Abstract

Hippocampal synaptic plasticity is expressed to very different extents in distinct rat strains in vivo. This may correlate with differences in learning ability. We investigated whether the metabotropic glutamate receptor mGluR5 contributes to differences in long-term potentiation (LTP) and learning in freely moving hooded Lister (HL) and Wistar rats. High-frequency tetanization (HFT) generated robust CA1 LTP in Wistar rats (> 24 h) and incremental potentiation in HL rats. The mGluR5 antagonist 2-methyl-6-(phenylethynyl) pyridine (MPEP; 1.8 μg), applied intracerebrally, impaired LTP from ∼60 min onwards in Wistar and from 24 h in HL rats. HFT generated LTP in the dentate gyrus (DG) of Wistar rats (> 24 h), which was blocked by MPEP, and MPEP-resistant short-term depression in HL rats. Training for 10 days in an eight-arm radial maze revealed no working memory differences, but better reference memory performance in Wistar compared with HL rats. Daily application of MPEP (1.8 μg) impaired working and reference memory in Wistar rats. In HL rats, working memory was impaired but reference memory was unaffected. Western blot analysis revealed lower expression of mGluR5 in HL compared with Wistar rats. MGluR1 expression was equivalent. These data reveal striking mGluR5-dependent differences in spatial learning in different rat strains, which correlate to synaptic plasticity and mGluR5 expression levels.

Introduction

Hippocampal synaptic plasticity, in the forms of long-term potentiation (LTP) and long-term depression (LTD), is believed to comprise the cellular mechanism of spatial learning in the mammalian brain (Bear, 1996; Kemp and Manahan-Vaughan, 2004). Observations that different rat strains express differing degrees of hippocampal synaptic plasticity (Manahan-Vaughan, 2000a) and spatial learning (Prior et al., 1997) provoke the question as to whether a correlation with spatial learning performance can be identified. The simplest hypothesis is that rats that express poor plasticity also exhibit a poor learning performance. However, the intrinsic ability of rodents to adapt to their environment and find effective survival strategies means that a direct relationship between, for example, poor plasticity and poor learning is often very difficult to detect.

Increasing the intellectual or environmental challenge may unmask learning deficits in poor plasticity expressers (D. Manahan-Vaughan and H. Schwegler, unpublished data). However, the cellular or molecular basis for such deficits has not yet been clarified. Strain-dependent differences in a multitude of cellular, biochemical and physiological mechanisms have been described in rodents (Prior et al., 1987; Strohl et al., 1997; Honda et al., 1990; Harris and Nestler, 1996), and it is clear that no single factor will explain the differences seen. On the other hand, differences in intrinsic molecular features of synaptic plasticity between rat strains may well play a very significant role in the degree of expression of plasticity and learning capability in differing strains.

In previous studies, we have found that very dramatic differences exist in the ability to express synaptic plasticity of hooded Lister and Wistar rats. In the CA1 region, low-frequency stimulation at 1 Hz generates LTD in Wistar and short-term depression (STD) in hooded Lister rats (Manahan-Vaughan and Braunewell, 1999; Manahan-Vaughan, 2000a,b). High-frequency stimulation at 100 Hz generates a much more robust and larger LTP in the CA1 region of Wistar compared with hooded Lister rats (Manahan-Vaughan and Braunewell, 1999). Differences in dentate gyrus plasticity in these strains have not yet been characterized; nor has the issue been addressed that a correlation may exist between learning performance and the expression of plasticity in the two strains. In the present study we investigated both of these aspects. In addition, we attempted to address the molecular basis for the differences seen. The role of group I metabotropic glutamate receptors (mGluRs) in hippocampal plasticity in vivo is well documented (Manahan-Vaughan, 1997; Manahan-Vaughan et al., 1998; Balschun et al., 1999). In particular, a role for the group I mGluR subtype mGluR5 in hippocampal synaptic plasticity and spatial learning is evident (Naie and Manahan-Vaughan, 2004a). We examined whether differences in the function or expression of this receptor subtype could contribute to differences in learning and plasticity seen in these strains. We discovered that mGluR5 may be of intrinsic importance in determining the learning proficiency and synaptic efficacy of the hippocampus.

Materials and Methods

Electrode Implantation

Seven- to eight-week-old male Wistar rats underwent electrode implantation into the dentate gyrus as described previously (Manahan-Vaughan et al., 1998; Kulla and Manahan-Vaughan, 2000). Briefly, under sodium pentobarbitone anaesthesia (‘Nembutal’, 40 mg/kg, i.p., Serva, Germany), animals underwent implantation of a monopolar recording and a bipolar stimulating electrode (made from 0.1 mm diameter Teflon-coated stainless steel wire). A cannula was implanted into the lateral cerebral ventricle (0.08 mm posterior to bregma, 1.6 mm lateral to the midline) through which drug or vehicle injections were subsequently made.

For CA1 implantations, a drill hole was made (1.0 mm diameter) for the recording electrode (2.8 mm posterior to bregma, 1.8 mm lateral to the midline) and a second drill hole was made for the stimulating electrode (3.1 mm posterior to bregma, 3.1 mm lateral to the midline). The dura was pierced through both holes and the recording and stimulating electrode lowered into the CA1 stratum radiatum and Schaffer collaterals respectively. For dentate gyrus implantations, a drill hole was made (1.0 mm diameter) for the recording electrode (2.8 mm posterior to bregma, 1.8 mm lateral to the midline) and a second drill hole (1.5 mm diameter, 6.9 mm posterior to bregma and 4.1 mm lateral to the midline) for the stimulating electrode. The dura was pierced through both holes and the recording and stimulating electrode lowered into the dentate gyrus granule cell layer and the medial perforant path respectively.

Recordings of evoked field potentials via the implanted electrodes were taken throughout surgery. A cannula was also implanted into the ipsilateral cerebral ventricle, through which drug application was made. Once verification of the location of the electrodes was complete, the entire assembly was sealed and fixed to the skull with dental acrylic (Paladur, Heraeus Kulzer GmbH, Germany). The animals were allowed between 7 and 10 days to recover from surgery before experiments were commenced. Experiments were carried out using 9- to 13-week-old rats. Throughout experiments the animals could move freely. Experiments were consistently conducted at the same time of day (commencing 9.00 a.m.). Baseline experiments to confirm stability of evoked responses were routinely carried out (at least 24 h) before LTP experiments were conducted. Where possible, the animals served as their own controls. Thus, basal synaptic transmission (in the absence of injection) was monitored over a 24 h period in all animals to confirm stability of evoked responses. Subsequently, a control experiment (e.g. assessment of basal synaptic transmission) was carried out in the presence of vehicle injection and ∼1 week later the same experiment was carried out in the same animal in the presence of a drug injection.

Measurement of Evoked Potentials

Responses were evoked by stimulating at low frequency (0.025 Hz, 0.2 ms stimulus duration, 10 000 Hz sample rate). For each time-point, five evoked responses were averaged.

