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We studied the role of γ-aminobutyric acid (GABA)ergic septohippocampal projections in medial septum (MS) self-stimulation of behaving mice. Self-stimulation was evoked in wild-type (WT) mice using instrumental conditioning procedures and in J20 mutant mice, a type of mouse with a significant deficit in GABAergic septohippocampal projections. J20 mice showed a significant modification in hippocampal activities, including a different response for input/output curves and the paired-pulse test, a larger long-term potentiation (LTP), and a delayed acquisition and lower performance in the MS self-stimulation task. LTP evoked at the CA3–CA1 synapse further decreased self-stimulation performance in J20, but not in WT, mice. MS self-stimulation evoked a decrease in the amplitude of field excitatory postsynaptic potentials (fEPSPs) at the CA3–CA1 synapse in WT, but not in J20, mice. This self-stimulation-dependent decrease in the amplitude of fEPSPs was also observed in the presence of another positive reinforcer (food collected during an operant task) and was canceled by the local administration of an antibody-inhibiting glutamate decarboxylase 65 (GAD65). LTP evoked in the GAD65Ab-treated group was also larger than in controls. The hippocampus has a different susceptibility to septal GABAergic inputs depending on ongoing cognitive processes, and the GABAergic septohippocampal pathway is involved in consummatory processes related to operant rewards.
In a seminal study, Olds and Milner (1954) reported that rats located in a Skinner box can learn to do a simple manipulation (i.e., to press a lever) in order to obtain a train electrical stimulation of selected brain sites. Presently, intracranial self-stimulation is a standardized operant conditioning paradigm by which animals are capable of self-administering electrical stimulation through electrodes chronically implanted in the brain (Carlezon and Chartoff 2007). Although it is generally assumed that the reinforcing electrical stimulus activates brain centers and/or pathways related with the neural processes involved in the recognition of natural rewards as water or food (Olds 1958; Wise 1996), no clear link has been yet established between the subsequent neural effects of internal (brain stimulation) and external (food, water) rewards on consummatory behavior. For example, both mesolimbic dopaminergic (Wise 2002) and γ-aminobutyric acid (GABA)ergic (Lassen et al. 2007; Leppä et al. 2011) systems have been reported to be involved in these processes.
Here, we studied the effects of medial septum (MS) self-stimulation on the synaptic strength of hippocampal CA3–CA1 synapses and compared these effects with those evoked by a food-rewarded operant task. It is known that rodents will work actively to obtain train electrical stimulations of this septal area (Ball and Gray 1971; Cazala et al. 1988), which have a triple (cholinergic, glutamatergic, and GABAergic) projection system to the hippocampus (Gulyás et al. 2003; Sotty et al. 2003; Habib and Dringenberg 2009). In addition, it has been recently reported that dorsal hippocampal CA3 connections with the ventral tegmental area (across the lateral septum) close a circuit putatively involved in the internal reward system (Luo et al. 2011).
Experiments were carried out in both wild-type (WT) and J20 mice (Mucke et al. 2000), which have been reported to have a severe deficit of GABAergic septohippocampal projections (Rubio et al. 2012). We tested the hypothesis that the decrease in GABAergic septal projection onto hippocampal interneurons could modify the functional properties of hippocampal circuits, including changes in input/output curves, paired-pulse facilitation, and experimentally evoked long-term potentiation (LTP). LTP was evoked by high-frequency stimulation (HFS) of Schaffer collaterals and recorded at the ipsilateral hippocampal CA1 area in the behaving animal. These functional changes could also have some putative consequences on MS self-stimulation. To this aim, and following procedures described elsewhere (Gruart et al. 2006), WT and J20 mice were prepared for the chronic recording of field excitatory postsynaptic potentials (fEPSPs) evoked at the CA3–CA1 synapse during the acquisition and performance of an operant self-stimulation task. Animals were rewarded either with a train of stimuli presented to the MS or with food pellets. In this regard, it has been already shown that the hippocampus is involved in the acquisition of food-rewarded operant tasks (Madroñal et al. 2010, Jurado-Parras et al. 2013). To determine further the role of GABAergic neurons in the decrease of fEPSPs evoked at the CA3–CA1 synapse by MS self-stimulation, we carried out local injections of the GAD65-specific monoclonal antibody b78, which specifically inhibit GAD65 enzyme activity. GAD65 is an isoform of GAD particularly prominent in axon terminals (Raju et al. 2005; Manto et al. 2011).
Results indicate that the GABAergic septohippocampal projection plays an important inhibitory role in the functional properties of hippocampal circuits, particularly on intrinsic neural mechanisms involved in consummatory behaviors of internal (MS self-stimulation) and external (food) rewards. In addition, the hippocampus seems to have a different susceptibility (i.e., synaptic potentiation) to ongoing cognitive processes, such as the acquisition or the performance of an already learned task, or during the display of specific behaviors, such as eating or resting (Jurado-Parras et al. 2013).
Materials and Methods
Histological and self-stimulation experiments (Figs 1–7) were carried out in mature (6/8-month old, 25–35 g) hemizygous transgenic male mice-expressing human amyloid precursor protein carrying both the Swedish and Indiana familial Alzheimer disease mutations (i.e., the J20 line; Mucke et al. 2000; Palop et al. 2003). These mice and their corresponding WT littermates were provided by the University of Barcelona Animal House (Barcelona, Spain) or by Drs Joaquín del Rio and Alberto Pérez-Mediavilla (CIMA Animal House, University of Navarra, Pamplona, Spain). Additional mature (6/7-month old, 24–35 g) WT male mice (C57BL/6J strain) obtained from an official supplier (University of Granada Animal House, Granada, Spain) were used in the operant conditioning test (Fig. 6G) and in the GAD65Ab-treatment study (Figs 8 and 9). Upon arrival to the Pablo de Olavide Animal House (Seville, Spain), animals were housed in shared cages (5 per cage), but they were switched to individual cages after surgery. Mice were kept on a 12-h light/dark cycle with constant ambient temperature (21.5 ± 1°C) and humidity (55 ± 8%), with food and water available ad libitum.
All of the experiments were performed in accordance with the guidelines of the European Union Council (2003/65/EU) and Spanish regulations (BOE 252/34367-91, 2005) for the use of laboratory animals in chronic studies, and approved by the local Ethics Committee of the Pablo de Olavide University. Unless otherwise indicated, a total of 10 successful animals were used per experimental group. We considered as successful those animals that finished experimental protocols presenting fEPSPs that did not deteriorate over time.
Detection of GABAergic Septohippocampal Projections
For the detection of GABAergic septohippocampal fibers (Fig. 1A,B), mice were deeply anesthetized with a mixture (10/1; 0.003 mL/g, intraperitoneally) of ketamine (Ketolar, Parke-Davis, Madrid, Spain) and xylazine (Rompun, Bayer UK Ltd., Suffolk, UK), and stereotaxically injected with 10% biotinylated dextran amine (BDA; 10 000 MW, Molecular Probes, Eugene, OR, USA) in the MS/diagonal band (MS/DB) complex. Each animal received 2 iontophoretic (7-µA positive direct current and 7-s on–off cycle) injections of the tracer into the MS/DB (0.0 mm lateral, 0.7 mm anterior, and 3.0 and 3.7 ventral to bregma; Paxinos and Franklin 2001). This protocol results in intense BDA labeling in the MS/DB, which contains the highest proportion of GABAergic septohippocampal neurons (Pascual et al. 2004). Five or 6 days later, animals were deeply reanesthetized (sodium pentobarbital, 50 mg/kg) and perfused with 4% paraformaldehyde in 0.12 M phosphate buffer. Brains were removed and frozen, and 30-µm sections were obtained. Coronal sections were stored in a cryoprotectant solution (30% glycerin, 30% ethyleneglycol, and 40% 0.1 M phosphate buffer) at −20°C until use. After blocking, BDA was visualized by incubating the sections overnight at 4°C with the ABC complex (Vectastain ABC Kit; Vector Labs, Burlingame, CA, USA) diluted 1/100. Peroxidase activity was developed with diaminobenzidine intensified with nickel ammonium sulfate and cobalt chloride (DAB/Ni–Co), and H2O2 yielding a black end-product in septohippocampal fibers. Thereafter, sections were mounted onto gelatinized slides, Nissl-stained, and coverslipped with Eukitt.
Double Immunodetection of BDA Tracer and Parvalbumin
Hippocampal sections from iontophoretically injected animals were processed for the double detection of BDA and parvalbumin (PARV; Fig. 1C–G). After blocking, free-floating sections were incubated overnight at 4°C with the ABC complex simultaneously with the rabbit polyclonal antibody against PARV (1/3000, Swant Antibodies, Fribourg, Switzerland). Next, BDA was revealed using DAB/Ni–Co producing a black end-product. Primary antibody was then visualized by sequential incubation with biotinylated secondary antibodies and the ABC complex (2 h each, Vector Labs). The peroxidase reaction was developed with DAB to produce a brown end-product. The sections were mounted onto gelatinized slides, dehydrated, and coverslipped with Eukitt. These double immunolabeled sections were used to perform subsequent quantifications.
For double-immunofluorescent detection of BDA and PARV, the sections were first blocked and then incubated overnight with anti-PARV antibody. The primary antibody was detected by simultaneous incubation with goat anti-rabbit IgG Alexa Fluor 568 (Invitrogen, Carlsbad, CA, USA) and streptavidin-fluorescein (1/200, Amersham Biosciences, Buckinghamshire, UK), that later of which allows the detection of BDA tracer. The sections were then mounted onto slides with Mowiol 4-88 (Merck Chemicals, Darmstadt, Germany) and viewed under a Leica SPE confocal microscope (Leica, Inc., Heidelberg, Germany). Images were then processed with ImageJ software (Rasband, W.S., ImageJ, NIH, Bethesda, MD, USA), and brightness and contrast were adjusted using Adobe Photoshop™ (Adobe, Systems, San Jose, CA, USA).
