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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2014 May 14;34(8):1340–1346. doi: 10.1038/jcbfm.2014.88

A subconvulsive dose of kainate selectively compromises astrocytic metabolism in the mouse brain in vivo

Anne B Walls 1, Elvar M Eyjolfsson 2, Arne Schousboe 1, Ursula Sonnewald 2,3, Helle S Waagepetersen 1,3,*
PMCID: PMC4126094  PMID: 24824917

Abstract

Despite the well-established use of kainate as a model for seizure activity and temporal lobe epilepsy, most studies have been performed at doses giving rise to general limbic seizures and have mainly focused on neuronal function. Little is known about the effect of lower doses of kainate on cerebral metabolism and particularly that associated with astrocytes. We investigated astrocytic and neuronal metabolism in the cerebral cortex of adult mice after treatment with saline (controls), a subconvulsive or a mildly convulsive dose of kainate. A combination of [1,2-13C]acetate and [1-13C]glucose was injected and subsequent nuclear magnetic resonance spectroscopy of cortical extracts was employed to distinctively map astrocytic and neuronal metabolism. The subconvulsive dose of kainate led to an instantaneous increase in the cortical lactate content, a subsequent reduction in the amount of [4,5-13C]glutamine and an increase in the calculated astrocytic TCA cycle activity. In contrast, the convulsive dose led to decrements in the cortical content and 13C labeling of glutamate, glutamine, GABA, and aspartate. Evidence is provided that astrocytic metabolism is affected by a subconvulsive dose of kainate, whereas a higher dose is required to affect neuronal metabolism. The cerebral glycogen content was dose-dependently reduced by kainate supporting a role for glycogen during seizure activity.

Keywords: anaerobic glycolysis, blood flow, 13C isotopes, glutamine synthesis, glycogen, lactate

Introduction

Seizure activity is characterized by hyperexcitability of brain tissue and is thought to arise as a consequence of disturbances in the balance of excitatory and inhibitory activity. Seizure activity may arise as a repercussion of trauma, brain tumors or infections, whereas epilepsy, which is the most prevalent seizure disorder, is defined by recurrent seizure activity evoked by an immediately unidentified cause (ILAE 1993). Approximately 50 million people worldwide are diagnosed with epilepsy and only ∼50% of these are adequately treated.1 Temporal lobe epilepsy is one of the most common forms of epilepsy and pharmacoresistance within these patients is particularly high accounting for up to 80% of patients.2 Hence, there is a persistent need for development of anticonvulsant drugs displaying improved efficacy and reduced adverse effects and to this end, a thorough understanding of the etiology of temporal lobe epilepsy and seizure activity is required.

Kainate is a well-established pharmacological tool used to induce acute seizure activity and the subsequent neuronal damage reflects that observed in human temporal lobe epilepsy;3 hence, kainate treatment is widely used as an animal model of mesial temporal lobe epilepsy. The convulsive effects of kainate are time and concentration dependent3, 4, 5 and the seizure propagation reflects the etiology of temporal lobe epilepsy observed in patients, i.e., it involves an initial precipitating injury followed by a latent phase before the chronic phase characterized by spontaneous seizures ensues.6 Metabolic changes in the cerebral cortex 24 hours after a systemic injection of a convulsive dose of kainate have been demonstrated to mainly involve the astrocyte compartment.7 Later, alterations within both astrocytes and neurons were observed in the latent phase while the chronic phase was characterized predominantly by neuronal changes.8, 9 However, little is known about the changes in cerebral metabolism immediately after kainate treatment, although this might provide valuable information with regard to determining the initial cause of seizure activity underlying subsequent propagation of epileptic disorders and may provide a unique diagnostic feature. An early diagnosis could potentially provide a therapeutic possibility during the latent phase, thereby preventing epileptogenesis from progressing into the chronic convulsive phase.

The classic definition of brain activity delineates the chemical communication between neurons and the origin of the synchronized neuronal hyperactivity involved in seizure activity is generally believed to rely on abnormalities intrinsic to neurons. However, the expression of a broad selection of ion channels, gap junctions, neurotransmitter transporters, and receptors enables astrocytes to respond to and modify neuronal activity and thus astrocytes are crucial partners in regulating neurotransmission.10 Moreover, a number of enzymes that are crucial for the maintenance of cerebral energy and amino-acid homeostasis are selectively distributed in either astrocytes or neurons.11 Thus, a vital dynamic interplay exists between neurons and astrocytes with regard to maintaining and adjusting neuronal activity. In line with this, there is an increasing amount of evidence for alterations in astrocyte metabolism being involved in the pathophysiology of epilepsy and seizure activity.9, 12 However, whether such changes in astrocyte metabolism are the cause or the consequence of excessive neuronal activity, i.e., seizure activity, is still a matter of uncertainty.

Here we aimed to identify the cellular origin of the metabolic changes appearing in a mouse model of temporal lobe epilepsy immediately after the injection of kainate. The onset of seizure activity was investigated using a subconvulsive and a convulsive dose of kainate and brain metabolism was mapped 15 minutes after kainate treatment by injecting the mice with a combination of [1-13C]glucose and the astrocyte-specific substrate [1,2-13C]acetate.13 Subsequently, changes in the metabolic pathways were revealed by nuclear magnetic resonance (NMR) spectroscopic analyses of cortical tissue extracts.

Materials and Methods

Materials

[1-13C]Glucose and [1,2-13C]acetate as well as D2O (99.9%) were purchased from Cambridge Isotope Laboratories (Woburn, MA, USA) while ethylene glycol was bought from Merck (Darmstadt, Germany). Kainate, amyloglucosidase, hexokinase, glucose-6-phosphate dehydrogenase, ATP, and NADP were from Sigma-Aldrich (St Louis, MO, USA). All other chemicals were of the purest grade available from commercial sources.

