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Keywords:

  • Status epilepticus;
  • Adenosine;
  • A1 receptor;
  • Dentate gyrus;
  • Perforant path stimulation

Summary

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Purpose:  Status epilepticus (SE) remains a potentially devastating condition that quickly becomes refractory to antiepileptic drug treatment and arises as a result of a failure of the brain’s endogenous antiepileptic mechanisms. Understanding these mechanisms and how they are disrupted in SE is necessary in order to identify novel therapeutic approaches. Adenosine is considered an endogenous anticonvulsant. Extracellular concentrations increase coinciding with seizure termination; activation of A1 receptors (A1Rs) reduces seizure-induced damage and epileptiform activity. The present study examines the effectiveness of focal drug delivery in a model of limbic SE that closely resembles the human condition and describes, for the first time, alterations in A1R signaling during prolonged seizures that may contribute to the progression from self-terminating seizures to self-sustaining SE (SSSE).

Methods:  We developed a rat perforant path stimulation model in which 50% of rats develop SSSE and tested whether modulation of A1Rs influenced SSSE development when drugs were infused to the dentate gyrus. We further determined the ability of A1Rs to modulate perforant path to granule cell transmission in hippocampal slices taken from sham-operated control and post-SE animals.

Key Findings:  Adenosine (3 μm) and the A1R-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 10 μm) reduced the severity of SSSE as measured by spike count, electroencephalography power and behavioral seizure score. In addition, CCPA suppressed the progression to SSSE. Surprisingly, the A1R-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 μm) had no effect on the severity of or progression to SSSE, suggesting a lack of intrinsic A1R activation. Immunohistochemistry revealed no alterations in total A1R expression. However, we observed a marked down-regulation of A1R modulation of neurotransmission in vitro, indicating acute A1R desensitization.

Significance:  These findings indicate that A1R activation can prevent the progression to SE and suggest that reduced A1R signaling promotes the transition of seizures to SSSE.

Status epilepticus (SE) is a medical emergency associated with considerable mortality and morbidity (Neligan & Shorvon, 2009). SE rapidly becomes refractory to drug treatment (Mazarati et al., 1998a) and first-line therapy is often ineffective (Treiman et al., 1998). New treatment approaches are, therefore, needed. Furthermore, the mechanisms by which seizures fail to terminate and progress to SE are unclear.

The neuromodulator adenosine (Latini & Pedata, 2001; Boison, 2008; Gomes et al., 2011) exerts a potent inhibitory influence on neuronal activity (Scholfield, 1978) and in regulating seizure susceptibility (Dunwiddie & Worth, 1982; Barraco et al., 1984; Dragunow et al., 1985; Alasvand et al., 2001; Gouder et al., 2003; Anschel et al., 2004b; Avsar & Empson, 2004; Vianna et al., 2005; Zuchora et al., 2005; Pagonopoulou et al., 2006; Yildirim & Marangoz, 2007; Boison, 2008; Kovac et al., 2008; Van Dycke et al., 2010). Increases in extracellular adenosine are thought to play a role in seizure termination (During & Spencer, 1992; Chin et al., 1995; Berman et al., 2000; Latini & Pedata, 2001) and depletion of cellular adenosine or antagonism of A1 receptors (A1Rs) lead to increased seizure duration and the development of SE (Murray et al., 1985; Dragunow & Robertson, 1987; Handforth & Treiman, 1994; Young & Dragunow, 1994; De Sarro et al., 1999; Avsar & Empson, 2004; Vianna et al., 2005; Zeraati et al., 2006). In addition, A1R knockout mice develop more severe SE after chemoconvulsant administration (Fedele et al., 2006) or traumatic brain injury (Kochanek et al., 2005). Previous work addressed whether targeting A1Rs before seizure initiation modifies seizure duration and severity in several seizure models. A confounder is that this intervention may reduce the severity of the ictogenic insult. Our objectives were to determine whether targeting A1Rs can influence the progression from established seizures to self-sustaining SE (SSSE) or modulate ongoing SE activity. In order to avoid secondary effects in other brain regions, we targeted drug administration to the dentate gyrus (DG), a region of the hippocampal formation that restricts the spread of epileptiform activity to the hippocampus proper (Behr et al., 1998; Gloveli et al., 1998; Hsu, 2007). We further determined the effects of prolonged activity on the efficacy of A1R modulation of neurotransmission in this region.

Here, we show that although administration of adenosine or the A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA) can modify SE, surprisingly, antagonism of A1Rs has no effect on the development or severity of SE following perforant path (PP) stimulation (PPS). We have found that this is not due to loss of receptors, but instead, 1-h of PPS reduces A1R-mediated modulation of transmission. This is the first demonstration of an acute down-regulation in A1R activity during prolonged seizure and provides a mechanism that may promote the progression to SE.