To assess the complexity of the baskets, the number of boutons per basket around the somata of parvoalbumin-positive interneurons was hand-counted in the same regions. The sections used for quantifications were collected from equivalent hippocampal levels (sections between 1.60 and 2.30 mm posterior to bregma; Paxinos and Franklin 2001). Histological data were processed for statistical analysis with Statgraphics Plus 5.1 (Statistical Graphics, Rockville, MD, USA). Two-tailed Student's t-test was used to examine differences between the experimental groups. The significance was set at α ≤ 0.05, except for some intragroup tests, where α ≤ 0.06 was accepted as significant due to the general data context.
Animal Preparation for Self-Stimulation and Electrophysiological Recordings
Animals were anesthetized with 0.8–1.5% isoflurane delivered by a mouse anesthesia mask (David Kopf Instruments, Tujunga, CA, USA). The anesthetic gas was supplied from a calibrated Fluotec 5 (Fluotec-Ohmeda, Tewksbury, MA, USA) vaporizer, at a flow rate of 1–2 L/min oxygen (AstraZeneca, Madrid, Spain). Animals were implanted with bipolar stimulating electrodes on the right MS (0.1 mm lateral and 0.6 mm anterior to bregma, and 3.8 mm from the brain surface; Paxinos and Franklin 2001) and in the ipsilateral Schaffer collateral/commissural pathway of the dorsal hippocampus (2 mm lateral and 1.5 mm posterior to bregma, and 1–1.5 mm from the brain surface; Paxinos and Franklin 2001). A recording electrode was aimed to the CA1 stratum radiatum (1.2 mm lateral and 2.2 mm posterior to bregma, and 1–1.5 mm from the brain surface; Paxinos and Franklin 2001). Electrodes were made from 50 μm, Teflon-coated, tungsten wire (Advent Research, Eynsham, UK). A bare silver wire was affixed to the bone as ground. All the implanted wires were soldered to one 6-pin socket (RS Amidata, Madrid, Spain) and fixed to the skull with dental cement (Fig. 2A–D; see Gruart et al. 2006 for details).
For the administration of the GAD65-specific monoclonal antibody b78, selected animals were also implanted chronically with a blunted, stainless steel, 26-G cannula (Plastic One, Reanoke, VA, USA) in the CA3–CA1 areas, nearby hippocampal stimulating and recording electrodes (1.6 mm lateral and 1.8 mm posterior to bregma, and 1 mm from the brain surface; Paxinos and Franklin 2001). The tip of the cannula was aimed to be located 0.25 mm above the infusion target.
Electrocortical Field Recordings, Input/Output Curves, and Paired-Pulse Facilitation
Recording sessions started 1 week after surgery. To determine the functional capabilities of hippocampal circuits in the 2 groups of mice, we performed input/output and a paired-pulse stimulation tests at the CA3–CA1 synapse. For this, the animal was placed in a small (5 × 5 × 10 cm) plastic chamber located inside a larger Faraday box (30 × 30 × 20 cm). fEPSP recordings were made with Grass P511 differential amplifiers through a high-impedance probe (2 × 1012 Ω, 10 pF).
For input/output curves, animals were stimulated at the CA3 area with 2 pulses (40 ms of interstimulus interval) of increasing intensity (∼0.05–1.0 mA) until reaching a maximum fEPSP response (see below). Data were normalized using as 100% the highest amplitude (average of 5 selected sweeps with same stimulation intensity) of the first fEPSP of each mouse as their own baseline (BL). Additionally, the ratio "2nd fEPSP/1st fEPSP × 100" and the total response "1st fEPSP + 2nd fEPSP" were evaluated (Fig. 3).
For the paired-pulse facilitation test, the intensity was fixed in accordance with the threshold of each mouse, within 30–40% of the intensity necessary for evoking a maximum fEPSP response. The effects of paired pulses of different (10, 20, 40, 100, 200, and 500 ms) interstimulus intervals were measured. Data are presented (the average of 5 selected sweeps with the same interval) using the same ratio as for the input/output test [(2nd/1st) × 100], but for every interstimulus interval (Fig. 4). The stimuli of these 2 tests (input/output and paired-pulse) were repeated ≥5 times with time intervals of 10 s, to avoid as much as possible interferences with slow short-term potentiation (Zucker and Regehr 2002). In all cases, we computed fEPSP amplitudes in a normalized way (Fig. 6A, inset), taking each mouse as its own BL in accordance with every experimental stage (Fig. 2E). We restricted the quantitative analysis to fEPSPs free of population spikes, noisy components, as well as of saturation signals, artifacts, or instability signs.
Self-Stimulation Procedures
Training took place in a Skinner box module measuring 12.5 × 13.5 × 18.5 cm (MED Associates, St. Albans, VT, USA) equipped with a lever. The shaping (Sh, Fig. 2E) protocol was carried out as follows: 1) The animal was placed for 5 min in a small box (5 × 5 × 10 cm) located besides the Skinner box. In this situation, the animal was stimulated at the CA3–CA1 synapse at a rate of 6 stimuli/min as BL (Fig. 2E) recordings; 2) afterwards, the animal was carefully placed for 20 min in the Skinner box where it was shaped applying the successive approximation method with the lever as a target until pressing it. The lever was programmed as a trigger to deliver a train of pulses (bipolar, 100 μs pulses at 100 Hz for 200 ms, with intensity ≤2 mA) in the MS using a fixed time interval of 5 s (FI5). This train was followed 40 ms after its end by a single pulse presented at the CA3–CA1 synapse (SB, Fig. 2E); and 3) finally, the animal was returned to the small box for a recovery (5 min) period, in which it was stimulated at the CA3–CA1 synapse at the initial rate of 6 stimuli/min (R, Fig. 2E).
The shaping protocol was applied for a maximum of 10 sessions, but when the animal reached criterion the shaping was completely stopped and switched to self-stimulation at the next day's session. The criterion was that the animal performed by itself a minimum of 20 lever presses during a 10-min period (Fig. 5A). In accordance with previous reports (Hodos and Valenstein 1962; Miliaressis and Rompre 1987), during the first 2 shaping sessions, the reward intensity threshold was adjusted for each animal using an additional behavioral criterion; this is, to reach the minimum current to get constant bar pressing (at least 1 lever press per min) in the absence of any observable arrest, general body reaction, or overt movements associated with the reward presentation. After this adjustment, the reward intensity remains without any change along all the experiment. Animals that did not reach the selected criterion during a maximum of 10 shaping sessions were discarded from the study. In this way, we could guarantee that all MS stimulation data included in the analysis came from rewarding phenomena.
Shaping sessions were followed by 7 self-stimulation sessions (Fig. 5B–D). Self-stimulation sessions were organized as described for shaping ones, but in this case, train stimulation of the MS (reward) was only carried out when the animal pressed the lever by itself. In all cases, self-stimulation rewards could be received along 20 min recording at a maximum rate of 1/5 s, that is, with the same fixed time interval (FI5) schedule. To evaluate self-stimulation performance in the different groups of animals (control, J20, and GAD65 mAb b78), we analyzed different behavioral parameters including the time expended in lever presses, the number of nonrewarded lever presses, and the latency to start pressing the lever. However, significant differences between groups were better represented by the number of reinforced obtained/the number of maximum available reinforcements (Fig. 5B), mostly when evaluating LTP effects on MS self-stimulation (Figs 7B and 9B).
Instrumental Conditioning for Food Reward
For this, an additional group of WT (C57BL/6J) mice was prepared with an identical surgical protocol (see above) but without stimulated electrodes in the MS. These mice were trained and tested in the already-mentioned Skinner box module. In this case, the Skinner box was equipped with a food dispenser from which pellets (MLabRodent Tablet, 20 mg; Test Diet, Richmond, IN, USA) could be delivered by pressing a lever. Before training, mice were handled daily for 7 days and food deprived to 85–80% of their free-feeding weight. Training took place for 20 min during successive days, in which mice were allowed to press the lever to receive pellets from the food tray using a fixed-interval 1-s (FI1) schedule. Animals were maintained on this FI1 schedule until they reached the selected criterion—namely, until they were able to obtain ≥20 pellets for 3 successive sessions. During this test, we recorded fEPSPs evoked in the CA3–CA1 synapse across the session in relation with selected ongoing behaviors. For this, single pulses were applied at the CA3–CA1 synapse, similar to those used during the self-stimulation protocol. All operant sessions were recorded with a synchronized video capture system (Sony HDR-SR12E, Tokyo, Japan).
Long-Term Potentiation
For LTP induction in behaving mice, we followed procedures described previously (Gruart et al. 2006). BL values for the amplitude of fEPSPs evoked at the CA3–CA1 synapse were collected 15 min prior to LTP induction using single 100 μs, square, biphasic pulses every 20 s. Pulse intensity was the same as during behavioral tests carried out with each mouse. BL values collected from the first day were selected as the normalization value (100%) for the next 2 days (illustrated in Figs 7A and 9A as a dotted horizontal line). For LTP induction, animals were presented with a HFS session consisting of five 200 Hz, 100 ms trains of pulses at a rate of 1/s repeated 6 times, at intervals of 1 min, that is, a total of 600 pulses were presented during the HFS session. To avoid evoking large population spikes and/or the appearance of cortical seizures, the stimulus intensity during HFS was set at the same intensity as that used for generating BL recordings. After the HFS session, exactly the same single-stimuli parameters as for BL recordings were presented for the following 30 min. On days 2 and 3, the same HFS session was repeated following a BL recording session lasting for 15 min and followed by a 30-min recording session. All HFS data were normalized using as 100% BL fEPSP values collected at the first day; in this way, we could evaluate early and long LTP (Figs 7 and 9).
