Pre- and Postsynaptic Components of the Altered Panx1^−/− LTP

Acute pharmacological blocking of Panx1 channels only partially emulated the Panx1^−/− LTP-phenotype and additional physiological alterations were considered. Since Panx1 is implicated in paracrine and autocrine signaling [35], [36], initiated by activity-dependent release of ATP [14], [15], [37], [38], [39], [40], it was reasonable to speculate that lack of Panx1 in adult mice prompted an ATP misbalance, where upon intracellular postsynaptic ATP levels increase and extracellular ATP, as well as its metabolic breakdown products, fall to critically low levels.

Testing this hypothesis directly, we found that extracellular application of adenosine (3 µM) [41] almost completely normalized LTP in Panx1^−/− (Fig. 3A, filled red circles) with values falling close to levels of Panx1^+/+ controls in ACSF (open circles). Early and late LTP responses were affected (Fig. 3A, B and Table S1; mean fEPSP early LTP: Panx1^+/+, 112.2±0.7%; n = 7; Panx1^−/−, 150.3±1.4%; n = 5; P<0.0001; late LTP mean fEPSP: Panx1^+/+, 127±0.7%; Panx1^−/−, 146.7±0.5%; P<0.001, all by ANOVA), and the oscillatory components of the fEPSP responses were abolished (Fig. 3D, arrow). This result is consistent with the anticonvulsant and anti-excitatory roles of adenosine, shifting the inhibitory function of A₁ adenosine receptors towards enhanced synaptic activity by a combination of actions [42], [43].

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Figure 3. Pre- and postsynaptic components of altered Panx1^−/− LTP.

a, Evoked LTP in presence of 3 µM adenosine: Panx1^−/− derived fEPSPs during early and persistent LTP phases are restored towards LTP levels of the untreated controls. Figure insets in (a) indicate ^−/− and ^+/+ original responses under blocking conditions before and 30 min post HFS (scale horizontal: 10 ms, vertical: 0.5 mV). b, Comparison for early phase (0–5 min) and persistent phase LTP (25–30 min). All conditions differ significantly from the Panx1^−/− (see Figure 1). c, IO- correlation of the adenosine-treated Panx1^−/− in comparison to Panx1^−/− and Panx1^+/+ under ACSF conditions. Adenosine treatment normalized the input sensitivity of the Panx1^−/− towards values of the untreated controls (Panx1^+/+). d, fEPSP sample traces of the Panx1^−/−, revealing a decrease in oscillatory activity (arrow) and amplitudes. e, NMDA dependence of ^−/− and ^+/+ derived LTP after application of 50 µM D-AP5. NMDA inhibition leads to decrease of the signal during early and persistent phase of LTP, as demonstrated in (f). f, All conditions differ significantly from the Panx1^−/−. Statistics: (b) ANOVA (early: _F3,64 = 157.9; P<0.0001; late: _F3,64 = 458.7; P<0.0001), (f) ANOVA (early: _F3,64 = 184.9; P<0.0001; late: _F3,64 = 547.4; P<0.0001).

https://doi.org/10.1371/journal.pone.0051767.g003

A postsynaptic component was anticipated because of altered presynaptic excitatory neurotransmitter release. Thus, the contribution of N-methyl-D-aspartate receptors (NMDAR) was tested. Bath application of the NMDAR antagonist, D-AP5 (50 µM), 10 min prior to LTP recordings, significantly reduced early and persistent LTP in Panx1^−/− slices to levels less than those of ACSF-treated Panx1^+/+ slices (Fig. 3E and Table S1; early: Panx1^+/+, 113.8±0.5%; n = 5; Panx1^−/−, 130.1±0.5%; n = 5; P<0.0001; late: Panx1^+/+, 126.2±0.2%; Panx1^−/−, 145.7±0.3%; P<0.0001, ANOVA). In general, application of D-AP5 led to significant prevention of LTP, as described for rats [44]. These results are in line with a chronic depletion of ATP/adenosine, causing sustained excitatory neurotransmitter release and increased postsynaptic excitability.