For the CA1 region, the field excitatory post-synaptic potential (fEPSP) slope was monitored and determined as the maximal slope through the five steepest points obtained on the first negative deflection of the potential. Both the fEPSP slope and the population spike (PS) amplitude were monitored for the dentate gyrus. The amplitude of PS was measured from the peak of the first positive deflection of the evoked potential to the peak of the following negative potential. The fEPSP slope was measured as the maximal slope through the five steepest points obtained on the first positive deflection of the potential. By means of input/output curve determination, the maximum PS amplitude was found for each individual animal, and all potentials employed as baseline criteria were evoked at a stimulus intensity which produced 40% of this maximum.

LTP in the CA1 region was induced by a high frequency tetanus (HFT) of 100 Hz (10 bursts of 10 stimuli, 0.1 ms stimulus duration, 10 s interburst interval). The stimulus amplitude for both protocols was the same as that used for test-pulse recordings. LTP in the dentate gyrus was induced by an HFT of 200 Hz (10 bursts of 15 stimuli, 0.2 ms stimulus duration, 10 s interburst interval) using an identical stimulus amplitude as that used for test-pulse recordings.

The cortical electroencephalogram (EEG) was monitored throughout the course of each experiment; however, no alteration in EEG was seen as a result of HFT or drug application.

Compounds and Drug Treatment

The metabotropic glutamate receptor antagonist 2-methyl-6-(phenylethynyl) pyridine (MPEP) was obtained from Tocris Cookson Ltd (Bristol, UK). For injection, MPEP was dissolved in 0.9% NaCl. MPEP or vehicle were injected in a 5 μl volume over a 6 min period via a Hamilton syringe. Antagonist injection was carried out 30 min prior to tetanization (or learning analysis in the case if radial maze experiments) to enable diffusion from the lateral cerebral ventricle to the hippocampus to occur (Manahan-Vaughan et al., 1998).

Throughout the experiments, injections were administered following measurement of the baseline for 30 min. In LTP experiments, a tetanus was applied 30 min following injection, with measurements then taken at t = 2, 5, 10 and 15 min, and then 15 min intervals up to 4 h, with additional measurements taken after 24 h.

Data Analysis

The baseline fEPSP or PS data were obtained by averaging the response to stimulating the perforant path, to obtain five sweeps at 0.025 Hz, every 5 or 15 min, as described above. The data were then expressed as mean percent pre-injection baseline reading ± SEM. Statistical significance was estimated using analysis of variance (ANOVA) with repeated measures, followed by post-hoc Student's t-tests. The probability level interpreted as statistically significant was P < 0.05.

As no differences in the direction of change or significance of effects was found with regard to dentate gyrus PS amplitude and fEPSP slope, the PS amplitude data were used for graphic representation.

Behavioural Experiments

The Radial Maze

Experiments were conducted as described previously (Naie and Manahan-Vaughan, 2004a). The radial maze consisted of a central octagonal platform (26 cm in diameter) from which eight arms (67 cm long, 20 cm deep, 10 cm wide) radiated. The floor of the maze was made of dark grey polyvinylchloride, whereas the walls were made of transparent Plexiglas. The maze was elevated 80 cm above floor level. At the end of each arm was a small circular indentation (1 cm deep, 3 cm diameter), ∼3.5 cm from the tip. In the centre of this indentation was a 3 mm deep ‘food cup’ (3 mm diameter) in which a 45 mg food pellet could be placed. The indentation served to prevent visibility of the food pellet from the centre of the maze. The sides but not the ends of the arms were walled.

The experimental room was brightly lit and had white walls which were decorated with conspicuous extra-maze cues. For example, on one of the white walls a black cross was placed (30 × 20 cm, with the arms of the cross having a diameter of 10 cm) while on another wall two large black rectangles were present (50 × 70 cm, 10 cm apart). The cues remained constant throughout the study. The maze was placed centrally in the room.

Experimental Procedure

Nine- to thirteen-week-old old male Wistar rats which had undergone implantation of an injection cannula were used for the behavioural study.

For 2 days before commencement of radial maze training, animals were habituated to the maze. Food pellets were placed at the end of each radial maze arm. Animals were taken individually from their home cages and placed in the centre of the maze for 15 min. Rats had access to all arms and could eat the pellets ad libitum. During these habituation days home cage food access was reduced so that animal weight decreased by 10–15% of it pre-habituation levels.

On training days four arms were baited with a single food pellet (‘Dustless Precision Pellet’, Bioserv, Frenchtown, NJ). For each animal a different constellation of baited arms was randomly chosen. This constellation remained constant throughout the 10 days of training. The trial commenced with placement of the animal on the central platform. A trial was deemed finished as soon as the food pellets had been found or when 15 min had elapsed: whichever occurred first. Once retrieved by the animals, the food pellets were not replaced. The number of arm entries was recorded until the trial was finished. The exact position of the entered arms was noted, together with the time spent in the maze, the frequency of freezing and number of rearings. At the end of each trial the number of fecal boli was counted and the maze was cleaned. In order to avoid the use of intramaze cues (odor trails etc) the maze was rotated by 45° after each training day.

Thirty minutes prior to the commencement of each trial, drug or vehicle was injected in a volume of 5 μl via the lateral cerebral ventricle using exactly the same procedure as for electrophysiological experiments.

Performance Scoring

Entry into an unbaited arm or entry into a baited arm without removing the food pellet was scored as a reference memory error. This reflects memory of information which remains constant across trials (Eichenbaum, 2001) and is a measure of long-term memory. Re-entry into a baited arm from which the food pellet had already been retrieved or re-entry into an unbaited arm was scored as a working memory error. This mirrors the ability of the animal to retain information for the duration of the trial, i.e. to temporarily hold information online and is a measure of short-term memory (Eichenbaum, 2001).

Animal activity (locomotion) was determined by a simple calculation based on the amount of time spent in the maze and the number of arms crossed: (no. of arms entered × 160)/(time (in s) spent in maze), where 160 equals the length of the maze from arm tip to opposite arm tip.

Rearing, grooming and fecal boli were quantitatively assessed. Thus, number of rears, grooms and fecal boli were counted and compared between drug- and vehicle-treated groups.

Data Analysis

The Mann–Whitney U-test was used to assess between group differences for individual trials. An ANOVA with repeated measures was used to assess behavior across days of training. The Wilcoxon matched pairs signed rank test was used to evaluate within-group differences. The probability level interpreted as statistically significant was P < 0.05.

Western Blot Analysis of mGluR Receptor Expression in the Hippocampus

Animals were anesthetized with ether and, following decapitation, both hippocampi were removed. The tissue was immediately placed in methyl butane at −40°C for 2 min, followed by refrigeration at −80°C until biochemical analysis took place. The expression of mGluR proteins was assessed by Western blot analysis using specific antibodies raised against synthetic peptides corresponding to the 21 carboxy-terminal residues of mGluR5 receptors (one-letter code: KSSPKYDTLIIRDYTNSSSSL) to label both mGluR5a and -b receptors, and the 21 carboxy-terminal residues of mGluR1a receptors (KPNVTYASVILRDYKQSSSTL) to label mGluR1a receptors (UpstateBiotechnology, Lake Placid, NY); and a monoclonal antibody to label β-actin (Sigma, St Louis, MO).