Histological Procedures for Electrode Location
Mice were perfused transcardially under deep pentobarbital anesthesia, with saline and then 4% paraformaldehyde in phosphate-buffered saline (PBS, 0.1 M, pH 7.4). Brains were cryoprotected with 30% sucrose in PB, and coronal sections (50 μm) were obtained with a sliding freezing microtome (Leica SM2000R, Nussloch, Germany) and stored at −20°C in 30% glycerol and 30% ethylene glycol in PB until used. Selected sections including the implanted (MS and hippocampus) sites were mounted on gelatinized glass slides and stained using the Nissl technique with 0.1% toluidine blue to determine the location of stimulating and recording electrodes (Fig. 2A–C).
Administration of Monoclonal Antibody b78
The human monoclonal antibody b78 specific to the 65kDa isoform of glutamate decarboxylase 65 (GAD65; Raju et al. 2005) was used in this experiment. Prior (30 min) to the sixth self-stimulation session, the b78 antibody was diluted in saline (1 μg/μL) and administered through the injection cannula. The injection cannula was 250 µm longer than the implanted cannula, which was used as a guide. The injection (1 µL) was administered with the help of a SP100i pump (WPI, Sarasota, FL, USA) at a rate of 0.2 µL/min.
Immunohistochemistry for b78 Detection
Selected brain slices, including the dorsal hippocampus, were processed "free-floating" for immunohistochemistry, and all of the sections studied passed through all procedures simultaneously to minimize any difference from immunohistochemical staining itself. After cooling down the sections to room temperature, they were incubated with 10% methanol and 0.003% H2O2 in PBS for 10 min to block endogenous peroxidase activity. After this, sections were treated for 1 h with 5% normal donkey serum (NDS; AbD Serotec, MorphoSys, Kidlington, UK) in PBS with 0.2% Triton X-100 (Sigma-Aldrich, Madrid, Spain), and then incubated overnight at room temperature with polyclonal rabbit antihuman lambda light chain (1:20; AbD Serotec) with PBS containing 0.2% Triton X-100 and 3% NDS. The following day, sections were incubated for 1 h with donkey anti-rabbit IgG conjugated to biotin (1:200; AbD Serotec) in PBS with 0.2% Triton-X-100 and 3% NDS. Then, sections were incubated with the avidin–biotin–peroxidase complex (Vector Labs) for 30 min in PBS. Color development was achieved by incubating with 3,3-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and 0.033% hydrogen peroxide in PB for 4 min. Finally, sections were mounted on slides, dried for 1 day at room temperature, dehydrated with ascending alcohols and rinsed in xylol. After this, sections were coverslipped using DPX mounting medium and viewed under a Leica light microscope (Leica, Madrid, Spain) for analysis.
Data Collection and Analysis
fEPSP and 5-V rectangular pulses corresponding to lever presses and brain stimulations were stored digitally on a computer through an analog/digital converter (CED 1401 mk II, Cambridge, UK). Filmed operant conditioning sessions were also stored with the help of the CED 1401 mk II. Extracellular recordings were analyzed using a home-modified MATLAB software (MathWorks, Madrid, Spain). Self-stimulation data were analyzed off-line for the quantification of each animal's performance in the Skinner box and fEPSP with the Spike 2 (CED) program. Five successive fEPSPs were averaged, and the mean value of the amplitude during the rise-time period (i.e., the period between the initial 10% and the final 10% of the fEPSP) was determined. Computed results were processed for statistical analysis using the Sigma Plot 11.0 package (SigmaPlot, San Jose, CA, USA). Data are always represented as mean ± SEM. Acquired data were analyzed with 2-tailed Student's t test or the 1-way or 2-way analysis of variance (ANOVA), with days as repeated measure and with a contrast analysis for a further study of significant differences.
Density of GABAergic Septohippocampal Contacts Is Decreased in J20 Mice
Following Rubio et al. (2012), in a first series of experiments, we checked whether J20 mice presented a lower density of GABAergic septohippocampal contacts and therefore, could be used as a suitable model to determine the role of the GABAergic septohippocampal pathway on the effects of MS self-stimulation. For this, we performed iontophoretic injections of the anterograde tracer BDA in the MS/DB of J20 (Mucke et al. 2000) and WT mice. This protocol results in intense BDA labeling in the MS/DB (Fig. 1A). As shown previously in WT mice (Freund and Antal 1988; Gulyás et al. 1990; Pascual et al. 2004), septohippocampal axons innervated all layers of the hippocampus and dentate gyrus (Fig. 1B). Two types of fibers were easily recognizable: Thin axons with "en passant" synaptic boutons, corresponding to cholinergic fibers, and abundant thick GABAergic axons forming complex baskets with large synaptic boutons around the perisomatic region of hippocampal interneurons (Fig. 1B, arrows). The GABAergic septohippocampal fibers were also recognizable in J20 transgenic mice, in which septal axons formed baskets on the perisomatic region of hippocampal interneurons.
To determine the density of GABAergic septohippocampal contacts in J20 mice, we performed double immunostaining of BDA tracer and PARV, which specifically label basket and axo-axonic interneurons largely contacted by GABAergic septohippocampal axons (Freund and Buzsaki 1996; Matyas et al. 2004). In agreement with previous results from our group (Rubio et al. 2012), our data showed that the complexity (i.e., the number of boutons per target neuron) of GABAergic septohippocampal contacts on PARV-positive interneurons was diminished in J20, compared with WT, mice (Fig. 1C–F). By confocal microscopy, we confirmed a substantial decrease in the complexity of GABAergic septohippocampal contacts on PARV-immunolabeled neurons in J20 mice (Fig. 1D,F). Quantitatively, the number of boutons per basket on PARV-positive cells was significantly reduced (38%) in transgenic mice compared with age-matched controls (P = 0.024, Student's t-test; Fig. 1G). In accordance with a recently published study (Rubio et al. 2012), the present data demonstrate a substantial reduction in the GABAergic septohippocampal connection affecting all interneuron types (immunodetected by GAD65/67, PARV, and calretinin) in J20 mice. The reduction affected mainly PARV-positive basket and axo-axonic hippocampal interneurons.
Differences in the Functional Properties of Hippocampal Circuits Between WT and J20 Mice
In the next experimental step, we tested the functional properties of hippocampal circuits in behaving WT and J20 mice (see Fig. 2 for details). During the input–output test, both groups separately [WT, F19,171 = 12.128, P < 0.001; J20, F19,171 = 2.050, P = 0.008] showed similar increases in the amplitude of the second fEPSP evoked at the CA1 area by the second of 2 pulses (40 ms of interstimulus interval) of increasing intensity presented to ipsilateral Schaffer collaterals (Fig. 3A,B). Interestingly, and as already described in behaving WT mice (Madroñal et al. 2009), the input–output facilitation in the second fEPSP evoked in both groups of mice at low intensities was reversed into a depression at higher intensities, but the J20 group showed a delay to switch the relationship between fEPSPs (Fig. 3C). Additionally, the total response obtained in this test (first + second fEPSPs) showed that J20 mice have significantly lower facilitation and depression (F19,171 = 4.731, P < 0.001), compared with WT mice (Fig. 3D).
Using the paired-pulse test, we evaluated the synaptic facilitation evoked by the contiguity of a pair of pulses. This is a typical presynaptic short-term plastic property of the hippocampal CA3–CA1 synapse at short (<60 ms) intervals. This phenomenon has been related to the process of neurotransmitter release (Zucker and Regehr 2002; Madroñal et al. 2009). As illustrated in Figure 4, both groups of mice showed a normal-like paired-pulse facilitation profile without significant differences between them (F5,45 = 1.542, P = 0.196). However, J20 animals showed (i.e., within their own group) a delayed facilitation, that is, the increase of the second fEPSP was only significant for 40 (P = 0.034) and 100 ms (P = 0.040) intervals, while for WT mice the paired-pulse facilitation was restricted to 10 (P = 0.01) and 40 ms (P = 0.007) interpulse intervals (Fig. 4B). These results suggest that the short-term facilitation process was delayed to longer intervals in J20 mice when compared with WT animals.
WT Mice Present Better Self-Stimulation Performance and Activity-Dependent Hippocampal Synaptic Depotentiations Than J20 Mice
As detailed in the Materials and Methods section and following a selection of individual stimulus intensities, animals were firstly shaped to associate lever presses with train stimulation of the MS. A maximum of ten 20-min sessions (1 per day) were allowed for each animal to reach criterion. For criterion, the animal was required to press the lever by itself a minimum of 20 times during a 10-min period, with pauses between self-stimulus <60 s. WT mice reached the selected criterion significantly sooner (P = 0.028, Mann–Whitney rank-sum test) than J20 animals (Fig. 5A). In addition, WT mice reached significantly (F9,72 = 2.637, P = 0.011) larger self-stimulation scores than J20 animals during most (5 of 7) self-stimulation sessions (Fig. 5B).
We checked the effects of manual train stimulation of the MS on field responses evoked in the hippocampal CA1 area. In WT mice, train stimulation of the MS evoked a positive–negative (0.24 ± 0.05 mV, peak-to-peak) extracellular field potential with a latency of 20 ± 5 ms, that lasted for 100 ± 12 ms (Fig. 6A1). The negative component was smaller and the positive component larger (0.41 ± 0.09 mV) in J20 mice than in the WT group (Fig. 6A1). This significant (P = 0.028; Student's t-test; Fig. 6A1) difference in the amplitude of field potentials evoked in CA1 by train stimulation of the MS was probably the result of the imbalance in the inhibitory direction between GABAergic and cholinergic septohippocampal projections observed in J20 mice (Palop et al. 2007; Rubio et al. 2012).