Upregulation of Metabotropic Glutamate Receptor 4 in Panx1^−/− Mice

Next, we investigated whether expression of 84 plasticity-related genes were altered in Panx1^−/− mice. The result was unexpected since transcriptional alterations were limited to upregulation of metabotropic glutamate receptor 4 (grm4) (Fig. 4A, and Fig. S4). All other candidates showed stable mRNA expression levels (Table S2). This transcriptional elevation was specific for adult Panx1^−/− mice, with no alterations found at younger ages (postnatal day 8; data not shown).

Application of the group III mGlu antagonist, UBP1112, in the grm4-sensitive dose of 100 µM [45] led to distinct changes in the LTP responses of Panx1^+/+ (n = 6) and Panx1^−/− (n = 7) mice (Fig. 4B). UBP1112 elicited a significant reduction of the persistent phase of LTP in Panx1^−/− (blue circles) starting at 15 min post-HFS, although the decreased LTP did not reach the level of late-LTP in untreated Panx1^+/+ controls (grey circles) (Fig. 4C; late: Panx1^+/+ in ACSF, 174.6±0.9%; Panx1^−/− +UBP, 210.1±1.4%; P<0.0001, ANOVA and Holm-Sidack post-hoc test). In contrast, the antagonist enhanced LTP in Panx1^+/+ slices. The latter finding is in line with an autoreceptor function for group III mGlu receptors [46]. Correspondingly, overexpression of grm4 may comprise an adaptive response to the enhanced excitability observed, caused by the putatively increased glutamate release.

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Figure 4. Upregulation of metabotropic glutamate receptor 4 in Panx1^−/− mice.

a, Volcano plot representing the expression of 84 genes relevant for synaptic plasticity (Panx1^+/+/Panx1^−/−, n = 4). b, Inhibition of grm4 by bath application of 100 µM UBP1112. Fifteen minutes post-HFS, UBP1112 induced impaired persistent LTP in Panx1^−/−s. Panx1^+/+-LTP steadily increased during the entire post-HFS period without reaching the level of untreated Panx1^−/−s. Insets in (b) indicate Panx1^−/− and Panx1^+/+ original responses under blocking conditions before and 30 min post-HFS (scale horizontal: 10 ms, vertical: 0.5 mV). c, No differences were found for Panx1^−/−s during the early LTP phase (0–5 min) compared to the ACSF treated Panx1^−/−s. Persistent phase analysis reveals alignment of the fEPSP levels of both the UBP1112-treated ^+/+ and ^−/−. Statistics: (c) ANOVA (early: _F3,64 = 637.1; P<0.0001; late: _F3,64 = 105.9; P<0.0001).

https://doi.org/10.1371/journal.pone.0051767.g004

Behavioral Dysfunctions of Panx1^−/− Mice

Subsequently, the consequences of the described molecular and physiological alterations were tested at the behavioral level. Pre-pulse inhibition of the acoustic startle response was used to assess anxiety levels and sensor-motor gating capabilities. Three different pre-pulse intensities (64, 68, 72 dB) were tested in Panx1^−/− (n = 8) and Panx1^+/+ mice (n = 6) [27]. An impoverished reduction in the amplitude of the startle response was evident in Panx1^−/− animals compared to Panx1^+/+ controls at all intensities tested, with a statistically significant effect emerging at the intensity of 72dB (Fig. 5A; 64 dB: F_(1,12) = 1.037; P = 0.3285; 68 dB: F_(1,12) = 1.28; P = 0.28; 72 dB: F_(1,12) = 4.933; P<0.05, One-way repeated ANOVA). This result is consistent with physiological alterations in the central nervous system and, experimentally, supports a tendency to stress and anxiety-related responses observed in the Panx1^−/− mouse colony.

