Electrographic seizures are significantly reduced by in vivo inhibition of neuronal uptake of extracellular glutamine in rat hippocampus

Chronic-phase procedures

During the chronic phase at 37–46 days after KA injection, we implanted EEG recording and grounding electrodes (Plastics One, Roanoke, VA, USA) and microdialysis guide cannula with a stylet (BioAnalytical systems, West Lafayette, IN, USA), as described previously (Kanamori and Ross, 2011). Three experimental protocols, with objectives described in section “Overview of experimental design”, were used in the present study. The surgical and experimental procedures for each protocol are described below.

EEG/microdialysis (Experiment I)

In Experiment I, EEG recordings were taken bilaterally with microdialysis only in the ipsilateral hippocampus, in overnight-fasted rats. The EEG electrode was implanted at AP = −5.6 mm, L = −4.5 mm and V = 5.5 mm in the contralateral hippocampus. In the ipsilateral hippocampus, the coordinates were AP = −5.6 mm, L = 4.5 mm and V = 5.5 mm for the electrode, and for the guide cannula attached to the electrode, V = 3.5 mm. This places the electrode tip in CA3 region and the tip of the microdialysis guide cannula, just above the CA1 region (Paxinos and Watson, 1997). For preliminary bilateral EEG recording from an awake rat one week after the surgery, the electrode contacts on the skull were connected through a commutator to an amplifier (A-M systems, Carlsborg, WA, USA). Recordings were taken wide-band 0.1 Hz to 1 KHz, sampled at 10 KHz/channel and with a gain of 10 K, and processed with DATAPAC 2K2 software (Run Technology, Mission Viejo, CA, USA). When EEG recordings taken for several hours showed chronic-phase recurrent seizures (Bragin et al., 1999, 2005), the rat was considered ready for the microdialysis/EEG experiment.

On the day of microdialysis, the rat was lightly anesthetized with pentobarbital and the guide stylet was replaced with the microdialysis probe 320 μm in OD and 2 mm in length (BioAnalytical systems, West Lafayette, IN, USA). The probe spans CA1–CA3 region, with the bottom of the probe in the CA3 region and the top of the probe in the CA1 region within 0.5 mm of the dentate gyrus. The probe collects extracellular fluid from hippocampal tissue ~700 μm in diameter and 2 mm in length at the site of KA injection. The probe was perfused at a rate of 2 μL/min with artificial cerebrospinal fluid (aCSF) containing the following equivalents of electrolytes (mM); 150 Na⁺, 3.0 K⁺, 1.4 Ca²⁺, 0.8 Mg²⁺, 1 PO₄³⁻ and 155 Cl⁻ at pH 7.4. The dialysis tubings were connected to a liquid swivel for collection from an awake animal. A 3-h stabilization period was allowed before the collection of dialysates for analysis. The rat usually woke within 1 h after probe insertion and hence 2 h before the start of dialysate collection and simultaneous EEG recording. The dialysates were collected every 2 min for 5 h, and stored at −20 °C till analysis. In correlating EEG activity with changes in GLU_ECF and GLN_ECF, we took into account the fact that it takes 180 s for dialysate to flow from rat brain to the collection vial under our experimental condition. The microdialysis time is reported at the center of each 2-min collection time.

EEG/microdialysis/i.v. infusion (Experiment II)

In Experiment II, dialysates were collected from both hippocampi with concominant EEG recording and i.v. infusion of glucose. Microdialysis guide cannula fixed to the recording electrode was implanted bilaterally at the coordinates used in Experiment I. Two days before the EEG/microdialysis experiment, an indwelling silastic catheter was placed in the right external jugular vein for i.v. infusion of glucose (Kanamori et al., 1998). The distal end of the catheter (90 cm long), exiting at the nape, was placed in a backpack worn by the rat (Kondrat et al., 2002).

On the day of the experiment, the EEG electrode contacts on the skull of the lightly anaesthetized rat were connected to a 90-cm cable which is mesh-covered on proximal end and equipped with solder lugs on the distal end for connection to the amplifier. The rat was placed in RATURN (BioAnalytical Systems) with its collar attached to a balance arm. The EEG cable, the inlet and outlet dialysis tubings and the i.v. infusion catheter were passed through an opening in the sensor of the RATURN. This arrangement permits free movement of the awake rat without twisting the EEG cable, dialysis tubings or the infusion catheter which was connected to a peristaltic infusion pump. Dialysates were collected bilaterally in 15-min fractions for 2 h without i.v. infusion, then every 5 min with concomitant EEG recording and i.v. infusion of glucose for 3 h. D-Glucose was given according to the protocol developed by (Fitzpatrick et al., 1990) and shown to achieve steady-state blood glucose concentration in 9 min and brain glucose concentration in 30 min (Kanamori and Ross, 2001). Thus, the total microdialysis time in Experiment II is 5 h as in Experiment I.

MeAIB perfusion (Experiment III)

In Experiment III, dialysates were collected from, and MeAIB perfused into, only the ipsilateral hippocampus with bilateral EEG recording in glucose-infused or fed rats, which were prepared as in Experiment II. 2-(Methylamino)isobutyric acid (MeAIB) (Sigma–Aldrich, St. Louis, MO, USA) was dissolved in aCSF at the concentration indicated in the “Result” section. At 175 mM (the optimized dose), the pH of this solution was 7.1, and the pH was adjusted to 7.4 (the same pH as that of aCSF) with a few drops of 25 mM NaOH before perfusion. After 88 min of microdialysis combined with i.v. infusion of glucose, MeAIB was perfused into ipsilateral hippocampus at a flow rate of 2 μL/min for 30 min.

