Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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SYNAPTIC STRENGTH AT THE TEMPOROAMMONIC INPUT TO THE HIPPOCAMPAL CA1 REGION IN VIVO IS REGULATED BY NMDA RECEPTORS, METABOTROPIC GLUTAMATE RECEPTORS AND VOLTAGE-GATED CALCIUM CHANNELS A. AKSOY-AKSEL AND D. MANAHAN-VAUGHAN *
elicited by test-pulse stimulation, DCG-IV did not affect LTP in a dose that inhibited LTP at pp-DG synapses in vivo. These data indicate that LTP at the pp-CA1 synapse of freely behaving animals is dually dependent on NMDAR and VGCCs, whereby group II mGlu receptor activation affect basal synaptic tonus, but not LTP. The lower frequencydependency of NMDA-VGCC LTP at pp-CA1 synapses compared to pp-DG synapses may comprise a mechanism to prioritize information processing at this synapse. This article is part of a Special Issue entitled: Hippocampus. Ó 2015 The Authors. Published by Elsevier Ltd. on behalf of IBRO. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Department of Neurophysiology, Medical Faculty, Ruhr University Bochum, Germany International Graduate School for Neuroscience, Ruhr University Bochum, Germany
Abstract—The hippocampal CA1 region receives cortical information via two main inputs: directly via the perforant (temporoammonic) path (pp-CA1 synapse) and indirectly via the tri-synaptic pathway. Although synaptic plasticity has been reported at the pp-CA1 synapse of freely behaving animals, the mechanisms underlying this phenomenon have not been investigated. Here, we explored whether long-term potentiation (LTP) at the pp-CA1 synapse in freely behaving rats requires activation of N-methyl-D-aspartate receptors (NMDAR) and L-type voltage-gated calcium channels (VGCCs). As group II metabotropic glutamate (mGlu) receptors are densely localized on presynaptic terminals of the perforant path, and are important for certain forms of hippocampal synaptic plasticity, we also explored whether group II mGlu receptors affect LTP at the pp-CA1 synapse and/or regulate basal synaptic transmission at this synapse in vivo. In adult male rats, high-frequency stimulation (200 Hz) given as 3, or 10 trains, resulted in robust LTP that lasted for at least 4 h in pp-CA1 or pp-dentate gyrus (DG) synapses, respectively. Pre-treatment with the NMDAR antagonist D-( )-2-amino-5-phosphopentanoic acid (D-AP5) partially inhibited LTP at pp-CA1, and completely prevented LTP at pp-DG synapses. Combined antagonism of NMDAR using D-AP5 and the VGCC inhibitor, ( )-methoxyverapamil hydrochloride elicited a further inhibition of the LTP response at pp-CA1 synapses. Whereas activation of group II mGlu receptors using (1R,2R)-3-((1S)-1-amino-2-hydroxy2-oxoethyl) cyclopropane-1,2-dicarboxylic acid (DCG-IV) dose-dependently reduced basal synaptic transmission
Key words: perforant path, temporoammonic, plasticity, hippocampus, CA1, in vivo.
synaptic
INTRODUCTION The hippocampal formation is a highly plastic structure that is reciprocally connected to cortical brain regions, and engages in information processing and storage (Martin et al., 2000) by means of processes such as long-term potentiation (LTP) and long-term depression (LTD) (Kemp and Manahan-Vaughan, 2007). The main cortical input to the hippocampus originates from the entorhinal cortex (EC), where layer II neurons give rise to the tri-synaptic pathway, comprising the dentate gyrus (DG), CA3 and CA1 regions. In addition, EC layer III neurons directly connect to the CA1 region via the temporoammonic path, often referred as temporoammonic or perforant path to CA1 (pp-CA1) synapse (Witter et al., 1988). Thus, the CA1 region encounters pre-processed information arising from the CA3 region that is transferred via the Schaffer collateral-CA1 (Sc-CA1) synapse, as well as un-edited information that reaches it directly via the ppCA1 synapse. This information might serve as an efference copy for newly stored information in the CA1 region (Lisman and Otmakhova, 2001) or subserve mismatch and/or error detection (Izumi and Zorumski, 2008). Various learning and memory forms are thought to be encoded via differential involvement of persistent synaptic plasticity mechanisms that require activation of specific molecular cascades (Martin and Morris, 2002; Kemp and Manahan-Vaughan, 2004; Lisman et al., 2012). Induction of LTP in the CA1 region typically involves
*Correspondence to: D. Manahan-Vaughan, Department of Neurophysiology, Medical Faculty, Ruhr University Bochum, MA 4/ 150, Universitaetsstr. 150, 44780 Bochum, Germany. Tel: +49-234322-2042; fax: +49-234-32-14490. E-mail address: [email protected] (D. Manahan-Vaughan). Abbreviations: ANOVA, analysis of variance; AP, anterioposterior; DAP5, D-( )-2-amino-5-phosphopentanoic acid; DCG-IV, (1R,2R)-3((1S)-1-amino-2-hydroxy-2-oxoethyl) cyclopropane-1,2-dicarboxylic acid; DG, dentate gyrus; EC, entorhinal cortex; fEPSP, field excitatory post-synaptic potential; HFS, high-frequency stimulation; LTD, long-term depression; LTP, long-term potentiation; mGlu, metabotropic glutamate; ML, mediolateral; NMDAR, N-methyl-Daspartate receptors; pp, perforant path; Sc-CA1, Schaffer collateralCA1; STP, short-term potentiation; VGCCs, voltage-gated calcium channels.
http://dx.doi.org/10.1016/j.neuroscience.2015.03.014 0306-4522/Ó 2015 The Authors. Published by Elsevier Ltd. on behalf of IBRO. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1
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postsynaptic calcium ion entry via the ionotropic, glutamatergic N-methyl-D-aspartate receptors (NMDAR) and subsequent activation of protein kinases (Luscher and Malenka, 2012). However, depending on the stimulation strength Sc-CA1 and pp-DG synapses are capable of expressing LTP in the presence of NMDARantagonists, in a process that involves L-type voltagegated calcium channels (VGCC) (Grover and Teyler, 1990; Manahan-Vaughan et al., 1998; Freir and Herron, 2003). In hippocampal slices it was also shown that ppCA1 synapses express NMDAR-dependent (Remondes and Schuman, 2002) as well as VGCC-dependent LTP (Remondes and Schuman, 2003). However, very little data are available that describes the processes that underlie LTP induction at the pp-CA1 synapse in the intact brain of freely behaving rats (Aksoy-Aksel and ManahanVaughan, 2013). Recently, we reported that compared to Sc-CA1 synapses, the threshold for induction of LTP is lower at pp-CA1 synapses in vivo and these synapses are comparatively resistant to strong stimulation protocols (Aksoy-Aksel and Manahan-Vaughan, 2013). In addition to ionotropic glutamate receptors, metabotropic glutamate (mGlu) receptors play an important role in hippocampal synaptic plasticity and in hippocampal basal synaptic tonus (Mukherjee and Manahan-Vaughan, 2013). Group II mGlu receptors (mGluR2/3) are G-protein receptors that are negatively coupled to adenylyl cyclase and are densely located in a perisynaptic manner on the perforant path and on mossy fiber boutons in the hippocampal formation (Shigemoto et al., 1997). Activation of group II mGlu receptors reduces basal synaptic transmission in the pp-DG and mossy fiberCA3 synapses in a dose-dependent manner in freely behaving rats (Kulla et al., 1999; Hagena and ManahanVaughan, 2010). In vitro, the pp-CA1 synapse exhibits a higher sensitivity to the activation of group II mGlu receptors compared to the Sc-CA1 synapse (Speed and Dobrunz, 2009) suggesting that they play a role in the regulation of synaptic plasticity thresholds. The capacity of a synapse to express different forms of potentiation offers insights into its role in behavioral phenomena such as learning and memory. Different demands for information encoding, retrieval or extinction, for example, might be mediated through differing requirements for NMDAR-dependent and VGCCdependent LTP (Borroni et al., 2000; Bauer et al., 2002; Moosmang et al., 2005; Davis and Bauer, 2012). Furthermore, very long-lasting forms of synaptic plasticity are linked to the activation of mGlu receptors and to longterm spatial memory (Mukherjee and Manahan-Vaughan, 2013). Considering that the temporoammonic input to CA1 is believed to play an important role in spatial recognition memory (Brun, 2002) and long-term memory maintenance (Remondes and Schuman, 2004) we investigated whether LTP at pp-CA1 synapses is NMDAR, VGCC or group II mGlu receptor dependent, and whether synaptic transmission is regulated by group II mGlu receptors. We observed that robust LTP at the pp-CA1 synapse in vivo is induced with less afferent stimulation than LTP at pp-DG synapses. Furthermore, LTP at pp-CA1 synapses is only partially affected by NMDAR antagonism, whereas LTP at the
pp-DG synapse is completely abolished. Moreover, NMDAR-independent LTP at the pp-CA1 synapse is sensitive to a VGCC antagonist. We also observed that prior agonist activation of group II mGlu receptors using a dose that does not affect basal synaptic transmission prevents persistent LTP at pp-DG synapses, but has had no effect on LTP at pp-CA1 synapses. These data suggest that synaptic plasticity at the pp-CA1 synapse is distinct to the pp-DG synapse. This may reflect its specific role in hippocampal information processing.