Animals

Mice of the C57BL/6 strain were obtained from Taconic (Ry, Denmark). After delivery to the animal facilities at the Norwegian University of Science and Technology, the mice were kept under standard conditions at ambient temperature of 20°C to 22°C, air humidity of 50% to 60%, and a 12-hour light–dark cycle (lights on at 0700 hours). The mice were acclimatized to these conditions for at least 1 week before they were used for experiments. Within this period as well as during the experiment, the animals had free access to food and water. Mice of both genders were used for experiments at the age of 15 to 23 weeks. All protocols were approved by the Norwegian National Animal Research Authority (07/58706) and animals were treated in compliance with the European Convention (ETS 123 of 1986).

Metabolic Studies

The mice were injected intraperitoneally with a low dose (3.75 mg/kg) or a high dose (15 mg/kg) of kainate while control animals were injected with 0.9% NaCl. The mice were observed for 30 minutes post injection of kainate or saline for incidence and severity of seizures. Seizure severity was graded according to a modified Racine scale.4, 14 Fifteen minutes after kainate injection, the mice were injected intraperitoneally with a combination of [1-13C]glucose (543 mg/kg) and [1,2-13C]acetate (504 mg/kg) and after another 15 minutes, the brains of the mice were subjected to microwave fixation at 4 kW for 1.7 seconds (model GA5013, Gerling Applied Engineering, Modesto, CA, USA). This is the most humane euthanization method instantly inactivating proteins in the brain. The cerebral cortices were dissected out and kept at −75°C until extraction employing 0.7% perchloric acid. In order to homogenize the samples, these were exposed to ultrasound using a Vibra Cell sonicator (model VCX 750, Sonics & Materials, Newtown, CT, USA). After centrifugation of the homogenous suspensions at 3,000 g and 4°C for 5 minutes, the precipitates were washed with distilled water and the centrifugation step was repeated. The precipitates were used for glycogen determination. The supernatants were pooled and pH was adjusted to 7.0±0.5 before lyophilization and NMR spectroscopic analyses.

Nuclear Magnetic Resonance Spectroscopy

The lyophilized brain tissue extracts were redissolved in 99% D2O containing 0.05% ethylene glycol as an internal standard and subsequently, the pH was readjusted to 7.0±0.5. The samples were transferred into 5 mm Shigemi NMR microtubes (Shigemi, Allison Park, PA, USA) and using a BRUKER DRX-600 spectrometer (BRUKER Analytik, Rheinstetten, Germany), the samples were analyzed for content of and 13C labeling in amino acids and lactate employing 1H and 13C-NMR spectroscopy, respectively. A pulse angle of 90° and a spectral width with 32 K data points were employed to obtain the 1H-NMR spectra. The acquisition time was 1.36 seconds and relaxation delay was 10 seconds. A low-power presaturation pulse at the water frequency was applied to suppress the water signal. The number of scans was 128 and the spectra were recorded at room temperature.

For proton decoupled 13C-NMR spectra of cortical extracts, a 30° pulse angle and 30 kHz spectral width with 64 K data points were employed. An acquisition time of 1.08 seconds and a relaxation delay of 0.5 seconds were used and the number of scans needed to obtain an appropriate signal-to-noise ratio was typically 30,000. Relevant peaks in the spectra were identified and integrated using XWINNMR software (BRUKER BioSpin).

The amounts of 13C labeling and the total amounts of metabolites were quantified by relating the integrals of the peak areas to that of ethylene glycol and correcting for brain tissue weight. Factors for the nuclear Overhauser and relaxation effects, obtained from several sets of spectra, i.e., one set obtained with decoupling, NOE, and short repetition time (the parameters used for all samples) and another set with 1H decoupling, no NOE, and 20 seconds delay time to allow for full relaxation of the relevant peaks, were applied to all 13C spectra. Singlets obtained in the 13C spectrum were corrected for naturally abundant 13C and amounts calculated from 1H spectra were corrected for 13C containing metabolites and number of protons.

13C Labeling Patterns Obtained from Metabolism of [1,2-13C]Acetate

Uptake and metabolism of [1,2-13C]acetate is confined to the astrocytic compartment13 and 13C labeling from acetate thus reflects astrocytic metabolism and potential subsequent interactions with neurons. [1,2-13C]Acetate is in astrocytes converted to [1,2-13C]acetyl co-enzyme A (CoA) and after entry into the TCA cycle [4,5-13C]α-ketoglutarate is generated. Owing to the high activity of mitochondrial transaminases,15 [4,5-13C]α-ketoglutarate is in rapid equilibrium with [4,5-13C]glutamate, which is a precursor for [4,5-13C]glutamine. If [4,5-13C]α-ketoglutarate remains in the astrocytic TCA cycle for further metabolism via this pathway, it will, after condensation with unlabeled acetyl CoA, give rise to [3-13C]- and [1,2-13C]α-ketoglutarate in the subsequent turn, which may give rise to glutamate and glutamine displaying the same labeling patterns. Based on the distinct 13C labeling in glutamine after the first and second turn of TCA cycle metabolism, a number indicative of astrocytic TCA cycle activity can be calculated by relating the amount of isotopomers generated in the second turn of TCA cycle metabolism to the amount of that generated in the first turn, i.e., ([1,2-13C]glutamine+[3-13C]glutamine)/[4,5-13C]glutamine (equation (1)).

Alternative to metabolism in successive turns of the astrocytic TCA cycle, [4,5-13C]glutamine may be transferred to the neuronal compartment where [4,5-13C]glutamate may be formed by the action of phosphate-activated glutaminase. In glutamatergic neurons, this [4,5-13C]glutamate may be released upon depolarization. Otherwise, [4,5-13C]glutamate may be converted to [4,5-13C]α-ketoglutarate for further TCA cycle metabolism or may in GABAergic neurons be converted to [1,2-13C]GABA. It should be noted that the largest pool of glutamate is situated in the glutamatergic neurons and the [4,5-13C]glutamate observed in the cortical extracts likely reflects this pool, although smaller glutamate pools exist in astrocytes and GABAergic neurons.16