In a next step, we studied the effects of train stimulation of the MS on fEPSPs evoked at the hippocampal CA1 area by single pulses presented to the ipsilateral Schaffer collaterals. Figure 6 illustrates several profiles of fEPSPs evoked at the CA3–CA1 synapse collected immediately (40 ms) after (Fig. 6A2, B, SB), or in the absence (Fig. 6B, BL and R) of a self-stimulation of the MS. As known, the first component (≍4 ms of latency) of fEPSPs evoked at the CA3–CA1 synapse (see inset at the right of Fig. 6A) corresponds to the activation of glutamate receptors (Collingridge et al. 1983a, 1983b; Bliss and Collingridge 1993). Similar fEPSPs have already been recorded in alert behaving mice (Gruart et al. 2006).
fEPSPs evoked at the CA3–CA1 synapse, recorded after MS stimulation (shaping or self-stimulation), presented a decreased amplitude during the shaping session stage, when compared with fEPSPs recorded without MS stimulation (BL) in WT mice (Figs 5C,D and 6A,B) with a decreasing tendency (y = −1.99x + 80.4, R2 = 0.78, Fig. 5C). This decrease disappeared during the recovery period (black triangles, Fig. 5C), that is, in the 5 min after the end of the MS stimulation session. In contrast, MS stimulation in J20 mice did not evoke a similar decrease in the amplitude of fEPSPs. In fact, after the first shaping session, the fEPSPs observed in J20 animals presented an increase in amplitude when compared with BL fEPSPs values with a decreasing tendency (y = −2.16x + 121.6, R2 = 0.64, Figs 5C,D and 6A,C). Interestingly, this increase returned to BL values, starting from the fourth self-stimulation session (white squares in Fig. 5C). The amplitude of fEPSPs in J20 mice during MS stimulation (shaping or self-stimulation) sessions returned to BL values during the recovery period (white triangles in Figs 5C and 6D). As a consequence of the different effects on synaptic strength evoked by MS self-stimulation in WT and J20 mice, fEPSPs evoked in WT mice during the successive self-stimulation sessions were significantly (F8,56 = 32.294, P < 0.001) smaller in amplitude than those evoked in J20 mice (asterisks in Figs 5C and 6D). Additionally, the data show for both groups a significant decrease throughout sessions, so that the last sessions are significantly (P ≤ 0.06) different from the first ones (circles in Fig. 5C).
The late negative components presented in the fEPSPs (Fig. 6A–C) correspond to the activation of GABAA and GABAB receptors (Schwartzkroin 1986). We also checked the effects of MS self-stimulation on these late components of fEPSP evoked at the CA3–CA1 synapse (see inset at the right of Fig. 6A) at 2 different times: First, at the beginning of the experiment, while placed in the small box (see Materials and Methods) and prior to any MS stimulation or behavioral test (insets in Fig. 5D) and secondly, during MS self-stimulation (Figs 5D and 6E,F). At the beginning of the experiment, a lower GABAA or B/glutamate (GLU) amplitude ratio was observed for J20 mice, but this difference was not significant (GABAA, P ≥ 0.138 and GABAB, P ≥ 0.194). Similar results were obtained during MS self-stimulation, whereas no significant differences (P ≥ 0.306) could be observed between the 2 groups of mice for GABAA and GABAB components of the field potential. These negative results indicate that (polysynaptic) hippocampal GABAergic circuits activated by the electrical stimulation of Schaffer collaterals were not differentially affected in the 2 groups of mice (WT and J20) by the preceding train of pulses applied to the MS nucleus.
The Amplitude of fEPSPs Evoked at the CA3–CA1 Was Decreased During Consummatory Behaviors in an Operant Conditioning Task
The fact that the amplitude of fEPSPs evoked at the CA3–CA1 synapse of WT animals was decreased by MS self-stimulation prompted us to determine whether this change in the strength of hippocampal synapses could be related to internal changes in the intrinsic value of the reward in the self-stimulated animal. As an indirect way of checking the intrinsic emotional state of the animal, we designed an instrumental test that allowed us to check the synaptic strength of the same hippocampal synapse during a positive reinforcement in an operant conditioning task. In a pilot study, we apply similar procedures reported elsewhere (Madroñal et al. 2010; Jurado-Parras et al. 2012), where C57Bl/6 (n = 12) mice were trained to press a lever to obtain a small pellet of food, using a fixed-interval 1-s (FI1) schedule. Once the animals reached criterion (i.e., pressing the lever ≥20 times/20 min session), we recorded for 3 additional (20 min) sessions fEPSPs evoked at the CA3–CA1 synapse during control behavior (i.e., when animals were resting in the Skinner box) and while the animals were eating the collected pellet. As illustrated in the top panel of Figure 6G,H, the amplitude of the GLU component of the fEPSPs evoked during the consummatory (eating) behavior was significantly (P = 0.001) reduced when compared with values collected during the control situation. No significant (P > 0.70) differences could be observed for the late GABAergic components of the evoked synaptic field potentials (data not shown). This negative result could be related to the high variability found in late (GABAergic) of the fEPSPs recorded in vivo.
To verify the above point, we decided to apply the same protocol in WT littermates and J20 mice (n = 10 per group). In this experiment, and following current reports (Jurado-Parras et al. 2013), the behavior "pressing the lever" (labeled as "L. press" in Fig. 6I–K) was used as a normalization value, in order to evaluate a pure appetitive behavior versus a consummatory one. For this experiment, the lever was programmed as a trigger to deliver a single-pulse stimulation of the CA3–CA1 synapse. Collected results shown a significant decrease during eating behavior in all of the components (GLU, F1,7 = 155.777, P < 0.001; GABAA, F1,7 = 18.572, P = 0.004; GABAB, F1,7 = 21.146, P < 0.002) of the evoked fEPSP in both WT and J20 mice (Fig. 6I–K). Importantly enough, we found in J20 mice that the GLU component was increased (P = 0.019) and the GABAB component decreased (P < 0.001) in relation to the amplitude values collected from their littermate WT mice (Fig. 6I,K).
In conclusion, both consummatory behaviors, MS self-stimulation and pellet-reward produced a similar decrease in the amplitude of fEPSPs evoked at the hippocampal CA3–CA1 synapse.
LTP Evoked at the Hippocampal CA3–CA1 Synapse in J20 Mice Presented Larger Values and Produced a Larger Depressing Effect on MS Self-Stimulation Than in WT Animals
We expected that the decrease in GABAergic septohippocampal projections observed in J20 mice could modify the excitability of hippocampal circuits. To check this possibility, we decided to evoke LTP at the hippocampal CA3–CA1 synapse and to check its effects on MS self-stimulation and food-reward in both J20 and WT mice.
For the initial 3 days, each animal underwent a set of BL recordings (see Materials and Methods). Afterwards, the HFS protocol was applied followed by 30 min of post-HFS recordings at the same stimulation rate and intensity as for BL records (Fig. 7A). The same recording session with same stimulation parameters was repeated 24 h later. BL recordings of the second were used to determine the remaining LTP. After this, a second HFS session was carried out, and a 30-min session of additional recordings was repeated as well. Finally, 48 h after the first HFS, we carried out a third BL recording that was followed by a third HFS session. As before, the third HFS session was followed by a post-HFS recording session. The BL of the first day was used as a normalization value (see dotted lines in Fig. 7A). With this experimental protocol, both WT and J20 mice presented a significant LTP for the 3 recording days (P ≤ 0.05). Notably, the LTP response presented by J20 mice was significantly (F26,104 = 1.765, P = 0.023) larger and longer-lasting than that presented by WT animals (Fig. 7A). The long-lasting effect is easily observable by the progressive increase in the second and third BL values collected from J20 animals, while BL values in WT mice remained without significant changes across the 3 recording sessions.
To check the effects of LTP on MS self-stimulation and food-reward, we calculated several indexes of performance, but food-reward did not show any significant difference (P = 0.89). In contrast, a daily efficiency coefficient ([(actual number of self-stimulation reinforcements/maximum number of reinforcements obtained during BL recordings) × 100]−100) showed differences in the MS self-stimulation performance, as illustrated in Figure 7B. Following the experimental induction of LTP, J20 mice were more affected in their performance of MS self-stimulation than WT animals. Indeed, the scores reached along 3 days, by J20 for the efficiency coefficient, were significantly (F1,5 = 129.792; P < 0.001) decreased, with respect to their control values following each of the 3 HFS sessions (see Fig. 7B for P-values quantified day by day). In contrast, values collected for WT mice indicated that self-stimulation performance was not significantly modified (F1,6 = 1.751; P = 0.234) by the LTP evoked at the hippocampal CA3–CA1 synapse. In addition, self-stimulation was not significantly modified (F1,4 = 2.683; P = 0.166) in those J20 mice that did not receive a HFS session. In conclusion, the larger LTP evoked in J20 mice seems to have a more deleterious effect on MS self-stimulation than the smaller LTP evoked in WT animals.
Hippocampal GABAergic Neurons Are Involved in the Decrease of fEPSPs Evoked at the CA3–CA1 by MS Self-Stimulation
Using the same criterion for selecting stimulus intensity (see Materials and Methods), and to determine the effect of reduced GABA levels in the hippocampus on the reported decrease in the amplitude of CA3–CA1 fEPSPs during the same MS self-stimulation protocol, we employed GAD65-specific monoclonal antibody b78. This antibody inhibits the conversion of glutamate into GABA catalyzed by the GAD65 enzyme, an isoform of GAD which is particularly prominent in many axon terminals (Esclapez et al. 1994; Ishida et al. 1999; Mitoma et al. 2003).