EXPERIMENTAL PROCEDURES Surgical procedures The present study was carried out in accordance with the European Communities Council Directive of September 22nd, 2010 (2010/63/EU) for care of laboratory animals and after approval of the local ethics committee (Bezirksamt Arnsberg, Germany). All efforts were made to reduce the number of animals used. Male Wistar rats (7–8 weeks old at the time of surgery) were implanted with recording and stimulation electrodes (polyurethane-coated stainless steel wire; 1 mm in diameter) into the right hippocampus and a cannula (length: 5.6 mm, diameter: 0.8 mm, Gu¨ndel Biomedical Instruments, Germany) into the right lateral cerebral ventricle, as described previously (Aksoy-Aksel and Manahan-Vaughan, 2013). The stereotaxic coordinates for electrode and cannula placements, were referenced to the bregma for anteroposterior (AP) and to midline for mediolateral (ML) distance. The coordinates were as follows: guiding cannula (AP: 0.5 mm; ML: +1.6 mm; DV: ca. 4 mm); recording electrode (AP: 3.0; ML: +2.0 mm) and stimulating electrode (AP: 6.9; ML: +4.1 mm) (Fig. 1). The accuracy of the electrode implantations was verified as described previously (Aksoy-Aksel and Manahan-Vaughan, 2013). In addition, another group of animals was implanted with a recording electrode in the granule cell layer of the DG (AP: 3.1, ML: +1.9 mm) and a stimulating electrode in the perforant path. Here, the same stimulating electrode coordinates were used as for the pp-CA1 preparation. For all synapses the depth of the electrodes was determined during the surgery by giving test-pulse stimulation to generate an field excitatory post-synaptic potential (fEPSP) while the recording electrode was lowered in stepwise decrements until a typical fEPSP was observed (AksoyAksel and Manahan-Vaughan, 2013). Electrophysiological recordings The in vivo experiments were carried out 7–10 days after the implantation of the electrodes. Synaptic transmission was recorded via test-pulse stimulation of the medial perforant path using a stimulation intensity that elicits ca. 40% of the maximum fEPSP obtained from a previously established input/output (I/O) relationship (100–900 lA) for each individual animal. Responses were evoked by stimulating at frequency of 0.025 Hz with a single biphasic square wave pulse of 0.2-ms stimulus duration and 10 kHz sample rate. For each time-point, five
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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Fig. 1. Potentials evoked in the CA1 region via perforant path stimulation exhibit distinct properties compared to perforant path-dentate gyrus synapses. Schema showing the position of the simulation electrode in the angular bundle (left) and the recording electrodes in either the Stratum lacunosum moleculare of the CA1 region or the granule cell layer of the dentate gyrus (middle). Right: Analog examples of field potentials evoked in the perforant path (pp)-CA1 synapse (top) or the pp-dentate gyrus synapse (bottom). The slope of the fEPSP elicited by perforant path stimulation was measured as the five steepest points obtained on the first negative deflection of the compound potential for the pp-CA1 synapse, or on the first positive deflection of the compound potential for the pp-dentate gyrus synapse (indicated by the line superimposed on the respective analog example).
sweeps of evoked responses were averaged. Changes in evoked potentials were assessed by measuring the fEPSP slope. For CA1 potentials this was measured as the maximum slope through the five steepest points from the beginning of the fEPSP and the first minimum located in the first deflection (Manahan-Vaughan, 1997) (Fig. 1). For DG potentials the fEPSP slope was measured as the maximum slope through the five steepest points from the beginning of the fEPSP and the first maximum (population spike onset) located in the first deflection (ManahanVaughan et al., 1998). Before being included in the study, the animals were assessed stability of their baseline responses during a ca. 5-h recording period. Instability of the responses was an exclusion criterion for the subsequent experiments. fEPSPs were recorded every 5 min for at least 30 min (six time points) before drug application and 30 min (six time points) after drug application. LTP at pp-CA1 synapses was induced using high-frequency stimulation (HFS) comprising 200-Hz stimulation given as three bursts of 15 stimuli (0.2-ms stimulus duration, 10-s interburst interval) as this protocol was previously shown to be effective in inducing LTP at pp-CA1 synapses in vivo (Aksoy-Aksel and Manahan-Vaughan, 2013). LTP at pp-DG synapses was induced with the stronger protocol of HFS comprising 200-Hz stimulation given as 10 bursts of 15 stimuli (0.2-ms stimulus duration, 10-s interburst interval). This protocol elicits LTP that lasts for over 24 h in freely behaving rats (Kulla et al., 1999) whereas the abovementioned protocol (for LTP induction at pp-CA1 synapses) induces short-term potentiation
(STP) at pp-DG synapses that lasts at most for ca. 2 h (Manahan-Vaughan et al., 1998). fEPSP recordings were made 5, 10 and 15 min postHFS followed by recordings every 15 min for a total of 4 h. Basal synaptic transmission was recorded in the same way but HFS was not applied. HFS was always given between 10 and 11 a.m. The cortical encephalogram (EEG) was monitored throughout the course of each experiment to verify that stimulation did not elicit neuronal disturbances such as epileptiform activity that could confound interpretation of the electrophysiological data. No cases of disturbances were observed. The animals’ arousal state was not affected by the stimulation. Animals were awake during HFS. Compounds and drug injection Pharmacological ligands were administered in a 5-ll volume to the lateral cerebral ventricle via the implanted cannula, and applied at a rate of 1 ll per min, control groups were injected with 5-ll vehicle. The NMDAR antagonist D-( )-2-amino-5-phosphopentanoic acid (DAP5, Biozol, Eching, Germany) was first dissolved in 5 ll of 1 N sodium hydroxide solution (NaOH), then 0.9% sodium chloride (NaCl) was added to made up a solution of 100-ll volume. The final amount injected was 100 nmol in an injection volume of 5 ll (Popkirov and Manahan-Vaughan, 2011). The L-type VGCC antagonist ( ) methoxyverapamil hydrochloride (methoxyverapamil) (Research Biochemicals International, Natick, USA) was dissolved in
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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distilled water and injected as 100 nmol/5 ll (ManahanVaughan et al., 1998; Po¨schel and Manahan-Vaughan, 2007). The group II mGluR agonist, (1R,2R)-3-((1S)-1-amino2-hydroxy-2-oxoethyl)cyclopropane-1,2-dicarboxylic acid (DCG-IV, Biozol, Eching, Germany) was dissolved in 0.9% NaCl solution and injected as 20 ng (low doseDCG-IV) or 40 ng (high dose-DCG-IV) (Klausnitzer and Manahan-Vaughan, 2008). The pH of the solutions was adjusted to 7. All compounds used had been previously assessed for their effects on synaptic plasticity at other hippocampal synapses in vivo (Manahan-Vaughan, 1997; Manahan-Vaughan et al., 1998; Popkirov and ManahanVaughan, 2011; Po¨schel and Manahan-Vaughan, 2007; Klausnitzer and Manahan-Vaughan, 2008). Data analysis For the analysis of the electrophysiological data, each time-point was calculated as the percentage of the average of the six recordings (pre-injection basal synaptic transmission values) for every subject individually. Between group statistics was done by a two-way analysis of variance with repeated measures (ANOVA) by comparing the whole train of measurements after the HFS. Post-hoc analysis with Fisher’s least significant difference (LSD) test was used to test the significance of individual values in plasticity experiments. P < 0.05 was considered significant.
RESULTS Antagonism of NMDAR significantly reduces synaptic potentiation at pp-CA1 synapses and abolishes synaptic potentiation at pp-DG synapses LTP induction requires activation of NMDAR at the Sc-CA1 (Nicoll and Malenka, 1999) and pp-DG synapses (Manahan-Vaughan et al., 1998). Here, we examined whether LTP at the pp-CA1 synapse of freely behaving rats depends on NMDAR in a similar manner to LTP at the pp-DG synapse. Stimulation of the perforant path to CA1 synapse with HFS at 200 Hz (three trains) resulted in LTP that lasted for at least 4 h (Fig. 2A). Intracerebroventricular (icv) injection of the NMDAR antagonist, D-AP5 30 min prior to HFS, significantly reduced the amplitude and the duration of synaptic potentiation (ANOVA, F(1,144) = 78.665; p < 0.001, n = 5; Fig. 2A). However, LTP was not fully abolished. Four hours after treatment a residual LTP was still evident, although this was significantly less compared to controls (Fig. 2A, p < 0.05). This dose of D-AP5 completely prevented LTP at the pp-DG synapse that was induced with a stronger stimulation protocol (200 Hz, ten bursts compared to three bursts used for pp-CA1 synapse) in agreement with previous findings (Manahan-Vaughan et al., 1998). Here, 4 h after HFS, LTP was completely absent in D-AP5-treated animals (n = 10) compared to vehicle-injected controls (n = 10, ANOVA, F(1,144) = 117.241; p < 0.001) (Fig. 2B). Thus, we further explored the neurotransmitter receptor requirements of the NMDAR-independent LTP at pp-CA1 synapses.
