post-synaptic sites on dentate granule cell dendrites, rather than at presynaptic locations like those seen in the CA1 and C A 3 regions (for review, see Huettner, 2003). In contrast to GluR5, GIuR6 subunits have been shown to be evident in the D G in high concentrations (Petralia et al., 1994). In addition, there is also some evidence for mixed GluR5/GluR6 subunit receptors (Paternain et al., 2000) and it may be that LY382884  16  A  Medial Perforant Path  -25 ^ -15  1  1  1  o  15  30  15  30  Time (minutes)  B  Lateral Perforant Path 150 T  -15  o Time (minutes)  Figure 8. LY382884 failed to block L T P induction in Mg -free A C S F . The 2+  administration of LTP-inducing conditioning stimuli to the medial (A) and lateral (B) perforant paths in the presence of LY382884 normally does not result in LTP (open diamonds). These same stimuli, when administered in Mg -free A C S F produce robust 2+  LTP in both paths in the presence of the Glur5 receptor antagonist LY382884 (filled diamonds).  17 is acting at these receptors as well. It may also be that receptors with both GluR5 and GluR6 subunits usually immunolabel as only being a GluR6 receptor, and not as GluR5 receptors, or that GluR5 receptors occupy key positions in the dendritic cytoarchitechture. We must also consider the possibility that LY382884 is acting at some site besides the GluR5 subunit, and that the response we are monitoring is in fact not kainate mediated. This seems unlikely given previous work indicating the high degree of specificity LY3 82884 has for receptors containing the GluR5 subunits (O'Neill et al., 1998). At present, the most parsimonious conclusion we can reach is that GluR5 subunits, located at postsynaptic sites in the DG, play an active role in synaptic transmission in this region. The other main finding of this study was that LY382884 was effective in blocking LTP at both LPP and M P P synapses. LTP in this region is normally thought of as being dependent upon the activation of NMDA-receptors, and indeed we have previously reported that this is the case in numerous experimental preparations (Christie and Abraham, 1992a,b; Christie et al., 1995; van Praag et al., 1999; Froc et al., 2003; Farmer et al., 2004). Although we have presented evidence for a GluR6 mediated form of NMDA-independent LTP in transgenic mice (Vissel et al., 2001), it should be stressed that this finding reflects a difference found in transgenic animals, rather than a phenomenon present in the normal population. These experiments directly indicate that GluR5 subunits can contribute to the induction of LTP in the DG. In addition, the experiments performed here indicate that one role K A receptors may play in the D G is to act as a source of post-synaptic depolarization, allowing for the subsequent activation of NMDA-receptors by alleviating the M g  2 +  blockade of this channel.  The role of K A receptors in the D G appears to differ from that they play in the cornu ammonis. It has been shown that LTP induction in GluR6 knockout mice is impaired in the CA1 and C A 3 subregions, while being relatively unaffected in the DG.  18 In contrast, while we have shown an involvement for GluR5 receptors in LTP in the DG, other researchers have failed to find a reduction in LTP in the CA1 and C A 3 regions of GluR5 knockout animals (Contractor et al., 2000). It is interesting that Bortolotto et al. (1999) have also shown that LY382884 can antagonize LTP induction in the CA3 region of the hippocampus, another region that does not normally show high GluR5 subunit density in any of the histological experiments to date (Contractor et al., 2001). This may indicate that some compensatory mechanism is engaged when this subunit is knocked out, leading to the discrepancy between the LTP seen in transgenic animals and the lack there of in the pharmacological experiments. 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