The b78 antibody was injected 30 min before the sixth self-stimulation session (n = 8) WT mice. Figure 8A illustrates the presence of the b78 antibody in hippocampal interneurons, including some interneurons in, or close to, the pyramidal cell layer, which correspond to the PARV-positive interneurons population (Freund and Buzsaki 1996; Matyas et al. 2004). Since b78 diffusion and effects were restricted to a rather small area of the dorsal hippocampus, its injection did not have any significant (P = 0.87) effect on MS self-stimulation performance of injected animals. Nevertheless, the local injection of b78 in the close proximity of the CA3–CA1 stimulating and recording electrodes produced a significant (P = 0.006) increase in the amplitude of evoked fEPSPs during the self-stimulation session when compared with fEPSPs collected during BL and recovery period (Fig. 8B). The collected data indicate that, after acute reduction in GABA levels, the expected decrease in fEPSP amplitude during self-stimulation was reversed into an increase, similar to what was observed in J20 mice.
Since the presence of experimentally evoked LTP in J20 mice significantly decreased MS self-stimulation (i.e., the efficiency coefficient; see Fig. 7B), we decided to repeat a similar study evoking LTP in GAD b78-injected mice. As shown in Figure 9A, the animals injected with the GAD65 antibody b78 presented a significantly (F26,130 = 7.180; P = 0.028) larger LTP than the controls. The efficiency coefficient calculated for both groups before and following the experimentally evoked LTP showed no significant differences (F1,4 = 0.0426; P = 0.847) for MS self-stimulation in the control group. In contrast, the GAD b78 group presented daily a significant (F1,5 = 16.550; P = 0.010) decrease in the performance of MS self-stimulation (Fig. 9B).
A Shared Neural Mechanism for Internal (Brain) and External (Natural) Rewards
We have shown here that train electrical stimulation of the MS can be rewarding for alert behaving WT mice, and that it can serve as an operant reinforcer—namely, the experimental animal will generate specific behaviors (lever presses) to obtain this internal reward (Olds and Milner 1954; Mora and Cobo 1990; Wise 1996). Indeed, the MS has been recognized for years as an important neural center involved in self-stimulation reward (Ball and Gray 1971; Buño and Velluti 1977; Grauer and Thomas 1982; Cazala et al. 1988). The present results support the notion that this internal reward system shares similar neural mechanisms with those activated by a natural reinforcer (e.g., food), that is, both of them produced a significant decrease in the amplitude of hippocampal fEPSPs evoked at the very moment of the reinforcement. The involvement of the hippocampus in these processes is probably related to the cognitive aspects, like attention and learning, of motivational processes. Further support of this model comes from recent reports of the presence of a hippocampal–septal–ventral tegmental area circuit involved in the relationships between reward-motivated behaviors and the learned value of contextual stimuli (Luo et al. 2011). The present results suggest that there are also septohippocampal feedback projections relating internal motivational states with their cognitive counterparts.
MS self-stimulation appears to be highly dependent on the proper functioning of the GABAergic septohippocampal pathway, since J20 mice characterized by decreased density of GABAergic terminals on hippocampal baskets and axo-axonic interneurons, showed a delayed acquisition and a lower performance of MS self-stimulation than their littermate controls. A further confirmation that hippocampal GABAergic circuits are involved in the decreased amplitude of hippocampal fEPSPs during self-stimulation reward was that local inhibition of the GABA-producing GAD65 enzyme (Ishida et al. 1999; Mitoma et al. 2003) also prevented these changes.
Functional Consequences of an Increased LTP in J20 Mice and Local Inhibition of GAD65
As reported here, J20 mice presented changes in hippocampal functional properties, including a larger hippocampal LTP. Similar findings were obtained following the local inhibition of GAD65 by monoclonal antibody b78 in the dorsal hippocampus, always without any motor disturbance. These effects could be ascribed to the imbalance between septohippocampal excitatory cholinergic (Krnjević and Ropert 1982; Markram and Segal 1990) and glutamatergic (Sotty et al. 2003; Habib and Dringenberg 2009) projections versus inhibitory GABAergic (Krnjević et al. 1988) projections. In particular, it has been described that septal cholinergic projections to the hippocampus increase the excitability and LTP of hippocampal circuits (Ovsepian et al. 2004; Palop et al. 2007; Dringenberg et al. 2008). As indicated below, this increased excitability and longer-lasting effects of HFS of the CA3–CA1 synapse have some important consequences on the reinforcing value of MS self-stimulation of J20 mice.
It has already been reported in a caloric restriction program that nonsaturating LTP evoked in hippocampal synapses (CA3–CA1) has no effect on the normal acquisition and/or performance of appetitive (lever press) and consummatory (food intake) behaviors evoked during operant conditioning tasks (Jurado-Parras et al. 2012). Here, nonsaturating LTP evoked at the hippocampal CA3–CA1 synapse did not have any noticeable effect on MS self-stimulation performance in WT mice. Nevertheless, when the evoked LTP reached high and long-lasting levels as those reported here in J20 mice —due probably to the specific decrease in GABAergic septohippocampal projections (present data and Rubio et al. 2012) or to an increased effect of CA3 feedback signals onto MS neurons (Gulyás et al. 2003; Colom 2006)—MS self-stimulation performance was significantly decreased. A putative reason for the above results is that the large facilitation of fEPSPs evoked during LTP in J20 and in mice injected with GAD65-inhibition antibody b78 undoes the internal rewarding effects of MS self-stimulation. These results further support our proposal that internal and/or natural rewards are related to a decrease in the amplitude of hippocampal fEPSP responses.
Role of GABAergic Septohippocampal Pathways in Self-Stimulation and Related Processes
It has been recently proposed that a complex network of electrically coupled GABAergic neurons widely distributed in the midbrain, hypothalamus, and thalamus could contribute to the brain stimulation reward system (Lassen et al. 2007). Indeed, there is additional evidence of the involvement of GABAergic pathways and receptors in diverse behavioral modalities, including emotional displays and motivational states (Macey et al. 2001; Leppä et al. 2011). Thus, GABAergic pathways could play a complementary role versus the widely accepted dopaminergic modulation of rewarding mechanisms (Liebman 1983; Gerhardt and Liebman 1985; Wise 2002). Indeed, it has been reported that septal self-stimulation is independent from the activation of dopaminergic terminals (Prado-Alcalá et al. 1984). Thus, and as shown here, septal GABAergic circuits play an important role in internal and external rewarding processes.
GABAergic septohippocampal projections terminate on hippocampal basket and axo-axonic interneurons (Freund and Buzsaki 1996; Matyas et al. 2004) and play an important regulatory role on the intrinsic excitability and rhythmic activities of hippocampal circuits (Buzsaki 2002; Ovsepian 2006). The deficit of the GABAergic septohippocampal innervation in J20 mice may result in the significant functional alterations affecting the potential role of MS self-stimulation as an internal rewarding agent. These results point to a particular role of MS-hippocampal circuits in the integration between internal motivational states, typical of the mesolimbic dopaminergic system (Wise 1996, 2002), and cognitive, learning, and memory processes characteristically ascribed to hippocampal circuits (Bliss and Collingridge 1993; Gruart et al. 2006). Early studies proposed that septal nuclei could play a definitive role in the integration between internal drives and learning and memory processes (Cazala et al. 1988), and that septal networks represent a nodal point for processing of information from brainstem and hypothalamic centers and archicortical and neocortical structures (Colom 2006). In addition, it has been already reported that MS self-stimulation is phase-locked to hippocampal theta rhythms, a fact that further supports the functional integration between motivational and cognitive activities taking place in septohippocampal circuits (Buño and Velluti 1977). Moreover, activation of the MS enhances the synchronized firing of hippocampal pyramidal cells and contributes to the fine tuning of hippocampal rhythmic activities (Ovsepian 2006).
The decreased fEPSP amplitudes observed in WT mice appears to contradict previous reports (Tóth et al. 1997). A putative explanation could be the different frequency of septal stimulation used as reward. This train used by us (100 Hz, 200 ms, 20 pulses) could excite in a recurrent manner: the MS-hippocampus-lateral septum-ventral tegmental area circuit, and the hippocamposeptal pathway, modifying the final effect on inhibitory hippocampal neurons (Risold and Swanson 1997; Rokers et al. 2002; Manseau et al. 2008). This is supported by a late inhibitory response recorded after every train of septal-stimulation (Fig. 6A1, WT mice). In fact, the local field potential recorded in the CA1 area of J20 mice after MS train stimulation was increased (Fig. 6A1) in comparison with values collected from WT mice. In addition, the GLU component of the fEPSP evoked at the CA3–CA1 synapse was also increased in J20 mice (Fig. 6A2,B–D). These 2 results clearly indicate a disturbance in the septohippocampal inhibitory mechanism present in J20 mice. Additionally, we decided to present the CA3 single-pulse stimulation at 40 ms after the end of the MS train stimulation, that is, at the moment during which the local field potential evoked by MS train stimulation was noticed (Fig. 6A).
Although the expected response on a GABAergic septohippocampal dysfunction would be a decreased GLU component of the CA3–CA1 response, some functional alterations cannot be discarded in the local interneuronal population of J20 mice that would try to counteract the GABA septohippocampal deficit. These compensatory mechanisms at the interneuronal level would lead to an increased response of the principal cell population, as described previously (Palop et al. 2007).