To assess cognition, Panx1^−/− (n = 7) and Panx1^+/+ mice (n = 9) were tested for their ability to discriminate between a known and a new object. In the first trial, animals were exposed to two objects (A) and (B) and allowed to explore them for 5 min. One hour later, the animals were re-exposed to object A and, exposed for the first time, exposed to a new object (C). No significant difference was seen found in the exploration of object A (P = 0.986) or object B (P = 0.968) when the animal groups were compared (Fig. 5B). Similarly, no significant difference was evident in the level of exploration of object A versus object B for either Panx1^+/+ or Panx1^−/− animals. When 1h-later, exploration of object A was compared with the novel object, C, the Panx1^+/+ animals explored the new object significantly more than the old object (Fig. 5C; P<0.05). By contrast, Panx1^−/− mice explored the new object significantly less than the old object (P<0.05), indicating that loss of Panx1 led to deficits in object recognition memory.

Finally, we tested spatial memory capabilities by investigating the ability to remember a place where a treat was hidden during consecutive training trials. Sequential reduction of cookie size and thereby odor cues trained mice to use a memory strategy in preference to olfaction. Both groups performed equally well during initial trials with no significant differences found (n = 9 each group, p>0.05, Student’s t-test). At trial 7, both groups found the cookie showing no significant difference, suggesting that olfaction is not impaired in Panx1^−/− mice (Fig. 5D, Panx1^+/+ = 64±21 s, Panx1^−/− = 45±10 s, P = 0.45). Finally, the cookie was removed to challenge the animals’ capability to remember the previously trained location. Panx1^+/+ mice remembered significantly better where the treat was hidden (Fig. 5E; path-length to correct location: Panx1^+/+, 8.4±1.3 cm; Panx1^−/−, 12.4±1.3 cm, P = 0.02). WT mice walking moved less and spent more time searching near the correct (former) cookie location (Movie S1). However, Panx1^−/− mice were not memory-deficient: they were capable of remembering, to some extent the previous training trials, as their mean path-length to the former location of the cookie (Panx1^−/−, 12.4±1.3 cm) was significantly lower compared to untrained littermates (Panx1^+/+: 18.7±1.5 cm, P<0.001, Student’s t-test). This test suggests that Panx1^−/− mice are memory-impaired but not memory-deficient.

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Figure 5. Behavioral dysfunctions of Panx1^−/− mice.

a, Pre-pulse inhibition of the acoustic startle response (PPI) showing a tendency towards lower levels in Panx1^−/− (n = 8) compared to Panx1^+/+ (n = 6) mice at the intensities of 62 and 64 dB. At the intensity of 72 dB, a statistically significant reduction of the PPI is detected. b, Object recognition was equivalent in both Panx1^+/+ and Panx1^−/− mice when the animals were allowed to explored two novel objects (A and B) for 5 minutes. c, One hour later control Panx1^+/+ mice explored the now familiar object A significantly less than the novel object C. By contrast, Panx1^−/− animals explored object C with significantly less intensity than object A. d, Cookie finding test: Training trials were performed on seven subsequent days, where in trial 1 and 2 a large cookie was used (500 mg), in trial 4 and 5 a smaller cookie (50 mg), and in trial 6 and 7 replaced by a very weak-odorous mouse chow. The time till the mice held the treat in their front paws is depicted in the bar diagram. Panx1^−/− performed equal to Panx1^+/+ mice in this experiment (P>0.05, Student’s t-test) e, A further trial was performed after training trails where no treat was hidden (Movie S1). Bar graphs depict the mean distance of the mice to the location where the cookie was hidden during training trials for the first 60 s. Error bars represent SEM. n = 9 for each mouse group. Panx1 knock out mice have an impaired memory, as they spent less time searching at the correct position (P = 0.02). However, they still remembered the former location to some extent, as they spent significantly more time searching at the correct position as untrained animals (P<0.001, Student’s t-test).

https://doi.org/10.1371/journal.pone.0051767.g005

Animals

Handling and housing of animals used in this study was performed in compliance with the German Animal Rights law and approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany (Permission No. 50.8735.1 Nr. 100/4). Animals were housed with a 12-hour light/dark cycle and free access to food and water. The generation of Panx1^+/+ mice (Panx1^fl/fl) with three LoxP consensus sequences integrated into the Panx1 gene, and knockout mice with global loss of Panx1 (Panx1^−/−; CMV-Cre/Panx1) have been described [21]. Animals in this study were 3–9 months old.