Materials and Methods

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Male Sprague-Dawley rats (290–350 g) (Harlan Laboratories Inc., Bicester, U.K.) were housed individually under a 12-h light–dark cycle with access to food and water ad libitum. Procedures were subject to local ethical approval and followed the United Kingdom Home Office Animal (Scientific Procedures) Act, 1986. Chemicals were purchased from Sigma-Aldrich, Gillingham, United Kingdom, with the exception of adenosine (50 mm stock in dimethyl sulfoxide, DMSO), 2-chloro-N6-cyclopentyladenosine (CCPA, 10 mm stock in DMSO), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 10 mm stock in DMSO), diazepam (100 mm stock in DMSO), and picrotoxin (100 mm stock in DMSO), which were purchased from Tocris, Bristol, United Kingdom.

PPS model

Electrodes, cannulas, and pedestals were purchased from Bilaney Consultants Ltd, Sevenoaks, United Kingdom.

We modified a well-established model of SE described in detail elsewhere (Errington et al., 1987; Walker et al., 1999). In brief, under isoflurane anesthesia a bipolar stimulating electrode and a unipolar recording electrode were implanted into the right angular bundle and DG, respectively. An earth electrode was positioned subcutaneously and electrodes were mounted into a six-channel plastic pedestal and secured using skull screws and dental cement (Kemdent, Swindon, United Kingdom). Stimulation of the PP and extracellular recordings from the DG were via a Neurolog system (Digitimer Ltd, Welwyn Garden City, United Kingdom). Recordings were amplified and bandpass-filtered (0.1–50 Hz), and then digitized and recorded at 100 Hz using a CED micro 1401 and SPIKE 2 software (both CED, Cambridge, United Kingdom). Buprenorphine (0.2 mg/kg) (Schering Plough, Milton Keynes, United Kingdom) was administered perioperatively. Animals were allowed to recover for a minimum of 7 days prior to SE induction.

In initial experiments, the duration of PPS (3.5 mA, 50 μs, 20 Hz) was varied (30 min to 2 h) to determine the effect of stimulation duration on SSSE development. Stimulus intensity was selected based on previous experiments carried out in our lab. These experiments determined a stimulation duration that reliably induced SSSE in 50% of animals, thereby allowing us to observe either an inhibition or enhancement of the progression from stimulus bound SE to SSSE in further experiments. During stimulation and the subsequent recording period, behavioral observations were noted every 10 min using a scale of 0–5, with 5 depicting maximal seizure severity during that period according to the Racine scale (Racine, 1972). Spike frequency and electroencephalography (EEG) power were analyzed offline using an analysis script within Spike 2 (designed in collaboration with Steven Clifford; CED Ltd.). EEG power was calculated as the ratio of root mean square (RMS) amplitude during treatment or seizure to baseline; this measure has been used to quantify seizure activity (Yang et al., 2003) and sedation (Ypparila et al., 2004). We focused on EEG power in the low gamma frequency range (30–50 Hz), which has been associated with seizures in humans and rodent models (Fisher et al., 1992; Medvedev, 2002; Willoughby et al., 2003). Activity in this range has been shown to be a useful measure with which to quantify seizure severity in SE (Lehmkuhle et al., 2009). Sections of EEG in which movement artifact was evident and correlated with movement were excluded from analysis. SSSE was defined as seizure activity persisting for ≥30 min after PPS. Animals displaying no behavioral seizures during PPS were excluded. Animals were sacrificed ≤2 h after PPS with CO2 overdose.

A second group of animals had a combined 22-gauge guide cannula implanted alongside the recording electrode. A subset of these animals was used to confirm recording stability and drug efficacy during focal drug delivery under anesthesia. Drugs were infused (2 μl over 4 min) via internal cannula, connected via thin-walled polyvinylchloride (PVC) tubing to a Hamilton Microliter syringe (Fischer Scientific, Loughborough, United Kingdom) driven by a micro-infusion pump (Harvard PHD 2000; Harvard Apparatus, Holliston, MA, U.S.A.). Cannulas were introduced ≤2 min before PPS cessation; drug infusion began at the point stimulation was terminated (0 min post-SE). The cannulas remained in place for an additional 10 min to prevent drug reflux. Only animals in which the total volume was infused were included in analysis.

The same protocol, without cannula implantation, was used in animals from which tissue was processed for histology and electrophysiology. These animals were sacrificed 30 min after PPS with pentobarbital overdose.

In vitro electrophysiology

Horizontal hippocampal slices (350 μm) were obtained from sham-operated controls and self-sustaining (SS) and nonsustaining (NS) SE rats 30 min after PPS. Slices were cut using a Leica VT 1200S (Leica Microsystems, Nussloch, Germany) at 0.16 mm/s in high-sucrose artificial cerebrospinal fluid (aCSF) containing (in mm): 87 NaCl, 2.5 KCl, 7 MgCl2, 75 sucrose, 1.25 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 25 glucose (316 mOsm), stored in a holding chamber at 37°C for 20 min then transferred to a gas–fluid interface chamber at room temperature. Slices were maintained in aCSF containing (in mm): 119 NaCl, 2.5 KCl, 1 NaH2PO4, 2.5 MgSO4, 2.5 CaCl2, 26.2 NaHCO3, and 22 glucose (296 mOsm), and experiments were carried out in a submerged recording chamber perfused with the same solution. All solutions were continuously gassed with 95% O2 and 5% CO2.