After the microdialysis experiment, the locations of the microdialysis probe and the electrodes were confirmed in each rat as described previously (Kanamori and Ross, 2011). The confirmed coordinates (mean ± SE) are shown in Table 1. The brain was then removed from the anesthetized rat and frozen in liquid nitrogen.

The protocol was approved by the Institute Animal Care and Use Committee and is in conformance with the US Public Health Service's Guide for the Care and Use of Laboratory Animals.

Identification of seizure and quantification of EEG activity

EEG seizure was identified according to the definition of (Bragin et al., 2005), as a period of consistent and repetitive changes in amplitude and frequency of electrical activity that was clearly different from interictal activity and which persisted for >10 s. Representative EEG recordings of ictal events in our KA rats were shown in Fig. 2 of (Kanamori and Ross, 2011). To examine possible correlation between EEG activity and changes in GLN_ECF, we quantified EEG activity in Experiment I by measuring (a) peak areas of EEG waves and (b) peak amplitudes of EEG spikes as described previously (Kanamori and Ross, 2011). Then peak areas in dialysate fractions (1–150) were consecutively added to determine cumulative peak areas for the 300-min observation period. The same method was used to obtain cumulative peak amplitudes of spikes. The height of a population spike, over a wide range, can be used as a measure of the number of discharging neurons (Andersen et al., 1971), and hence as an approximate measure of the quantity of neurotransmitter GLU that is released into ECF as hypersynchronized population bursts in electrographic seizures.

Open in a separate window

Figure 2

The time-course of GLU_ECF in the ipsilateral hippocampus of the Seizure Groups of KA rats in Experiments I and II. GLU_ECF is expressed as percentage of the initial concentration and shown as the mean ± SE for n = 6 (Experiment I) and n = 5 (Experiment II). The initial concentration was measured at t = 6 min for Exp. I, and for Exp. II (microdialysis concomitant with glucose infusion) at t = 112 min (section “EEG/microdialysis/i.v. infusion (Experiment II)”). As the initial concentrations were set at 100%, there is no error bar at these data points.

HPLC assay of extracellular GLU, GLN and TAU

Amino acids in brain dialysate were assayed, after pre-column derivatization with ortho-phthaldehyde (OPA) and 2-mercaptoethanol and separation on a reverse-phase column, by fluorometric detection as described previously (Kanamori and Ross, 2011), with the following minor modification. To achieve baseline separation of GLU, GLN and TAU from adjacent peaks, the following chromatographic program was used; elution with 25% methanol and 75% aqueous sodium phosphate buffer (50 mM, pH 5.38) for 10 min followed by increase in the percentage of methanol to 49.6% in 15 min and to 100% in 8 min.

Tissue concentrations of GLU, GLN and GABA in the hippocampal region sampled by microdialysis

Hippocampal tissue was sectioned at −10 °C from the brain that had been snap frozen at the end of the microdialysis experiment and stored in liquid nitrogen. The tissue was homogenized and intracellular metabolites were extracted as described previously (Kanamori and Ross, 2011). Tissue concentrations of GLU and GLN were measured by HPLC as described above. For measurement of GABA, the chromatographic program was modified as follows for baseline separation of GABA from the adjacent peaks; elution with 25% methanol and 75% aqueous sodium phosphate buffer (50 mM, pH 5.38) for 10 min, followed by increase in the percentage of methanol to 40% in 29.5 min, to 84% in 22.3 min and to 100% in 3 min.

K⁺ assay

K⁺ concentration in the dialysate was measured with an ion-selective electrode in the Clinical Laboratory at Huntington Hospital, Pasadena, California.

Overview of experimental design

Table 1 shows EEG characteristics of KA rats in three Experimental Groups used in the present study. Experiments I and II address the question of possible correlation between seizure activity and changes in GLN_ECF as outlined in the Introduction. In Experiment I, GLU_ECF and GLN_ECF were collected only from the ipsilateral hippocampus (site of KA injection) for 5 h. In Experiment II, after 2 h of microdialysis, intravenous infusion of glucose was started and dialysates were collected from both the ipsilateral and contralateral hippocampi for additional 3 h. Experiment II was designed (1) to examine whether the seizure-induced decrease of GLN_ECF observed in Experiment I was dependent on energy status and (2) to compare their occurrence in the seizure-prone ipsilateral vs seizure-free contralateral hippocampus. Experiment III was designed to address the question of whether the observed decrease of GLN_ECF reflects increased neuronal uptake of GLN_ECF, as outlined in the Introduction. Dialysates were collected from, and uptake inhibitor 2-(methylamino)isobutyrate (MeAIB) perfused into, the ipsilateral hippocampus of glucose-infused or fed KA rats and the effect of inhibition on seizure frequency was examined.