Two additional experiments reported here reinforce this integrative role of septohippocampal circuits regarding motivation and learning and memory processes. First, both consummatory internal (self-stimulation) and external (food) rewards depressed in a similar way, fEPSPs evoked in hippocampal CA3–CA1 synapses and, secondly, that this functional depression of fEPSPs is prevented by the local inhibition of the GAD65 enzyme by monoclonal antibody b78 (Esclapez et al. 1994; Raju et al. 2005; Manto et al. 2011). Previous studies using slice-patch recordings have shown that b78 acts on the terminals of GABAergic neurons to suppress the release of GABA, thereby depressing the inhibitory transmission with a gradual and long-lasting time course. Administration of b78 antibody was also associated with an increase of glutamate concentrations after its administration (Mitoma et al. 2003; Manto et al. 2011). Both activity-dependent gradual functional loss of GABAergic neurons and the increase in glutamate concentrations seem to be confirmed here by the larger LTPs evoked in b78-injected mice.
In conclusion, the hippocampus can play a dual role in cognitive processes underlying internal and external rewards where septohippocampal pathways represent an important path of this intrinsic network. One of these roles could be during the acquisition process when the hippocampus is more active and, in addition, less susceptible to inhibitory effects. The other role could be during consummatory behaviors when the MS inhibitory inputs seem to be more effective.
This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU2008-00899 and BFU2008-03390) and Junta de Andalucía (Spain, BIO-122, CVI-02487, and P07-CVI-02686 to J.M.D.G. and A.G. and BFU2008-3980 to E.S.), and Spanish Fondo de Investigaciones Sanitarias (PI11/00704) to M.P. The research leading to these results also received funding from the European Community's seventh Framework Program (FP7/2007-2013) under grant agreement n° 201714 (DEVANX). M.A.G.C. was supported by a JCI-2011-09013 post-doctoral fellowship from the Spanish Ministry of Science and Innovation.
The authors thank Dr R. Sánchez-Campusano for adapting MATLAB software, Mr. J.A. Santos for help in the experimental set-up, Natalia Ruiz for histological support, and Mr. R. Churchill for his help in manuscript editing. Conflict of Interest: None declared.


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off your little training bra Don't you understand me? I'm gonna take off you little maroon hot pants I'm gonna get down on my knees, baby, dont'cha
You better make up your mind We're gonna leave you behind (repeat) RAP SPEARHEAD We're gonna move on up Five by five Say no to drugs Keep
No, I'll never give the hunt up, And I won't muck it up. Somehow this is it, I knew. Maybe fate wants you dead, too We've come together in
You'se a baffoon, caught up in your own cocoon Leave your head rest maroon Drunk heavy in the side street saloon Til' I figured it out, to the 3rd
(New words & music by DeCormier/Stookey/Yarrow/Travers) Children go where I send thee: how shall I send thee? Well, I'm gonna send thee one by one
leave you behind Make up your mind Stop marching in time Get in line You better make up your mind We're gonna get on up Five by five And this
leave you behind Make up your mind Stop marching in time Get in line You better make up your mind We're gonna get on up Five by five And this
love her Deep down I said don't ever Because it's gonna keep him hanging around Five years going by Everyone is standing on their side Never Deep down
you're down Either way I'm gonna be around You should've known that I would stay by your side You should've known your girl was gonna ride or die And it
stood at the door Five by five, five for the five that stayed alive Six by six, six for the six that never got fixed Seven by seven, seven for
They say baby how you do it? Never question greatness Never question greatness Tell 'em never question greatness I just do it I'm so high I feel
sweet throat That's been touched by the Tingler But in the night cold as ice Hi-five co-ed mingler It leaves no marks in spite of sparks And so
from your sweet throat That's been touched by the Tingler But in the night cold as ice Hi-five co-ed mingler It leaves no marks in spite of sparks
about four or five months Samantha Brown lived in my house for about four or five months Don't know how it looked to other people I never slept with
Holy man get you hands down out of twenty five (some strange yelling type thing) aaa! Go ahead and leave ya When she said to see me Said ya gone
Looking for a soul to take You're gonna see me coming by the selfish things that you did I'm gonna leave you guessing how this funnel is gonna hit I'm
See the faulty now See the dawn See clear Five six it's gonna be narrow By written law For right of hollow form By thy all night Figure
down Either way I'm gonna be around You should've known that I would stay by your side You should've known your girl was gonna ride or die And it
And the means to it all will get a ski mask involved And you winning or you swimming in a flood of the maroon type Sheets by the dozen and you covered by a boom
10:00 I was gonna write a letter but at 1:00 is when I opened up my eyes. 3:00 turned into 5:00. I watched the day pass me by. 6:00 I was changing
and it's all gonna roll your way Workin' 9 to 5, what a way to make a livin' Barely gettin' by, it's all takin' and no givin' They just use your mind
send thee? I'm gonna send thee six by six Six for the six that never got fixed Five for the five that stayed alive Four for the four that stood
to believe Feeling like Maroon 5 without ya girl its harder to breath I thought we'd make it through the coldest weather We was supposed to grow old
nobody sting me but you I'll be buzzin' 'round your hive Ev'ry day at five And I'm never gonna leave once I arrive I'm done, ah-ha, I got stung I
the five I looked him in his eyes, grabbed the dice Son I'll five 'em better twice for ya fuckin' life Money feed good, all downs is a bet Meanin', any

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It hasn't been a happy new year for gasoline prices in Connecticut.
The price of a gallon of regular unleaded gas hit $3.77 in the Norwich-New London area Friday, a nearly 4 percent increase since the first of the year, according to a new report by the American Automobile Association.
"Prices remained pretty steady for most of January, but then in the last week they increased about 10 and a half cents," said Aaron Kupec, public affair manager for AAA in West Hartford.
Kupec pointed out that the Norwich-New London region experienced a much bigger bump in gas prices than the rest of Connecticut. The state as a whole saw about a 3 percent rise in prices.
Kupec pointed out that Connecticut - with the fourth-highest gas prices in the nation - actually had a smaller increase in fuel costs than the United States as a whole, which saw a 4 percent spike over the past month.
But he warned that prices may escalate further, especially after a planned closure at Hess Corp.'s 70,000-barrel-a-day Port Reading, N.J., refinery is completed by the end of this month. The facility provides 7.5 percent of the gasoline consumed in the Northeast.
"The next few months may be difficult," Kupec said.
Kupec said zone pricing could be responsible for some of the price discrepancies seen this month among the various regions of Connecticut. Under a zone pricing system, according to a state Office of Legislative Research report issued in 2006, suppliers of motor fuel set their wholesale price according to certain boundaries that they refuse to identify for competitive reasons and which Kupec said could be as many as several dozen.
"Each supplier decides for itself whether and where to have price zones," the report said. "Where price zones exist, suppliers consider such factors as local competition among retailers and geography."
While zoning worked out in favor of the Norwich-New London region around Thanksgiving, when the area had among the lowest gas prices in Connecticut, the cost of motor fuel now is closer to the statewide average.
Though gas prices have spiked in the past month, they are not significantly higher than last year at this time, when they averaged $3.74 a gallon, only 3 cents lower than today.
The average national price for unleaded gas is $3.35, 7 cents cheaper than a year ago.
"The retail price of gasoline has now increased for 11 consecutive days, the longest such streak since prices rose for 21 straight days July 28-Aug. 18 (of last year)," according to AAA.
Driver dies after his car rolls over in Groton early Monday morning
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Latest Comments
"I like this vintage style Santa graphic, but I read that the designer used a cutting machine to cut a red vinyl decal to afix to the cream painted wood board. I don't know what kind of cutting machine she is talking about and I wonder how pricey it is? I'd like an alternative to getting that Santa on there."
"This wall decor is very chic with any message you write on it! And it couldn't be easier. However, another option to using foam core board would be to actually buy an old thrift store bulletin board and spray paint it black. I think it would give the wall art a little bit more of a lived-in rustic look as the cork board adds texture and interest."
"This DIY holiday project is something that I would like to do at home myself. This craft is not for everyone's taste, but if you like a certain minimalist decorating approach and you've got a creative eye then this is for you! At first I did not even notice the picture frame in the background. It's used to attach the branch to it. Cute idea!"
Linen and Lace Summer Scarf
We are adding the craft to your Craft Projects.
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Scarves aren't just for fall and winter -- a lace-trimmed linen scarf is the perfect accessory for warmer weather. If you want to add a touch of color to your summer fashion, this easy-to-make scarf from Jenya of MyEvaForeva fits the bill.
- Good quality pure linen, 1-1/2 to 2 yds. x 50" to 60" wide
- Flat lace, 2-7/8 to 3-3/8 yds.
- Sewing machine
- Serger (optional)
- Thread
- Scissors
- Serge the shorter edges and use a fancy picot edging stitch on longer sides. If you don't have a serger you can use your sewing machine and do a running stitch on a narrow folded edge.
- Attach the lace to your shorter edges using a sewing machine. To secure exposed sides of the lace trim I just used a zig-zag stitch.
- Now submerge the whole scarf in cool water and them squeeze the water out. Let the scarf dry a bit. When it's still slightly wet twist it really well like shown below and let it dry completely. Once it's dry, open it up and give it a good shake.
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Apr 30, 2016
I agree arodriguez. This scarf is so beautiful. So simple yet so elegant. And the choice of color is just so cool and classy. Thanks for the sewing instructions.
arodriguez 5298921
Jan 29, 2016
I absolutely love the color of this scarf as well as the tiny lace detail. It is a spring scarf that everyone should have in their closet.
antoinettegrussell 0668209
Apr 14, 2014
This is a great idea for people (like me) who live in hot countries and need sun protection of the neck and decolletege while keeping cool. I like this scarf and will try to make it. It could be used as a wrap for cooler summer evenings as well.
magginkat 4915793
Jun 18, 2013
Too....too bulky. 58-60" wide? 2 yds long? I'm a fan of skinny scarves. This looks more like a blanket of some sort.