Field excitatory postsynaptic potentials (fEPSPs) were recorded using glass microelectrodes filled with aCSF (approximately 1 MΩ) positioned in the middle molecular layer of the DG. A bipolar stainless steel stimulating electrode (approximately 8–10 MΩ) was used to deliver <1 mA, 50 μs pulses to the medial perforant path (MPP) every 20 s. Electrode position and stimulus intensity were adjusted to maximize the fEPSP. Slices were discarded when the response obtained was <1 mV. Stimulus intensity was reduced to elicit approximately 75% of the maximal response (similar in test and control slices, p = 0.871, Student’s t-test), and paired-pulses were applied at a 50 ms interstimulus interval. Paired-pulse ratio (PPR) was calculated as the amplitude of the test pulse expressed as a percentage of the amplitude of the conditioning pulse. Stimulation of the PP with paired stimuli induced paired-pulse inhibition (PPI), confirming that the MPP was targeted (Hanse & Gustafsson, 1992). Recordings were acquired using an Axopatch 1D amplifier (Axon Instruments, Molecular Devices, Sunnyvale, CA, U.S.A.) low pass filtered at 500 Hz and digitized at 2 kHz using a Labview 5.0 (National Instruments, Austin, TX, U.S.A.).

fEPSP measurements were taken when responses had been stable for 10 min (10–30 min after drug addition). Average fEPSP slope and amplitude were measured over 10 min (30 sweeps). Data were excluded if responses did not return to ≥80% of baseline upon washout.

Immunohistochemistry

Brains from sham-operated control, SS, and NS rats were immersed in 4% paraformaldehyde (PFA) in 0.01 m phosphate-buffered saline (PBS), pH 7.4. Brains were fixed overnight at 4°C and then stored at 4°C in PBS. Horizontal slices (50 μm) were cut in PBS, permeabilized, and blocked for 1 h in 0.01 m PBS with 0.1% Triton-X and 5% neonatal goat serum (Invitrogen, Paisley, United Kingdom) and incubated with a combination of the following antibodies: rabbit anti-adenosine A1 (1:100; Sigma-Aldrich), mouse anti-synaptophysin (1:200; Sigma-Aldrich), and mouse anti-PSD-95 (1:200; Abcam, Cambridge, United Kingdom) overnight at 4°C. Previous work demonstrated the selectively of the anti-A1R antibody (Schindler et al., 2001) and the reproducibility of staining using this protocol (Avsar, 2005). Preliminary experiments validated the use of this antibody to visualize A1Rs in the DG (see Data S1 and Fig. S1). Slices were washed and incubated with secondary antibodies; goat anti-rabbit Alexa 488 (1:500; Invitrogen), and goat anti-mouse Alexa 546 (1:500; Invitrogen). Control experiments confirmed the absence of nonspecific binding. A minimum of four slices per animal were prepared for each antibody combination or antibody-omitted controls.

Confocal microscopy and staining intensity and colocalization quantification

Using a modification of the method described by Jensen et al. (2007), preparations were examined on an LSM510 Meta Confocal with a 20× air or 63× oil immersion objective (NA 0.75 and 1.40, respectively) and using LSM 510 Expert Mode (Carl Zeiss Ltd., Welwyn Garden City, United Kingdom). Eight-bit images were taken (1,024 × 1,024 resolution, 1 pixel = 0.45 or 0.14 μm) avoiding superficial regions of tissue. This procedure aimed to decrease the variability between tissue sections by normalizing for antibody penetration. Laser power, gain, and black level were optimized to obtain the full dynamic range while avoiding saturation for excitation wavelengths. The pinhole was set to the Airy disk diameter at the shortest excitation wavelength. Settings were consistent while imaging the control and test tissue. Images represent Kalman scans (three times), collected separately at each excitation wavelength (488 or 543 nm) to prevent bleed-through of the fluorescent signal, and from which false-color and composite images were created.

Mean signal intensity and protein colocalization measured as Pearson’s correlation coefficient (a standard method to quantify colocalization) were calculated within LSM 510 Expert Mode (Carl Zeiss Ltd) using three images from each slice. Pearson’s coefficient (Equation 1) is defined as the covariance of two variables divided by the product of their standard deviations. This method of assessing colocalization is independent of pixel intensity.

  • image

All images were thresholded to exclude the lower 30% of the signal in order to reduce the contribution from background fluorescence to the colocalization score.

Statistics

Sigmoidal curves were fitted using a 20 iteration logged data fit function (ORIGINPRO Version 8; OriginLab Corporation, Northampton, MA, U.S.A.) and with constraint of the baseline to 100% half maximal effective concentration (EC50) or half maximal inhibitory concentration (IC50) values and minimal or maximal responses were calculated for each experiment. Values expressed in text represent the mean EC50 or IC50 and minimal or maximal values averaged across all experiments. Data are plotted as curves fitted to the mean values for all experiments and weighted by standard errors. Aside from Racine scores, which are presented as median and interquartile range (IQR), all data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were made using one-way analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) post hoc test, paired or unpaired Student’s t-test, Mann-Whitney U test, or Fisher’s exact test and were performed in SPSS12.0.1 (SPSS Inc., Chicago, IL, U.S.A.). Statistical significance was taken as p < 0.05. The n values given are the number of animals used in each experiment. Power calculations were performed using G*Power 2 (Department of Psychology, Bonn University, Germany).

