Chen, G. Colquhoun, D. Sakmann and E. Neher, eds. Connors, B. Corssen, G. Cleveland 53 — Courtney, K. Croucher, M. Curtis, D. London — Davis, R. Dingledine, R. Duchen, M. Fagg, G. Flatman, J.
Foster, A. Gage, P. Gurney, A. Hablitz, J. Harrison, N. Hayashi, T. Hayes, B. Herron, C. Hille, B. Honey, C. Jahr, C. Johnston, D. Johnston, G. Lacey, M. Little, H. Previous 13 C-labelling studies in temporal lobe epilepsy patients [ 16 , 17 ] and in rodent models [ 18 , 19 ] have focused on the effect of seizures on the 13 C enrichment of GLU and other neurochemicals in hippocampal tissue, which is predominantly intracellular , using 13 C NMR or gas-chromatography mass-spectrometry GCMS to quantify 13 C-metabolites in brain extracts.
For the first time in vivo, the present study shows how the flux of the excitatory neurotransmitter GLU from neuron to ECF is increased during frequent seizures in awake, freely behaving kainate-treated rats. The advantages, limitations and the potential for improvement of this 13 C labelling approach for pre-clinical and clinical studies are discussed. GLN ECF is taken up into neurons by the sodium-coupled neutral amino acid transporter subtypes 1 and 2 SNAT 1 and 2 reviewed by [ 25 ] , where it is hydrolyzed by glutaminase to replenish the metabolic and neurotransmitter pools of GLU.
South Bend, IN, U. Sodium kainate Sigma-Aldrich, St. The rat, which awoke from anesthesia within 1 h, was continuously monitored for behavioral seizures for 6 h after injection acute phase. The grounding electrode a stainless-steel wire 0. The recording electrode a pair of stainless steel wires of the same dimension with tips 0. The recording electrodes and adjacent anchor screws were then fixed to the skull with acrylic cement.
The sockets one from the grounding electrode and two pairs from the recording electrodes were inserted into the bottom contacts of a 6-pin plastic pedestal, which was then cemented to the skull and capped. Several days after the surgery, a preliminary EEG recording was taken. The EEG electrode contacts on the skull were connected to a cm cable, which was mesh-covered on the proximal end and equipped with solder lugs on the distal end for connection to the amplifier.
Recordings were taken wide-band 0. Preliminary EEG recordings were taken for several hours with the observation of behavioral seizures. Two days before the experiment, an indwelling silastic catheter was placed in the right external jugular vein for i. The distal end of the catheter 90 cm long , exiting at the nape, was placed in a backpack worn by the rat [ 26 ].
The EEG cable, the inlet and outlet dialysis tubings and the i. Pentobarbital was used for consistency with our previous studies [ 14 , 26 ]. The necessity of connecting the inlet and outlet tubings to the brain microdialysis probe precludes the use of isoflurane anesthesia for this experiment.
A 3-h stabilization period was allowed before the start of dialysate collection for analysis. Dialysates were collected bilaterally in min fractions for 1 h without i. Thus, the interval between pentobarbital injection and the start of 13 C glucose infusion for enrichment analysis was approximately 4 h 1 h until waking, additional 2 h for the stabilization of extracellular neurochemicals and 1 h for basal dialysate collection. Subsequently, a constant infusion rate of 1. This protocol, initially developed by Fitzpatrick et al.
For controls, the same procedure was applied to normal rats given an injection of saline instead of kainic acid. After the experiment, the locations of the microdialysis probe and the electrodes were confirmed in each rat as described previously [ 14 ].
Excitatory amino acid transporter 2
Briefly, the probe was removed and the guide stylet was reinserted. The cement fixing the guide cannula and the EEG electrodes to the skull could be lifted from the skull of the anesthetized rat by inserting a flat spatula between the cement and the nasal bone. Accordingly, the cement with the electrodes and the guide cannula still attached could be examined for vertical coordinates in every rat. The lateral coordinates, too, could be confirmed from the distance between the right and left electrodes and the location of the burr holes on the exposed skull.
The confirmed coordinates of the microdialysis probe and the EEG electrode are shown in Results. The brain was then removed from the anesthetized rat and frozen in liquid nitrogen. This procedure, instead of fixing the brain for histology, was adapted at the start of the project because, in our previous studies on the same model, it was informative to measure the intracellular glutamine concentration in the relevant hippocampal region of the end-point brain for interpretation of the effect of seizure on GLN ECF [ 14 , 26 ].