Reverse Transcriptase-PCR

The Message Sensor TM RT Kit was used to synthesize cDNA from total RNA obtained from dissected tissues. RT-PCR was performed with Panx1 exon4-specific primers. PCR conditions were: 95°C, 15 minutes; 40 cycles at 94°C, 30 seconds; 58°C, 30 seconds; 72°C, 1 minute. Amplified PCR products were analyzed by gel electrophoresis. Forward primer was F 5′- CCCTCTGGTCTGCTCTGTGTC-3′ and reverse primer was 5′- GGGGGTCCAGGTCCGTCTCT-3′. The exon4 specific amplicon has a size of 287 bp.

Western Blot

Tissues were dissected, frozen instantly in liquid nitrogen, and homogenized in T-PER buffer lysis buffer (Tissue Protein Extraction Reagent by Thermo Scientific, Inc.) supplemented with complete protease inhibitor (Roche). After debris removal by low speed centrifugation and protein concentration measurements using BCA kit (Pierce), equal amounts of total protein from each sample were resolved on SDS-PAGE gradient 4–12% Bis-Tris gels and transferred to PVDF membrane (both from Invitrogen). To visualize Panx1, homogenates were extracted with 0.1% Triton X-100 and the supernatant supplemented with the loading buffer (Invitrogen). Blots were blocked in 5% milk in Tris-buffered saline (TBS, pH 7.6), probed with the primary antibody overnight, washed in 0.15% Tween20 in TBS, and incubated for 1 h with secondary antibody (1∶1,000, Amersham Biosciences, NJ, USA) diluted in TBS. Affinity purified rabbit anti-Panx1 CT-395 (Px-34) antibody was provided by Dr. D.W. Laird (University of Western Ontario, Canada) [22]. A 1∶5,000 dilution was used for western blot analysis. Anti-actin antibody was used to control protein loading. Protein bands were visualized using SuperSignal (Pierce) and quantified using the FUJIFILM software.

RNA-expression Profiling

SABiosciences RT² First Strand Kits was used for cDNA synthesis from 1 µg total RNA isolated from freshly dissected hippocampi of adult mice (n = 4 for each genotype; 6–9 month old). The kit contains an effective genomic DNA elimination step and a built-in External RNA Control for real-time PCR-based gene expression analysis with SABiosciences’ RT² Profiler™ PCR Mouse Synaptic Plasticity PCR Array (all from Qiagen Inc., Toronto, Ontario M5J 2T3, Canada). All procedures were performed according to the manufacturers protocol using a DNA Engine Opticon 2 Real-Time Cycler PCR detection system (Bio-Rad Laboratories, CA, USA), with the integrated web-based software package for this PCR Array System was used for all ΔΔC_t based fold-change calculations from raw threshold cycle data. Data are presented as Vulcano plot, with thresholds set to: fold difference >1.5, p<0.01 (Student t-test).

Fluorescence Immunohistochemistry

Sections (10 µm) from adult brain tissue of transcardially perfused mice (4% paraformaldehyde) were fixed with 3% paraformaldehyde for 15 min, permeabilized with 1% Triton X-100 for 10 min, and blocked for 1 h with 3% normal horse serum, 1% BSA in PBS, pH 7.4. A chicken anti-Panx1 antibody provided by Dr. G. Dahl (University of Miami Miller School of Medicine, Miami, FL, USA) was diluted 1∶100 [23]. Secondary antibodies were Alexa 488 nm coupled (1∶3,000). Specimen were finally stained with Hoechst 33342 (Invitrogen) and mounted with Prolong Antifade Gold (Invitrogen). Confocal image analysis was performed using the LSM 510 META system (Carl Zeiss MicroImaging GmbH,), equipped with argon and HeNe lasers, 63×(NA 1.4) oil objectives, and the LSM 510 META software as described previously [24], [25].