Results

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Development of SSSE model

We adapted a PPS model of SSSE (Walker et al., 1999) in order to test the efficacy of compounds targeting A1Rs. We varied the duration of PPS from 30 min to 2 h and determined the percentage of animals that developed SSSE and the severity of SSSE, measured by the maximal Racine score noted in each 10 min period, and spike count over 30 min (Table 1).

Table 1.   Influence of stimulation duration on the development of self-sustaining status epilepticus
Duration of stimulationN%SSMedian Racine scoreMean spike count (SS)Median Racine score (SS)
30 min8250 (IQR 0)4,320 ± 2,7302 (IQR 3.5)
1 h8502 (IQR 3)5,167.5 ± 1,532.62 (IQR 1)
1.5 h8752 (IQR 2)4,487 ± 1,012.62 (IQR 1)
2 h887.52 (IQR 1)4,581.6 ± 657.22 (IQR 0.25)

In agreement with previous work (Mazarati et al., 1998b; Wasterlain et al., 2000), we found that SSSE development is dependent upon the duration of PPS (Table 1). However, varying the stimulation duration did not affect the severity of SSSE that ensued (Table 1). One-hour PPS induced SSSE in 50% of animals (Table 1), a suitable paradigm with which to test either the inhibition or enhancement of progression of stimulus-bound SE to SSSE through the use of pharmacologic agents.

We next determined the effect of focal drug delivery to the DG on SSSE development. Focal delivery of diazepam inhibits the appearance of SE when administered prior to the epileptogenic insult (Anschel et al., 2004a). It is unclear whether it has an effect if administered later. We tested whether administration of 2 μl diazepam (40 mm) into the DG after 1 h PPS could also prevent the development of SSSE. Diazepam reduced the amplitude of an evoked population spike (to 18.1 ± 10.1% of that obtained during the infusion of saline, n = 4, p = 0.005, paired Student’s t-test; Fig. 1A) in anesthetized animals. This dose of diazepam also inhibited SSSE development in all animals when administered after 1 h PPS (p = 0.014, Fisher’s exact test, Fig. 1, Table 2) confirming that the progression to SSSE can be halted by targeting drug administration to this region and validating the use of the model and experimental paradigm to investigate the potential of local administration of other compounds to modulate the progression from stimulus-bound SE to SSSE.

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Figure 1.   Local infusion of diazepam to the DG inhibits population spikes and the progression to SSSE. (A1) Population spikes were recorded in the stratum granulosum of the DG of anesthetized rats and were elicited by PPS. Recordings were made after the infusion of either 2 μl saline or 2 μl, 40 mm diazepam to the DG. Traces were averaged over 10 sweeps (a total of 10 s). Stimulus artifacts have been removed for clarity. (A2) Summary graph of population spike amplitudes in the presence of saline and diazepam, demonstrating a significant reduction after focal infusion of diazepam (n = 4). (B) EEG traces were recorded prior to and post 1 h continuous PPS in vivo and after the infusion of either saline (n = 8) or diazepam (DZP, n = 8) to the DG. Traces are from two separate experiments. The saline trace is representative of experiments in which animals developed SSSE; the diazepam trace is representative of all recordings from the diazepam treatment group. Values are plotted as raw data (open circles) or mean ± SEM (closed circles). **p < 0.005 (paired Student’s t-test).

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Table 2.   Influence of intrahippocampal delivery of diazepam, adenosine, CCPA, and DPCPX on the progression to self-sustaining status epilepticus
TreatmentN% SSMean spike countMedian Racine scoreMedian Racine score (SS)
Vehicle16508,510.94 ± 2,440.121 (IQR 1–2)3.5 (IQR 3–5)
Diazepam8000 (IQR 0)0 (IQR 0)
Adenosine8506,903.34 ± 2,849.800 (IQR 0–1)2.5 (IQR 1.75–3.25)
CCPA8252,395 ± 1,581.10 (IQR 0–1)1.5 (1.25–1.75)
DPCPX8508,742.88 ± 2,387.961 (IQR 0–3)5 (IQR 4.5–5)

Focal delivery of compounds targeting A1Rs modulates transmission at the PP to DG synapse

Two microliters of either adenosine (3 μm) or the A1R-selective agonist CCPA (10 μm) reduced the amplitude of an evoked dentate granule cell (DGC) population spike in anesthetized rats (adenosine: 40.9 ± 11.3% of control values, n = 3, p = 0.035, paired Student’s t-test; CCPA: 26.8 ± 14.8% of control values, n = 3, p = 0.018, paired Student’s t-test; Fig. 2A). The A1R-selective antagonist DPCPX (1 μm) increased the amplitude of an evoked population spike to 201.0 ± 27.9% of baseline (n = 4, p = 0.036, paired Student’s t-test, Fig. 2A). This finding indicates that under control conditions there is tonic A1R receptor activation. These doses were selected to infuse focally after PPS.

