Involvement of group I mGluRs in LTP induced by strong high frequency stimulation in the dentate gyrus in vitro

Neuroscience Letters 436 (2008) 235–238

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Involvement of group I mGluRs in LTP induced by strong high frequency stimulation in the dentate gyrus in vitro Jianqun Wu a , Sarah Harney a , Michael J. Rowan b , Roger Anwyl a,∗ a b

Department of Physiology, Trinity College, Dublin 2, Ireland Pharmacology and Therapeutics, Trinity College, Dublin 2, Ireland

a r t i c l e

i n f o

Article history: Received 13 February 2008 Received in revised form 11 March 2008 Accepted 12 March 2008 Keywords: LTP mGluR5 Ryanodine Dentate gyrus

a b s t r a c t The involvement of group I metabotropic glutamate receptors (mGluRs) and ryanodine receptors was investigated in the induction of LTP induced either by application of one standard high frequency stimulation (HFS) or by strong multiple HFS in the medial perforant path to granule cell synapse of the rat dentate gyrus. Whilst a standard brief HFS induced LTP close to 50%, strong stimulation consisting of multiple HFS induced a much larger LTP. mGluR5 was found to be partially involved in the induction of the enhanced LTP induced by the strong HFS but not in the standard LTP induced by the brief HFS. Thus the mGluR5 antagonists LY341495 and MPEP partially inhibited the induction of LTP induced by strong HFS but did not inhibit LTP induced by a standard HFS. Ryanodine was found to partially inhibit LTP induced by the strong HFS but not to inhibit the standard LTP induced by the brief HFS, demonstrating the involvement of Ca-induced Ca release from ryanodine-sensitive Ca stores in the former. These studies demonstrate that the large amplitude LTP induced by strong stimulation involves additional mechanisms to the LTP induced by brief HFS, in particular involving activation of mGluR5 and RyR-sensitive Ca stores. © 2008 Elsevier Ireland Ltd. All rights reserved.

Long-term potentiation (LTP) is a long-term increase in synaptic transmission induced by high frequency stimulation (HFS) and which is widely studied as a cellular model for learning and memory [4,5]. The involvement of mGluRs in the induction of LTP has been controversial. Initial studies supported a role for a role of group I mGluRs in induction of LTP. Thus an mGluR agonist, ACPD, enhanced LTP induced by HFS in CA1 [12] and an mGluR antagonist, MCPG, blocked induction of LTP by a brief HFS in CA1 [3] and dentate gyrus [8]. However, certain other studies failed to demonstrate a block of brief-HFS induced LTP by either MCPG [11] (reviewed [1]) or more recently, by newly developed mGluR antagonist such as LY341495 [10,18]. In the present studies, we have investigated the involvement of group I mGluRs in LTP induced by strong multiple spaced HFS in the medial perforant path of the adult dentate gyrus. We show that such strong HFS induces an additional component of LTP to that induced by the standard brief stimulation. The additional LTP, unlike that produced by brief HFS, involves activation of group I mGluRs. All experiments were carried out on transverse slices of the rat (Bioresources Unit, Trinity College, Dublin) hippocampus (age 3–4 weeks, weight 40–80 g). Animal use was approved by the Bioresources Committee, Trinity College. The brains were rapidly removed after decapitation and placed in cold oxygenated (95%

∗ Corresponding author. Tel.: +353 1 8961624; fax: +353 1 679 3545. E-mail address: [email protected] (R. Anwyl). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.03.027