Fig. 2. LTP at the pp-CA1 synapse is significantly but incompletely prevented by NMDAR antagonism. LTP at pp-dentate synapses is completely abolished by NMDAR antagonism and curtailed by activation of group II mGlu receptors. (A) High-frequency stimulation (HFS) at 200 Hz (three bursts) elicited LTP at the pp-CA1 synapse of vehicle-treated freely behaving animals that endured for over 4 h. Treatment with an NMDAR antagonist (D-AP5) prior to HFS resulted in reduced synaptic potentiation. The graphs represent the mean value of the percentage ± standard error of the mean (SEM). Inset: Analog traces evoked from vehicle-treated (left) or D-AP5-treated animals (right), (1) before HFS, (2) 5 min after HFS and (3) 4 h after HFS. Vertical scale bar = 3 mV, horizontal scale bar = 5 ms. (B) HFS at 200 Hz given as ten trains induced very robust LTP at ppdentate gyrus synapses that was still present 4 h after HFS was given. Treatment of the animals with the same dose of D-AP5 30 min prior to HFS, completely abolished LTP. Treatment with a low dose of the group II mGluR agonist (DCG-IV) prior to HFS significantly prevented the late phase of LTP. Inset: Analog traces evoked from vehicle-treated animals, (1) before HFS and (2) 4 h after HFS. Vertical scale bar = 5 mV, horizontal scale bar = 5 ms.
Combined antagonism of NMDA receptors and L-type VGCC further reduces synaptic potentiation at ppCA1 synapses L-type VGCC contribute to LTP at the pp-DG synapse (Manahan-Vaughan et al., 1998) and Sc-CA1 synapse (Grover and Teyler, 1990) when elicited by substantially higher and stronger afferent stimulation (400 Hz for DG and 200 Hz for CA1). At the pp-CA1 synapse VGCCdependent LTP can be induced in vitro (Remondes and
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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Schuman, 2003). Thus, VGCC are likely to contribute to the NMDA-independent LTP at the pp-CA1 synapse in vivo. Application of the VGCC antagonist, methoxyverapamil in the presence of D-AP5 further diminished LTP at the pp-CA1 synapse compared to vehicle injection (ANOVA, F(1,143) = 49.582; p < 0.001, n = 5; Fig. 3). Thus, LTP at the pp-CA1 synapse depends on activation of both NMDAR and L-type VGCCs, whereby the frequency-dependency of the pp-CA1 synapse on NMDAR and L-type VGCCs is lower than that observed at pp-DG synapses in vivo (Manahan-Vaughan et al., 1998).
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at pp-CA1 synapses, we examined the effect of DCG-IV treatment on evoked potentials that were elicited by test-pulse stimulation. Administration of the low dose of DCG-IV (n = 5) (20 ng) had no effect on basal synaptic transmission compared to vehicle-treated controls (n = 5) (Fig. 4A). Increasing the dose of DCG-IV (40 ng) resulted in a transient depression of basal synaptic responses (n = 5) (Fig. 4A) (ANOVA, F(1,230) = 39.00; p < 0.001). The depression began about 10 min after injection (p < 0.05), peaked at about 1 h (p < 0.05), and had returned to pre-injection baseline values by ca. 2 h.
Activation of group II mGlu receptors dosedependently affects basal synaptic transmission at pp-CA1 synapses Previous findings show that group II mGlu receptors facilitate the expression of robust LTD at pp-DG synapses using doses that do not elicit significant changes in basal synaptic responses (ManahanVaughan, 1998), although a dose-dependent depression of basal synaptic transmission is also observed. This suggests that these receptors play a dual role in the DG, regulating basal tonus on the one hand, and engaging in metaplastic regulation (Abraham and Tate, 1997) on the other hand. To assess whether group II mGlu receptors contribute to the regulation of basal excitatory tonus
Fig. 3. LTP at the pp-CA1 synapse is significantly prevented by combined NMDAR and VGCC antagonism. High-frequency stimulation (HFS) at 200 Hz elicited LTP at the pp-CA1 synapse of vehicle-treated freely behaving animals that endured for over 4 h. Prior treatment with an NMDAR antagonist (D-AP5) together with an L-type VGCC antagonist (methoxyverapamil) potently inhibited LTP at the pp-CA1 synapse. Effects were significantly greater than those elicited with D-AP5 alone. The graphs represent the mean value of the percentages ± standard error of the mean (SEM). Inset: Analog traces evoked from vehicle-treated (left) or D-AP5/methoxyverapamil-treated animals (right), (1) before HFS, (2) 5 min after HFS and (3) 4 h after HFS. Vertical scale bar = 3 mV, horizontal scale bar = 5 ms.
Fig. 4. Agonist activation of group II metabotropic glutamate receptors dose-dependently depresses basal synaptic transmission at the pp-CA1 synapses in vivo, but does not affect LTP. (A) Basal synaptic transmission evoked by test-pulse stimulation was unaffected by intracerebroventricular application of the group II mGluR agonist, DCG-IV given as a dose (DCG-IV low) (at the time-point marked by the arrow). In contrast, a higher dose of DCG-IV (DCG-IV high) significantly depressed evoked responses. Effects were transient, and by ca. 2 h after DCG-IV-treatment, evoked potentials were equivalent to those obtained in vehicle-treated animals. Inset in: Analog traces evoked from vehicle-treated animals (left) or animals treated with high dose DCG-IV-treated (right), (1) prior to injection, (2) 5 min after injection and (3) 4 h after injection. Vertical scale bar = 3 mV, horizontal scale bar = 5 ms. (B) High-frequency stimulation (HFS) at 200 Hz (three trains) elicited LTP at the pp-CA1 synapse of vehicle-treated freely behaving animals that endured for over 4 h. Treatment with the low dose of the group II mGluR agonist (DCG-IV) prior to HFS did not affect LTP at the pp-CA1 synapse. Inset: Analog traces evoked from vehicle-treated animals (left) or animals treated with DCG-IV-treated (right), (1) prior to HFS, (2) 5 min after HFS and (3) 4 h after HFS Vertical scale bar = 3 mV, horizontal scale bar = 5 ms.
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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Activation of group II mGlu receptors does not affect LTP at pp-CA1 synapses but prevents the late phases of DG LTP Using the dose of DCG-IV that did not affect basal synaptic transmission at pp-CA1 synapses, and is known to have no effect on basal synaptic transmission at pp-DG synapses in vivo (Manahan-Vaughan, 1998), we explored if group II mGlu receptors directly contribute to LTP at pp-CA1 or pp-DG synapses. Administration of the low dose of DCG-IV significantly prevented the late phase of LTP (elicited by 200 Hz HFS given as 10 trains) at pp-dentate synapses (n = 10) compared to vehicle-treated controls (n = 10, ANOVA, F(1,144) = 87.221; p < 0.001) (Fig. 2B). Here, it should be noted that whereas LTP at pp-CA1 synapses can be induced by three trains of 200 Hz (Fig. 3B), this protocol elicits short-term potentiation at pp-DG synapses (Manahan-Vaughan et al., 1996). Robust LTP at these synapses requires afferent stimulation in the form of at least 200 Hz given as 10 trains (Kulla et al., 1999). By contrast, agonist activation of group II mGlu receptors (DCG-IV; low dose) had no significant effect on LTP at pp-CA1 synapses compared to vehicle controls (ANOVA, F(1,144) = 3.089; p > 0.05, n = 5; Fig. 4B). This suggests that the pp-CA1 synapse is distinct to the pp-DG synapse in terms of LTP regulation by group II mGlu receptors.