As shown in Table 1, all KA rats underwent microdialysis/EEG experiments during the chronic phase between 44 and 58 days post KA. Out of the 30 KA rats that showed recurrent seizures during preliminary EEG recording (section “EEG characteristics of KA rats”), those that showed frequent spontaneous seizures during the 5 h microdialysis experiments are called Seizure Group and those that did not are called No-Seizure Group. In the Seizure Group of Experiment I (n = 6), the number of seizures ranged from 5 to 15, with mean ± SE of 9 ± 1.9. The No-Seizure Group (n = 6) had no seizure during the microdialysis experiment. In Seizure Group of Experiment II (n = 5), the number of seizures (mean ± SE) observed during 3 h of EEG/microdialysis/glucose infusion was 5 ± 1, while the No-Seizure Group (n = 4) showed no seizure. For Experiment III (n = 9), the result is shown later in Table 3. As shown in Table I, the coordinates of the EEG electrodes and the microdialysis probes, confirmed after the experiment in every rat, as described previously (Kanamori and Ross, 2011), were virtually the same in all groups.

GLU_ECF during resting and seizure periods

Fig. 2 shows the time-course of GLU_ECF in KA rats of Experiment I. The concentration of dialysate GLU (in μM) at each time point was measured and expressed as percentage of the basal concentration measured at the start of microdialysis in each rat. Then the mean ± SE of the percentage change at each time point was calculated for n = 6. With significance defined as p < 0.05, these mean ± SE values did not differ significantly from the initial value, indicating that GLU_ECF in Seizure Group of Experiment I underwent no significant change during the 5-h microdialysis experiment. The figure also shows the time-course of GLU_ECF for glucose-infused KA rats of Experiment II (n = 5) during the last 3 h of microdialysis. In these rats too, the mean ± SE values did not differ significantly from the initial value, indicating that there was no significant change in GLU_ECF.

Effect of EEG seizure on GLN_ECF in the ipsilateral hippocampus

As shown in Table 1, the basal concentration of dialysate GLN in KA rats of Seizure Group of Experiment I, 31.5 ± 3.8 μM, was not significantly different from that, 30.3 ± 5.0 μM in the No-Seizure Group.

Fig. 3A (top) shows, for one rat (R1051) of Seizure Group, the time of occurrence of EEG seizures (arrows). The seizures consisted of low-voltage fast (LVF) onset and hypersynchronous (HYP) onset seizures that are characteristic of chronic-phase seizures in this model (Bragin et al., 1999, 2005). Representative EEG recordings are shown later (Figs. 5, ​,66 and ​and10).10). The top graph shows cumulative peak areas of EEG waves (left coordinate) and cumulative peak amplitudes of EEG spikes (right coordinate), determined as described in section “Identification of seizure and quantification of EEG activity”. Six seizures were detected during the 300 min observation period, with frequent seizures occurring between 180 and 253 min. During this period, the cumulative spike amplitude increased sharply. Fig. 3A (bottom) shows, for the same rat, the time course of GLN_ECF expressed as dialysate GLN concentration. Comparison of the two time-courses shows that GLN_ECF decreases significantly, when seizures occur frequently and cumulative spike amplitude increases sharply. When seizures ceased (t > 253 min), GLN_ECF increased slightly. During t = 231–295 min, GLN_ECF was 23.8 ± 1.4 μM which is significantly different from the initial concentration of 37.5 μM with p < 0.0001. Fig. 3B shows the result for another rat (R1052) of Seizure Group, which underwent 7 seizures (LVF and HYP). Seizures between 85 and 125 min were accompanied by gradual increase in cumulative peak areas and spike amplitudes (top). During this period, GLN_ECF decreased slightly (bottom). More dramatic is the effect of frequent seizures between 237 and 275 min which caused sharp increase in cumulative peak areas and spike amplitudes. During this period, GLN_ECF decreased significantly (bottom figure); GLN_ECF was 14.7 ± 0.5 μM during t = 237–295 min, which is significantly different from the initial concentration of 26.4 μM with p < 0.0001. Taken together, these results show that (a) the timing of GLN_ECF decrease correlates with the frequency and magnitude of seizure activity, and (b) when seizures occur frequently, persistent reduction in GLN_ECF is observed. Similar results were observed in other KA rats of the Seizure Group.

Open in a separate window

Figure 3

The top figures show the time of occurrence of EEG seizures (arrows) and the cumulative peak areas and spike amplitudes (section “Identification of seizure and quantification of EEG activity”) for comparison with the time-course of GLN_ECF change, shown as dialysate GLN concentration (μM) in the bottom figures, for KA rats of Seizure Group of Experiment I; (A) R1051 and (B) R1052.

Open in a separate window

Figure 5

Effect of EEG seizure on GLN_ECF in the seizure-prone ipsilateral vs seizure-free contralateral hippocampus. (A) EEG recording of electrographic seizure in the ipsilateral (KA injected) hippocampus, compared to the contralateral hippocampus, in a KA rat (R1119) of Experiment II. (B) The time-course of GLN_ECF from the ipsi- and contra-lateral hippocampus, expressed as percentage of the initial concentration measured at t = 7.5 min. The EEG seizures shown in (A) occurred during t = 220–295 min as shown at the top of (B).

Open in a separate window

Figure 6

High-amplitude ictal discharges in the ipsilateral hippocampus compared to the contralateral in a KA rat (R1111) of Experiment II. The time-course of GLN_ECF shows significant decrease in the ipsilateral hippocampus and no change in the contralateral hippocampus during 3 h of dialysate collection concomitant with i.v. glucose infusion. The initial concentration refers to that collected 7.5 min before this 3 h period.