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Figure 2.   Influence of intrahippocampal delivery of adenosine, CCPA, and DPCPX on DGC population spikes and the progression to SSSE. (A1) Sample traces showing population spikes elicited by PPS and recorded in the stratum granulosum of the DG of anesthetized rats, following the infusion of 2 μl vehicle, 3 μm adenosine (Ado, n = 3), 10 μm CCPA (n = 3), or 1 μm DPCPX (n = 4) to the DG. Traces are averages over 10 sweeps. Stimulus artifacts have been removed for clarity. (A2) Summary data demonstrating that granule cell population spike amplitudes decreased after the focal infusion of adenosine, or CCPA, and increased after the infusion of DPCPX to the DG. (B) Sample EEG traces recorded prior to and after 1 h continuous PPS in vivo and after the infusion of vehicle (n = 16), adenosine (n = 8), CCPA (n = 8), or DPCPX (n = 8) to the DG. Traces are from four separate experiments and are representative of experiments in which animals developed SSSE. (C) EEG power in the low gamma frequency band (30–50 Hz) analyzed in all animals and in only those that self-sustained was reduced after the focal infusion of adenosine (Ado, n = 8) or CCPA (n = 8), but was unaltered by infusion of DPCPX (n = 8) to the DG. (D) Spike frequency in EEG recordings from self-sustaining animals was reduced after the focal infusion of adenosine (n = 4) or CCPA (n = 2), but remained unaltered after the infusion of DPCPX (n = 4) to the DG. Values are plotted as mean ± SEM. *p < 0.05 **p < 0.005 (Tukey’s HSD post hoc tst).

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A1R activation reduces the propensity to develop SSSE and its severity

Forty rats underwent 1 h PPS. Following CCPA infusion, only 25% of animals progressed to SSSE (n = 2/8, Table 2) and the severity of SSSE experienced by SS animals was reduced (Fig. 2, Table 2). Both adenosine and CCPA reduced EEG power ratio in the low gamma frequency band (p = 0.021, one way ANOVA, Fig. 2C). EEG power ratio was reduced from 3.79 ± 0.67 in vehicle controls to 1.59 ± 0.27 and 1.32 ± 0.26 after adenosine (n = 8, p = 0.038, Tukey’s HSD post hoc test) and CCPA (n = 8, p = 0.016, Tukey’s HSD post hoc test) administration, respectively (Fig. 2C).

Infusion of either adenosine or CCPA reduced the total spike count (Table 2) and mean spike frequency recorded over 1 h in SS animals (p = 0.009, one-way ANOVA, Fig. 2D). Spike frequency decreased from 4.71 ± 0.52 Hz in controls (n = 8) to 3.27 ± 0.68 Hz after adenosine infusion (n = 4, p = 0.048, Tukey’s HSD post hoc test) and 2.35 ± 0.61 Hz after CCPA infusion (n = 2, p = 0.030, Tukey’s HSD post hoc test, Fig. 2D). In addition, behavioral scores, recorded as the maximal noted in each 10 min period, tended to decrease after adenosine and CCPA infusion (Table 2).

A1R blockade neither increases the severity of SE nor increases the progression to SSSE

Surprisingly, the infusion of DPCPX did not enhance the progression to SSSE (Table 2). No alteration was observed in the low gamma frequency band (20–50 Hz) when comparing EEG power ratio in all animals treated (3.54 ± 0.91, n = 8, Fig. 2C). Spike frequency was unaltered in SS animals after DPCPX infusion (5.20 ± 0.77 Hz, n = 4, Fig. 2D), and although there was a tendency for behavioral scores to increase (Table 2) this difference was not statistically significant.

The inability of DPCPX to enhance the progression to SSSE or to increase seizure severity was surprising considering the results of previous studies (Dragunow & Goddard, 1984; Murray et al., 1985; Handforth & Treiman, 1994; Young & Dragunow, 1995; De Sarro et al., 1999; Vianna et al., 2005) and the influence of DPCPX on evoked population spikes in the naive DG (Fig. 2A). A possible explanation is that adenosine concentrations in the DG following stimulus-bound SE are too low to be detected by A1Rs. Alternative explanations are that there is a down-regulation of A1Rs (Hettinger et al., 1998; Rebola et al., 2005; Roman et al., 2008; Mundell & Kelly, 2011) or that there has been an interruption in the intracellular signaling cascade (Green et al., 1990; Ciruela et al., 1997; Gao et al., 1999; Jajoo et al., 2010). We explored these possibilities further.