O2 /5% C02 ) media. Slices were cut at a thickness of 350 ␮m using a Campden vibroslice, and submerged in a storage container containing oxygenated media at room temperature (20–22 ◦ C). The slices were then transferred as required to a recording chamber for submerged slices and continuously superfused at a rate of 6–7 ml/min at 30–32 ◦ C. The control media contained: (mM) NaCl, 120; KCl 2.5, NaH2 P04 , 1.25; NaHC03 26; MgS04 , 2.0; CaCl2 , 2.0; d-glucose 10. All solutions contained 50 ␮M picrotoxin (Sigma) to block (-aminobutyric acid (GABA)A -mediated activity. Standard electrophysiological techniques were used to record field potentials. Presynaptic stimulation was applied to the medial perforant pathway of the dentate gyrus, and field excitatory postsynaptic potentials (EPSPs) were recorded at a control test frequency of 0.01 Hz from the middle one-third of the molecular layer of the dentate gyrus. In each experiment, an input–output curve (afferent stimulus intensity versus EPSP amplitude) was plotted at the test frequency and the amplitude of the test EPSP was adjusted to one-third of maximum, usually 1–1.2 mV. The baseline was considered to be stable if no change in the EPSP occurred for 30 min prior to experimentation. A standard or a strong stimulation protocol was used to induce LTP. Standard stimulation was a single HFS consisting of eight trains, each of eight stimuli at 200 Hz, intertrain interval 2 s, with the stimulation voltage increased during the HFS amplitude so as to elicit an EPSP of double the normal test EPSP amplitude. Strong stimulation was two or three HFS applied at 10 min interval, with each HFS identical to that used in the brief HFS except for the use of 20 trains rather than eight trains. EPSP of

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Fig. 1. Strong HFS induces a larger LTP than a single HFS. (A) A single brief HFS induced LTP of ∼50% at 60 min post-HFS. (B) Strong HFS consisting of two standard HFS at a 10 min interval induced LTP of ∼double the amplitude of that induced by a single HFS. (C) Strong HFS consisting of three standard HFS at 10 min intervals induced a large amplitude LTP similar to that induced by two spaced HFS.

double the normal test EPSP amplitude. Recordings were analysed using p-CLAMP (Axon Instruments). Values are the mean ± S.E.M. for n slices and two-tailed dependent student’s t-test was used for statistical comparison. We initially compared the amplitude of LTP induced by a standard HFS with that induced by strong HFS. Standard HFS, which consisted of a single HFS (see above) induced LTP which measured 153 ± 8%, n = 6, p < 0.005 at 60 min respectively following HFS (Fig. 1A). The strong stimulation, which consisted of two or three HFS induced a larger amplitude LTP than that induced by a standard HFS. Thus two spaced HFS induced LTP measuring 193 ± 13%, n = 5, p < 0.005 (Fig. 1B), whilst three spaced HFS induced LTP measuring 187 ± 5%, n = 5, p < 0.005 (Fig. 1C). The amplitude of LTP produced by the strong stimulation was significantly higher than that induced by the standard stimulation (p < 0.005). Two spaced HFS produced a maximal LTP as three spaced HFS did not produce a larger LTP than two spaced HFS. We therefore used two spaced HFS for further studies. Additional interleaved control experiments involving the induction of LTP by multiple HFS trains were carried out throughout the study in order to ensure that the two spaced multiple HFS consistently evoked a large amplitude LTP. Such multiple HFS trains resulted in the induction of LTP of 192 ± 6%, n = 17, p < 0.005. We found that mGluR5 is involved in LTP induced by multiple but not single HFS. Thus the LTP induced by a single HFS was not inhibited by the global mGluR antagonist LY341495 (50 ␮M) (Fig. 2A), or the mGluR5 antagonist MPEP (1 ␮M) (Fig. 2B), LTP measuring 144 ± 12%, n = 5, p > 0.05 and 139 ± 6%, n = 5, p > 0.05

respectively, not significantly different from control LTP. In contrast, the LTP induced by multiple trains was inhibited by mGluR5 antagonists, although only partially. Thus LTP induced by multiple HFS measured 145 ± 13%, n = 5, p < 0.01 in the presence of LY341495 (Fig. 2C) and 146 ± 7%, n = 5, p < 0.01 in MPEP (Fig. 2D). The baseline response was not affected by 30 min perfusion of either LY341495 (101% ± 2%, n = 5, p > 0.05) or MPEP (98% ± 2%, n = 5, p > 0.05). Group 1 mGluRs are known to cause Ca mobilization from internal ryanodine-sensitive stores [14]. In order to block Ca release from such stores, we applied ryanodine, an agent which inhibits the ryanodine receptor and which blocks Ca release from the Ca stores at concentrations of 10–100 ␮M [20]. Ryanodine (20 ␮M) did not inhibit the induction of standard LTP induced by a brief HFS (154 ± 9% p > 0.1; n = 6) (Fig. 3A). However, ryanodine partially inhibited induction of LTP induced by multiple HFS, LTP measuring 134 ± 7%, p > 0.1; n = 6 (Fig. 3B). The present studies have shown that strong HFS induces a much larger amplitude LTP than that induced by a single HFS in the medial perforant path of the dentate gyrus. Importantly, we have shown that the induction mechanisms of the additional LTP evoked by the strong HFS differ from that evoked by the brief HFS. In particular, the enhanced additional LTP induced by the strong HFS, unlike the standard LTP induced by a brief HFS, involves activation of mGluR5, RyR-sensitive Ca stores. The lack of effect of group I mGluR antagonist on the induction of LTP induced by a brief HFS shown in the present studies in the dentate gyrus is in agreement with our previous studies in the dentate gyrus [18] and also with