DISCUSSION In this study we report that LTP at the pp-CA1 synapse in vivo depends on NMDAR and L-type VGCC. Antagonism of NMDAR alone results in an incomplete inhibition of LTP at pp-CA1 synapses compared to completely abolished LTP at pp-DG synapses. Additionally, activation of group II mGlu receptors does not affect LTP at pp-CA1 synapses, but prevents LTP maintenance at pp-DG synapses at doses that do not affect basal transmission. Strikingly, however the ppCA1 synapse responds with a depression of evoked responses to higher doses of the agonist, as is the case for the DG (Manahan-Vaughan, 1998). The distinct characteristics of the neurotransmitter receptor-dependency of both LTP and synaptic transmission at pp-CA1 synapses may reflect the unique postulated role of this synapse in information processing. The CA1 region, as the last module in the network of the hippocampus proper, has been subjected to particular scrutiny in terms of the mechanisms that underlie basal synaptic tonus and synaptic plasticity. However, most of the information regarding CA1 plasticity has been gathered through studies of the Sc-CA1 synapse and relatively little is known about plasticity at the pp-CA1 synapse in intact brain. The main constraint to study the pp-CA1 synapse in vivo is possible signal contamination from the surrounding hippocampal synapses (pp-DG and Sc-CA1), as it is technically impossible to completely isolate the synapse in vivo in a similar way to approaches used in in vitro preparations. However, the distinct profile of the fEPSP elicited in the CA1
region via perforant path stimulation, along with its specific and distinct response to long term plasticity protocols distinguishes this synapse from the Sc-CA1 (Aksoy-Aksel and Manahan-Vaughan, 2013) or the ppDG synapse (the present study) in freely behaving animals. A persistent change in synaptic strength is thought to be the leading mechanism underlying the physiological basis of learning and memory (Martin and Morris, 2002) and has been studied extensively in relation to NMDAR activation (Luscher and Malenka, 2012). All hippocampal synapses, with the exception of the mossy fiber-CA3 synapse, express NMDAR-dependent LTP (4 h) in vivo. Although a small degree of short-term potentiation still remained under these conditions, it is unlikely that the concentration of the drugs used was insufficient to block either the NMDAR or VGCCs, as it was previously shown that higher concentrations of the NMDAR antagonist or the VGCC-blocker used does not further affect LTP expression (at least not for LTP induced by the 400 Hz stimulation at pp-DG synapses) in freely behaving rats (Manahan-Vaughan et al., 1998). The potentiation that remained after combined block of NMDAR and VGCCs might involve other receptor types such as Ni-sensitive R- and T-type VGCCs that are thought to contribute to the calcium influx at the distal CA1 dendrites (Magee et al., 1998; Isomura et al., 2002), mGlu receptors, as shown previously for the ppDG synapse in vivo (Manahan-Vaughan et al., 1998) or calcium permeable AMPA receptors (Yuste et al., 1999). The contribution of VGCCs to hippocampal LTP is highly frequency-dependent. In the DG, HFS at 400 Hz
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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is needed to induce LTP that depends on VGCCs, whereas NMDAR-dependent LTP is elicited by means of HFS at 200 Hz (Manahan-Vaughan et al., 1998; current study). In this context, it was also quite striking that LTP that endures for over 4 h could be induced with the HFS protocol we used here. This protocol elicits short-term potentiation at pp-DG synapses (Altinbilek and Manahan-Vaughan, 2009) and at Sc-CA1 synapses causes epileptiform activity in the cortical electroencephalogram that is coupled with a temporary loss of evoked potentials (Manahan-Vaughan, unpublished data). Thus, it was not possible to study NMDARindependent LTP in vivo at Sc-CA1 synapse since it was shown in vitro to require HFS at 200 Hz (Grover and Teyler, 1990; Morgan and Teyler, 1999; Freir and Herron, 2003), whereby typically LTP (that is wholly NMDAR-dependent) is elicited by HFS of up to 100 Hz (Manahan-Vaughan, 1997). This suggests that the ppCA1 synapse is less vulnerable than the Sc-CA1 synapse and has a lower threshold for NMDA-independent LTP induction than the pp-DG synapse. Why does the pp-CA1 synapse require VGCC activation to induce LTP? Transgenic mice deficient of a subunit of VGCC have impaired hippocampusdependent memory and late-LTP (Moosmang et al., 2005). Similarly, VGCC antagonism results in impaired long-term spatial memory (Borroni et al., 2000; Woodside et al., 2004; Da Silva et al., 2013). In Sc-CA1 and pp-DG synapses VGCCs only become involved in LTP when very strong stimulation patterns are used (Grover and Teyler, 1990; Manahan-Vaughan et al., 1998) in contrast to the pp-CA1 synapse. Disturbances of information transfer to the hippocampus via the temporoammonic pathway impair long-term memory (Remondes and Schuman, 2004; Nakashiba et al., 2008; Suh et al., 2011). The lower threshold for VGCCdependent LTP at this pathway may relate to the putative role of the pp-CA1 synapse in providing an efference copy of information that should undergo synaptic storage within the tri-synaptic circuit (Lisman and Otmakhova, 2001) or in error-detection (Izumi and Zorumski, 2008). It is conceivable that the lower threshold facilitates rapid and robust encoding of new information that is then used as a ‘‘blueprint’’ against which information that is encoded by LTP that originates in the tri-synaptic circuit, and which ultimately occurs at the Sc-CA1 synapse. Agonists of group II mGlu receptors inhibit LTP at the Sc-CA1 in vivo (Holscher et al., 1997) and pp-DG synapses both in vitro (Huang et al., 1997) and in vivo (present study). On the other hand, LTD at pp-DG and Sc-CA1 synapses requires these receptors (ManahanVaughan, 1997; Altinbilek and Manahan-Vaughan, 2009). Interestingly, metaplastic effects (Abraham and Tate, 1997) of group II mGlu receptors were not evident at the pp-CA1 synapse. In the present study we observed that in pp-DG synapses, priming of group II mGlu receptors using agonist doses that do not affect basal synaptic transmission have potent effects on the outcome of a subsequent attempt to induce LTP. The pp-CA1 synapse did not respond to this kind of agonist priming, suggesting
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that this receptor does not contribute to synaptic plasticity at this synapse in a similar way to pp-DG synapses. This observation is very striking in light of the relative resistance of the pp-CA1 synapses in vivo to expression of LTD (Aksoy-Aksel and Manahan-Vaughan, 2013). At other hippocampal synapses in vivo, group II mGlu receptors are critically involved in the expression and persistency of LTD (Manahan-Vaughan, 1997; Altinbilek and Manahan-Vaughan, 2009) and in specific forms of hippocampus-dependent spatial learning (Altinbilek and Manahan-Vaughan, 2009; Pitsikas et al., 2012), and regulate the thresholds for LTP induction (Kulla et al., 1999). The lack of involvement of group II mGlu receptors in LTP at pp-CA1 synapses set this synapse apart in a functional sense, from the synapses of the trisynaptic circuit. Activation of group II mGlu receptors reduces basal synaptic transmission in the Sc-CA1, pp-DG and mossy fiber-CA3 synapses in a dose-dependent manner in freely behaving rats (Manahan-Vaughan, 1997; Kulla et al., 1999; Hagena and Manahan-Vaughan, 2009). The mossy fiber-CA3 synapse has a particularly high sensitivity to the activation of group II mGlu receptors compared to the pp-DG synapse, a characteristic that is successfully used to identify the mossy fiber-CA3 synapse in vivo (Hagena and Manahan-Vaughan, 2010). A similar approach was used in vitro to identify the highly sensitive pp-CA1 synapse from the Sc-CA1 synapse (Kew et al., 2001, 2002; Speed and Dobrunz, 2009). In the present study we also observed that the group II mGlu receptor agonist reduces the basal synaptic transmission in a dose-dependent manner (ManahanVaughan, 1998; Klausnitzer and Manahan-Vaughan, 2008). This property implies that lack of effect of group II mGlu receptor activation on pp-CA1 LTP is synapse specific and not due to general lack of drug activity.
CONCLUSIONS This study suggests that the pp-CA1 synapse of freely behaving rats is quite distinct from other hippocampal synapses. It exhibits a lower LTP induction threshold than the pp-DG synapse (current study) and the Sc-CA1 synapse (Aksoy-Aksel and Manahan-Vaughan, 2013), and exhibits a dependency on both NMDAR and voltagedependent calcium channels that normally only emerges at higher afferent stimulation frequencies for LTP at ScCA1 (Freir and Herron, 2003) and pp-DG synapses (Manahan-Vaughan et al., 1998; and current study). Furthermore, basal synaptic transmission is dose dependently sensitive to regulation by group II mGlu receptors, however, these receptors do not contribute to LTP at the pp-CA1 synapse, at least from the perspective of metaplasticity, in contrast to the pp-DG synapse. The lower LTP induction threshold of the pp-CA1 synapse compared to the Sc-CA1 synapse, combined with its distinct neurotransmitter profile may comprise mechanisms whereby information processing at this synapse is distinguished from processing by the synapses of the other hippocampal subregions.
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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CONFLICT OF INTEREST None. Acknowledgments—We gratefully acknowledge the technical assistance of Jens Colitti-Klausnitzer, Beate Krenzek and Lena Fabritius. We thank Nadine Kollosch for animal care. This work was funded by the German Research Foundation (DFG, SFB 874/B1).