Open in a separate window

Figure 10

(A) EEG recordings from the ipsilateral hippocampus of a representative KA rat (R1156) of Experiment III during (a) pre-perfusion period (b) MeAIB perfusion and (c) post-perfusion period. The frequency of seizure is significantly reduced during MeAIB perfusion. The amplitude of ictal spikes often exceeded 1.6 mV, but the expanded plot (inset) shows a wave pattern characteristic of hypersynchronous population bursts from glutamatergic neuron. (B) An EEG recording of a hypersynchronous seizure from the ipsilateral hippocampus of another KA rat (R1155) of Experiment III during post-perfusion period, processed with a faster sweep time.

Fig. 4A compares the time-course of GLN_ECF between the Seizure Group (n = 6) and the No-Seizure Group (n = 6) of Experiment I, in which the rats were fasted overnight. Dialysate concentration at each time point during the microdialysis experiment was first expressed as the percentage of initial concentration in each rat, then the mean ± SE (n = 6) was calculated. The result clearly shows that, in rats of Seizure Group, GLN_ECF decreased progressively and at t = 295 min, was 62 ± 4.4% of the initial value. This is significantly different from the initial value with p < 0.01. By contrast, in rats that underwent no seizure during microdialysis, the mean ± SE values were not significantly different from the initial value, indicating that there was no change in GLN_ECF. Fig. 4B shows changes in GLN_ECF in the ipsilateral hippocampus of KA rats of Experiment II which underwent i.v. glucose infusion for the last 3 h. As shown in Fig. 4B, GLN_ECF in the Seizure Group (n = 5) decreased progressively and at t = 295 min, was 75 ± 2.6% (significantly different from the initial value with p < 0.01). By contrast, in the No-Seizure group (n = 4), the mean ± SE values were not significantly different from the initial value, indicating that there was no change in GLN_ECF. These results clearly show that the decrease of GLN_ECF in response to seizure activity occurs in glucose-infused as well as in overnight-fasted rats. The difference in the mean percentage decrease of 62 ± 4.4% in Experiment I and 75 ± 2.6% in Experiment II was not statistically significant.

Open in a separate window

Figure 4

(A) The time-course of change in GLN_ECF, expressed as percentage of the initial concentration for KA rats of Seizure Group of Experiment I (filled square; mean ± SE for n = 6). The initial concentration refers to that measured in the first fraction after the start of microdialysis. The value at t = 275 min (shown by *) is significantly different from the initial value with p < 0.01. The time-course for KA rats of No-Seizure Group (mean ± SE for n = 6) is shown by filled triangle. (B) The time-course for Seizure Group of KA rats of Experiment II (mean ± SE for n = 5). The value at t = 295 min (shown by *) is significantly different from the initial value with p < 0.01. The No-Seizure Group (n = 4) showed no change. The initial value for the No-Seizure Group was measured at the same time point as that for the Seizure Group, but the filled triangle symbol overlaps with, and is hidden under, the filled square symbol for the Seizure Group. In both A and B, the error bar is smaller than the symbol (<2.5%) where not shown.

Effect of seizure on GLN_ECF in ipsilateral vs contralateral hippocampus

For KA rats (n = 5) of Experiment II, basal dialysate GLN concentration was 32.2 ± 4.4 μM in the ipsilateral hippocampus and 25.3 ± 4.0 μM in the contralateral hippocampus in the Seizure Group, while in the ipsilateral hippocampus of the No-Seizure Group, it was 30.3 ± 8%. Dialysate GLN in the normal rat was 28.4 ± 1.5 μM (n = 6 for two hippocampi from 3 rats). There was no statistically significant difference among those mean values.

Fig. 5 shows a representative result for changes in GLN_ECF in the ipsilateral vs contralateral hippocampus. Fig. 5A shows EEG recordings from the ipsilateral (top trace) and the contralateral hippocampus (bottom trace) in a KA rat of this group (R1119). The ipsilateral hippocampus showed two seizure episodes with high-amplitude ictal spikes; each episode lasting about 20 min. These were electrographic seizures detected by EEG but not accompanied by behavioral or motor seizures. By contrast, the contralateral hippocampus showed little EEG activity. As shown at the top of Fig. 5B, these seizures in the ipsilateral hippocampus occurred during 220–295 min of microdialysis. Fig. 5B shows changes in GLN_ECF collected by microdialysis from the ipsilateral and contralateral hippocampi before and during the seizure, and expressed as percentage of the initial concentration. In the ipsilateral hippocampus, GLN_ECF decreased to ~70% of the initial concentration during seizure, while in the seizure-free contralateral hippocampus, GLN_ECF remained at 88–100%. The mean GLN_ECF level in percentage in the ipsilateral hippocampus during t = 257–287 min when the level has stabilized, 70.4 ± 0.8%, is significantly different from the level, 87.2 ± 0.9%, in the contralateral hippocampus during the same period with p < 0.01.

Fig. 6 compares the effect of hypersynchronous seizures on GLN_ECF in the two hippocampi in another KA rat (R1111) of Experiment II. In the ipsilateral hippocampus, the seizures caused decrease of GLN_ECF to 70–80% of the initial value while GLN_ECF was unchanged in the seizure-free contralateral hippocampus. The mean level of GLN_ECF in percentage in the ipsilateral hippocampus, 78.7 ± 1.8%, is significantly different from that in the contralateral hippocampus, 99.8 ± 0.9%, with p < 0.01. A similar result was observed in another KA rat (result not shown). Table 2 summarizes changes in GLN_ECF in response to seizure activity in KA rats of Experiments I and II. Taken together, the results strongly suggest that significant decrease in hippocampal GLN_ECF is associated with electrographic epileptiform activity in those KA rats that show no detectable change in GLU_ECF.