Hippocampi were homogenized at 4°C in Tris–HCl buffer (20 mM, pH 7.4) containing 10% sucrose. Homogenates were centrifuged at 1000 g for 5 min and the resulting supernatant was centrifuged at 16 000 g for 30 min to obtain the P2 fraction. Pellets were resuspended in ice-cold Tris–HCl buffer, pH 7.4, containing protease inhibitors (Boehringer Mannheim, Germany). Proteins were resuspended in Laemmli buffer and an aliquot was used for protein determinations with Amido black stained cellulose acetate stripes. Western blot analysis was carried out using 8% sodium dodecylsulfate–polyacrylamide minigels which were electroblotted on PVDF membranes (Roth, Germany) for 1 h. Filters were blocked for 1 h in TBST buffer (100 mM Tris–HCl; 0.9% NaCl, 1% Tween 20, pH 7.4) containing 2% non-fat dry milk. Blots were incubated overnight at 4°C with specific primary polyclonal antibodies and the immunoreactivity was visualized using HRP-coupled goat anti-rabbit or goat anti-mouse secondary antibodies (Dianova, Hamburg, Germany) and the enhanced ECL detection system (Amersham Biosciences, Freiburg, Germany). Quantification of Western blots was done by densitometric analysis using the NIH Image program v1.61. The ratio of the densities for mGluR1 or mGluR5 and β-actin in controls was set to 100%. The relative expression level of mGluR1 and 5 was expressed as the ratio of the densities for mGluR1 or mGluR5 and β-actin in the experimental conditions in % of control ratio. The relative expression levels are expressed as mean values ± SD. The statistical significance of the differences was determined by Student's t-test. Values of P < 0.05 were considered as statistically significant.

Results

Long-term Potentiation is Significantly Different in the CA1 Region of Wistar and Hooded Lister Rats

High-frequency tetanization at 100 Hz (10 bursts of 15 stimuli, 0.2 ms stimulus duration) in the presence of vehicle resulted in robust LTP in the CA1 region of Wistar rats in vivo (n = 8, Fig. 1). Responses persisted for longer than the 25 h observation period. Hooded Lister rats responded to HFT with a slowly developing potentiation which reached maximal levels by ∼90 min post-HFT (n = 10, Fig. 1) and was persistent over the 25 h recording period. The magnitude of the initial potentiation was significantly smaller than that seen in Wistar rats (P < 0.05). Basal synaptic transmission was stable in both Wistar (n = 6) and hooded Lister (n = 6) rat strains throughout the course of the 25h experiment (data not shown). These data are consistent with previously described results (Manahan-Vaughan, 2000a).

Figure 1.

Wistar and hooded Lister rats express different types of LTP in the CA1 region in response to high-frequency stimulation. Antagonism of mGluR5 dose-dependently impairs long-term potentiation in both rat strains. (A) 100 Hz HFS in the presence of vehicle (n = 8) results in a robust long-term potentiation in the CA1 region of Wistar rats. Administration of the mGluR5 antagonist MPEP (1.8 μg, n = 5) to Wistar rats inhibits LTP compared with vehicle injected controls (A, B). Effects become evident ∼60 min post-HFT. Administration of MPEP (3.6 μg, n = 4) abolishes LTP compared with vehicle injected controls (A, B). (B) 100 Hz HFS elicits incremental LTP in hooded Lister rats. Maximal LTP is reached by ∼60 min post-HFS (n = 10). Administration of MPEP (1.8 μg, n = 4) to hooded Lister rats has no effect on LTP induction but an impairment of LTP 24 h after HFS is evident. Administration of MPEP (3.6 μg, n = 6) inhibits LTP compared with vehicle injected controls (A, B). Effects become evident ∼3 h post-HFT. (C) Original analog traces showing the field potentials evoked from the CA1 pre-HFS, 5 min, 4 h and 24 h following HFT in the presence of (i) vehicle or (ii) MPEP (3.6 μg) in Wistar rats. (D) Original analog traces showing the field potentials evoked from the CA1 pre-HFS, 5 min, 4 h and 24 h following HFT in the presence of (i) vehicle or (ii) MPEP (3.6 μg) in hooded Lister rats. Vertical scale bar corresponds to 5 mV, horizontal scale bar to 4 ms.

Figure 1.

Wistar and hooded Lister rats express different types of LTP in the CA1 region in response to high-frequency stimulation. Antagonism of mGluR5 dose-dependently impairs long-term potentiation in both rat strains. (A) 100 Hz HFS in the presence of vehicle (n = 8) results in a robust long-term potentiation in the CA1 region of Wistar rats. Administration of the mGluR5 antagonist MPEP (1.8 μg, n = 5) to Wistar rats inhibits LTP compared with vehicle injected controls (A, B). Effects become evident ∼60 min post-HFT. Administration of MPEP (3.6 μg, n = 4) abolishes LTP compared with vehicle injected controls (A, B). (B) 100 Hz HFS elicits incremental LTP in hooded Lister rats. Maximal LTP is reached by ∼60 min post-HFS (n = 10). Administration of MPEP (1.8 μg, n = 4) to hooded Lister rats has no effect on LTP induction but an impairment of LTP 24 h after HFS is evident. Administration of MPEP (3.6 μg, n = 6) inhibits LTP compared with vehicle injected controls (A, B). Effects become evident ∼3 h post-HFT. (C) Original analog traces showing the field potentials evoked from the CA1 pre-HFS, 5 min, 4 h and 24 h following HFT in the presence of (i) vehicle or (ii) MPEP (3.6 μg) in Wistar rats. (D) Original analog traces showing the field potentials evoked from the CA1 pre-HFS, 5 min, 4 h and 24 h following HFT in the presence of (i) vehicle or (ii) MPEP (3.6 μg) in hooded Lister rats. Vertical scale bar corresponds to 5 mV, horizontal scale bar to 4 ms.

The mGluR5 Receptor Antagonist MPEP Dose-dependently Inhibits LTP in the CA1 Region of Wistar and Hooded Lister Rat Strains

MPEP (1.8 μg, n = 5) when applied 30 min prior to HFT in Wistar rats had no effect on the initial magnitude of potentiation (5 min post-HFT) when compared with vehicle-injected animals (n = 8). However, by roughly 60 min after tetanization the fEPSP slope and the PS amplitude had significantly decreased compared with LTP levels in vehicle-injected controls (t-test, P < 0.001, Fig. 1) [ANOVA: between-factor, F(1,35) = 26.854 P < 0.0001]. Raising the concentration of MPEP to 3.6 μg (n = 4) caused a complete abolition of LTP evoked by HFT compared with vehicle-injected controls [ANOVA: between-factor, F(1,35) = 41.169 P < 0.0001; Fig. 1].