13C Labeling Patterns Obtained from Metabolism of [1-13C]Glucose

One molecule of [1-13C]glucose is by way of glycolysis converted to two molecules of pyruvate; one being 13C labeled in the third carbon atom and one being unlabeled. [3-13C]Pyruvate can be converted into [3-13C]lactate or [3-13C]alanine or can via [2-13C]acetyl CoA enter the TCA cycle and give rise to [4-13C]α-ketoglutarate, which is a precursor for [4-13C]glutamate. In astrocytes, [4-13C]glutamate may be converted into [4-13C]glutamine that can be transferred to neurons where it is reconverted to [4-13C]glutamate by phosphate-activated glutaminase. This [4-13C]glutamate may in glutamatergic neurons be packaged into vesicles and used for neurotransmitter release, and in GABAergic neurons, [4-13C]glutamate may be decarboxylated to [2-13C]GABA. In all cell types, [4-13C]glutamate may be converted to [4-13C]α-ketoglutarate and metabolized via the TCA cycle for energy generation. A measure of TCA cycle activity can be calculated on the basis of the 13C labeling pattern in glutamate obtained after metabolism of [1-13C]glucose via glycolysis and successive turns of TCA cycle metabolism. [1-13C]Glucose generates equal amounts of [2-13C]- and [3-13C]glutamate in the 2nd turn of TCA cycle metabolism but as carboxylation of [3-13C]pyruvate also leads to [2-13C]glutamate after TCA cycle metabolism of oxaloacetate, the amount of [2-13C]glutamate cannot be used for calculation of TCA cycle activity. Metabolism of [1,2-13C]acetate gives rise to equal amounts of [1,2-13C]glutamate and [3-13C]glutamate in the 2nd turn of TCA cycle metabolism but the amount of [3-13C]glutamate can be corrected for the amount originating from [1,2-13C]acetate by subtracting the amount of [1,2-13C]glutamate. Hence, the contribution of [1-13C]glucose to glutamate in the 2nd turn over that of 1st turn of TCA cycle metabolism can be calculated as (2*([3-13C]glutamate–[1,2-13C]glutamate))/[4-13C]glutamate (equation (2), ref. 17).

After vesicular release of [4-13C]glutamate, it is predominantly accumulated into astrocytes18 and may subsequently be converted to [4-13C]glutamine, which can be transferred to the neurons and reconverted to [4-13C]glutamate, a process generally referred to as the glutamate–glutamine cycle. As neurons do not express the quantitatively most important anaplerotic enzyme, pyruvate carboxylase, they are highly dependent upon glutamine transfer from astrocytes to compensate for the carbon skeleton lost when neurotransmitter glutamate is taken up into astrocytes. However, the glutamate–glutamine cycle does not operate in a stoichiometric fashion in the sense that the carbon skeleton of glutamate released is the same as that of the glutamine returned to the neuron. A part of the glutamate taken up by astrocytes is metabolized via the TCA cycle and accordingly astrocytic glutamine biosynthesis and subsequent transfer to neurons is required to compensate for the neuronal loss of neurotransmitter after astrocytic uptake.19

As [1-13C]glucose is metabolized in both neurons and astrocytes, 13C is incorporated into amino acids in both compartments. However, as glutamate and aspartate constitute large amino-acid pools, and the majority of these are located in neurons, the 13C labeling in glutamate and aspartate predominantly reflects the neuronal compartment. Glutamine is an astrocytic product and it is 13C labeled from [1-13C]glucose via astrocyte glycolysis and TCA cycle metabolism but also partly from [4-13C]glutamate and [2-13C]GABA subsequent to neurotransmission. Using fluorocitrate to inhibit astrocytic TCA cycle activity, it was demonstrated that almost 40% of the [4-13C]glutamine labeled from [1-13C]glucose originates from [4-13C]glutamate, which was initially labeled in the neuronal compartment.20 Thus, although glutamine biosynthesis is confined to astrocytes,21 a substantial part of the [4-13C]glutamine being labeled from [1-13C]glucose is likely to reflect neuronal metabolism.

Glycogen Determination

The precipitates generated after perchloric acid extraction were used for determination of cortical glycogen content. The amount of glycosyl units in glycogen was assessed by employing a coupled enzymatic assay. Glycogen was first degraded to glucose and subsequently, the amount of NADPH was measured in the conversion of glucose-6-phosphate to 6-phosphogluconolactone as previously described.22 Briefly, the precipitates were reconstituted in 0.1 mol/L sodium acetate (pH 4.5) containing amyloglycosidase 0.435 U/ml and agitated at 37°C for 1 hour. The soluble glucose units were separated from insoluble remnants by centrifugation at 20,000 g for 10 minutes and the supernatants were transferred to a black microtiter plate. Tris buffer containing MgCl2 (12.1 mmol/L), ATP (0.81 mmol/L), and NADP (0.16 mmol/L) was added to ensure pH optimum (7 to 8) for hexokinase and glucose-6-phosphate dehydrogenase. After measuring background fluorescence using excitation and emission wavelengths of 360 and 415 nm, respectively, the reaction was initiated by the addition of hexokinase (final concentration 1.52 U/mL) and glucose-6-phosphate dehydrogenase (final concentration 0.54 U/mL) in a Tris buffer containing bovine serum albumin. The plate was left at room temperature for 40 minutes for the reaction to proceed to completion, and subsequently, the glucose content was determined by measuring NADPH autofluorescence. Glucose in the concentration range 20 to 200 μmol/L was used as standards.

Data Analysis

Results are presented as averages±s.e.m. Data obtained from NMR analyses are normalized at letting the control group treated with saline represent 100%. The effect of different doses of kainate on total amounts of and 13C labeling in organic- and amino acids was tested employing one-way analysis of variance followed by least significant difference post hoc test. These parametric statistical analyses were performed employing SPSS–PASW statistics 18. Glycogen data were plotted using GraphPad Prism 5 and the statistical analysis of the slope deviating from zero was also calculated using this software. Data were taken to be significantly different when P<0.05. The data obtained from 1H and the 13C-NMR spectroscopy, including the calculated TCA cycle activities, have previously been published in a different context evaluating metabolism in a GAD65 knockout mouse.23

Results

Seizure Severity After Injection of Saline and Kainate

Increasing doses of kainate were used to investigate cerebral metabolism during the onset of seizure activity in mice. A low (3.75 mg/kg) and a higher (15 mg/kg) dose of kainate were used, and the mice were observed for occurrence and severity of seizures for 30 minutes post injection, i.e., until microwave fixation. Seizure severity was graded according to a modified Racine scale.4, 14 After injection of saline and the low dose of kainate, the mice showed no abnormal behavior. In contrast, treatment with the high dose of kainate led to head nodding and face washing within 5 minutes after injection corresponding to stage 2 seizures on a modified Racine scale4, 14 and the mice remained in this seizure stage throughout the experiment. Thus, treatment with the two doses of kainate represented a subconvulsive and a mildly convulsive dose.