Tissue concentrations of GLU, GLN and GABA in the ipsilateral hippocampal region sampled by microdialysis

To examine whether the observed decrease of GLN_ECF is due to lower concentration of glial GLN which can decrease the rate of SNAT3-mediated efflux of GLN to ECF (Fig. 1), we examined the tissue concentration of GLN, as well as those of GLU and GABA, in the ipsilateral hippocampal tissue that contained the CA1 and CA3 subfields sampled by microdialysis and the KA injection site. The weight of tissue (mean ± SE) was 10.7 ± 0.99 mg and 9.5 ± 1.5 mg for the Seizure and No-Seizure Groups of Experiment I respectively. The tissue concentrations are shown in Fig. 7. In KA rats of Seizure Group, GLN concentration was 4.3 ± 0.27 mM which was not significantly different from that in No-Seizure Group, 4.4 ± 0.47 mM with significance defined as p < 0.05. GLU concentration in this region was 7.4 ± 0.55 mM in Seizure Group and 7.3 ± 0.55 mM in No-Seizure Group, while GABA concentration was 1.77 ± 0.43 mM in the Seizure Group and 1.38 ± 0.39 mM in the No-Seizure Group. The difference in the mean values between Seizure and No-Seizure Groups was not statistically significant for either metabolite, with significance defined as p < 0.05.

Open in a separate window

Figure 7

Tissue concentrations of GLN, GLU and GABA in the ipsilateral hippocampus (in the region sampled by microdialysis and the site of KA injection) of Seizure (S) and No-Seizure (N–S) Groups of KA rats of Experiment I. The data are shown as the mean ± SE for n = 6. Tissue conc. in mM was calculated from the measured weight of hippocampal tissue and metabolite concentration in μmol, based on the usual assumption that 80% of tissue weight is water.

The results strongly suggest that the observed decrease of GLN_ECF in the Seizure Group is unlikely to be due to reduced concentration of glial GLN which lowers the rate of GLN efflux to ECF. (On the basis of immunocytochemical studies, at least 80% of brain tissue GLN is estimated to be in glia (Ottersen et al., 1992; Nagelhus et al., 1996; Kanamori and Ross, 1997)). Because the result raised an intriguing possibility that the observed decrease of GLN_ECF reflects its increased uptake into neurons, we inhibited neuronal uptake of GLN_ECF by perfusion of the competitive, non-metabolizable and reversible inhibitor MeAIB in vivo, and examined the effect on frequency of electrographic seizures.

Inhibition of neuronal uptake of GLN_ECF by MeAIB perfusion: Optimization of dose and duration

Our previous study in normal rat hippocampus showed that perfusion of 50 mM MeAIB elevates GLN_ECF 1.6 fold in vivo in 2 h (Kanamori and Ross, 2004); this concentration is close to that of 12.5 and 50 mM used respectively in hippocampal slices and neuronal cultures (Bacci et al., 2002). For the present study, a more rapid elevation by this nonmetabolizable and non-toxic inhibitor (section “Inhibition of neuronal uptake of GLN_ECF in vivo”) was required to study its effect on seizure frequency. The minimum concentration of MeAIB that achieves in vivo inhibition of neuronal uptake of GLN_ECF within 10 min, as indicated by elevation of GLN_ECF, was examined by intrahippocampal perfusion at MeAIB concentrations of 125, 175 and 250 mM. At 125 mM, no elevation was observed within this period. At 175 and 250 mM, GLN_ECF was elevated by 1.7–2 fold. Accordingly, a concentration of 175 mM was used in subsequent experiments. The duration of MeAIB perfusion that elevates GLN_ECF without change in extracellular taurine (TAU_ECF), an indicator of osmotic stress, was examined by perfusion for 30 or 55 min. Perfusion of 175 mM MeAIB for 30 min achieved GLN_ECF elevation without elevation of TAU_ECF during that period. Accordingly, this experimental condition was used in all subsequent experiments.

Elevation of GLN_ECF by MeAIB perfusion

Fig. 8 shows a representative time course of GLN_ECF during in vivo inhibition of its neuronal uptake by MeAIB perfusion. In this rat (R1156), GLN_ECF was elevated 1.8 fold when the perfusate was changed from aCSF to 175 mM MeAIB for 30 min. When the perfusate was changed back to aCSF, GLN_ECF returned to the initial concentration. GLU_ECF remained at low concentrations throughout the experiment. TAU_ECF did not change significantly during the 30-min MeAIB perfusion, showed a slight elevation after the perfusate was changed back to aCSF but eventually returned to the basal concentration.

Open in a separate window

Figure 8

The time-course of GLU_ECF, GLN_ECF and TAU_ECF in the ipsilateral hippocampus of a representative KA rat (R1156) of Experiment III. The bottom abscissa shows microdialysis time t. The top abscissa shows perfusion time T. At T = 0, the perfusate was changed from aCSF to MeAIB (an inhibitor of neuronal uptake of GLN_ECF) for 30 min, resulting in 1.8 fold elevation of GLN_ECF. Upon changing the perfusate to aCSF, GLN_ECF returned to basal value. GLU_ECF did not change and TAU_ECF showed only transient and modest change. The arrows show the time of occurrence of EEG seizures (see section “Elevation of GLN_ECF by MeAIB perfusion” for details).