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- 2-3 Flare
- Delayed Forward
- Downshift clunk
- No Forward
- Poor servo timing
Weak or broken servo spring.
Install Sonnax servo spring for better servo timing.
Low/Intermediate Servo Spring
Part No.
Sonnax low/intermediate servo spring 96995A replaces the OE spring found in Ford AX4S, AXOD and AXODE units.
- Upgraded spring is longer and has a spring rate approximately 12 lbs. lighter than the OE, reducing delayed Forward engagements
- Spring is stiffer than the common OE replacement spring to eliminate downshift clunk complaints
Related Parts
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BARACK OBAMA BIRTH CERTIFICATE: Suit contesting Obama's citizen...
There are 237681 comments on the Chicago Tribune story from Jan 8, 2009, titled BARACK OBAMA BIRTH CERTIFICATE: Suit contesting Obama's citizen.... In it, Chicago Tribune reports that:The U.S. Supreme Court will consider Friday whether to take up a lawsuit challenging President-elect Barack Obama 's U.S. citizenship, a continuation of a New Jersey case embraced by some opponents of Obama's ...
Join the discussion below, or Read more at Chicago Tribune.
#172438 Sep 21, 2013
What's the difference between Obama and Osama?
Just a little bs.
"zero nuclear weapons"
Since: Sep 08
#172439 Sep 21, 2013
Zimmerman saw Martin. Zimmerman followed Martin. Zimmerman confronted Martin. Martin tried to defend himself Zimmernan shot and killed Martin.
Zimmerman lied about what happened. What he told the police changed 2 or 3 times like none of Zimmerman's blood or DNA was found on Martin's hands or his clothing. Zimmerman told the police that Martin had knocked him down, broken his nose, and covered his face with his bare hands while he slammed Zimmerman's head into a concrete sidewalk.
The lie: Zimmerman told his friend Mark Ostermanwho has himself been a cop for over 20 yearsthat he was in a desperate struggle with Trayvon for Zimmerman's gun. Zimmerman never said this to police. But Osterman wrote in a book, and it was entered into evidence, that Zimmerman said,somehow I broke his grip on the gun where the guy grabbed it between the rear site and the hammer. I got the gun in my hand, raised it towards the guys chest, and pulled the trigger.
The truth: DNA evidence ruled out any real possibility that Trayvon ever touched Zimmerman's gun. The only DNA evidence on the gun's grip was that of George Zimmerman. None was Trayvon's. Zimmerman also claimed that Trayvon went for Zimmerman's holster. Anthony Gorgone, a DNA lab analyst for the Florida Department of Law Enforcement, testified as an expert witness that Zimmerman's holster tested positive for only Zimmerman's DNA as well.
The lie: Zimmerman claimed that Trayvon bashed,slammed, and wailed his head into the sidewalk. One of Zimmerman's lawyers claimed that the sidewalk was Trayvon's deadly weapon. Pictures of Zimmerman's alleged injuries have been shown repeatedly on tv.
The truth: There is no way that Zimmerman's head was repeatedly slammed into the sidewalk. As Dr. Valerie Rao, a medical examiner in Florida, testified, Zimmerman's injuries were insignificant,so minor that all they required was an ordinary band-aid.
The lie: Zimmerman claimed that Trayvon jumped out of bushes and hit him, knocking him to the ground.
The truth: There are no bushes to jump out of where Zimmerman claims he was assaulted. This is clearly evident on the video where Zimmerman walked through his version of what happened with some Sanford police
"zero nuclear weapons"
Since: Sep 08
#172440 Sep 21, 2013
Get this though that empty space between your ears people that the ones from that sight are religious fanatics. people like that NUTS!!!! Do you realize that they said saddam hussein was the anti christ
If knew anything the name of the antichrist is MABUS so stop beliving sites like Twelvebooks and get the mental help you need
#172441 Sep 21, 2013
Speaking of fanatics! Kook!
#172442 Sep 21, 2013
Quit your damn whining. The case is over. The Jury has rendered its verdict! Do you understand what that means? Get over it already.
#172443 Sep 21, 2013
Sort of like two national elections and more than 200 court decisions. Obama is POTUS. Birfoons need to get over it and find a new fable
#172444 Sep 21, 2013
The gum-flappers are the only ones that bring it up. You don't hear us bytchin' and moanin' about it. Guess you've got to have a peg to hang your hat on, huh? Besides, 2 elections and 200 cases really don't compare to Path's individual record of 0 - 316 for false accusations. LMAO
Think ND has a shot? I don't know anything about either team.
#172445 Sep 21, 2013
Out for a bit. Cya
#172446 Sep 21, 2013
Jercque has been on record pace. And I will keep your comment in mind the next time you paste in one of your Birfoon fables
Who is ND playing? As I've told you before - not everyone in Indiana is a ND fan. I appreciate the fact that they are a good school that graduates their players and fields a competitive team. So does Stanford but that doesn't mean I'm a Stanford fan either
#172447 Sep 21, 2013
Your referring to Rachel Jeantel, who admitted she can't read (product of the union sponsored public schools) and also admitted in a post trial interview that Trayvon thought that Zimmerman was a "creepy cracker" that could possibly be a gay, male rapist. You see, according to Jeantel, Trayvon profiled Zimmerman and concluded that he needed a "beat down" to make sure he did not know which unit Trayvon and his brother were staying, because he was protecting his younger brother and anyone else in the complex from the "creepy cracker". The miscalculation on Trayvon's part was not informing Zimmerman that he was only going to beat his ass and not kill him. Obviously George interpreted Trayvon's actions as aggressive enough to put a slug in the little 6'+, 17 year old "boys" chest that was kicking his ass before Trayvon could clue him in. It was simply a matter of poor communication and a misunderstanding.
"Bonjour Hello Buongiorno Hola"
Since: Feb 12
#172448 Sep 21, 2013
At no times did I dispute the jury's decision. Not even once. I did say Z killed Martin. I'm right. I did say you were there along with LRS ,(the way you two described how it all unravelled, you MUST have been), I was wrong.
#172449 Sep 21, 2013
It also could have been that Trayvon was just playing a quick round of the knock out game too.
"Bonjour Hello Buongiorno Hola"
Since: Feb 12
#172450 Sep 21, 2013
ah,I forgot , you were there too. He's dead, whatever the reason, and you insist, without knowing what exactly happened, though you insist you KNOW how it went, that the late Trayvon Martin is "the annoying lil pryck "? Remind me : You talk with God daily, right? Have you told him about this?
"Bonjour Hello Buongiorno Hola"
Since: Feb 12
#172451 Sep 21, 2013
You wrote : "Act like a punk and you just might get shot. Pretty simple lesson. "
Hmmm, how come you haven't been shot dead yet?
"Bonjour Hello Buongiorno Hola"
Since: Feb 12
#172452 Sep 21, 2013
Still haven't figured out what Notre-Dame means, have you? Google it. Google translator. Quel CON.
#172453 Sep 21, 2013
Quit your damn whining republican House members. Obamacare is the law. The case is over. The Supreme Court has rendered its verdict! Do you understand what that means? Not even a 43rd defund vote will change that. Get over it already.
#172454 Sep 21, 2013
Well played sir. Well played
#172455 Sep 21, 2013
Her reading and writing skills are a moot point.
The racist uneducated jurors did not believe her testimony and used her speech impediment against her.
It is sad that Martin did not possess a knock out punch.
It would have been only two hits.
Martin hitting Zimmerman and Zimmerman hitting the floor.
What you reap you sow and Zimmerman will get his at the civil trial.
"Terrorism is Part of Our History": Angela Davis on 63 Church Bombing, Growing up in "Bombingham" September 16, 2013 / http://tinyurl.com/maovl66
Sunday marked the 50th anniversary of the bombing of the Sixteenth Street Baptist Church in Birmingham, Alabama, a watershed moment in the civil rights movement. On Sept. 15, 1963, a dynamite blast planted by the Ku Klux Klan killed four young girls in the church Denise McNair, age 11, and Carole Robertson, Cynthia Wesley and Addie Mae Collins, all 14 years old. Twenty other people were injured. No one was arrested for the bombings for 14 years. We hear an address by world-renowned author, activist and scholar Angela Davis, professor emerita at University of California, Santa Cruz. She spoke last night in Oakland, California, at an event organized by the Civil Rights and Restorative Justice Project at Northeastern University School of Law.
#172456 Sep 21, 2013
I hear you loud and clear.
I am always flummoxed by rightwingers un-appreciation for Obama, Pelosi, and Reid for not impeaching Bush and Cheney.
Rightwingers dislike Obama for continuing over 90% of all Bush/Cheney failed policies. Only the wealthy benefits.
Rightwing healthcare, Rightwing Tax Cuts for the wealthy, Rightwing Wars, Rightwing spying on Americans, Rightwing everything in the Obama administration.
We just had 8 years of Bush/Cheney and another 8 years of Obama/Bush.
2016 looks very favorable for another Clinton vs Bush matchup.
Hillary and Jeb.
Very sad, but very true, unfortunately.
Bush's disapproval rating worst of any president in 70 years
Apr 22, 2008 / http://tinyurl.com/5bwryk
WASHINGTON President Bush has set a record he'd presumably prefer to avoid: the highest disapproval rating of any president in the 70-year history of the Gallup Poll.
In a USA TODAY/Gallup Poll taken Friday through Sunday, 28% of Americans approve of the job Bush is doing; 69% disapprove. The approval rating matches the low point of his presidency, and the disapproval sets a new high for any president since Franklin Roosevelt.
The previous record of 67% was reached by Harry Truman in January 1952, when the United States was enmeshed in the Korean War.