Cortical Content of Metabolites

The effect of different doses of kainate on the cortical content of glutamate, glutamine, GABA, aspartate, lactate, and alanine were determined from tissue extracts using 1H-NMR spectroscopy (Figure 1). Employing the subconvulsive dose of kainate, the contents of glutamate, glutamine, GABA, and aspartate were unaltered compared with control mice, whereas the contents of lactate and alanine were increased by ∼50%. This implies a pronounced amplification of glycolytic activity in mice treated with the subconvulsive dose of kainate. Treatment with the convulsive dose of kainate also led to a significant increase in the content of alanine of almost 50% compared with control mice while the lactate content was unaltered. A reduction of ∼20% was observed in the contents of glutamate, glutamine, GABA, and aspartate in mice treated with the convulsive dose of kainate compared with control mice (Figure 1), indicating that catabolism of amino acids was necessary to sustain the cellular energy demand.

Figure 1.

Figure 1

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Metabolite content in the cerebral cortex of control mice (black bars), mice treated with a subconvulsive dose of kainate (3.75 mg/kg; gray bars) and others treated with a convulsive dose of kainate (15 mg/kg; white bars). The amounts of the metabolites were determined by 1H-NMR spectroscopy and data are normalized letting the amount of each metabolite in control mice represent 100%. The numbers represent the absolute amount (μmol/g tissue) of each metabolite in control mice. Values are averages±s.e.m. (n=3–8) and the asterisk indicates a statistically significant difference between controls and kainate-treated animals (P<0.05). The number sign indicates statistically significant difference between the mice treated with the subconvulsive and those treated with the convulsive dose of kainate (P<0.05).

13C Labeling in Metabolites from [1,2-13C]Acetate

The astrocyte-specific substrate [1,2-13C]acetate was employed to map astrocytic metabolism. The subconvulsive dose of kainate significantly lowered the amount of [4,5-13C]glutamine labeled from [1,2-13C]acetate by almost 25% (Figure 2). The amounts of [4,5-13C]glutamate and [1,2-13C]GABA were lowered by ∼15% and 30%, respectively, although these apparent decrements were not statistically significant (P=0.261 and 0.132, respectively). Treatment with the convulsive dose of kainate led to a similar reduction in the amount of [4,5-13C]glutamine as observed with the subconvulsive dose. Moreover, when employing the convulsive dose of kainate, the amount of [4,5-13C]glutamate was reduced to a similar extent as that of its precursor [4,5-13C]glutamine. Also, the amount of [1,2-13C]GABA was reduced by almost 30%, although this was not statistically different from that observed in control animals. The extent of astrocytic TCA cycle metabolism was calculated based on the different 13C labeling patterns in glutamine obtained after successive turns of TCA cycle metabolism of [1,2-13C]acetate. The subconvulsive dose of kainate significantly increased astrocytic TCA cycle activity by almost 20% whereas the convulsive dose lowered the TCA cycle activity by ∼20% (Figure 3A).

Figure 2.

Figure 2

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Metabolism of [1,2-13C]acetate in the cerebral cortex of control mice (black bars), mice treated with a subconvulsive dose of kainate (3.75 mg/kg; gray bars) and mice treated with a convulsive dose of kainate (15 mg/kg; white bars). The amounts of [4,5-13C]glutamate, [4,5-13C]glutamine, and [1,2-13C]GABA in the extracts of cerebral cortex were determined by 13C-NMR spectroscopy and data are normalized letting the amount of each isotopomer in control mice represent 100%. The numbers represent the amount of 13C labeling in each amino acid in control expressed as nmol/g tissue. Values are averages±s.e.m. (n=3–8) and the asterisk indicates a statistically significant difference between controls and kainate-treated animals (P<0.05). Glu, glutamate; Gln, glutamine.

Figure 3.

Figure 3

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The calculated TCA cycle activity in control mice (black bars), mice treated with a subconvulsive dose of kainate (3.75 mg/kg; gray bars) and others treated with a convulsive dose of kainate (15 mg/kg; white bars). (A) The relative TCA cycle activity in astrocytes calculated based on the isotopomers of glutamine originating from successive turns of TCA cycle metabolism of [1,2-13C]acetate using equation (1), i.e., ([1,2-13C]glutamine+[3-13C]glutamine)/[4,5-13C]glutamine. (B) The relative TCA cycle activity in neurons calculated based on the isotopomers of glutamate originating from successive turns of TCA cycle metabolism of 13C originating from [1-13C]glucose using equation (2), i.e., (2*([3-13C]glutamate–[1,2-13C]glutamate))/[4-13C]glutamate. Values are averages±s.e.m. (n=3–8) and the asterisk indicates a statistically significant difference between controls and kainate treated animals (P<0.05). The number sign indicates statistically significant difference between the mice treated with the subconvulsive and those treated with the convulsive dose of kainate (P<0.05).