However, the frozen brain was not used for this purpose in the present study, because the time-course of 13 C enrichment of extracellular GLN, unlike that of extracellular GLU, turned out to show little dependence on seizure activity as described in Results. The microdialysis time was reported at the center of each collection time. Amino acids in the brain dialysate were assayed after precolumn derivatization with ortho-phthaldehyde OPA and 2-mercaptoethanol and separation on a reverse-phase column by fluorometric detection as described previously [ 26 ].
After eluting neutral and cationic metabolites with water 1. Meticulous care was taken to clean the column and the resin and all glassware to avoid contamination by trace [ 12 C]GLU, as described in detail previously [ 21 ]. Louis, MO, U. The eluted GLN ECF in the neutral fraction of the anion-exchange column was further purified by cation-exchange chromatography. After adjusting the pH to 2. Accordingly, glutamine was converted to pyroglutamic acid, which is a cyclic compound that contains all five carbons of glutamine and forms a very stable t BDMS-derivative [ 32 ].
The pH was then adjusted to 2. Separation was achieved on a 30 m x 0. For MS, the operating conditions were 70 eV electron-impact ionization and 13 C enrichment analyses by selected ion monitoring. The structures of the fragment ions are shown in Results. The injection liner and the septum were changed frequently and the injection needle was rinsed 12 times with acetonitrile after each injection.
Between each biological sample, a blank consisting of the solvent acetonitrile and MTBSTFA was run to ensure no carry-over of 13 C enriched derivative. Biological 13 C enrichment resulting from i. Correction for the contribution of naturally occurring heavy isotopes was performed as follows. The corresponding correction factors were 0. In this study, the expression 13 C enrichment instead of 13 C labelling is used to denote enrichment above the natural abundance 1.
Our previous results showed that i. To quantify the low enrichment at C2, C3 and C4 relative to the enrichment at C5, 13 C NMR spectra were taken of the perchloric acid extracts of the brain after 2 and 3. For GLU, this ratio was only 0. As shown in S3A Fig , this ratio increased linearly with time with a slope of 0. The increase was biphasic with a small further increase at 3. The validity of this approach in relation to reports from other laboratories is discussed in Discussion.
Data analyses, including post-hoc tests to determine which group, among the three, differs significantly from another, were performed by the statistical software of QI Macros KnowWare International Inc. At the low dose of kainate, 0. As shown in Table 1 , those kainate-treated KA rats were examined 49—56 days after unilateral kainate injection. The numbers of spontaneous recurrent seizures that occurred during 3 hours of glucose infusion are shown.
As shown in Table 1 , the locations of the EEG electrodes and microdialysis probes, implanted stereotaxically and confirmed at the end of in vivo experiments, were very close. Fig 1 shows EEG recordings from the hippocampi of an awake freely behaving KA rat R from the frequently seizing group during the chronic phase. The top trace of Fig 1A shows a recording from the kainate-injected ipsilateral hippocampus.
A quiescent period characterized by a single inter-ictal spike IIS is followed by a seizure Box , which, by definition see section Identification of seizure , is a period of consistent and repetitive changes in amplitude and frequency of electrical activity that is clearly different from inter-ictal activity.
This seizure occurred in the ipsilateral hippocampus top but was absent from the contralateral hippocampus bottom. Fig 1B top shows an expanded plot of the seizure, with a time-scale of 10 s. The inset shows an expanded plot of the peak in the box that shows a wave pattern characteristic of a population burst from glutamatergic neurons [ 37 ]. The occurrence of these two types of spontaneous seizures, with the hypersynchronous onset as the major type, during the chronic phase of KA rats, is consistent with a previous report [ 13 ].
A Top : The kainate-injected ipsilateral hippocampus shows a quiescent period with a single inter-ictal spike IIS followed by a seizure enclosed in box. Bottom : The contralateral hippocampus was seizure-free.
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B Top : Expanded plot of the seizure with a time-scale of 10 s. The inset shows an expanded plot of the peak in the box with a time scale of 10 ms; the wave pattern is characteristic of a hypersynchronous population burst from glutamatergic neurons. Bottom : Corresponding recording from the seizure-free contralateral hippocampus.