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(Accepted 5 March 2015) (Available online xxxx)
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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SYNAPTIC STRENGTH AT THE TEMPOROAMMONIC INPUT TO THE HIPPOCAMPAL CA1 REGION IN VIVO IS REGULATED BY NMDA RECEPTORS, METABOTROPIC GLUTAMATE RECEPTORS AND VOLTAGE-GATED CALCIUM CHANNELS A. AKSOY-AKSEL AND D. MANAHAN-VAUGHAN *
elicited by test-pulse stimulation, DCG-IV did not affect LTP in a dose that inhibited LTP at pp-DG synapses in vivo. These data indicate that LTP at the pp-CA1 synapse of freely behaving animals is dually dependent on NMDAR and VGCCs, whereby group II mGlu receptor activation affect basal synaptic tonus, but not LTP. The lower frequencydependency of NMDA-VGCC LTP at pp-CA1 synapses compared to pp-DG synapses may comprise a mechanism to prioritize information processing at this synapse. This article is part of a Special Issue entitled: Hippocampus. Ó 2015 The Authors. Published by Elsevier Ltd. on behalf of IBRO. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Department of Neurophysiology, Medical Faculty, Ruhr University Bochum, Germany International Graduate School for Neuroscience, Ruhr University Bochum, Germany
Abstract—The hippocampal CA1 region receives cortical information via two main inputs: directly via the perforant (temporoammonic) path (pp-CA1 synapse) and indirectly via the tri-synaptic pathway. Although synaptic plasticity has been reported at the pp-CA1 synapse of freely behaving animals, the mechanisms underlying this phenomenon have not been investigated. Here, we explored whether long-term potentiation (LTP) at the pp-CA1 synapse in freely behaving rats requires activation of N-methyl-D-aspartate receptors (NMDAR) and L-type voltage-gated calcium channels (VGCCs). As group II metabotropic glutamate (mGlu) receptors are densely localized on presynaptic terminals of the perforant path, and are important for certain forms of hippocampal synaptic plasticity, we also explored whether group II mGlu receptors affect LTP at the pp-CA1 synapse and/or regulate basal synaptic transmission at this synapse in vivo. In adult male rats, high-frequency stimulation (200 Hz) given as 3, or 10 trains, resulted in robust LTP that lasted for at least 4 h in pp-CA1 or pp-dentate gyrus (DG) synapses, respectively. Pre-treatment with the NMDAR antagonist D-( )-2-amino-5-phosphopentanoic acid (D-AP5) partially inhibited LTP at pp-CA1, and completely prevented LTP at pp-DG synapses. Combined antagonism of NMDAR using D-AP5 and the VGCC inhibitor, ( )-methoxyverapamil hydrochloride elicited a further inhibition of the LTP response at pp-CA1 synapses. Whereas activation of group II mGlu receptors using (1R,2R)-3-((1S)-1-amino-2-hydroxy2-oxoethyl) cyclopropane-1,2-dicarboxylic acid (DCG-IV) dose-dependently reduced basal synaptic transmission
Key words: perforant path, temporoammonic, plasticity, hippocampus, CA1, in vivo.
synaptic
INTRODUCTION The hippocampal formation is a highly plastic structure that is reciprocally connected to cortical brain regions, and engages in information processing and storage (Martin et al., 2000) by means of processes such as long-term potentiation (LTP) and long-term depression (LTD) (Kemp and Manahan-Vaughan, 2007). The main cortical input to the hippocampus originates from the entorhinal cortex (EC), where layer II neurons give rise to the tri-synaptic pathway, comprising the dentate gyrus (DG), CA3 and CA1 regions. In addition, EC layer III neurons directly connect to the CA1 region via the temporoammonic path, often referred as temporoammonic or perforant path to CA1 (pp-CA1) synapse (Witter et al., 1988). Thus, the CA1 region encounters pre-processed information arising from the CA3 region that is transferred via the Schaffer collateral-CA1 (Sc-CA1) synapse, as well as un-edited information that reaches it directly via the ppCA1 synapse. This information might serve as an efference copy for newly stored information in the CA1 region (Lisman and Otmakhova, 2001) or subserve mismatch and/or error detection (Izumi and Zorumski, 2008). Various learning and memory forms are thought to be encoded via differential involvement of persistent synaptic plasticity mechanisms that require activation of specific molecular cascades (Martin and Morris, 2002; Kemp and Manahan-Vaughan, 2004; Lisman et al., 2012). Induction of LTP in the CA1 region typically involves
*Correspondence to: D. Manahan-Vaughan, Department of Neurophysiology, Medical Faculty, Ruhr University Bochum, MA 4/ 150, Universitaetsstr. 150, 44780 Bochum, Germany. Tel: +49-234322-2042; fax: +49-234-32-14490. E-mail address: [email protected] (D. Manahan-Vaughan). Abbreviations: ANOVA, analysis of variance; AP, anterioposterior; DAP5, D-( )-2-amino-5-phosphopentanoic acid; DCG-IV, (1R,2R)-3((1S)-1-amino-2-hydroxy-2-oxoethyl) cyclopropane-1,2-dicarboxylic acid; DG, dentate gyrus; EC, entorhinal cortex; fEPSP, field excitatory post-synaptic potential; HFS, high-frequency stimulation; LTD, long-term depression; LTP, long-term potentiation; mGlu, metabotropic glutamate; ML, mediolateral; NMDAR, N-methyl-Daspartate receptors; pp, perforant path; Sc-CA1, Schaffer collateralCA1; STP, short-term potentiation; VGCCs, voltage-gated calcium channels.
http://dx.doi.org/10.1016/j.neuroscience.2015.03.014 0306-4522/Ó 2015 The Authors. Published by Elsevier Ltd. on behalf of IBRO. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1
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postsynaptic calcium ion entry via the ionotropic, glutamatergic N-methyl-D-aspartate receptors (NMDAR) and subsequent activation of protein kinases (Luscher and Malenka, 2012). However, depending on the stimulation strength Sc-CA1 and pp-DG synapses are capable of expressing LTP in the presence of NMDARantagonists, in a process that involves L-type voltagegated calcium channels (VGCC) (Grover and Teyler, 1990; Manahan-Vaughan et al., 1998; Freir and Herron, 2003). In hippocampal slices it was also shown that ppCA1 synapses express NMDAR-dependent (Remondes and Schuman, 2002) as well as VGCC-dependent LTP (Remondes and Schuman, 2003). However, very little data are available that describes the processes that underlie LTP induction at the pp-CA1 synapse in the intact brain of freely behaving rats (Aksoy-Aksel and ManahanVaughan, 2013). Recently, we reported that compared to Sc-CA1 synapses, the threshold for induction of LTP is lower at pp-CA1 synapses in vivo and these synapses are comparatively resistant to strong stimulation protocols (Aksoy-Aksel and Manahan-Vaughan, 2013). In addition to ionotropic glutamate receptors, metabotropic glutamate (mGlu) receptors play an important role in hippocampal synaptic plasticity and in hippocampal basal synaptic tonus (Mukherjee and Manahan-Vaughan, 2013). Group II mGlu receptors (mGluR2/3) are G-protein receptors that are negatively coupled to adenylyl cyclase and are densely located in a perisynaptic manner on the perforant path and on mossy fiber boutons in the hippocampal formation (Shigemoto et al., 1997). Activation of group II mGlu receptors reduces basal synaptic transmission in the pp-DG and mossy fiberCA3 synapses in a dose-dependent manner in freely behaving rats (Kulla et al., 1999; Hagena and ManahanVaughan, 2010). In vitro, the pp-CA1 synapse exhibits a higher sensitivity to the activation of group II mGlu receptors compared to the Sc-CA1 synapse (Speed and Dobrunz, 2009) suggesting that they play a role in the regulation of synaptic plasticity thresholds. The capacity of a synapse to express different forms of potentiation offers insights into its role in behavioral phenomena such as learning and memory. Different demands for information encoding, retrieval or extinction, for example, might be mediated through differing requirements for NMDAR-dependent and VGCCdependent LTP (Borroni et al., 2000; Bauer et al., 2002; Moosmang et al., 2005; Davis and Bauer, 2012). Furthermore, very long-lasting forms of synaptic plasticity are linked to the activation of mGlu receptors and to longterm spatial memory (Mukherjee and Manahan-Vaughan, 2013). Considering that the temporoammonic input to CA1 is believed to play an important role in spatial recognition memory (Brun, 2002) and long-term memory maintenance (Remondes and Schuman, 2004) we investigated whether LTP at pp-CA1 synapses is NMDAR, VGCC or group II mGlu receptor dependent, and whether synaptic transmission is regulated by group II mGlu receptors. We observed that robust LTP at the pp-CA1 synapse in vivo is induced with less afferent stimulation than LTP at pp-DG synapses. Furthermore, LTP at pp-CA1 synapses is only partially affected by NMDAR antagonism, whereas LTP at the
pp-DG synapse is completely abolished. Moreover, NMDAR-independent LTP at the pp-CA1 synapse is sensitive to a VGCC antagonist. We also observed that prior agonist activation of group II mGlu receptors using a dose that does not affect basal synaptic transmission prevents persistent LTP at pp-DG synapses, but has had no effect on LTP at pp-CA1 synapses. These data suggest that synaptic plasticity at the pp-CA1 synapse is distinct to the pp-DG synapse. This may reflect its specific role in hippocampal information processing.