In hooded Lister rats, the application of MPEP (1.8 μg, n = 4) did not affect the profile of LTP induction or intermediate maintenance compared with vehicle-injected controls (Fig. 1). However, a significant impairment of LTP 24 h after induction was seen in MPEP-treated animals. Application of the higher concentration of 3.6 μg had an effect on intermediate and late-LTP (n = 6). Thus, by 3 h post-HFT, a significant impairment of LTP occurred (t-test, P < 0.01) compared with vehicle-injected controls (Fig. 1) [ANOVA: between-factor, F(1,35) = 23.109, P < 0.0001].

MPEP (1.8 or 3.6 μg) had no effect on basal synaptic transmission in Wistar (n = 6) or hooded Lister rats (n = 6) compared with vehicle-injected Wistar (n = 5) or hooded Lister (n = 6) controls (data not shown).

Long-term Potentiation is Significantly Different in the Dentate Gyrus of Wistar and Hooded Lister Rats

HFT at 200 Hz (10 bursts of 15 stimuli, 0.2 ms stimulus duration) resulted in robust LTP in the dentate gyrus of Wistar rats in vivo (n = 6, Fig. 2). Responses persisted for longer than the 25 h observation period. However, hooded Lister rats responded with short-term potentiation (STP) to an identical stimulation protocol (n = 6, Fig. 2). In addition, the magnitude of the initial potentiation was significantly smaller than that seen in Wistar rats (t-test, P < 0.05 for both PS amplitude and fEPSP slope). Approximately 2.5 h after tetanization the fEPSP slope and the PS amplitude of hooded Lister rats had decreased back to basal levels. Basal synaptic transmission was stable in both Wistar (n = 6) and hooded Lister (n = 6) rat strains throughout the course of the 25 h experiment (data not shown).

Figure 2.

Wistar and hooded Lister rats express different degrees of potentiation in the dentate gyrus in response to high-frequency stimulation. Antagonism of mGluR5 impairs long-term potentiation in Wistar but not short-term potentiation in hooded Lister rats. (A) 200 Hz HFT in the presence of vehicle (n = 6) results in a robust long-term potentiation of both PS (A) and fEPSP (not shown) in the dentate gyrus of Wistar rats. Administration of the mGluR5 antagonist MPEP (1.8 μg, n = 6) to Wistar rats inhibits LTP compared with vehicle injected controls (A, B). Effects become evident ∼2.5 h post-HFT. (B) 200 Hz HFT elicits short-term potentiation of both PS (B) and fEPSP (not shown) in hooded Lister rats (n = 6). Administration of MPEP (1.8 μg, n = 6) to hooded Lister rats has no effect on STP. (C) Original analog traces showing the field potentials evoked from the CA1 pre-HFS, 5 min, 4 h and 24 h following HFT in the presence of (i) vehicle or (ii) MPEP (1.8 μg) in Wistar rats. (D) Original analog traces showing the field potentials evoked from the CA1 pre-HFS, 5 min, 4 h and 24 h following HFT in the presence of (i) vehicle or (ii) MPEP (1.8 μg) in hooded Lister rats. Vertical scale bar corresponds to 5 mV, horizontal scale bar to 4 ms.

Figure 2.

Wistar and hooded Lister rats express different degrees of potentiation in the dentate gyrus in response to high-frequency stimulation. Antagonism of mGluR5 impairs long-term potentiation in Wistar but not short-term potentiation in hooded Lister rats. (A) 200 Hz HFT in the presence of vehicle (n = 6) results in a robust long-term potentiation of both PS (A) and fEPSP (not shown) in the dentate gyrus of Wistar rats. Administration of the mGluR5 antagonist MPEP (1.8 μg, n = 6) to Wistar rats inhibits LTP compared with vehicle injected controls (A, B). Effects become evident ∼2.5 h post-HFT. (B) 200 Hz HFT elicits short-term potentiation of both PS (B) and fEPSP (not shown) in hooded Lister rats (n = 6). Administration of MPEP (1.8 μg, n = 6) to hooded Lister rats has no effect on STP. (C) Original analog traces showing the field potentials evoked from the CA1 pre-HFS, 5 min, 4 h and 24 h following HFT in the presence of (i) vehicle or (ii) MPEP (1.8 μg) in Wistar rats. (D) Original analog traces showing the field potentials evoked from the CA1 pre-HFS, 5 min, 4 h and 24 h following HFT in the presence of (i) vehicle or (ii) MPEP (1.8 μg) in hooded Lister rats. Vertical scale bar corresponds to 5 mV, horizontal scale bar to 4 ms.

The mGluR5 Receptor Antagonist MPEP Inhibits LTP in the Dentate Gyrus of in Wistar Rats and Has No Effect on STP in Hooded Lister Rat Strains

MPEP (1.8 μg, n = 6), when applied 30 min prior to HFT in Wistar rats, had no effect on the initial magnitude of potentiation (5 min post-HFT) when compared with vehicle-injected animals (n = 6). However, by roughly 3 h after tetanization the fEPSP slope and the PS amplitude had significantly decreased compared with LTP levels in vehicle-injected controls (t-test, P < 0.01, Fig. 2) [ANOVA: between-factor, F(1,35) = 37.966, P < 0.001 for PS; F(1,35) = 40.021, P < 0.001 for fEPSP]. These findings are consistent with previous observations from our laboratory (Naie and Manahan-Vaughan, 2004a).

In hooded Lister rats, the application of MPEP (1.8 μg, n = 5) had no effect on the profile of STP seen compared with vehicle-injected controls (n = 6, Fig. 2), although a tendency towards improvement of synaptic strength was evident [ANOVA: between-factor, F(1,35) = 2.617 P > 0.05 for PS; F(1,35) = 0.414, P > 0.05 for fEPSP].

MPEP (1.8 or 3.6 μg) had no effect on basal synaptic transmission in Wistar (n = 6) or hooded Lister rats (n = 6) compared with vehicle-injected Wistar (n = 5) or hooded Lister (n = 6) controls (data not shown).

Behavioral activity, but not spatial learning performance, is significantly different in Wistar and hooded Lister rats. A tendency towards differences in reference memory performance is evident

Learning performance in an eight-arm radial maze was assessed for the Wistar (n = 8) and hooded Lister (n = 11) rat strains (Fig. 3). Overall working memory performance was equivalent in both strains (Mann–Whitney U-analysis; Table 1a). Whereas Wistar rats exhibited a clear learning curve for reference memory, performance appeared unchanged throughout trial days for hooded Lister rats, however (Fig. 3).

Figure 3.

Working memory performance is comparable in Wistar and hooded Lister rats. Reference memory performance reveals differences occurring on the final trial day. (A) Animals were placed in an eight-arm radial maze with four baited arms. (B) Reference memory performance improved in Wistar rats across trial days. Reference memory performance remained relatively constant for hooded Lister rats throughout the trial days. On day 10, a significantly better reference memory performance was evident in Wistar rats. Whereas Wistar rats show a better reference memory performance when day 10 is compared with day 1 or day 2, hooded Lister rats did not improve performance across trial days. Graph shows average reference memory errors on each day of the study. (C) Working memory performance was not significantly different in Wistar and hooded Lister rats across trial days. Graph shows average working memory errors on each day of the study.