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Fig. 2. mGluR5 antagonists partially inhibit LTP induced by a strong HFS but not LTP induced by a single brief HFS. (A) The group I/II mGluR antagonist LY341495 did not inhibit LTP induced by a single brief HFS. (B) The mGluR5 antagonist MPEP did not inhibit LTP induced by a single brief HFS. (C) The group I/II mGluR antagonist LY341495 partially inhibited LTP induced by strong HFS. (B) The mGluR5 antagonist MPEP partially inhibits LTP induced by a strong HFS.

that in CA1 [6]. There is therefore now convincing evidence that the standard LTP induced by brief HFS is independent of activation of group I mGluRs. However, we have shown in the present studies that the enhanced additional component of the LTP induced by strong HFS, is generated by activation of mGluR5, as such addi-

tional LTP is inhibited by the global mGluR antagonist LY341495 and the selective mGluR5 antagonist MPEP. Group I mGluRs are located perisynaptically [16], and previous studies have shown that they are activated most strongly by spillover of glutamate evoked by a strong stimulus [7]. We therefore postulate that the appli-

Fig. 3. LTP induced by strong stimulation, but not standard stimulation, is mediated via activation of ryanodine receptors. (A) Blocking the ryanodine receptor with ryanodine does not inhibit the induction of LTP by a single HFS. (B) Blocking the ryanodine receptor with ryanodine partially inhibits the induction of LTP by strong HFS.

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cation of strong HFS, but not brief HFS, causes sufficient release and spillover of glutamate to activate the perisynaptically located mGluR5. Such activation of mGluR5 results in the induction of an additional component of LTP which summates with that induced by brief HFS. Activation of group 1 mGluR has also been shown to be involved in the priming of LTP in CA1 [13] and in the very late (>24 h) component of LTP in the dentate gyrus in vivo [15]. We have also shown using the RyR receptor antagonist ryanodine that the induction of the additional LTP by a strong HFS, although not the standard LTP induced by a brief HFS, requires release of Ca from RyR-sensitive stores. The RyR-sensitive stores are known to be linked to group I mGluRs [9], and we suggest that activation of mGluR5 results in Ca release from the RyR-sensitive Ca stores and thereby induction of additional LTP. Such an effect of ryanodine in the dentate gyrus differs from that in CA1 where deletion or inhibition of the ryanodine receptor type did not affect robust LTP induced by strong stimulation but did cause inhibition of both the weak LTP induced by mild stimulation [2,17] and the priming enhancement of LTP [13]. Acknowledgement This work was supported by the Science Foundation Ireland. References [1] R. Anwyl, Metabotropic glutamate receptors: electrophysiological properties and role in plasticity., Brain Res. Brain Res. Rev. 29 (1999) 83–120. [2] D. Balschun, D.P. Wolfer, F. Bertocchini, V. Barone, A. Conti, W. Zuschratter, L. Missiaen, H.P. Lipp, J.U. Frey, V. Sorrentino, Deletion of the ryanodine receptor type 3 (RyR3) impairs forms of synaptic plasticity and spatial learning, EMBO J. 18 (1999) 5264–5273. [3] Z.I. Bashir, Z.A. Bortolotto, C.H. Davies, N. Berretta, A.J. Irving, A.J. Seal, J.M. Henley, D.E. Jane, J.C. Watkins, G.L. Collingridge, Induction of LTP in the hippocampus needs synaptic activation of glutamate metabotropic receptors, Nature 363 (1993) 347–350. [4] T.V. Bliss, G.L. Collingridge, A synaptic model of memory: long-term potentiation in the hippocampus, Nature 361 (1993) 31–39.

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