Electrophysiology in vitro

Mice were sacrificed by cervical dislocation and horizontal hippocampal slices (350 µm) cut with a Leica VT1000 vibratome (Leica Microsystems, Wetzlar, Germany). Slices were kept in ice-cold artificial cerebrospinal fluid (ACSF) containing in mM: 124 NaCl, 2.69 KCl, 1,25 KH₂PO₄, 2 MgSO₄, 10 Glucose, 2 CaCl₂, 26 NaHCO₃. Slices were incubated for >2 h prior to recording in ACSF at room temperature (RT) for recovery. External solutions were continuously gassed with 95% O₂/5% CO₂ and applied at a flow rate of 8 ml per minute. During procedures temperature was kept constant at 23±0.5°C). Field excitatory postsynaptic potentials (fEPSPs) were recorded by extracellular placement of a metal recording microelectrode (impedances 0, 5–0, 8 MΩ) (TREC-SE, Multi Channel Systems; Reutlingen, Germany) into the stratum radiatum of the hippocampal CA1 region. A concentric SNEX1200 Wolfram electrode (Hugo Sachs Elektronik, Harvard Apparatus, March-Hugstetten, Germany), was placed into the Schaffer collateral fibers to stimulate hippocampal CA1 pyramidal neurons. The fEPSP responses were driven by bipolar stimuli (50–600 µA) with a STG 1200 (Multichannel systems, Reutlingen, Germany). Before recordings were commenced, input to output correlations were determined to identify the optimum stimulation intensity, which was adjusted to 50% of the evoked maximal response amplitude. To elicit fEPSP response for long-term potentiation (LTP) recordings, 10 min of baseline stimulation at 5 Hz was performed. LTP was evoked by using high frequency stimulation (HFS) with four trains of 10 shocks at 100 Hz every 1 sec. Signals were amplified and filtered by an DAM80 extracellular amplifier (World Precision Instruments, Sarasota FL, USA), digitized at 20 kHZ and displayed, stored and analyzed using WinWCP software (Strathclyde; Biologic, Knoxville TN, USA). A 16bit analog to digital converter (BNC 2110 connected to Ni-PCi 6229; National Instruments; Munich, Germany) was used to digitize the signals.

Pharmacology

Pharmacological agents were: Mefloquine (MEQ; Bioblocks, CA, USA), α-Methyl-3-methyl-4-phosphonophenylglycine (UBP1112; Tocris Bioscience, Ellisville, Missouri, USA), Adenosine (Sigma Aldrich, Deisenhofen, Germany), D (−)-2-Amino-5-phosphonopentanoic acid (D-AP5, Sigma Aldrich, Deisenhofen, Germany). All other chemicals were obtained from Sigma Aldrich. Pharmacology was applied 10 min before the onset of LTP recordings. Pharmacology exposure was kept stabile during the entire recording period.

Electophysiological Data Analysis

fEPSP data were normalized as a percentage of control, based on average amplitudes from the 10 min recording immediately before LTP protocol application. For each time point, consecutive responses at 20 sec intervals were averaged and the results were expressed as the mean percentage ± standard error of the mean (s.e.m.) and summarized in table S1. LTP responses were analyzed as described [26]. All values are expressed as mean ± standard error of the mean (s.e.m.). The level of significance was set at p<0.01 [p<0.01 = *; p<0.001 = **; p<0.0001 = ***]. fEPSP amplitude data, summarized in Box- and whisker-plots in figures 1,2,3 are represented as sample minimum, lower quartile, median, sample maximum, upper quartile and considered outliers.