13C Labeling in Metabolites Labeled from [1-13C]Glucose

[1-13C]Glucose is taken up into both neurons and astrocytes and accordingly glucose metabolism takes place in both compartments.24 Hence, mice were injected with [1-13C]glucose to investigate glycolytic activity as well as TCA cycle metabolism in astrocytes and neurons. As the injected [1-13C]glucose is dispersed in a pool of endogenous glucose, and as the amount of this may vary with kainate treatment that occurs 15 minutes before the injection of 13C substrates, the amount and the percentage 13C enrichment in cerebral glucose was determined. Neither the amount of glucose nor the percentage of glucose being 13C labeled was altered by kainate injection (results not shown), and accordingly, the amount of 13C labeling in metabolites can be compared directly without correcting for possible differences in the labeling of the precursor. In mice treated with the subconvulsive dose of kainate, the incorporation of 13C labeling from [1-13C]glucose into all measured metabolites was similar to that of controls (Figure 4). In contrast, treatment with the convulsive dose of kainate led to a reduction of ∼50% in the amounts of [4-13C]glutamate, [4-13C]glutamine, [2-13C]GABA, [2-13C]aspartate, [3-13C]aspartate, [3-13C]alanine, and [3-13C]lactate compared with control mice, indicating that neuronal metabolism was altered. The neuronal TCA cycle activity calculated based on the differential 13C labeling in glutamate after metabolism of 13C originating from [1-13C]glucose in successive turns of the TCA cycle showed that the subconvulsive dose of kainate did not alter neuronal TCA cycle activity, while it was decreased by ∼30% in mice treated with the convulsive dose of kainate (Figure 3B).

Figure 4.

Figure 4

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Metabolism of [1-13C]glucose in the cerebral cortex of control mice (black bars), mice treated with a subconvulsive dose of kainate (3.75 mg/kg; gray bars) and others treated with a convulsive dose of kainate (15 mg/kg; white bars). The amounts of [4-13C]glutamate, [4-13C]glutamine, [2-13C]GABA, [2-13C]aspartate, [3-13C]aspartate, [3-13C]lactate and [3-13C]alanine in extracts from cerebral cortex were determined by 13C-NMR spectroscopy and data are normalized letting the amount of each isotopomer in control mice represent 100%. The numbers represent the amount of 13C labeling in each amino acid in control expressed as nmol/g tissue. Values are averages±s.e.m. (n=3–8) and the asterisk indicates a statistically significant difference between controls and kainate-treated animals (P<0.05). The number sign indicates statistically significant difference between the mice treated with the subconvulsive and those treated with the convulsive dose of kainate (P<0.05). Glu, glutamate; Gln, glutamine; Asp, aspartate; Lac, lactate; Ala, alanine.

Glycogen Content

The glycogen content was plotted against the dose of injected kainate (Figure 5), and this gave rise to a straight line (R2=1.000) with a slope that is statistically different from zero (P=0.0388). Hence, the glycogen content, which is exclusively located in astrocytes, appears to be concentration dependently reduced by kainate, indicating that glycogen is involved in the maintenance of cerebral energy and/or amino-acid homeostasis during seizure activity.

Figure 5.

Figure 5

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Glycogen content in the cerebral cortex of control mice, mice treated with a subconvulsive dose of kainate (3.75 mg/kg) and others treated with a convulsive dose of kainate (15 mg/kg). Glycogen was determined using a coupled enzymatic assay (see Materials and Methods for details). Values are averages±s.e.m. (n=3–8). The glycogen content was dose-dependently reduced and plotting glycogen content as a function of the dose of kainate gave rise to a straight line (r2=1.000) with a slope significantly different from zero (P=0.0388).

Discussion

Investigations of the pathophysiological mechanisms underlying seizure activity are complicated owing to the intricate network of different cell types and the interdependent substrate transfer among these. Moreover, the broad constellation of structural and functional changes in the brain of epileptic patients and animal models of epilepsy obscures the initial cause of malfunction.9, 12 Accordingly, the origin of the excessive neuronal activity related to seizure activity remains a matter of uncertainty. In this study, we demonstrate that astrocytic metabolism is affected by a subconvulsive dose of kainate while neuronal metabolism is not. This is evident from the lower amount of [4,5-13C]glutamine labeled from the astrocyte-specific substrate [1,2-13C]acetate and the increased TCA cycle activity in mice treated with the subconvulsive dose of kainate compared with control animals. In contrast, incorporation of 13C from [1-13C]glucose into glutamate, aspartate and GABA, reflecting neuronal metabolism, was unaltered by the subconvulsive dose of kainate. Thus, although kainate is widely used as a pharmacological tool to induce neuronal depolarization and seizure activity, it appears to exert a significant effect on astrocyte metabolism even at a dose unable to affect neuronal metabolism.

Although only a small fraction of systemically injected kainate reaches the brain,3 even the subconvulsive dose is able to alter astrocytic metabolism. In addition to the decrease in the amount of [4,5-13C]glutamine, it appears that anaerobic glycolytic activity is augmented after treatment with the subconvulsive dose of kainate as observed by the 50% increase in the cortical content of lactate. As neuronal metabolism was not affected by this dose of kainate, the increased glycolytic metabolism is most likely an astrocytic event. Such increase in anaerobic glycolysis may be a consequence of lower oxygen availability in line with a previous study demonstrating that kainate reduced heart rate and blood flow in rats.25 In that study, both heart rate and blood flow returned to initial values 20 minutes after injection of kainate,25 i.e., the period of restrained oxygen availability appears to be relatively brief. This may be in line with our findings, as the extensive lactate production appears to take place before injection of [1-13C]glucose as observed by the unaltered production of [3-13C]lactate as response to the subconvulsive dose of kainate, indicating that glycolytic activity was normalized in the time period when the 13C-labeled substrates were available for metabolism. However, in the period subsequent to that of enhanced anaerobic glycolysis, astrocytic metabolism continues to be altered by the subconvulsive dose of kainate. This is seen from the lower amount of [4,5-13C]glutamine generated from [1,2-13C]acetate in combination with an augmented TCA cycle activity in astrocytes of ∼20%. It should be noted that the lactate produced during the initial phase of anaerobic glycolysis may originate not only from glucose but also from glycogen, as the glycogen stores are mobilized during kainate treatment. The lactate produced in the initial phase provides a large pool of an unlabeled substrate that can dilute the 13C labeling in the astrocytic acetyl CoA pool. Thus, although TCA cycle activity is increased by the subconvulsive dose of kainate a lower amount of [4,5-13C]glutamine may be generated owing to the concomitant metabolism of unlabeled lactate. In agreement with this, it has previously been proposed that astrocytes consume at least as much lactate as neurons if not more.26, 27 Despite the increase in cortical lactate content in mice injected with the subconvulsive dose of kainate, the neuronal lactate consumption seems to be unaltered, as the 13C labeling of the pyruvate pool in neurons does not appear to be diluted by unlabeled lactate. This is observed from the sustained incorporation of 13C from [1-13C]glucose into glutamate, aspartate and GABA, reflecting neuronal metabolism. Hence, it appears that the relative extent of anaerobic and aerobic metabolism in astrocytes, but not that of neurons, is altered by the subconvulsive dose of kainate. In the initial phase, there is an increase in anaerobic glycolytic activity leading to lactate production followed by a period of enhanced oxidative metabolism in astrocytes.