Figure 3.

Working memory performance is comparable in Wistar and hooded Lister rats. Reference memory performance reveals differences occurring on the final trial day. (A) Animals were placed in an eight-arm radial maze with four baited arms. (B) Reference memory performance improved in Wistar rats across trial days. Reference memory performance remained relatively constant for hooded Lister rats throughout the trial days. On day 10, a significantly better reference memory performance was evident in Wistar rats. Whereas Wistar rats show a better reference memory performance when day 10 is compared with day 1 or day 2, hooded Lister rats did not improve performance across trial days. Graph shows average reference memory errors on each day of the study. (C) Working memory performance was not significantly different in Wistar and hooded Lister rats across trial days. Graph shows average working memory errors on each day of the study.

Table 1

Mann–Whitney U-test analysis of working and reference memory performance in Wistar and hooded Lister rats in an eight-arm radial maze

Trial day
 
n1
 
n2
 
U
 
P
 
(a) Comparison of working memory errors in Wistar and hooded Lister rats across the 10 days of trials in the eight-arm radial maze     
10 32 0.315 
10 22 0.065 
10 23 0.079 
10 25 0.113 
10 22 0.065 
10 24.5 0.095 
10 40 0.719 
10 35.5 0.447 
10 21 0.053 
10 10 38 0.603 
(b) Comparison of reference memory errors in Wistar and hooded Lister rats across the 10 trial days in the eight-arm radial maze     
11 30 0.272 
11 36.5 0.545 
11 35.5 0.492 
11 21 0.062 
11 36.5 0.272 
11 41.5 0.840 
11 37.5 0.599 
11 28.5 0.206 
11 36.5 0.272 
10
 
8
 
11
 
17
 
< 0.05
 
Trial day
 
n1
 
n2
 
U
 
P
 
(a) Comparison of working memory errors in Wistar and hooded Lister rats across the 10 days of trials in the eight-arm radial maze     
10 32 0.315 
10 22 0.065 
10 23 0.079 
10 25 0.113 
10 22 0.065 
10 24.5 0.095 
10 40 0.719 
10 35.5 0.447 
10 21 0.053 
10 10 38 0.603 
(b) Comparison of reference memory errors in Wistar and hooded Lister rats across the 10 trial days in the eight-arm radial maze     
11 30 0.272 
11 36.5 0.545 
11 35.5 0.492 
11 21 0.062 
11 36.5 0.272 
11 41.5 0.840 
11 37.5 0.599 
11 28.5 0.206 
11 36.5 0.272 
10
 
8
 
11
 
17
 
< 0.05
 

n1 = number of hooded Lister rats; n2 = number of Wistar rats; U = U-test result; P = statistical significance.

Whereas Wistar rats displayed a clear improvement in reference memory performance by the end of the study (P < 0.05, day 1 versus day 10; P < 0.05, day 2 versus day 10, Mann–Whitney U-analysis; Table 1b), no clear evidence of long-term learning was apparent in hooded Lister rats, with the number of reference memory errors remaining very consistent across all trial days. In fact, on the final trial day (day 10) a significantly lower number of reference memory errors was evident in Wistar compared with hooded Lister rats.

Locomotory behavior was taken as a measure of mobility in the rat strains. Hooded Lister rats appeared more mobile than Wistar rats on all days of the study (t-test P < 05) with the exception of days 1 and 10 (Fig. 4). These effects were not confirmed as significant by ANOVA, however [between-factor, F(1,9) = 1.451 P = 0.1699].

Figure 4.

Antagonism of mGluR5 significantly impairs working memory in both rat strains. (A) Daily application of MPEP (1.8 μg) caused a significant impairment of reference memory performance in Wistar rats. Effects were significant from day 6 of the study. (B) Daily application of MPEP (1.8 μg) caused a significant impairment of working memory performance in Wistar rats. Effects were significant from day 5 of the study. (C) Daily application of MPEP (1.8 μg) had no effect on reference memory performance in hooded Lister rats. (D) Daily application of MPEP (1.8 μg) caused a significant impairment of working memory performance in hooded Lister rats. Effects were significant from day 8 of the study.

Figure 4.

Antagonism of mGluR5 significantly impairs working memory in both rat strains. (A) Daily application of MPEP (1.8 μg) caused a significant impairment of reference memory performance in Wistar rats. Effects were significant from day 6 of the study. (B) Daily application of MPEP (1.8 μg) caused a significant impairment of working memory performance in Wistar rats. Effects were significant from day 5 of the study. (C) Daily application of MPEP (1.8 μg) had no effect on reference memory performance in hooded Lister rats. (D) Daily application of MPEP (1.8 μg) caused a significant impairment of working memory performance in hooded Lister rats. Effects were significant from day 8 of the study.

Wistar rats groomed more than hooded Lister rats on all days of the study (Fig. 5) [ANOVA: between-factor, F(1,9) = 3.345 P < 0.001]. Lister rats reared more than Wistar rats on days 1–3 of the study [t-test, P < 0.001; ANOVA for days 1–3: between factor, F(1,2) = 0.16.044, P < 0.0001]; thereafter no differences in rearing behavior were seen between both rats strains (Fig. 5) [ANOVA for days 4–10, between-factor, F(1,6) = 0.774 P = 0.5921]. Defecation was negligible in both groups (data not shown).

Differences in Spatial Learning Performance in Wistar and Hooded Lister Rats are Revealed by Application of the mGluR5 Antagonist MPEP

Intracerebral application of MPEP 30 min before training on each day of the 10 day study revealed considerable differences in learning performance in the rat strains.

In Wistar rats administration of MPEP (1.8 μg, n =12) caused a significant impairment of working memory performance compared with vehicle-treated controls (n = 9) which became evident from day 6 onwards (Fig. 4) (Mann–Whitney U-analysis; Table 2a).

Figure 5.

Wistar rats move less, rear less and groom more than hooded Lister rats. (A) With the exceptions of days 1 and 10, Wistar rats moved less throughout the radial maze than hooded Lister rats. Activity was measured as distance covered in cm/s. Hooded Lister rats covered significantly more distance throughout the maze than Wistar rats. Graph shows average locomotion represented as cm/s on each day of the study. (B) Wistar rats groomed significantly more on all days of the study compared with hooded Lister rats. Graph shows average number of grooms on each day of the study. C: Wistar rats reared significantly less on days 1–3 of the study compared with hooded Lister rats. Graph shows average number of rears on each day of the study.

Figure 5.

Wistar rats move less, rear less and groom more than hooded Lister rats. (A) With the exceptions of days 1 and 10, Wistar rats moved less throughout the radial maze than hooded Lister rats. Activity was measured as distance covered in cm/s. Hooded Lister rats covered significantly more distance throughout the maze than Wistar rats. Graph shows average locomotion represented as cm/s on each day of the study. (B) Wistar rats groomed significantly more on all days of the study compared with hooded Lister rats. Graph shows average number of grooms on each day of the study. C: Wistar rats reared significantly less on days 1–3 of the study compared with hooded Lister rats. Graph shows average number of rears on each day of the study.