EXPERIMENTAL PROCEDURES Surgical procedures The present study was carried out in accordance with the European Communities Council Directive of September 22nd, 2010 (2010/63/EU) for care of laboratory animals and after approval of the local ethics committee (Bezirksamt Arnsberg, Germany). All efforts were made to reduce the number of animals used. Male Wistar rats (7–8 weeks old at the time of surgery) were implanted with recording and stimulation electrodes (polyurethane-coated stainless steel wire; 1 mm in diameter) into the right hippocampus and a cannula (length: 5.6 mm, diameter: 0.8 mm, Gu¨ndel Biomedical Instruments, Germany) into the right lateral cerebral ventricle, as described previously (Aksoy-Aksel and Manahan-Vaughan, 2013). The stereotaxic coordinates for electrode and cannula placements, were referenced to the bregma for anteroposterior (AP) and to midline for mediolateral (ML) distance. The coordinates were as follows: guiding cannula (AP: 0.5 mm; ML: +1.6 mm; DV: ca. 4 mm); recording electrode (AP: 3.0; ML: +2.0 mm) and stimulating electrode (AP: 6.9; ML: +4.1 mm) (Fig. 1). The accuracy of the electrode implantations was verified as described previously (Aksoy-Aksel and Manahan-Vaughan, 2013). In addition, another group of animals was implanted with a recording electrode in the granule cell layer of the DG (AP: 3.1, ML: +1.9 mm) and a stimulating electrode in the perforant path. Here, the same stimulating electrode coordinates were used as for the pp-CA1 preparation. For all synapses the depth of the electrodes was determined during the surgery by giving test-pulse stimulation to generate an field excitatory post-synaptic potential (fEPSP) while the recording electrode was lowered in stepwise decrements until a typical fEPSP was observed (AksoyAksel and Manahan-Vaughan, 2013). Electrophysiological recordings The in vivo experiments were carried out 7–10 days after the implantation of the electrodes. Synaptic transmission was recorded via test-pulse stimulation of the medial perforant path using a stimulation intensity that elicits ca. 40% of the maximum fEPSP obtained from a previously established input/output (I/O) relationship (100–900 lA) for each individual animal. Responses were evoked by stimulating at frequency of 0.025 Hz with a single biphasic square wave pulse of 0.2-ms stimulus duration and 10 kHz sample rate. For each time-point, five
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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Fig. 1. Potentials evoked in the CA1 region via perforant path stimulation exhibit distinct properties compared to perforant path-dentate gyrus synapses. Schema showing the position of the simulation electrode in the angular bundle (left) and the recording electrodes in either the Stratum lacunosum moleculare of the CA1 region or the granule cell layer of the dentate gyrus (middle). Right: Analog examples of field potentials evoked in the perforant path (pp)-CA1 synapse (top) or the pp-dentate gyrus synapse (bottom). The slope of the fEPSP elicited by perforant path stimulation was measured as the five steepest points obtained on the first negative deflection of the compound potential for the pp-CA1 synapse, or on the first positive deflection of the compound potential for the pp-dentate gyrus synapse (indicated by the line superimposed on the respective analog example).
sweeps of evoked responses were averaged. Changes in evoked potentials were assessed by measuring the fEPSP slope. For CA1 potentials this was measured as the maximum slope through the five steepest points from the beginning of the fEPSP and the first minimum located in the first deflection (Manahan-Vaughan, 1997) (Fig. 1). For DG potentials the fEPSP slope was measured as the maximum slope through the five steepest points from the beginning of the fEPSP and the first maximum (population spike onset) located in the first deflection (ManahanVaughan et al., 1998). Before being included in the study, the animals were assessed stability of their baseline responses during a ca. 5-h recording period. Instability of the responses was an exclusion criterion for the subsequent experiments. fEPSPs were recorded every 5 min for at least 30 min (six time points) before drug application and 30 min (six time points) after drug application. LTP at pp-CA1 synapses was induced using high-frequency stimulation (HFS) comprising 200-Hz stimulation given as three bursts of 15 stimuli (0.2-ms stimulus duration, 10-s interburst interval) as this protocol was previously shown to be effective in inducing LTP at pp-CA1 synapses in vivo (Aksoy-Aksel and Manahan-Vaughan, 2013). LTP at pp-DG synapses was induced with the stronger protocol of HFS comprising 200-Hz stimulation given as 10 bursts of 15 stimuli (0.2-ms stimulus duration, 10-s interburst interval). This protocol elicits LTP that lasts for over 24 h in freely behaving rats (Kulla et al., 1999) whereas the abovementioned protocol (for LTP induction at pp-CA1 synapses) induces short-term potentiation
(STP) at pp-DG synapses that lasts at most for ca. 2 h (Manahan-Vaughan et al., 1998). fEPSP recordings were made 5, 10 and 15 min postHFS followed by recordings every 15 min for a total of 4 h. Basal synaptic transmission was recorded in the same way but HFS was not applied. HFS was always given between 10 and 11 a.m. The cortical encephalogram (EEG) was monitored throughout the course of each experiment to verify that stimulation did not elicit neuronal disturbances such as epileptiform activity that could confound interpretation of the electrophysiological data. No cases of disturbances were observed. The animals’ arousal state was not affected by the stimulation. Animals were awake during HFS. Compounds and drug injection Pharmacological ligands were administered in a 5-ll volume to the lateral cerebral ventricle via the implanted cannula, and applied at a rate of 1 ll per min, control groups were injected with 5-ll vehicle. The NMDAR antagonist D-( )-2-amino-5-phosphopentanoic acid (DAP5, Biozol, Eching, Germany) was first dissolved in 5 ll of 1 N sodium hydroxide solution (NaOH), then 0.9% sodium chloride (NaCl) was added to made up a solution of 100-ll volume. The final amount injected was 100 nmol in an injection volume of 5 ll (Popkirov and Manahan-Vaughan, 2011). The L-type VGCC antagonist ( ) methoxyverapamil hydrochloride (methoxyverapamil) (Research Biochemicals International, Natick, USA) was dissolved in
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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distilled water and injected as 100 nmol/5 ll (ManahanVaughan et al., 1998; Po¨schel and Manahan-Vaughan, 2007). The group II mGluR agonist, (1R,2R)-3-((1S)-1-amino2-hydroxy-2-oxoethyl)cyclopropane-1,2-dicarboxylic acid (DCG-IV, Biozol, Eching, Germany) was dissolved in 0.9% NaCl solution and injected as 20 ng (low doseDCG-IV) or 40 ng (high dose-DCG-IV) (Klausnitzer and Manahan-Vaughan, 2008). The pH of the solutions was adjusted to 7. All compounds used had been previously assessed for their effects on synaptic plasticity at other hippocampal synapses in vivo (Manahan-Vaughan, 1997; Manahan-Vaughan et al., 1998; Popkirov and ManahanVaughan, 2011; Po¨schel and Manahan-Vaughan, 2007; Klausnitzer and Manahan-Vaughan, 2008). Data analysis For the analysis of the electrophysiological data, each time-point was calculated as the percentage of the average of the six recordings (pre-injection basal synaptic transmission values) for every subject individually. Between group statistics was done by a two-way analysis of variance with repeated measures (ANOVA) by comparing the whole train of measurements after the HFS. Post-hoc analysis with Fisher’s least significant difference (LSD) test was used to test the significance of individual values in plasticity experiments. P < 0.05 was considered significant.
RESULTS Antagonism of NMDAR significantly reduces synaptic potentiation at pp-CA1 synapses and abolishes synaptic potentiation at pp-DG synapses LTP induction requires activation of NMDAR at the Sc-CA1 (Nicoll and Malenka, 1999) and pp-DG synapses (Manahan-Vaughan et al., 1998). Here, we examined whether LTP at the pp-CA1 synapse of freely behaving rats depends on NMDAR in a similar manner to LTP at the pp-DG synapse. Stimulation of the perforant path to CA1 synapse with HFS at 200 Hz (three trains) resulted in LTP that lasted for at least 4 h (Fig. 2A). Intracerebroventricular (icv) injection of the NMDAR antagonist, D-AP5 30 min prior to HFS, significantly reduced the amplitude and the duration of synaptic potentiation (ANOVA, F(1,144) = 78.665; p < 0.001, n = 5; Fig. 2A). However, LTP was not fully abolished. Four hours after treatment a residual LTP was still evident, although this was significantly less compared to controls (Fig. 2A, p < 0.05). This dose of D-AP5 completely prevented LTP at the pp-DG synapse that was induced with a stronger stimulation protocol (200 Hz, ten bursts compared to three bursts used for pp-CA1 synapse) in agreement with previous findings (Manahan-Vaughan et al., 1998). Here, 4 h after HFS, LTP was completely absent in D-AP5-treated animals (n = 10) compared to vehicle-injected controls (n = 10, ANOVA, F(1,144) = 117.241; p < 0.001) (Fig. 2B). Thus, we further explored the neurotransmitter receptor requirements of the NMDAR-independent LTP at pp-CA1 synapses.