Total A1R expression following PPS

We first determined the distribution of A1Rs in the DG from control animals. A1R staining appeared punctate throughout all layers of the DG (Fig. 3A), suggesting that the receptor is synaptically located. To clarify the synaptic localization, we examined colocalization with the presynaptic protein synaptophysin (Calakos & Scheller, 1994) and the postsynaptic protein PSD-95 (Chetkovich et al., 2002) in the molecular layer of the DG. This method has been used successfully to distinguish between pre- and postsynaptic sites (Jensen et al., 2007). Staining for both synaptophysin (Fig. 3A, synaptophysin staining) and PSD-95 (Fig. 3A, PSD-95 staining) also appeared punctate, with slightly larger puncta visualized in slices stained for synaptophysin (possibly attributable to variability in antibody binding to individual protein targets and epitope availability for antibody binding). These staining patterns were comparable with previously published data (Jensen et al., 2007). A1R puncta colocalized extensively with those of synaptophysin (r = 0.591 ± 0.019, n = 4, Fig. 3A, synaptophysin staining, lower panel), but not with PSD-95 (r = 0.060 ± 0.045, n = 4, Fig. 3A, PSD-95 staining, lower panel) in the molecular layer of the DG, indicating a predominantly presynaptic localization (i.e., on the terminals of the perforant path, Fig. 3A).

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Figure 3.   Total adenosine A1R density in the DG is unaltered during the development of SSSE. (A) Immunohistochemistry confirmed the synaptic localization of adenosine A1Rs in the DG. Fifty-micrometer slices were stained with antibodies directed against either the presynaptic protein synaptophysin (Syn) or the postsynaptic protein PSD-95 (PSD) (both red, first column) and the adenosine A1 receptor (A1R, green, second column). Images were taken at 20× magnification of the entire DG and 63× magnification of the molecular layer, where perforant path terminals synapse with the dendrites of dentate granule cells, and colocalization is indicated in the overlaid panels as yellow pixels (where green and red channels overlap). The A1R colocalized extensively with synaptophysin (n = 4), indicating the presence at the active zone of the perforant path fibers and exhibited little or no colocalization with PSD-95 (n = 4), present at the postsynaptic densities of granule cell dendrites indicating a presynaptic localization of the protein in the molecular layer. (B) The staining intensity of the A1R was unaltered in slices taken from sham-operated unstimulated controls (n = 5), animals in which SE progressed to SSSE (self-sustainers, “SS,” n = 5) or animals in which SE was stimulus bound (nonsustainers, “NS,” n = 6). Staining intensity of the A1R (green, second column) was compared with control slices prepared under the same conditions and staining intensity of synaptophysin (red, first column) was used as an internal control. Overlaid images are displayed in the third column. Analyses were performed on values obtained from high magnification images (Fig. S2). Images are representative of all images collected.

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We next took slices from sham-operated control, NS, and SS animals and stained for the A1Rs and synaptophysin. Staining intensity was examined at 63× magnification (Fig. S2) and no alteration in staining intensity of either protein was noted in slices from NS or SS animals prepared alongside slices from sham-operated controls (p = 0.408, one-way ANOVA, n ≥ 5, Fig. 3B), lending no support to the hypothesis that a loss of A1Rs in this region is responsible for the transition to SSSE. Using the variance from our experiments, we calculated that we would have been able to detect a 25% difference in staining intensity at p < 0.05 with a power of >95%.

A1R-mediated inhibition of transmission is reduced during the development of SSSE

Rapid down-regulation of A1R activity has been described during hypoxic–ischemic conditions (Coelho et al., 2006; Castillo et al., 2008), so we asked whether a similar activity-driven phenomenon occurs in SE.

Slices were prepared from sham-operated controls, and NS and SS animals and were maintained in aCSF. fEPSPs were recorded from the middle molecular layer of the DG. Blocking γ-aminobutyric acid (GABA)A receptor–mediated inhibition with picrotoxin had no effect on fEPSP slope or PPR (Fig. S3), indicating that the fEPSP slope in the molecular layer was a reliable measure of glutamatergic transmission (Andersen et al., 1966).

A1R-mediated inhibition of transmission at MPP terminals has been described previously (Prince & Stevens, 1992; Kahle et al., 1993). We confirmed the influence of targeting A1Rs at this synapse (see Fig. S4). Recordings were then made from sham-operated controls and NS and SS animals. Under basal conditions, fEPSP slope and PPR were not altered by 1 h SE in vivo (Fig. 4A). The mean fEPSP slope observed in slices from controls (−0.25 ± 0.05 mV/ms, n = 5), NS animals (−0.17 ± 0.04 mV/ms, n = 6), and SS animals (−0.19 ± 0.04 mV/ms, n = 5) were similar (p = 0.774, one-way ANOVA, Fig. 4A). There was no difference in the PPR observed in slices taken from controls (0.55 ± 0.07, n = 5), NS animals (0.60 ± 0.05, n = 6), or SS animals (0.56 ± 0.09, n = 5, p = 0.366, one-way ANOVA, Fig. 4B).