Table 2

Mann–Whitney U-test analysis of working and reference memory performance in Wistar rats following treatment with MPEP or vehicle

Trial day
 
n1
 
n2
 
U
 
P
 
(a) Comparison of working memory errors in Wistar rats treated with either MPEP or vehicle, across the 10 days of trials in the eight-arm radial maze     
12 43.5 0.464 
12 36.5 0.219 
12 34 0.169 
12 43.5 0.464 
12 30 0.095 
12 22.5 < 0.05 
12 24 < 0.05 
12 26.5 < 0.05 
12 23.5 < 0.05 
10 12 8.5 < 0.01 
(b) Comparison of reference memory errors in Wistar rats treated with either MPEP or vehicle, across the 10 days of trials in the eight-arm radial maze     
12 37.5 0.247 
12 27.5 0.058 
12 33.5 0.148 
12 28.5 0.277 
12 22.5 < 0.05 
12 21 < 0.05 
12 23.5 < 0.05 
12 15.5 < 0.01 
12 23.5 < 0.01 
10
 
12
 
9
 
17.5
 
< 0.01
 
Trial day
 
n1
 
n2
 
U
 
P
 
(a) Comparison of working memory errors in Wistar rats treated with either MPEP or vehicle, across the 10 days of trials in the eight-arm radial maze     
12 43.5 0.464 
12 36.5 0.219 
12 34 0.169 
12 43.5 0.464 
12 30 0.095 
12 22.5 < 0.05 
12 24 < 0.05 
12 26.5 < 0.05 
12 23.5 < 0.05 
10 12 8.5 < 0.01 
(b) Comparison of reference memory errors in Wistar rats treated with either MPEP or vehicle, across the 10 days of trials in the eight-arm radial maze     
12 37.5 0.247 
12 27.5 0.058 
12 33.5 0.148 
12 28.5 0.277 
12 22.5 < 0.05 
12 21 < 0.05 
12 23.5 < 0.05 
12 15.5 < 0.01 
12 23.5 < 0.01 
10
 
12
 
9
 
17.5
 
< 0.01
 

n1 = number of MPEP-treated rats; n2 = number of vehicle-treated rats; U = U-test result; P = statistical significance.

Furthermore, in Wistar rats, administration of MPEP (1.8 μg, n = 12) caused a significant impairment of reference memory performance compared with vehicle-treated controls (n = 9) which became evident from day 5 onwards (Fig. 4) (Mann–Whitney U-analysis; Table 2b).

In hooded Lister rats administration of MPEP (1.8 μg, n = 10) caused a significant impairment of working memory performance compared with vehicle-treated controls (n = 11) which only became apparent from day 8 onwards (Fig. 4) (Mann–Whitney U-analysis; Table 3a). No effect of MPEP was seen on reference memory performance in hooded Lister rats (Fig. 4) (Mann–Whitney U-analysis; Table 3b).

Table 3

Mann–Whitney U-test analysis of working and reference memory performance in hooded Lister rats following treatment with MPEP or vehicle

Trial day
 
n1
 
n2
 
U
 
P
 
(a) Comparison of working memory errors in hooded Lister rats treated with either MPEP or vehicle, across the 10 days of trials in the eight-arm radial maze     
10 11 52.5 0.863 
10 11 50 0.756 
10 11 49.5 0.705 
10 11 36.5 0.197 
10 11 46 0.557 
10 11 33 0.132 
10 11 42.5 0.387 
10 11 22.5 < 0.05 
10 11 20.5 < 0.05 
10 10 11 26 < 0.05 
(b) Comparison of reference memory errors in hooded Lister rats treated with either MPEP or vehicle, across the 10 days of trials in the eight-arm radial maze     
10 11 45 0.512 
10 11 44.5 0.468 
10 11 54.5 0.973 
10 11 28.5 0.061 
10 11 49.5 0.705 
10 11 35.5 0.173 
10 11 48.5 0.654 
10 11 53.5 0.918 
10 11 44.5 0.467 
10
 
10
 
11
 
45
 
0.512
 
Trial day
 
n1
 
n2
 
U
 
P
 
(a) Comparison of working memory errors in hooded Lister rats treated with either MPEP or vehicle, across the 10 days of trials in the eight-arm radial maze     
10 11 52.5 0.863 
10 11 50 0.756 
10 11 49.5 0.705 
10 11 36.5 0.197 
10 11 46 0.557 
10 11 33 0.132 
10 11 42.5 0.387 
10 11 22.5 < 0.05 
10 11 20.5 < 0.05 
10 10 11 26 < 0.05 
(b) Comparison of reference memory errors in hooded Lister rats treated with either MPEP or vehicle, across the 10 days of trials in the eight-arm radial maze     
10 11 45 0.512 
10 11 44.5 0.468 
10 11 54.5 0.973 
10 11 28.5 0.061 
10 11 49.5 0.705 
10 11 35.5 0.173 
10 11 48.5 0.654 
10 11 53.5 0.918 
10 11 44.5 0.467 
10
 
10
 
11
 
45
 
0.512
 

n1 = number of MPEP-treated rats; n2 = number of vehicle-treated rats; U = U-test result; P = statistical significance.

The differences in locomotion which were evident in control animals were sustained when MPEP was applied. Hooded Lister rats were thus more mobile than Wistar rats on all days of the study (t-test, P < 05), with the exception of day 1 (Fig. 6). These effects were not confirmed as significant by ANOVA, however [between-factor, F(1,9) = 1.509 P = 0.1492].

Figure 6.

Antagonism of mGluR5 does not affect locomotion, rearing or grooming in either rat strain. (A–C) Daily application of MPEP (1.8 μg) had no effect on locomotion (A), grooming (B) or rearing (C) in Wistar rats. (D–F) Daily application of MPEP (1.8 μg) had no effect on locomotion (D), grooming (E) or rearing (F) in hooded Lister rats.

Figure 6.

Antagonism of mGluR5 does not affect locomotion, rearing or grooming in either rat strain. (A–C) Daily application of MPEP (1.8 μg) had no effect on locomotion (A), grooming (B) or rearing (C) in Wistar rats. (D–F) Daily application of MPEP (1.8 μg) had no effect on locomotion (D), grooming (E) or rearing (F) in hooded Lister rats.

MPEP did not affect the greater degree of grooming seen in Wistar rats compared with hooded Lister rats on all days of the study (Fig. 6) [ANOVA: between-factor, F(1,9) = 2.345 P < 0.05]. Hooded Lister rats reared more than hooded Lister rats on days 1–3 of the study in the presence of MPEP [ANOVA for days 1–3: between factor, F(1,2) = 3.826, P < 0.05]; thereafter no differences in rearing behavior were seen (Fig. 6) [ANOVA for days 4–10: between-factor, F(1,6) = 0.095 P = 0.9967]. Defecation was negligible in both groups in the presence of MPEP (data not shown).