The fact that astrocytic metabolism appears to be distinctively affected by the subconvulsive dose of kainate indicates that the effect is mediated directly by receptors in the astrocytic membrane. In keeping with this, it has repeatedly been shown that astrocytes express a selection of glutamate receptors including kainate and AMPA receptors both of which are sensitive to kainate.10, 28, 29 This was supported by several studies carried out in cultured astrocytes demonstrating alterations in cellular ion homeostasis after exposure to kainate including complex changes in intracellular concentrations of Na+ and H+ (see ref. 30) and increases in the extracellular K+ concentration likely due to increased astrocytic K+ efflux.31 As K+ is cleared from the extracellular space predominantly by ATP-dependent exchangers in the astrocytic membrane, K+ homeostasis relies on proper energy metabolism in astrocytes.32 The depolarizing nature of an elevated extracellular K+ concentration links increased neuronal activity to disruptions in K+ homeostasis suggesting that impairments in astrocytic metabolism may precede the neuronal hyperactivity related to seizures. It should be noted that increased neuronal activity or EEG seizures that did not lead to visible seizure activity could be present in the brains of the subconvulsive mouse model.

In contrast to the subconvulsive dose of kainate that eventually led to enhanced astrocytic TCA cycle activity, treatment with the convulsive dose lowered the calculated TCA cycle activity in astrocytes and neurons by 20% and 30%, respectively. This, in combination with a 50% reduction in the amounts of metabolites being 13C labeled from [1-13C]glucose metabolized via glycolysis and the TCA cycle (i.e., [4-13C]glutamate, [4-13C]glutamine, [2-13C]GABA, [2-13C]- and [3-13C]aspartate) suggests that treatment with the convulsive dose of kainate leads to hypometabolism of glucose affecting both astrocytes and neurons. When employing this dose, reduced oxidative metabolism of glucose is not associated with enhanced anaerobic glycolysis, as observed using the subconvulsive dose, as the lactate content was not increased. Actually, hypometabolism appeared to include glycolytic activity as [3-13C]lactate was also reduced by 50% in these mice.

We have previously suggested that kainate-induced hypometabolism is mediated by an increase in the action of GABA, specifically on extrasynaptic GABA receptors, i.e., by augmenting tonic inhibition.23 This was based on the observation that kainate did not lead to hypometabolism in a GAD65 knockout mouse model exhibiting impairments in tonic inhibition.23, 33 As alterations in tonic inhibition are expected to rely on changes in neuronal GABA release, a convulsive dose of kainate, which affects both neurons and astrocytes, is required to mediate an augmented tonic inhibition and the associated hypometabolism. Moreover, the effect of tonic inhibition appears to be dramatic, as it is able to override the substantial metabolic changes observed in astrocytes induced by the subconvulsive dose. Hence, our results demonstrate a broad spectrum of metabolic effects mediated by kainate, and these effects are different depending on the cell type(s) being affected, which is again dose dependent. This is in line with previous findings demonstrating a biphasic effect of kainate on GABA release, i.e., lower concentrations of kainate enhances GABA release while higher concentrations of kainate attenuate GABA release (reviewed in Lerma and Marques5). The convulsive dose used in our study appears to be within the concentration range, which enhances GABA release reflected in the immobility of the animal in this seizure stage and the increased tonic inhibition leading to hypometabolism of glucose. Hence, it appears that although the amount of [4,5-13C]glutamine is lowered to a similar extent after treatment with the two different doses of kainate, the metabolic alterations leading to this reduction seem to be distinct, i.e., relying on dilution of the 13C labeling in the acetyl CoA pool in combination with increased TCA cycle metabolism and hypometabolism mediated by increments in tonic inhibition for the subconvulsive and convulsive dose, respectively.

While the convulsive dose of kainate leads to hypometabolism of glucose, it appears that other substrates are metabolized to maintain energy homeostasis. This is observed from the significant reduction in the cortical contents of glutamate, glutamine, GABA, and aspartate of almost 20% implying increased catabolism of amino acids in combination with attenuated amino-acid synthesis (hypometabolism) as discussed above. Utilization of aspartate and GABA for energy production is dependent upon conversion to TCA cycle intermediates by the action of aminotransferase activity concomitantly forming glutamate from α-ketoglutarate. Also, glutamine may in a reaction catalyzed by phosphate-activated glutaminase be converted to glutamate. In order to gain a net formation of TCA cycle intermediates from the amino acids, a subsequent oxidative deamination of glutamate to α-ketoglutarate by glutamate dehydrogenase is necessary. In line with this, an increased activity of glutamate dehydrogenase has been reported in the epileptogenic (sclerotic) hippocampus.34 Alternative to glutamate dehydrogenase, alanine aminotransferase catalyzing the transfer of the amino group from glutamate to pyruvate to generate alanine and α-ketoglutarate, may be increased for short-term anaplerosis of TCA cycle intermediates. This enzyme seems to account for at least some of the conversion of glutamate to α-ketoglutarate, as the amount of alanine was increased by almost 50% after treatment with the convulsive dose of kainate compared with controls. As this increase in alanine is not reflected in the 13C labeling of alanine, it is indicated that alanine aminotransferase activity occurs before injection of labeling. Amino-acid degradation likely precedes the enhanced alanine aminotransferase activity and therefore, the period of amino-acid degradation occurs before injection of 13C-labeled substrates.