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Figure 4.   A1R-mediated inhibition of fEPSPs is reduced during the development of SE. (A1) Example fEPSPs from stimulation of the MPP recorded in the middle molecular layer of the dentate gyrus. Recordings were made in slices taken from sham-operated unstimulated controls, animals in which SE progressed to SSSE (self-sustainers, “SS”) or animals in which SE was stimulus bound (nonsustainers, “NS”) in the presence of 1 nm–100 μm of CCPA (only the response in the presence of 100 μm CCPA is shown). (A2) The A1R-mediated inhibition of fEPSP slope observed in sham-operated controls (black circles, n = 5) is reduced after 1 h SE in vivo. No difference was observed between slices taken from SS (open squares, n = 5) or NS (open circles, n = 6) animals. EC50 values were not altered. (A3) Summary data demonstrating that the maximal A1R-mediated reduction in fEPSP slope is reduced after 1 h SE in vivo. (B1) Paired-pulse recordings (50 ms interstimulus interval) from sham-operated unstimulated controls, self-sustaining (SS), and nonsustaining (NS) animals and recordings were made in the presence of 1 nM–100 μm of CCPA. (B2) The CCPA-mediated increase in paired pulse ratio (PPR) observed in sham-operated controls (black circles, n = 5) is reduced after 1 h SE in vivo. No difference was observed between slices taken from SS (open squares, n = 5) or NS (open circles, n = 6) animals. EC50 values were not altered. (B3) The maximal A1R-mediated increase in PPR is reduced after 1 h SE in vivo. All data are plotted as mean ± SEM. Representative traces are averaged over 30 sweeps (10 min). Stimulus artifacts have been removed for clarity. *p < 0.05 (one-way ANOVA followed by Tukey’s HSD post hoc test).

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Slices were exposed to increasing concentrations of CCPA in order to assess whether A1R-mediated inhibition of transmission was altered during the development of SE (Fig. 4). In control slices, CCPA maximally reduced the slope of the fEPSP from −0.26 ± 0.03 mV/ms to −0.04 ± 0.02 mV/ms (28.3 ± 3.10% of control), with an IC50 of 0.37 ± 0.19 μm (n = 5, Fig. 4A). The decrease in fEPSP slope was less in slices taken from NS and SS animals (p = 0.007, one-way ANOVA), decreasing from −0.19 ± 0.01 mV/ms to −0.09 ± 0.03 mV/ms (52.6 ± 21.5% decrease, n = 5) in SS slices and from −0.16 ± 0.01 mV/ms to −0.10 ± 0.02 mV/ms (47.2 ± 5.02% decrease, n = 6, Fig. 4A). The IC50 values, 0.58 ± 0.73 μm (n = 5) in SS slices and 0.42 ± 0.22 μm (n = 6) in NS slices, were similar to those observed in control slices (p = 0.356, one-way ANOVA).

Increasing concentrations of CCPA led to an increase in PPR from 0.54 ± 0.09 to 2.41 ± 0.22 (353.6 ± 72.8% control), with an EC50 of 0.83 ± 0.86 μm (n = 5, Fig. 4B). The increase in PPR in post-SE slices was less than that observed in controls (p = 0.009, one-way ANOVA, Fig. 4B). The PPR increased from 0.55 ± 0.05 to 1.13 ± 0.52 (181.2 ± 28.3% control, n = 5) in slices form SS animals (p = 0.01, Tukey’s HSD post hoc test) and from 0.60 ± 0.06 to 1.57 ± 0.24 (216.8 ± 37.2% control, n = 6) in slices form NS animals (p = 0.029, Tukey’s HSD post hoc test, Fig. 4B). The EC50 values, 1.84 ± 2.91 μm in SS slices and 0.82 ± 0.46 μm in slices from NS animals were similar to those observed in control slices (p = 0.663, one-way ANOVA).

The finding of decreased efficacy but maintained potency indicates no change in kinetics but rather a decrease in the number of A1Rs able to mediate an effect.

Discussion

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

This is the first study to investigate the effect of intrahippocampal infusion of compounds acting at A1Rs and to examine alterations in A1R function during the development of limbic SE. The main findings are that prolonged seizures result in a loss of A1R function, although not expression, and that targeting the adenosinergic system within the DG can reduce the severity of established SE. Although several studies have assessed the alteration in A1R expression in the postseizure or post-SE brain (Ochiishi et al., 1999; Ekonomou et al., 2000; Vanore et al., 2001; Rebola et al., 2005) and alterations in A1R function have been described in the chronically epileptic brain (Psarropoulou et al., 1994; Doriat et al., 1999; Rebola et al., 2003), this is the first demonstration of an acute reduction in A1R function during the development of SE.