Fig. 2. LTP at the pp-CA1 synapse is significantly but incompletely prevented by NMDAR antagonism. LTP at pp-dentate synapses is completely abolished by NMDAR antagonism and curtailed by activation of group II mGlu receptors. (A) High-frequency stimulation (HFS) at 200 Hz (three bursts) elicited LTP at the pp-CA1 synapse of vehicle-treated freely behaving animals that endured for over 4 h. Treatment with an NMDAR antagonist (D-AP5) prior to HFS resulted in reduced synaptic potentiation. The graphs represent the mean value of the percentage ± standard error of the mean (SEM). Inset: Analog traces evoked from vehicle-treated (left) or D-AP5-treated animals (right), (1) before HFS, (2) 5 min after HFS and (3) 4 h after HFS. Vertical scale bar = 3 mV, horizontal scale bar = 5 ms. (B) HFS at 200 Hz given as ten trains induced very robust LTP at ppdentate gyrus synapses that was still present 4 h after HFS was given. Treatment of the animals with the same dose of D-AP5 30 min prior to HFS, completely abolished LTP. Treatment with a low dose of the group II mGluR agonist (DCG-IV) prior to HFS significantly prevented the late phase of LTP. Inset: Analog traces evoked from vehicle-treated animals, (1) before HFS and (2) 4 h after HFS. Vertical scale bar = 5 mV, horizontal scale bar = 5 ms.
Combined antagonism of NMDA receptors and L-type VGCC further reduces synaptic potentiation at ppCA1 synapses L-type VGCC contribute to LTP at the pp-DG synapse (Manahan-Vaughan et al., 1998) and Sc-CA1 synapse (Grover and Teyler, 1990) when elicited by substantially higher and stronger afferent stimulation (400 Hz for DG and 200 Hz for CA1). At the pp-CA1 synapse VGCCdependent LTP can be induced in vitro (Remondes and
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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Schuman, 2003). Thus, VGCC are likely to contribute to the NMDA-independent LTP at the pp-CA1 synapse in vivo. Application of the VGCC antagonist, methoxyverapamil in the presence of D-AP5 further diminished LTP at the pp-CA1 synapse compared to vehicle injection (ANOVA, F(1,143) = 49.582; p < 0.001, n = 5; Fig. 3). Thus, LTP at the pp-CA1 synapse depends on activation of both NMDAR and L-type VGCCs, whereby the frequency-dependency of the pp-CA1 synapse on NMDAR and L-type VGCCs is lower than that observed at pp-DG synapses in vivo (Manahan-Vaughan et al., 1998).
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at pp-CA1 synapses, we examined the effect of DCG-IV treatment on evoked potentials that were elicited by test-pulse stimulation. Administration of the low dose of DCG-IV (n = 5) (20 ng) had no effect on basal synaptic transmission compared to vehicle-treated controls (n = 5) (Fig. 4A). Increasing the dose of DCG-IV (40 ng) resulted in a transient depression of basal synaptic responses (n = 5) (Fig. 4A) (ANOVA, F(1,230) = 39.00; p < 0.001). The depression began about 10 min after injection (p < 0.05), peaked at about 1 h (p < 0.05), and had returned to pre-injection baseline values by ca. 2 h.
Activation of group II mGlu receptors dosedependently affects basal synaptic transmission at pp-CA1 synapses Previous findings show that group II mGlu receptors facilitate the expression of robust LTD at pp-DG synapses using doses that do not elicit significant changes in basal synaptic responses (ManahanVaughan, 1998), although a dose-dependent depression of basal synaptic transmission is also observed. This suggests that these receptors play a dual role in the DG, regulating basal tonus on the one hand, and engaging in metaplastic regulation (Abraham and Tate, 1997) on the other hand. To assess whether group II mGlu receptors contribute to the regulation of basal excitatory tonus
Fig. 3. LTP at the pp-CA1 synapse is significantly prevented by combined NMDAR and VGCC antagonism. High-frequency stimulation (HFS) at 200 Hz elicited LTP at the pp-CA1 synapse of vehicle-treated freely behaving animals that endured for over 4 h. Prior treatment with an NMDAR antagonist (D-AP5) together with an L-type VGCC antagonist (methoxyverapamil) potently inhibited LTP at the pp-CA1 synapse. Effects were significantly greater than those elicited with D-AP5 alone. The graphs represent the mean value of the percentages ± standard error of the mean (SEM). Inset: Analog traces evoked from vehicle-treated (left) or D-AP5/methoxyverapamil-treated animals (right), (1) before HFS, (2) 5 min after HFS and (3) 4 h after HFS. Vertical scale bar = 3 mV, horizontal scale bar = 5 ms.
Fig. 4. Agonist activation of group II metabotropic glutamate receptors dose-dependently depresses basal synaptic transmission at the pp-CA1 synapses in vivo, but does not affect LTP. (A) Basal synaptic transmission evoked by test-pulse stimulation was unaffected by intracerebroventricular application of the group II mGluR agonist, DCG-IV given as a dose (DCG-IV low) (at the time-point marked by the arrow). In contrast, a higher dose of DCG-IV (DCG-IV high) significantly depressed evoked responses. Effects were transient, and by ca. 2 h after DCG-IV-treatment, evoked potentials were equivalent to those obtained in vehicle-treated animals. Inset in: Analog traces evoked from vehicle-treated animals (left) or animals treated with high dose DCG-IV-treated (right), (1) prior to injection, (2) 5 min after injection and (3) 4 h after injection. Vertical scale bar = 3 mV, horizontal scale bar = 5 ms. (B) High-frequency stimulation (HFS) at 200 Hz (three trains) elicited LTP at the pp-CA1 synapse of vehicle-treated freely behaving animals that endured for over 4 h. Treatment with the low dose of the group II mGluR agonist (DCG-IV) prior to HFS did not affect LTP at the pp-CA1 synapse. Inset: Analog traces evoked from vehicle-treated animals (left) or animals treated with DCG-IV-treated (right), (1) prior to HFS, (2) 5 min after HFS and (3) 4 h after HFS Vertical scale bar = 3 mV, horizontal scale bar = 5 ms.
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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Activation of group II mGlu receptors does not affect LTP at pp-CA1 synapses but prevents the late phases of DG LTP Using the dose of DCG-IV that did not affect basal synaptic transmission at pp-CA1 synapses, and is known to have no effect on basal synaptic transmission at pp-DG synapses in vivo (Manahan-Vaughan, 1998), we explored if group II mGlu receptors directly contribute to LTP at pp-CA1 or pp-DG synapses. Administration of the low dose of DCG-IV significantly prevented the late phase of LTP (elicited by 200 Hz HFS given as 10 trains) at pp-dentate synapses (n = 10) compared to vehicle-treated controls (n = 10, ANOVA, F(1,144) = 87.221; p < 0.001) (Fig. 2B). Here, it should be noted that whereas LTP at pp-CA1 synapses can be induced by three trains of 200 Hz (Fig. 3B), this protocol elicits short-term potentiation at pp-DG synapses (Manahan-Vaughan et al., 1996). Robust LTP at these synapses requires afferent stimulation in the form of at least 200 Hz given as 10 trains (Kulla et al., 1999). By contrast, agonist activation of group II mGlu receptors (DCG-IV; low dose) had no significant effect on LTP at pp-CA1 synapses compared to vehicle controls (ANOVA, F(1,144) = 3.089; p > 0.05, n = 5; Fig. 4B). This suggests that the pp-CA1 synapse is distinct to the pp-DG synapse in terms of LTP regulation by group II mGlu receptors.