To examine the time-courses of these extracellular metabolites in all KA rats of Experiment III, the following analysis was performed. As shown in Fig. 9, the start of MeAIB perfusion was set at perfusion time T = 0 (top abscissa) in all rats. The basal concentration of metabolites was measured at 40 ± 3 min (mean ± SE for n = 7) before the start of MeAIB perfusion, which corresponds to 48 ± 3 min of microdialysis time t (bottom abscissa). For GLN_ECF and TAU_ECF, the concentration at each subsequent time point was expressed as percentage of the basal concentration in each rat, then the mean ± SE at each time-point was calculated for n = 7 (the left coordinate). For GLU_ECF, the mean ± SE of the dialysate GLU concentration (as shown on the right coordinate) was calculated at each time point for n ranging from 4 to 7 (n was less than 7 at some time points because a 2-μL dialysate aliquot, necessary for accurate measurement of the low concentration GLU_ECF, was not available from all rats of this group). The basal concentrations (mean ± SE) were 32.5 ± 3.0 μM for GLN_ECF and 0.64 ± 0.24 μM for GLU_ECF (Table 1), and 5.9 ± 0.33 μM for TAU_ECF.

Open in a separate window

Figure 9

The time-courses of GLN_ECF and TAU_ECF, each expressed as percentage of pre-perfusion concentration (mean ± SE for n = 7) for KA rats of Experiment III (left coordinate). The time-course of GLU_ECF is expressed as dialysate concentration (mean ± SE for n = 7), as shown on the right coordinate, on a scale of 0–5 so that the symbols do not overlap with those of GLN_ECF and TAU_ECF. During MeAIB perfusion, GLN_ECF was elevated while GLU_ECF and TAU_ECF remained stable. TAU_ECF was transiently elevated after the perfusate was changed back to aCSF, but returned to basal value by T = 73 min. At the top, time periods shown as pre-, MeAIB, and post- (30-min each) refer to those used for comparison of the frequency of EEG seizures (see section “Elevation of GLN_ECF by MeAIB perfusion”).

As shown in Fig. 9, upon MeAIB perfusion, GLN_ECF was rapidly elevated 1.6 fold in 10 min and achieved a maximum elevation of 1.8 ± 0.17 fold (significantly different from the basal value with p < 0.01). Upon changing the perfusate back to aCSF, GLN_ECF returned to the basal value. TAU_ECF was unchanged during the 30-min MeAIB perfusion, but upon changing the perfusate to aCSF, was transiently elevated 1.6 fold (significantly different from the basal value with p < 0.05). By T = 73 min, TAU_ECF returned to the basal concentration. This interval of 43 (=73 – 30) min corresponds to the time required for the non-metabolizable inhibitor, MeAIB, to be cleared from ECF, as shown by our previous results and described in section “Inhibition of neuronal uptake of GLN_ECF in vivo”. For dialysate GLU, the mean concentration remained low without significant change (p > 0.05) throughout the experiment.

There was no difference in the time-courses of these extracellular metabolites between KA rats that were overnight-fasted then given i.v. infusion of glucose during microdialysis (n = 6) and those KA rats that were fed ad libitum (n = 3). Blood glucose concentrations at end point were 152 ± 9.6 mg/dL for the former and 137 mg/dL for the latter. K⁺ concentration in the effluxing dialysate during MeAIB perfusion was 2.7 mM compared to 2.9 mM in pre-perfusion period; both are close to the normal concentration of 3 mM in CSF.

Seizure-induced decrease of GLN_ECF

In KA rats of Seizure Group of Experiment I, GLN_ECF decreased progressively during the 5-h microdialysis experiment to 62 ± 4.4% of the initial value (Fig. 4A; n = 6), while in KA rats of Experiment II which were given i.v. infusion of glucose, the final GLN_ECF concentration was 75 ± 2.6% (Fig. 4B and Table 2). Hence, seizure-induced decrease of GLN_ECF occurs in both glucose-infused and overnight-fasted rats. The timing of GLN_ECF decrease correlated with the frequency and magnitude of seizure activity (Fig. 3A and B). Further evidence that the decrease in GLN_ECF is associated with epileptic activity is provided by the observation (Fig. 5) that after intense electrographic seizures in the ipsilateral hippocampus, GLN_ECF decreased to 70%, while it showed little change in the seizure-free contralateral hippocampus. Whether a single seizure associated with release of modest quantities of glutamate into ECF causes detectable change in GLN_ECF under our experimental condition is a question that must await future investigation.

It is informative to consider whether the observed seizure-induced GLN_ECF decrease during frequent epileptiform activity is reversible. SNAT3 mediates the co-transport of GLN with Na⁺ in exchange for H⁺ (Chaudhry et al., 1999). In the intact brain, the trans-membrane GLN gradient, GLN_glia/GLN_ECF, is estimated to be 17–20 (Bröer and Brookes, 2001; Kanamori and Ross, 2005), and an increase in the ratio caused by GLN_ECF decrease is expected to induce efflux. Hence, during long seizure-free period when the rate of neuronal uptake of GLN_ECF is normal, increased release of glial GLN into ECF may restore GLN_ECF to equilibrium concentration. In our study, KA rats which showed recurrent seizures in preliminary EEG recording (section “EEG/microdialysis (Experiment I)”) were subsequently used for microdialysis (section “EEG characteristics of KA rats”). These rats were lightly anesthetized on the day of microdialysis for probe insertion, woke in about 1 h and were allowed additional 2 h for stabilization of extracellular metabolites before the start of simultaneous EEG recording and the collection of dialysates for analysis (section “EEG/microdialysis (Experiment I)”). Restoration to equilibrium concentration during this quiescent period may explain the finding that GLN_ECF concentrations measured at the start of microdialysis experiment (after the stabilization period) do not differ significantly between those that subsequently exhibited frequent seizures and those that did not (Table 1).