Evaluation of the Expression of mGluR5 in Wistar and Hooded Lister Rats

Western blots performed with specific peptide antibodies against mGluR5 receptors revealed major immunoreactivity in the region of 150 kDa, corresponding to mGluR5a and -b receptor monomers. Similarly, antibodies against mGlu1a receptors showed a single protein band around 150 kDa in the extracts of both strains. Comparison of Wistar with hooded Lister rats demonstrated that all examined Wistar rats (n = 4) have a significantly higher expression of mGluR5 receptor, whereas the control protein β-actin did not show significant differences between the strains (Fig. 7B, mGluR5 and actin). Densitometric analysis of the relative amount of receptor protein in the immunoblots performed with mGluR1 and mGluR5 receptor antibodies revealed that hooded Lister rats express only 67% of the amount of mGluR5 receptor protein seen in Wistar rats (Fig. 7A, mGluR5). The amount of mGluR1 protein did not show any significant changes in the Wistar and hooded Lister strains (n = 5). In contrast to the mGluR5 receptor, immunoreactivity for mGluR1 protein showed some interindividual hetergeneity with increased or decreased expression levels in the hooded Lister strain (Fig. 7A, mGluR1). Therefore, these results indicate that differences in the expression level of the group I metabotropic receptor mGluR5 may contribute to the physiological differences observed between hooded Lister and Wistar rats.

Figure 7.

Expression of mGluR5 is significantly lower in hooded Lister compared with Wistar rats, whereas no difference in the expression levels of mGluR1 is evident. (A) The relative mGluR expression level was assessed in Wistar and hooded Lister rats by Western blot analysis. Homogenates of hippocampi of four Wistar (lanes 1–4) and four hooded Lister rats (lanes 5–8) were immunostained using specific antibodies against mGluR5, mGluR1 (data not shown) and β-actin. A representative Western blot out of five experiments is shown. Sizes of relevant marker proteins in kDa are shown in the left margin. (B) Quantification of Western blot analysis of homogenates of Wistar and hooded Lister rat hippocampi. Densities of mGluR1, mGluR5 and actin immunoreactive bands were measured using the NIH Image 1.61 program. Hooded Lister rats (grey bars) expressed significantly less mGluR5 compared with Wistar rats (black bars). No significant difference in the expression of mGluR1 was found. Mean values ± SD for mGluR5 were calculated from four different animals (n = 4) in five experiments and for mGluR1 from five different animals (n = 5) in 3–5 experiments. The relative expression level of mGluR1 and 5 was expressed as the ratio of the densities for mGluR-1 and β-actin or mGluR5 and β-actin. Control expression without treatment was set to 100%. Asterisks (*) show statistically significant values with P < 0.05.

Figure 7.

Expression of mGluR5 is significantly lower in hooded Lister compared with Wistar rats, whereas no difference in the expression levels of mGluR1 is evident. (A) The relative mGluR expression level was assessed in Wistar and hooded Lister rats by Western blot analysis. Homogenates of hippocampi of four Wistar (lanes 1–4) and four hooded Lister rats (lanes 5–8) were immunostained using specific antibodies against mGluR5, mGluR1 (data not shown) and β-actin. A representative Western blot out of five experiments is shown. Sizes of relevant marker proteins in kDa are shown in the left margin. (B) Quantification of Western blot analysis of homogenates of Wistar and hooded Lister rat hippocampi. Densities of mGluR1, mGluR5 and actin immunoreactive bands were measured using the NIH Image 1.61 program. Hooded Lister rats (grey bars) expressed significantly less mGluR5 compared with Wistar rats (black bars). No significant difference in the expression of mGluR1 was found. Mean values ± SD for mGluR5 were calculated from four different animals (n = 4) in five experiments and for mGluR1 from five different animals (n = 5) in 3–5 experiments. The relative expression level of mGluR1 and 5 was expressed as the ratio of the densities for mGluR-1 and β-actin or mGluR5 and β-actin. Control expression without treatment was set to 100%. Asterisks (*) show statistically significant values with P < 0.05.

Discussion

Our data demonstrate that very distinct differences in synaptic plasticity in the dentate gyrus of hooded Lister and Wistar rats occur and that these differences may relate to the learning capability of these rats. Most striking was the observation that whereas Wistar rats express robust late-LTP and exhibit clear formation of reference memory, hooded Lister rats express only STP in the dentate gyrus and do not appear to create reference memory. These differences may relate to the very differing degrees of expression of mGluR5 in these rats strains.

We found that the group I mGlu receptor mGluR5 is intrinsically involved in the regulation of both hippocampal synaptic plasticity and learning performance. The ability to respond to mGluR5 antagonism related to the degree of expression of mGluR5 in the hippocampus. Thus, Wistar rats that express a higher level of hippocampal mGluR5 than hooded Lister rats responded with a greater inhibition of both LTP and spatial learning in the presence of an mGluR5 antagonist. Hooded Lister rats, on the other hand responded to mGluR5 antagonism with weak or no inhibition of LTP and impairment of working but not reference memory performance. These data provide evidence on the one hand that LTP may correlate to long-term memory, whereas STP correlates to short-term memory. On the other hand, these data strongly support an important role for mGluR5 in learning ability and the expression of synaptic plasticity in the hippocampus.

It has long been speculated that LTP corresponds to the cellular mechanism of spatial learning. Recently, we showed that whereas LTP appears to encode space itself, LTD most likely encodes the contextual features of space (Kemp and Manahan-Vaughan, 2004). We focused in this study on evaluations of LTP and spatial learning in the radial maze, as this behavioral paradigm depends on learning of space and orientation within space: aspects of spatial learning which appear to best correlate with LTP. Our observations in the past, that different rat strains express very different degrees of synaptic plasticity in the CA1 region (Manahan-Vaughan and Braunewell, 1999; Manahan-Vaughan, 2000a), provoked the question as to whether clear correlations could be found between the ability to express hippocampal synaptic plasticity and spatial learning performance. We approached this issue with a degree of scepticism that results would be found. It is clear that rodents possess a broad spectrum of survival strategies, any of which could be employed to compensate for an inadequate spatial memory. Intensive, randomized search strategies as opposed to systematic learning of food locations could be used to locate food, and indeed observation of exploration strategies in the different rat strains we have studied certainly suggest that this may in fact occur. Thus, despite clear differences in synaptic plasticity, differences in spatial learning may be difficult to detect.

On a general behavioral level, hooded Lister rats display a more frenetic exploration strategy than Wistar rats — they are far more mobile, rear more and appear less fearful (groom less). Wistar rats, on the other hand, display a more focused exploration strategy: they rotate in the center of the maze and stretch their bodies into almost every arm (without entering) before deciding to explore one. Their slower movements appear to occur less due to fear than due to deliberate action. Wistar rats groom more than hooded Lister rats, which may be indicative of an absence of anxiety but may also derive from a form of displacement behavior arising from stress. It has been demonstrated that hooded Lister rats have a lower basal level of serum corticosterone than Wistar rats, and that Wistar rats show a higher relative increase of serum corticosterone in response to stressful challenges (Manahan-Vaughan and Braunewell, 1999).