The time of occurrence of seizures is shown by arrows at the top. Fig 3B shows the corresponding time course in one rat R from group II that showed intermittent and fewer seizures at the times indicated by arrows. The mean time course of 13 C enrichment was substantially faster in frequently seizing rats compared to those in control or in infrequently seizing rats. Fig 3D shows an example of the least squares line through the plots for a rat from each group; the slope is similar for the control and the infrequently seizing rat, but substantially higher for the frequently seizing rat.
These rates are shown in Table 2 the middle column. The rate in frequently seizing rats, 0. A representative time course in a frequently seizing KA rat R black square compared to control open square. In this rat, the seizures occurred at the times shown by arrows. Time course black diamond in a representative KA rat with rare seizures R; shown by arrows compared to control open square. The 13 C enrichment was substantially faster in frequently seizing rats compared to both the control and the infrequently seizing rats.
Likely explanations for the observed multi-phase increases in the frequently seizing KA rats are described in Discussion. At the bottom are shown representative mass spectra of t BDMS-pyroGLU derived from extracellular glutamine GLN ECF , which was collected by microdialysis from the ipsilateral hippocampus of a frequently seizing rat R during intravenous infusion of [2,5- 13 C]glucose.
The time courses were very similar among the three groups, but in the frequently seizing rats, the mean enrichment of 0. In the control and infrequently seizing rats, the 13 C enrichment increased linearly up to min then levelled off. The results are shown in Table 2 last column. The rate in infrequently seizing rats 0. The error bar, when invisible, is smaller than the symbol. As shown in Table 1 , the concentration of dialysate GLN before infusion was Although ANOVA was performed on three groups simultaneously, the time-course data for groups II and III, compared to the control, are shown in separate panels to avoid overlap of symbols and error bars.
In infrequently seizing rats inverted black triangle , the concentration decreased significantly while controls open diamond showed little change. Time course for frequently seizing rats black triangle , showing a more pronounced decrease. The difference in the mean number of seizures between infrequently seizing rats group II and frequently seizing rats group III Table 1 is due to spontaneous nature of chronic-phase recurrent seizures [ 12 — 14 ].
The procedure for determining the locations of EEG electrodes and microdialysis probes in this study is the same as that used in our previous publications [ 14 , 26 , 28 ]. The elevation was probably caused by synchronous firing of glutamatergic neurons, such as is observed in the ictal discharge of Fig 1A top trace. These findings suggest the following sequence of events.
This, in turn, overstimulated GLU receptors in the CA3 region, which is highly populated with glutamatergic neurons with recurrent networks [ 38 ].
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Another proposed explanation for GLU ECF elevation is the reduction of glutamine synthetase GS observed in the hippocampal formation of mesial temporal lobe epilepsy patients [ 39 ]. In the present study, there was no statistically significant difference in the pre-infusion concentrations of dialysate GLU among the three groups, although the concentration was highest in the frequently seizing rats Table 1 and Fig 4. A likely explanation is that when seizures are rare, the flux of GLU from the neuron to ECF returns to near-normal levels in the inter-ictal period.
To detect these short time changes, 13 C enrichment analyses at higher temporal resolution will be needed, as discussed in Section MS methodology—advantages, limitations and potential for improvement. Likewise, the similarity of the time-courses of total GLU ECF between infrequently seizing and control rats can most reasonably be attributed to the reversal of elevation of total GLU ECF during quiescent inter-ictal period, which was reported in our previous study [ 14 ] and is described below.
It is informative to ask why the GLU ECF concentration measured before the start of the 3-h [2,5- 13 C]glucose infusion Table 1 is not significantly higher in the frequently seizing rats compared to those with rare seizures and controls. A higher concentration is expected if the seizure patterns are the same before and during the infusion.
Chronic-phase recurrent seizures occur spontaneously [ 12 , 13 ] and the time of occurrence is unpredictable on a short time scale. As shown in Fig 5a, 5b and 5c of that study, the elevation of GLU ECF does correlate with the frequency and magnitude of seizure activity, but the pattern of seizure activity during the first 2. This reversal of GLU ECF elevation during quiescent inter-ictal period accounts, at least in part, for the observation that basal GLU ECF concentration is not significantly different between KA rats that subsequently showed frequent seizures and those that did not.
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Excitatory Amino Acids and Epilepsy
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