DISCUSSION In this study we report that LTP at the pp-CA1 synapse in vivo depends on NMDAR and L-type VGCC. Antagonism of NMDAR alone results in an incomplete inhibition of LTP at pp-CA1 synapses compared to completely abolished LTP at pp-DG synapses. Additionally, activation of group II mGlu receptors does not affect LTP at pp-CA1 synapses, but prevents LTP maintenance at pp-DG synapses at doses that do not affect basal transmission. Strikingly, however the ppCA1 synapse responds with a depression of evoked responses to higher doses of the agonist, as is the case for the DG (Manahan-Vaughan, 1998). The distinct characteristics of the neurotransmitter receptor-dependency of both LTP and synaptic transmission at pp-CA1 synapses may reflect the unique postulated role of this synapse in information processing. The CA1 region, as the last module in the network of the hippocampus proper, has been subjected to particular scrutiny in terms of the mechanisms that underlie basal synaptic tonus and synaptic plasticity. However, most of the information regarding CA1 plasticity has been gathered through studies of the Sc-CA1 synapse and relatively little is known about plasticity at the pp-CA1 synapse in intact brain. The main constraint to study the pp-CA1 synapse in vivo is possible signal contamination from the surrounding hippocampal synapses (pp-DG and Sc-CA1), as it is technically impossible to completely isolate the synapse in vivo in a similar way to approaches used in in vitro preparations. However, the distinct profile of the fEPSP elicited in the CA1
region via perforant path stimulation, along with its specific and distinct response to long term plasticity protocols distinguishes this synapse from the Sc-CA1 (Aksoy-Aksel and Manahan-Vaughan, 2013) or the ppDG synapse (the present study) in freely behaving animals. A persistent change in synaptic strength is thought to be the leading mechanism underlying the physiological basis of learning and memory (Martin and Morris, 2002) and has been studied extensively in relation to NMDAR activation (Luscher and Malenka, 2012). All hippocampal synapses, with the exception of the mossy fiber-CA3 synapse, express NMDAR-dependent LTP (4 h) in vivo. Although a small degree of short-term potentiation still remained under these conditions, it is unlikely that the concentration of the drugs used was insufficient to block either the NMDAR or VGCCs, as it was previously shown that higher concentrations of the NMDAR antagonist or the VGCC-blocker used does not further affect LTP expression (at least not for LTP induced by the 400 Hz stimulation at pp-DG synapses) in freely behaving rats (Manahan-Vaughan et al., 1998). The potentiation that remained after combined block of NMDAR and VGCCs might involve other receptor types such as Ni-sensitive R- and T-type VGCCs that are thought to contribute to the calcium influx at the distal CA1 dendrites (Magee et al., 1998; Isomura et al., 2002), mGlu receptors, as shown previously for the ppDG synapse in vivo (Manahan-Vaughan et al., 1998) or calcium permeable AMPA receptors (Yuste et al., 1999). The contribution of VGCCs to hippocampal LTP is highly frequency-dependent. In the DG, HFS at 400 Hz
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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is needed to induce LTP that depends on VGCCs, whereas NMDAR-dependent LTP is elicited by means of HFS at 200 Hz (Manahan-Vaughan et al., 1998; current study). In this context, it was also quite striking that LTP that endures for over 4 h could be induced with the HFS protocol we used here. This protocol elicits short-term potentiation at pp-DG synapses (Altinbilek and Manahan-Vaughan, 2009) and at Sc-CA1 synapses causes epileptiform activity in the cortical electroencephalogram that is coupled with a temporary loss of evoked potentials (Manahan-Vaughan, unpublished data). Thus, it was not possible to study NMDARindependent LTP in vivo at Sc-CA1 synapse since it was shown in vitro to require HFS at 200 Hz (Grover and Teyler, 1990; Morgan and Teyler, 1999; Freir and Herron, 2003), whereby typically LTP (that is wholly NMDAR-dependent) is elicited by HFS of up to 100 Hz (Manahan-Vaughan, 1997). This suggests that the ppCA1 synapse is less vulnerable than the Sc-CA1 synapse and has a lower threshold for NMDA-independent LTP induction than the pp-DG synapse. Why does the pp-CA1 synapse require VGCC activation to induce LTP? Transgenic mice deficient of a subunit of VGCC have impaired hippocampusdependent memory and late-LTP (Moosmang et al., 2005). Similarly, VGCC antagonism results in impaired long-term spatial memory (Borroni et al., 2000; Woodside et al., 2004; Da Silva et al., 2013). In Sc-CA1 and pp-DG synapses VGCCs only become involved in LTP when very strong stimulation patterns are used (Grover and Teyler, 1990; Manahan-Vaughan et al., 1998) in contrast to the pp-CA1 synapse. Disturbances of information transfer to the hippocampus via the temporoammonic pathway impair long-term memory (Remondes and Schuman, 2004; Nakashiba et al., 2008; Suh et al., 2011). The lower threshold for VGCCdependent LTP at this pathway may relate to the putative role of the pp-CA1 synapse in providing an efference copy of information that should undergo synaptic storage within the tri-synaptic circuit (Lisman and Otmakhova, 2001) or in error-detection (Izumi and Zorumski, 2008). It is conceivable that the lower threshold facilitates rapid and robust encoding of new information that is then used as a ‘‘blueprint’’ against which information that is encoded by LTP that originates in the tri-synaptic circuit, and which ultimately occurs at the Sc-CA1 synapse. Agonists of group II mGlu receptors inhibit LTP at the Sc-CA1 in vivo (Holscher et al., 1997) and pp-DG synapses both in vitro (Huang et al., 1997) and in vivo (present study). On the other hand, LTD at pp-DG and Sc-CA1 synapses requires these receptors (ManahanVaughan, 1997; Altinbilek and Manahan-Vaughan, 2009). Interestingly, metaplastic effects (Abraham and Tate, 1997) of group II mGlu receptors were not evident at the pp-CA1 synapse. In the present study we observed that in pp-DG synapses, priming of group II mGlu receptors using agonist doses that do not affect basal synaptic transmission have potent effects on the outcome of a subsequent attempt to induce LTP. The pp-CA1 synapse did not respond to this kind of agonist priming, suggesting
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that this receptor does not contribute to synaptic plasticity at this synapse in a similar way to pp-DG synapses. This observation is very striking in light of the relative resistance of the pp-CA1 synapses in vivo to expression of LTD (Aksoy-Aksel and Manahan-Vaughan, 2013). At other hippocampal synapses in vivo, group II mGlu receptors are critically involved in the expression and persistency of LTD (Manahan-Vaughan, 1997; Altinbilek and Manahan-Vaughan, 2009) and in specific forms of hippocampus-dependent spatial learning (Altinbilek and Manahan-Vaughan, 2009; Pitsikas et al., 2012), and regulate the thresholds for LTP induction (Kulla et al., 1999). The lack of involvement of group II mGlu receptors in LTP at pp-CA1 synapses set this synapse apart in a functional sense, from the synapses of the trisynaptic circuit. Activation of group II mGlu receptors reduces basal synaptic transmission in the Sc-CA1, pp-DG and mossy fiber-CA3 synapses in a dose-dependent manner in freely behaving rats (Manahan-Vaughan, 1997; Kulla et al., 1999; Hagena and Manahan-Vaughan, 2009). The mossy fiber-CA3 synapse has a particularly high sensitivity to the activation of group II mGlu receptors compared to the pp-DG synapse, a characteristic that is successfully used to identify the mossy fiber-CA3 synapse in vivo (Hagena and Manahan-Vaughan, 2010). A similar approach was used in vitro to identify the highly sensitive pp-CA1 synapse from the Sc-CA1 synapse (Kew et al., 2001, 2002; Speed and Dobrunz, 2009). In the present study we also observed that the group II mGlu receptor agonist reduces the basal synaptic transmission in a dose-dependent manner (ManahanVaughan, 1998; Klausnitzer and Manahan-Vaughan, 2008). This property implies that lack of effect of group II mGlu receptor activation on pp-CA1 LTP is synapse specific and not due to general lack of drug activity.
CONCLUSIONS This study suggests that the pp-CA1 synapse of freely behaving rats is quite distinct from other hippocampal synapses. It exhibits a lower LTP induction threshold than the pp-DG synapse (current study) and the Sc-CA1 synapse (Aksoy-Aksel and Manahan-Vaughan, 2013), and exhibits a dependency on both NMDAR and voltagedependent calcium channels that normally only emerges at higher afferent stimulation frequencies for LTP at ScCA1 (Freir and Herron, 2003) and pp-DG synapses (Manahan-Vaughan et al., 1998; and current study). Furthermore, basal synaptic transmission is dose dependently sensitive to regulation by group II mGlu receptors, however, these receptors do not contribute to LTP at the pp-CA1 synapse, at least from the perspective of metaplasticity, in contrast to the pp-DG synapse. The lower LTP induction threshold of the pp-CA1 synapse compared to the Sc-CA1 synapse, combined with its distinct neurotransmitter profile may comprise mechanisms whereby information processing at this synapse is distinguished from processing by the synapses of the other hippocampal subregions.
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
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CONFLICT OF INTEREST None. Acknowledgments—We gratefully acknowledge the technical assistance of Jens Colitti-Klausnitzer, Beate Krenzek and Lena Fabritius. We thank Nadine Kollosch for animal care. This work was funded by the German Research Foundation (DFG, SFB 874/B1).
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(Accepted 5 March 2015) (Available online xxxx)
Please cite this article in press as: Aksoy-Aksel A, Manahan-Vaughan D. Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.014