Inhibition of neuronal uptake of GLN_ECF in vivo

Neuronal uptake of GLN_ECF was inhibited in vivo by perfusion of MeAIB, as demonstrated by 1.8 fold elevation of GLN_ECF (Figs. 8 and ​and9).9). This maximal elevation is in good accord with our earlier result that, in the cortex of normal rat, maximum elevation was 1.8 fold regardless of the concentration of perfused MeAIB over 50–250 mM range (Kanamori and Ross, 2004). A possible cause for this upper limit is that elevation of GLN_ECF reduces or suppresses SNAT3-mediated GLN efflux from glia, because the rate of efflux depends on [GLN]_glia/[GLN]_ECF ratio (Chaudhry et al., 1999; Kanamori and Ross, 2005).

The concentration of 175 mM used for in vivo inhibition appears to be high but was necessary to achieve inhibition within 10 min, as explained in section “Inhibition of neuronal uptake of GLN_ECF by MeAIB perfusion: Optimization of dose and duration”. Upon perfusion through the dialysis membrane, MeAIB diffuses through the hippocampal interstitial space, and is transported by SNAT1/SNAT2 into glutamatergic and GABAergic neurons, resulting in inhibition of GLN_ECF uptake. Because MeAIB has a lower affinity for these transporters (Albers et al., 2001; Su et al., 1997) than GLN (Mackenzie et al., 2003; Varoqui et al., 2000; Yao et al., 2000), a high concentration is required for effective inhibition. However, MeAIB is non-metabolizable and was non-toxic when administered intraperitoneally to rats at a dose of 10 mmol/kg (Christensen and Cullen, 1968) for study of its effect on hepatic retention of amino acids. Furthermore, in vivo inhibition occurs in a very small volume of the hippocampus, estimated to be 700 μm in diameter and 2 mm in length (Kanamori and Ross, 2004). The rat in the present study, observed during microdialysis, appeared to be in good physiological condition and showed no abnormal behavior. This was also true in our previous study (Kanamori and Ross, 2005) when 500 mM MeAIB was perfused through a 4 mm probe for 2 h. Taken together, these results strongly suggest that local perfusion of MeAIB in a small volume of the hippocampus achieves rapid inhibition without toxic or adverse physiological effect. It is worth noting that, in hippocampal slices, the frequency of spontaneous epileptiform discharges was decreased by MeAIB-induced inhibition of GLN uptake, but the frequency was restored after MeAIB washout (Bacci et al., 2002). The same work also shows that MeAIB-induced reduction of the amplitude of population excitatory post-synaptic potential was reversible. Taken together, these results strongly suggest that the effect of this non-metabolizable inhibitor on neuronal activity is reversible.

During MeAIB perfusion for 30 min, TAU_ECF, a major osmoregulatory metabolite in the brain (Estevez et al., 1999; Gullans and Verbalis, 1993) remained at the basal level (Figs. 8 and ​and9).9). The result is in agreement with our previous report that, upon perfusion of 250 mM MeAIB in the cerebral cortex of normal rats which caused 1.8 fold elevation of GLN_ECF, TAU_ECF, measured 30 min after the start of perfusion, remained at the basal value (Kanamori and Ross, 2004). These results show that MeAIB-induced elevation of GLN_ECF in vivo is due to inhibition of neuronal uptake of GLN_ECF, and not due to osmoregulatory efflux of organic osmolytes. After MeAIB saturates SNAT1/SNAT2, residual MeAIB is in ECF. Upon change of perfusate to aCSF, MeAIB in ECF diffuses back into dialysate. In a previous study, we measured the concentration of MeAIB diffusing back into dialysate as function of CSF perfusion time; at 6 min after switch from 500 mM MeAIB to aCSF, MeAIB concentration in the dialysate was 6 mM and MeAIB was cleared from ECF in 30 min (Kanamori and Ross, 2006; Fig. 4). In the present study, during this clearance of MeAIB, TAU_ECF was transiently elevated, but returned to basal value in 30–40 min (Fig. 9). TAU efflux probably occurs mainly from glia (Junyent et al., 2011). However, the mechanism of the observed transient TAU_ECF elevation during MeAIB clearance is beyond the scope of the present paper, because the nature of the volume-activated taurine efflux pathway is still controversial (Shennan, 2008). The important points for the present study are (1) elevation of GLN_ECF resulting from MeAIB perfusion is due to inhibition of neuronal uptake of GLN_ECF and (2) the frequency of EEG seizure in the post-perfusion period is analyzed after MeAIB is cleared from ECF and TAU_ECF, as well as GLN_ECF, has returned to the basal value.