Bush's rating has worsened amid "collapsing optimism about the economy," says Charles Franklin, a political scientist at the University of Wisconsin-Madison who studies presidential approval. Record gas prices and a wave of home foreclosures have fueled voter angst. http://www.usatoday.com/printedition/news/200...
The Constitution 101:
In the Federalist Papers it said: We were not going to have a monarchy, and we were not going to have a democracy.$$$$$$$
And to this day we have neither. For two hundred years we have had an oligarchical system in which men of property can do well (Dumbya Bush), and others are on their own.$$$$$$$
American politics is essentially a family affair, as are most oligarchies. When the father of the Constitution, James Madison, was asked how on earth any business could get done in Congress when the country contained 100 million people whose representatives would number half a thousand, Madison took the line that oligarchy's iron law always obtains: A few people invariably run the show; and keep it, if they can, in the family.$$$$$$$
Our founding fathers had such a fear and loathing of democracy that they invented the Electoral College so that the popular voice of the people could be throttled, much as the Supreme Court throttled the Floridians on December 12, 2000.$$$$$$$
We were to be neither a democracy, subject to majoritarian tyranny, nor a dictatorship, subject to Caesarean folly.$$$$$$$
As American media is controlled by that corporate America which provides us with political candidates a well-informed electorate is not possible.$$$$$$$
#172459 Sep 21, 2013
Yes, I have. He's cool with it.
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ONTARIO -- A Memphis Grizzlies forward allegedly got into a fight with the head coach of the New York Knicks and, of course, it involved the Los Angeles Lakers.�
But after the reported altercation between Matt Barnes and Derek Fisher, the player who knows both of them very well had nothing to say.�
"I'm not getting into that one," he said.�
Fisher was Bryant's teammate for all five of the championships won by the Lakers under Phil Jacakson. Barnes joined the Lakers following the title in 2010, and was a member of the roster until 2012. He spent the next two seasons with the Clippers, before being shipped out in the deal that brought Lance Stephenson to L.A.�
On Thursday, Barnes told reporters in Santa Barbara, where the Grizzlies are holding training camp, that he went to his old house to check on his kids, not to confront Fisher as was commonly reported. Fisher is reportedly involved in a relationship with Barnes' estranged wife.�
A few other notes from the Lakers 105-97 loss to the Toronto Raptors...�
"And at the other guard..." are some of the most famous words in Lakers history. Staples Center public address announcer Lawrence Tanter has been introducing Bryant that way for 19 seasons.�
This week in Hawaii, however, with D'Angelo Russell and Jordan Clarkson starting in the backcourt, he had to change it up and start introducing Bryant as a forward.�
"Messing with my routine," he joked Thursday.�
So what's it like to have to make an adjustment?
"Very weird," Tanter said. "After 20 years?" �
Bryant got a kick out of the change, at least when he was asked about it. Up to that point, he said he hadn't noticed his introduction being any different.
"Honestly I haven't paid too much attention to it," he said. "Guess I will now, though." �
For the third straight year, Bryant's rank in ESPN's annual countdown of all NBA players turned heads. This year, he checked in at No. 93.�
By now, however, Bryant doesn't even bother to fight back.�
"I don't need to defend that," he said. "Nobody does really. ... Please don't ask me about silly stuff like that."
A year ago he called ESPN "a bunch of idiots" for ranking him 40th overall, and a year earlier, he called being ranked No. 25 "silly" and "laughable."�
Julius Randle was really impressive for a second straight game. He scord 17 points on 7-of-10 shooting, and after getting into early foul trouble, stayed on the court for 23 highly effective minutes.�
"I'm just very happy with the way he's playing," Byron Scott said. "He's competing, he's getting a lot of things right that we've talked about on both ends of the floor.
"And he's a worker, he's had to work hard to get to this point, and I think he has a lot of ability to do a lot of different things on the basketball court. And we're starting to see some of those things come to light and want him to keep going."�
Randle said the key to his success -- and a disclaimer to all that this is the preseason we're talking about here -- is "just making easy, simple plays."�
"Letting the game come to me,' he said. "Just trusting everything I worked on in the summer and staying confident."
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The Journey Home
What Near-Death Experiences and Mysticism Teach Us About the Gift of Life
By (author) Phillip L. Berman
Format: Paperback
Normal Price: $36.06
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Journey Home by Phillip L. Berman
Book DescriptionHarvard theologian Phillip Berman examines how near-death and mystical experiences can give our lives new meaning. Universal truths culled from the author's and others' experiences allow readers to see that these messages have immediate relevance to everyone's lives, and show how to let these lessons transform one's own sense of worth and meaning.
Buy Journey Home book by Phillip L. Berman from Australia's Online Bookstore, Boomerang Books.
Book DetailsISBN: 9780671502379
(209mm x 134mm x 17mm)
Pages: 224
Imprint: Pocket Books
Publisher: Simon & Schuster
Publish Date: 1-Jan-1998
Country of Publication: United States
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Melanie Marshall was left in tears after she crashed out of the 200m freestyle when she finished last in the opening semi-final.
The 22-year-old from Lincolnshire went out with all guns blazing and took control of the race to lead at the 150m mark. However, she ran out of steam in the final 50 metres and was overtaken by every other swimmer, recording a very disappointing time of 2 minutes 01.06 seconds.
Marshall is the fastest woman in the world this year in the event, and was fancied for a medal, but the Olympic dream is now over for the


text:
Sale Agreed
Savills is delighted to present 12 Douglas House, a superb two bedroom first floor apartment in excellent decorative order. The apartment has its own door access and has been thoughtfully designed to maximise the amount of natural light getting into both the living and bedroom accommodation.
The apartment has been finished to a high standard and includes features such as ash solid wood floors, polished black granite worktops and a Jacuzzi style bath. A balcony area off the living room offers a lovely westerly aspect to enjoy the evening sun.
The grounds of the development have been well landscaped which has complimented the mature site, this has resulted in the creation of quite seating areas and rolling lawn spaces.
The location is ideal with all of Douglas village's amenities such as shops, bars and restaurants within strolling distance. Whilst Cork city, the link road network and Cork International airport are located close-by.
Hallway An inviting and private space with Ash solid wood floor, storage closet complete with hangers and recessed lighting.
Living/Kitchen/Dining Room Bright open plan room with an Ash solid wood floor in the living area and a tiled floor and splashback in the kitchen/dining space. The living room provides direct access onto the west facing balcony. The kitchen provides eye and floor level units, integrated gas hob and electric oven. Recessed lighting.
Bedroom 1 Double room with built-in wardrobes and a dual aspect.
En Suite WC. Whb. Shower unit. Tiled throughout.
Bedroom 2 Double room with built-in wardrobes.


text:
Fair Promotes New Products and Services along with Business Cooperation
Around a hundred corporate and individual innovators here in China and abroad are now demonstrating new products and services while seeking business cooperation during a two day creative event that ended today in Shekou. In 2006 the Maker Faire started in the US is an event organized by people that are interested in experimentation and innovation in the fields of engineering, performance arts and craft and science which enables them to showcase and share their works.
The Maker Faire was co-organized by CSDN, Leiphone.com and Seeed Studio. The fair is said to be the largest gathering of do it yourself professionals and hobbyists and it's the first the city has hosted such an event. Shenzhen residents enjoyed looking at new innovative products and were also able to experience the latest technologies at the fair.
Several technologies that were presented in the fair and it something to look forward to is the new remote control technology developed for cameras. The new remote technology will allow people to shout in front of their cameras to take their picture. Called the triggertrap which users can download for free allows them to connect their phone to any camera
Another technology is a smart bicycle handlebar that has a built in SIM card with Bluetooth and GPS capabilities that allows the rider to connect their mobile phones wirelessly to the handlebar and gather navigation information.


text:
Call 0333 200 2024 lines open Mon to Fri - 9am to 6pm
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Westminster Liberty White Dining Table
Model Number:
Westminster Roma Platinum Armchair
United Kingdom based Westminster produce what is considered to be some for the finest outdoor furniture on the market. All of the Westminster range is handmade by skilled craftsman using only the highest grade raw materials. As well as this, their outdoor furniture comes with a 10 year guarantee on frames and a 5 year guarantee on fabrics.
View the whole Westminster range.
Usually dispatched in 7-10 days, deliveries normally arrive within 12 working days.
For more information please call us on 0333 200 2024. Lines are open from Monday to Friday - 9am to 6pm. Alternatively please email us at firstname.lastname@example.com.


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Get ready to hail the Master Chief!
Enter to win a new, XBOX ONE Halo: The Master Chief Collection Gaming Bundle!
Hosted by The Link Fairy
Get ready to celebrate one of the biggest games in XBOX history. BUNGIE brings Halo's Master Chief and all of his amazing adventures back to life in one place with the new XBOX ONE Halo: The Master Chief Collection Gaming Bundle! My husband is a self-proclaimed big kid, and he would absolutely LOVE to be the owner of this awesome gaming bundle.
Gaming bundle includes:
- Xbox One Console with 500GB hard drive***
- Halo Master Chief Collection Game Download Code
- Wireless Controller
- Chat Headset
- HDMI cable
- Power Supply
- Full download of all four Master Chief Halo games at 60fps
- 45 campaign missions and over 100 multiplayer maps, including 6 re-imagined maps from Halo 2: Anniversary
- Access to Halo: Nightfall, the live action digital series from executive producer Ridley Scott
This giveaway is in no way associated with facebook, twitter, pinterest or other social media. Newly Crunchy Mama of 3 is not responsible for the fulfillment or shipment of prizes. If you'd like to have your product hosted through a Link Fairy giveaway, please contact Rachel at firstname.lastname@example.com.