The reduction in the contents of GABA, glutamate, and aspartate, which are present predominantly in neurons, points to alterations in neuronal energy metabolism upon kainate treatment. However, the metabolic effect of kainate also involves the astrocytic compartment, as the cerebral glycogen content, which is localized predominantly in astrocytes, is dose-dependently reduced by kainate. This suggests that glycogen contributes to maintenance of energy homeostasis during seizure activity. Such role of glycogen has previously been suggested based on measurements of glycogen content in the brain tissue from humans with epilepsy.35 Also, it was demonstrated that glycogen is involved in limiting the rate of propagation of spreading depression in brain slices.36 Glycogen was suggested to be essential for maintenance of the homeostasis of extracellular depolarizing agents such as glutamate and K+ as previously demonstrated.37, 38 Similarly, glycogen may be involved in the regulation and possibly restriction of neuronal activity when mice are injected with kainate that also involves an elevated extracellular K+ concentration. It should be noted, however, that although glycogen is confined to astrocytes,39 it is also able to sustain energy homeostasis in neurons via conversion to lactate and transfer to the neuronal compartment for reconversion to pyruvate entering the TCA cycle for energy production. In addition, it has been demonstrated that glycogen is an important precursor for de novo synthesis of glutamate and glutamine,40 i.e., for maintenance of cerebral amino-acid homeostasis. Accordingly, glycogen may be important as an energy substrate and/or as a precursor for amino-acid biosynthesis during seizure activity.

Conclusion

Altogether, this study provides evidence for astrocytic metabolism being compromised by a subconvulsive dose of kainate while a higher dose of kainate was required to affect neuronal metabolism in mice. The subconvulsive dose of kainate leads to a biphasic metabolic response in astrocytes comprising an initial phase of extensive anaerobic glycolysis leading to lactate production and a subsequent phase of enhanced TCA cycle metabolism. The convulsive dose of kainate gave rise to mild convulsions and led to general cerebral hypometabolism of glucose likely mediated by an increased tonic inhibition. Hypometabolism of glucose is accompanied by degradation of amino acids and mobilization of glycosyl units from glycogen, which serve to support energy homeostasis when glucose metabolism is attenuated. Altogether these results suggest that kainate alters cerebral metabolism by a multifaceted action, which is dose dependent.

The authors declare no conflict of interest.

Footnotes

This study was supported by grants from Manufacturer Vilhelm Pedersen and Wife Memorial Legacy, a support granted upon recommendation from the Novo Nordisk Foundation, as well as the Danish Medical Research Council (grant no. 10-094362/0602-01660B) and the Novo Nordisk, the Hørslev, and the Lundbeck Foundations.