Are Good LTP Expressers also Good Learners?

In this study we show for the first time that although Wistar rats respond to HFT of the medial perforant path with robust very persistent LTP in the dentate gyrus, the same stimulation produces very weak STP in hooded Lister rats. In keeping with previous reports (Manahan-Vaughan and Braunewell, 1999), we found that HFT of the Schaffer collaterals generate persistent LTP in Wistar and incremental LTP in hooded Lister rats. Thus, although the level of LTP 24 h post-HFS was similar in both rat strains, the initial potentiation (STP) is significantly smaller in hooded Lister rats, and increases gradually over the period of roughly 1 h, to equivalent LTP levels in Wistar rats. Interestingly, although working memory performance appeared the same in the two rats stains, differences in reference memory performance were apparent. Whereas Wistar rats demonstrated a very clear learning curve, and had fewer reference memory errors than hooded Lister rats on the final trial day, hooded Lister rats exhibited the same number of reference memory errors at the start of the trial compared with at the end. This suggests that the hooded Lister strain was using a strategy independent of reference memory formation to locate the food pellets in the radial maze.

Taken together, these findings offer the intriguing possibility that hooded Lister rats that express only STP in the dentate gyrus are not capable of forming reference (i.e. long-term) memory. On the other hand, Wistar rats that express persistent LTP also form reference memory traces.

Does mGluR5 Receptor Contribute to the Molecular Mechanisms of Strain-dependent Learning and Plasticity?

It has been widely documented that mGluRs contribute to synaptic plasticity and memory formation in the hippocampus. Whereas group II mGluRs are specifically required for persistent LTD (Manahan-Vaughan, 1997), group III mGluRs regulate the threshold for LTP induction, facilitate LTD (Manahan-Vaughan, 2000b; Klausnitzer et al., 2004) and may be involved in short-term memory formation (Holscher et al., 2004). Group I mGluRs appear to be especially significant, however, as they are required for both persistent LTP and LTD, as well as for spatial memory. Thus, mGluR1, a group I mGluR, regulates both LTP induction and reference memory formation (Aiba et al., 1994; Naie and Manahan-Vaughan, 2004b). On the other hand, pharmacological antagonism of mGluR5 prevents spatial working and reference memory formation, as well as dentate gyrus LTP in vivo (Naie and Manahan-Vaughan, 2004a).

A particularly important role for mGluR5 in memory and plasticity is becoming evident: induction of LTP in vivo leads to an enhanced expression of mGluR5, but no upregulation of any other mGluR (Manahan-Vaughan et al., 2003). Pharmacological activation of mGluR5 leads to protein synthesis-dependent synaptic plasticity in the CA1 region (Huber et al., 2001), stimulation of protein synthesis to enable prolongation of LTP (Raymond et al., 2000) and induction of plasticity-relevant proteins (Braunewell et al., 2003). Furthermore, animals with targeted genetic mutations which lack mGluR5 show impaired LTP and spatial learning (Jia et al., 1998; Lu et al., 1997). Thus, differences in mGluR5 expression or function comprise a possible mechanism for the differences in strain-dependent plasticity and learning seen in rat strains. This possibility is supported by our data. Intriguingly, hooded Lister rats expressed roughly only 67% of the mGluR5 receptor protein compared with Wistar rats. Given the importance of mGluR5 for LTP maintenance (Naie and Manahan-Vaughan, 2004a), this could explain why in the dentate gyrus hooded Lister rats express STP and not LTP. In the CA1 region, initial STP was far less in hooded Lister rats compared with Wistar rats. Mannaioni et al. (2001) have demonstated that activation of mGluR5 can enhance currents through NMDA receptors in the CA1 region of Sprague–Dawley rats, whereas Rae and Irving (2004) showed that mGluR5 enhances Ca2+ inward currents in the CA1 region of the same rat strain. These factors very likely contribute to LTP induction. The lower level of mGluR5 in hooded Lister rats may explain why STP (which comprises the NMDA receptor dependent phase of LTP) was less than that seen in Wistar rats.

Treatment with the mGluR5 antagonist MPEP revealed that whereas CA1 LTP could be blocked in both strains in a dose-dependent manner, Wistar rats were more sensitive to the drug. In the dentate gyrus, LTP in Wistar but not STP in hooded Lister rats was blocked by MPEP. These findings are consistent with the expression levels of mGluR5 in the two rat strains. Whereas daily application of MPEP impaired both reference and working memory performance in Wistar rats, only working memory performance was impaired by MPEP in hooded Lister rats. The lack of effect of MPEP on reference memory in hooded Lister rats may relate to the low level of mGluR5 expression in this strain, or to the fact that reference memory performance was weak (ostensibly absent) in these rats. Consistent with findings by others (Tatarczynska et al., 2001; Kinney et al., 2004), antagonism of mGluR5 with MPEP had no effect on locomotion. High doses of MPEP (1–20 mg/kg, i.p.) have been shown to reduce anxiety in rats (Tatarczynska et al., 2001; Pilc et al., 2002), but alterations in grooming or rearing behavior were not evident in our study — perhaps due to the decidedly lower drug concentration or behavioral tests used.

It is tempting to speculate that, given the fact that MPEP impaired LTP in the CA1 region as well as working memory of hooded Listers, the CA1 region may be intrinsically involved in hippocampal working memory. On the other hand, the finding that reference memory is absent and LTP is not expressed in the dentate gyrus of these rats, suggests a particular role for the dentate gyrus in the processing of long-term memory traces.

Conclusions

Our data demonstrate that expression of poor LTP may relate to poor reference memory performance in rats. Furthermore, mGluR5, a group I mGluR, may comprise a significant molecular component underlying these deficits. Thus, the mGluR5-dependency of LTP and learning performance related to the degree of expression of mGluR5 in the hippocampus. These data provide new evidence that LTP may correlate to long-term memory formation, and strongly support an important role for mGluR5 in learning ability and the expression of hippocampal synaptic plasticity in vivo.

We are very grateful for the excellent technical assistence of Jens Klausnitzer, Beate Krenzek and Mirja Petri. This work was supported by a Deutsche Forschungsgemeinschaft (German Research Foundation) grant Ma 1843 to D.M.-V.

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Author notes

1Learning and Memory Research, International Graduate School for Neuroscience, Ruhr University Bochum, FNO 1/116, Universitaetsstrasse 150, 44780 Bochum, Germany, 2Institute of Physiology of the Charite, Synaptic Plasticity Research Group, Humboldt University, Tucholskystrasse 2, 10117 Berlin, Germany and 3Neuroscience Research Center of the Charite, Signal Transduction Research Group, Humboldt University, Tucholskystrasse 2, 10117 Berlin, Germany