Prepulse Inhibition (PPI) of the Acoustic Startle Response

All animals were subjected to 4 behavioral test sessions investigating PPI of ASR as described previously [27]. Prior to experiments animals had been individually handled and habituated to the test apparatus [28]. PPI of the ASR was measured in a sound-attenuated isolation chamber (41×41×41 cm) using a movement-sensitive piezoelectric measuring platform connected to a personal computer with an analogue to digital (AD) converter (Startle Response System, TSE, Bad Homburg, Germany) [27], [29]. During test sessions, animals were placed in a wire mesh cage (22.5 cm×8 cm×8.5 cm) mounted on the transducer-platform. For acoustic stimulation, two loudspeakers were used, mounted on both sides of the test cage at a distance of 4 cm. On the day of PPI testing, the animals were transported to the startle-box room and left undisturbed for 30 min. The experiment consisted of a 5 min acclimatization phase and a test session. During the acclimatization time, animals received background noise (60 dB sound pressure level (SPL), white noise) followed by 10 initial startle stimuli (100 dB SPL, white noise) lasting each for 20 ms (0 ms rise/fall times). The test session consisted of seven different trial types given in a pseudorandom order: (1) pulse alone (100 dB SPL white noise, 20 ms duration); (2) control (no stimulus); (3–4) pre-pulse alone (72 or 68 dB, pure tone, 10 Hz, 20 ms duration); (5–7) pre-pulse (72, 68, or 64 dB) each followed by a pulse with an inter-stimulus interval of 100 ms. A total of 10 presentations of each type was given with an inter-trial interval randomized between 20,000 ms and 30,000 ms [30]. Background noise intensity during the whole experiment was 60 dB SPL. The entire test session took about 40 min.

PPI was calculated according to the formula 100–100% 3 (PPx/PA), in which PPx is the mean acoustic startle response (ASR) of the 10 PPI trials (separate for each individual pre-pulse intensity) and PA is the mean ASR to the pulse alone trials [28]. Analysis of variance (ANOVA) was used for all comparisons (treatment, prepulse-intensity). A probability level (P) of less than 0.05 was considered statistically significant.

Object Recognition Test

In a chamber (40×40×40 cm) that is familiar to the animals, two novel objects (i.e. A and B) were presented for 5 min. After a delay of 60 min, one familiar and one novel object (i.e. A and C) were presented to test for object recognition memory. The presentation of objects lasted for 5 min, where the animals were left to explore freely, and were removed from the recording chamber after the presentation.

The objects and the recording chambers were cleaned thoroughly between task trials to ensure the absence of olfactory cues. The objects were distinctly different from one another and heavy so that the mice could not move them. Several copies of each object were available.

Behavioral data were recorded from cameras positioned above the chambers, and digitally stored. Exploration of the objects was then analyzed post-hoc using the within-object area scoring system, which was defined as sniffing of the object (with nose contact or head directed to the object) within ∼2 cm radius of the object [31]. Standing, sitting or leaning on the object was not scored as object exploration. OR data were expressed as a percentage of the total exploration time for each object per experiment. [32], [33]. The results across animals were expressed in terms of mean ± s.e.m. The data were then statistically assessed using the Student’s t-test by comparing group means with the fixed value of 50%, which represents no differentiation between objects. The significance level was set at p<0.05 [32].

Cookie Finding Test

Mice were trained in cages marked with letter “X” as visual cue. In trials 1–3 a cookie was buried beneath ∼6 cm (400 g) of woodchip bedding in their home cage. Reducing the cookie size in trials 4 and 5 reduced the olfactory component in this test. Finally, in trials 6 and 7 weakly odorous mouse chow replaced the original cookie. Training was performed on subsequent days and the treat was always hidden at the same position, with animals allowed to locate the treat in a 10 min. period. The time till the mice held the treat in their front paws was defined as finding time. After seven training trials animals were exposed to the test trial with no hidden treat. The movements were recorded and tracked with the EthoVision XT7 Software from Noldus (Wageningen, Netherland). The walking distances to the place where the cookie was hidden during the training trials were used to quantify the spatial memory of the mice. Statistical significance was tested by Student’s T-test. The significance level was set at P<0.05.