The concentrations of electrolytes Na⁺ and K⁺ in ECF are unlikely to be perturbed by MeAIB perfusion for the following reasons. K⁺ concentration in the effluxing dialysate during MeAIB perfusion was 2.7 mM compared to 2.9 mM in the basal pre-perfusion dialysate (Unpublished result in this Laboratory), and is quite close to the normal K⁺ concentration of 3 mM in CSF. Na⁺ concentration in MeAIB perfusate, prepared as described in section “MeAIB perfusion (Experiment III)”, is at most 0.5% higher than the normal Na⁺ concentration of 150 mM in ECF (Kanamori and Ross, 2004).

Effect of inhibition on seizure frequency

Inhibition of neuronal uptake of GLN_ECF in the ipsilateral hippocampus by MeAIB perfusion decreased the frequency of electrographic seizures to 35 ± 7% of the frequency observed in the pre-perfusion period (Fig. 10 and Table 3). After clearance of this non-metabolizable and reversible inhibitor from ECF, the frequency of seizures returned to 88 ± 9% of the initial frequency. These results strongly suggest that electrographic seizures are significantly reduced when pyramidal neurons in the CA3/CA1 region are deprived of GLN_ECF as potential provider of neurotransmitter GLU. To the best of our knowledge, this is the first study that reports the effect of inhibition of neuronal uptake of GLN_ECF on seizure frequency in vivo.

It is informative to briefly summarize (a) novel aspects of this study in relation to our previous study, (b) suggested biochemical bases for our findings, and (c) remaining issues for future investigation. In our previous study (Kanamori and Ross, 2011), we focused on those KA rats which showed significant elevation of GLU_ECF, possibly due to impairment or down-regulation of glial glutamate transporter EAAT2. Such impairment of GLU uptake into glia reduced the availability of substrate GLU for GLN synthesis (Fig. 1), as suggested by the observed reduction of intracellular GLN in the relevant hippocampal region in the Seizure Group compared to the No-Seizure Group (Table 2, Kanamori and Ross, 2011). Hence, the observed seizure-induced decrease in GLN_ECF could reasonably be attributed to reduced GLN efflux from glia to ECF, although there remained an intriguing possibility that seizure-induced decrease of GLN_ECF also reflects its increased uptake into neurons to replenish neurotransmitter glutamate during enhanced epileptiform activity. Subsequently, we found that seizure-induced decrease in GLN_ECF also occurs in those KA rats that exhibit no significant elevation of GLU_ECF, which is the subject of the present study. A probable cause for the low and steady level of GLU_ECF is that glial glutamate transporter EAAT2 was not significantly impaired by KA injection (section “GLU_ECF”). We hope to examine the cause of the difference in these two groups of KA rats by measuring the levels of EAAT2 proteins by Western blotting. GLU_ECF, taken up into glia serves as an important substrate for glutamine synthesis. In support of this concept, the intracellular hippocampal GLN in the Seizure Group was virtually the same as that in the No Seizure group (Fig. 7). Accordingly, it is reasonable to assume that the rates of GLN efflux into ECF were similar in the two groups and unlikely to account for the observed seizure-induced decrease in GLN_ECF. Hence, in the present study, we examined more closely the possibility that the observed GLN_ECF decrease reflects increased uptake into neurons. We inhibited neuronal uptake of GLN_ECF in vivo and found that this causes significant reduction of seizure frequency. The novel findings in this study are (a) seizure-induced decrease in GLN_ECF occurs in those KA rats that exhibit no significant elevation of GLU_ECF, (as well as in those that do), and (b) neuronal uptake of GLN_ECF plays an important role in sustaining recurrent epileptiform activity.

Another factor that can influence the concentrations of GLU_ECF and GLN_ECF and of hippocampal intracellular GLU and GLN is the activity and concentration of glutamine synthetase (GS) (Fig. 1). In the hippocampal formation of patients with mesial temporal lobe epilepsy (MTLE), GS protein and activity are severely reduced (Eid et al., 2004), and this has been proposed as a possible mechanism for the elevated GLU_ECF reported in these patients (section “Role of GLN_ECF in sustaining epileptiform activity in vivo”). Eid et al. also report that in a rat model of MTLE, inhibition of GS activity by chronic perfusion of methioninesulfoxime into the hippocampal formation induces recurrent seizures (Eid et al., 2008); among proposed mechanisms (Eid et al., 2012) is elevation of astrocyte GLU due to GS inhibition which impairs glial uptake of GLU_ECF resulting in its elevation. These findings can have important implications for epileptogenesis in MTLE patients and provide a reasonable explanation for the occurrence of seizures in their animal model. However, in chronic KA rats, it remains to be investigated whether changes in GS activity contribute to the seizure-induced GLN_ECF decrease reported in our study. In KA rats, proliferation of astroglia (gliosis) in the chronic phase can complicate interpretation of GS activity measurements (Hammer et al., 2008).

KK and BDR are Visiting Associates in the Division of Chemistry and Chemical Engineering at California Institute of Technology. We are very grateful to Dr. Anatol Bragin, Department of Neurology, University of California, Los Angeles, California, for very valuable practical advice on how to prepare chronic kainate rat model and to perform EEG recording. We thank Ms Susan Lee in the Clinical Laboratory at Huntington Hospital, Pasadena, California, for performing K⁺ assay.

Grants

This work was supported by Research Grant RO1-NS048589 from the National Institute of Neurological Disorders and Stroke, the US Public Health Service, and Institute fund from Huntington Medical Research Institutes. KK is grateful for funds donated by Ms Rita Pudenz. BDR thanks Rudi Schulte Research Institute for financial support.