We targeted the DG because of its role in the propagation of limbic seizures. However, we cannot be certain of its role in seizure generation. Because the infusion of diazepam to the DG was sufficient to inhibit the progression to SSSE in all animals tested, it can be assumed that targeting focal drug delivery to the DG is a suitable means by which to treat limbic SE. Previous work has indicated that focal infusion of compounds acting at A1Rs can terminate spontaneous or evoked single seizures or that preapplication can inhibit the development of SSSE (Alasvand et al., 2001; Zuchora et al., 2001; Anschel et al., 2004b; Zeraati et al., 2006; Yildirim & Marangoz, 2007). That targeting the A1R during prolonged seizures does not completely inhibit the progression to SSSE with drug doses that reduce DGC activation to a similar degree as diazepam in control animals, suggests a loss of A1R function during the development of SSSE, and this is supported by a lack of an effect of the A1R antagonist DPCPX. Several studies have demonstrated a proconvulsant effect of A1R antagonism given prior to seizure initiation (Dragunow & Goddard, 1984; Murray et al., 1985; Dragunow & Robertson, 1987; Handforth & Treiman, 1994; Young & Dragunow, 1995; De Sarro et al., 1999; Vianna et al., 2005). Although the concentration of DPCPX tested here was sufficient to increase excitability in the naive DG, it did not increase the propensity to develop SSSE following PPS. We cannot discount the possibility that the lack of effect of DPCPX is due to depletion of extracellular adenosine and so minimal A1R activation at this time point. Previous studies have indicated that adenosine levels can increase with brief seizures (Berman et al., 2000; Latini & Pedata, 2001), but it is unknown whether this also occurs during SE. To our knowledge, no histopathologic studies have determined hippocampal A1R density during the development of SE. A loss of A1R expression at the level of the plasma membrane of presynaptic terminal has been shown after 60 min hypoxia–ischemia (Domanska-Janik et al., 1993; Coelho et al., 2006), indicating that a loss of receptors can occur over a short space of time. In these studies, at least a 30% decrease in A1R expression was noted. In our experiments, we had a 95% power of detecting a significant change of at least 25%. Although we cannot rule out internalization of A1Rs at the presynaptic terminal (Saura et al., 1998; Escriche et al., 2003), using a protocol previously used to demonstrate alterations in the expression of a synaptic protein within the hippocampus (Jensen et al., 2009), we show that total adenosine A1R content is not altered after 1 h SE in vivo, which is consistent with [3H]-CHA binding studies carried out using a similar model (Young & Dragunow, 1994). If it is the case that A1Rs are not internalized, then a loss of function would explain the lack of inhibition to SSSE seen during focal infusion experiments and also explain the inability of DPCPX to enhance the development of SSSE.

We found a reduction in A1R-mediated inhibition of at the PP to DGC synapse to in vitro after 1 h SE in vivo. The A1R-mediated inhibition of fEPSP slope and increase in PPR was reduced in slices taken from post-SE animals. As these variables were not altered under basal conditions, the alteration can be attributed to a reduction in A1R-mediated signaling. This enhanced excitability is most likely the result of reduced A1R-mediated inhibition of presynaptic Ca2+ channels and enhanced release of glutamate (Dolphin et al., 1986; Macdonald et al., 1986; Wu & Saggau, 1994; Gundlfinger et al., 2007). As no difference was observed between slices taken from NS and SS animals, it can be concluded that 1 h stimulus-bound SE is sufficient to induce down-regulation in A1R signaling. However, the loss of A1R signaling alone is not sufficient to enhance the progression to SSSE.

Whether the desensitization is specific to the A1R, or reflects a wider disruption of Gi-mediated signaling remains to be established. Additional studies should aim to elucidate the relative involvement of the pathways that may be involved in the SE-induced down-regulation of A1R-mediated signaling.

Conclusions

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

We have shown that presynaptic A1R-mediated inhibition of transmission at the PP to DGC synapse is reduced acutely after prolonged seizures. This is associated with a decrease in the sensitivity of the EPSP to the A1R agonist CCPA and a reduction in the inhibition of PPI. This finding cannot be explained by a loss of total A1Rs at PP terminals, but may be due to desensitization of A1Rs. Reduced A1R receptor–mediated inhibition of transmission during SE may play a role in the maintenance of self-sustaining seizure activity, although alone it is not sufficient to enhance the progression to SSSE. The present study allows us to conclude that SE causes a rapid functional desensitization of A1Rs that control excitatory transmission in the DG, providing evidence that prolonged seizures can cause a rapid compromise of the neuroprotective adenosine A1R system.

Acknowledgments

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

NH was funded by a St George’s/Royal Holloway University of London joint studentship. This work was supported by a grant from ERUK. We gratefully acknowledge Professor D. Kullmann, Dr. C. Henneberger, Dr. I. Pavlov, and Dr. S. Sylantyev for their valuable comments during the preparation of this manuscript.

Disclosure

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

References

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Data S1. Methods.

Figure S1. Confirmation of specificity of antibody used in this study.

Figure S2. Analysis of adenosine A1 receptor staining intensity in slices taken from sham-operated animals and animals that underwent electrical stimulation was performed using 63× magnification images taken in the molecular layer of the dentate gyrus.

Figure S3. Electrophysiologic recordings made in the middle molecular layer were not under the influence of tonic GABAergic inhibition.

Figure S4. Adenosinergic modulation of paired-pulse depression at the perforant path to granule cell synapse.

FilenameFormatSizeDescription
EPI_3340_sm_FigS1.tif243KSupporting info item
EPI_3340_sm_FigS2.tif2487KSupporting info item
EPI_3340_sm_FigS3.tif165KSupporting info item
EPI_3340_sm_FigS4.tif392KSupporting info item
EPI_3340_sm_Supplementary-Methods.docx16KSupporting info item

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