References

  1. Madsen KK, Clausen RP, Larsson OM, Krogsgaard-Larsen P, Schousboe A, White HS. Synaptic and extrasynaptic GABA transporters as targets for anti-epileptic drugs. J Neurochem. 2009;109 (Suppl 1:139–144. doi: 10.1111/j.1471-4159.2009.05982.x. [DOI] [PubMed] [Google Scholar]
  2. Semah F, Picot MC, Adam C, Broglin D, Arzimanoglou A, Bazin B, et al. Is the underlying cause of epilepsy a major prognostic factor for recurrence. Neurology. 1998;51:1256–1262. doi: 10.1212/wnl.51.5.1256. [DOI] [PubMed] [Google Scholar]
  3. Sperk G. Kainic acid seizures in the rat. Prog Neurobiol. 1994;42:1–32. doi: 10.1016/0301-0082(94)90019-1. [DOI] [PubMed] [Google Scholar]
  4. Ben-Ari Y. Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy. Neuroscience. 1985;14:375–403. doi: 10.1016/0306-4522(85)90299-4. [DOI] [PubMed] [Google Scholar]
  5. Lerma J, Marques JM. Kainate receptors in health and disease. Neuron. 2013;80:292–311. doi: 10.1016/j.neuron.2013.09.045. [DOI] [PubMed] [Google Scholar]
  6. Mathern GW, Babb TL, Vickrey BG, Melendez M, Pretorius JK. The clinical-pathogenic mechanisms of hippocampal neuron loss and surgical outcomes in temporal lobe epilepsy. Brain. 1995;118 (Pt 1:105–118. doi: 10.1093/brain/118.1.105. [DOI] [PubMed] [Google Scholar]
  7. Qu H, Eloqayli H, Muller B, Aasly J, Sonnewald U. Glial-neuronal interactions following kainate injection in rats. Neurochem Int. 2003;42:101–106. doi: 10.1016/s0197-0186(02)00051-7. [DOI] [PubMed] [Google Scholar]
  8. Alvestad S, Hammer J, Eyjolfsson E, Qu H, Ottersen OP, Sonnewald U. Limbic structures show altered glial-neuronal metabolism in the chronic phase of kainate induced epilepsy. Neurochem Res. 2008;33:257–266. doi: 10.1007/s11064-007-9435-5. [DOI] [PubMed] [Google Scholar]
  9. Alvestad S, Hammer J, Qu H, Haberg A, Ottersen OP, Sonnewald U. Reduced astrocytic contribution to the turnover of glutamate, glutamine, and GABA characterizes the latent phase in the kainate model of temporal lobe epilepsy. J Cereb Blood Flow Metab. 2011;31:1675–1686. doi: 10.1038/jcbfm.2011.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Parpura V, Heneka MT, Montana V, Oliet SH, Schousboe A, Haydon PG, et al. Glial cells in (patho)physiology. J Neurochem. 2012;121:4–27. doi: 10.1111/j.1471-4159.2012.07664.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Waagepetersen HS, Sonnewald U, Schousboe A.Energy and amino acid neurotransmitter metabolism in astrocytesIn: Parpura V, Haydon PG, (eds). Astrocytes in (Patho)Physiological of the Nervous System Springer: New York; 2009177–200. [Google Scholar]
  12. Eid T, Williamson A, Lee TS, Petroff OA, de Lanerolle NC. Glutamate and astrocytes -key players in human mesial temporal lobe epilepsy. Epilepsia. 2008;49 (Suppl 2:42–52. doi: 10.1111/j.1528-1167.2008.01492.x. [DOI] [PubMed] [Google Scholar]
  13. Sonnewald U, Westergaard N, Schousboe A, Svendsen JS, Unsgard G, Petersen SB. Direct demonstration by [13C]NMR spectroscopy that glutamine from astrocytes is a precursor for GABA synthesis in neurons. Neurochem Int. 1993;22:19–29. doi: 10.1016/0197-0186(93)90064-c. [DOI] [PubMed] [Google Scholar]
  14. Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr. Clin Neurophysiol. 1972;32:281–294. doi: 10.1016/0013-4694(72)90177-0. [DOI] [PubMed] [Google Scholar]
  15. Mason GF, Rothman DL, Behar KL, Shulman RG. NMR determination of the TCA cycle rate and alpha-ketoglutarate/glutamate exchange rate in rat brain. J Cereb Blood Flow Metab. 1992;12:434–447. doi: 10.1038/jcbfm.1992.61. [DOI] [PubMed] [Google Scholar]
  16. Ottersen OP, Zhang N, Walberg F. Metabolic compartmentation of glutamate and glutamine: morphological evidence obtained by quantitative immunocytochemistry in rat cerebellum. Neuroscience. 1992;46:519–534. doi: 10.1016/0306-4522(92)90141-n. [DOI] [PubMed] [Google Scholar]
  17. Walls AB, Eyjolfsson EM, Smeland OB, Nilsen LH, Schousboe I, Schousboe A, et al. Knockout of GAD65 has major impact on synaptic GABA synthesized from astrocyte-derived glutamine. J Cereb Blood Flow Metab. 2011;31:494–503. doi: 10.1038/jcbfm.2010.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schousboe A. Transport and metabolism of glutamate and GABA in neurons are glial cells. Int Rev Neurobiol. 1981;22:1–45. doi: 10.1016/s0074-7742(08)60289-5. [DOI] [PubMed] [Google Scholar]
  19. Schousboe A, Bak LK, Waagepetersen HS. Astrocytic control of biosynthesis and turnover of the neurotransmitters glutamate and GABA. Front Endocrinol. 2013;4:102. doi: 10.3389/fendo.2013.00102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hassel B, Bachelard H, Jones P, Fonnum F, Sonnewald U. Trafficking of amino acids between neurons and glia in vivo. Effects of inhibition of glial metabolism by fluoroacetate. J Cereb Blood Flow Metab. 1997;17:1230–1238. doi: 10.1097/00004647-199711000-00012. [DOI] [PubMed] [Google Scholar]
  21. Martinez-Hernandez A, Bell KP, Norenberg MD. Glutamine synthetase: glial localization in brain. Science. 1977;195:1356–1358. doi: 10.1126/science.14400. [DOI] [PubMed] [Google Scholar]
  22. Walls AB, Sickmann HM, Brown A, Bouman SD, Ransom B, Schousboe A, et al. Characterization of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) as an inhibitor of brain glycogen shunt activity. J Neurochem. 2008;105:1462–1470. doi: 10.1111/j.1471-4159.2008.05250.x. [DOI] [PubMed] [Google Scholar]
  23. Walls AB, Nilsen LH, Eyjolfsson EM, Vestergaard HT, Hansen SL, Schousboe A, et al. GAD65 is essential for synthesis of GABA destined for tonic inhibition regulating epileptiform activity. J Neurochem. 2010;115:1398–1408. doi: 10.1111/j.1471-4159.2010.07043.x. [DOI] [PubMed] [Google Scholar]
  24. Qu H, Haberg A, Haraldseth O, Unsgard G, Sonnewald U. (13)C MR spectroscopy study of lactate as substrate for rat brain. Dev Neurosci. 2000;22:429–436. doi: 10.1159/000017472. [DOI] [PubMed] [Google Scholar]
  25. Cremer JE, Seville MP, Cunningham VJ. Tracer 2-deoxyglucose kinetics in brain regions of rats given kainic acid. J Cereb Blood Flow Metab. 1988;8:244–253. doi: 10.1038/jcbfm.1988.55. [DOI] [PubMed] [Google Scholar]
  26. Zielke HR, Zielke CL, Baab PJ, Tildon JT. Effect of fluorocitrate on cerebral oxidation of lactate and glucose in freely moving rats. J Neurochem. 2007;101:9–16. doi: 10.1111/j.1471-4159.2006.04335.x. [DOI] [PubMed] [Google Scholar]
  27. Gandhi GK, Cruz NF, Ball KK, Dienel GA. Astrocytes are poised for lactate trafficking and release from activated brain and for supply of glucose to neurons. J Neurochem. 2009;111:522–536. doi: 10.1111/j.1471-4159.2009.06333.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Bowman CL, Kimelberg HK. Excitatory amino acids directly depolarize rat brain astrocytes in primary culture. Nature. 1984;311:656–659. doi: 10.1038/311656a0. [DOI] [PubMed] [Google Scholar]
  29. Kettenmann H, Backus KH, Schachner M. Aspartate, glutamate and gamma-aminobutyric acid depolarize cultured astrocytes. Neurosci Lett. 1984;52:25–29. doi: 10.1016/0304-3940(84)90345-8. [DOI] [PubMed] [Google Scholar]
  30. Rose CR, Ransom BR. Mechanisms of H+ and Na+ changes induced by glutamate, kainate, and D-aspartate in rat hippocampal astrocytes. J Neurosci. 1996;16:5393–5404. doi: 10.1523/JNEUROSCI.16-17-05393.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. MacVicar BA, Baker K, Crichton SA. Kainic acid evokes a potassium efflux from astrocytes. Neuroscience. 1988;25:721–725. doi: 10.1016/0306-4522(88)90272-2. [DOI] [PubMed] [Google Scholar]
  32. Hertz L, Peng L, Dienel GA. Energy metabolism in astrocytes: high rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis. J Cereb Blood Flow Metab. 2007;27:219–249. doi: 10.1038/sj.jcbfm.9600343. [DOI] [PubMed] [Google Scholar]
  33. Kubo K, Nishikawa K, Ishizeki J, Hardy-Yamada M, Yanagawa Y, Saito S. Thermal hyperalgesia via supraspinal mechanisms in mice lacking glutamate decarboxylase 65. J Pharmacol Exp Ther. 2009;331:162–169. doi: 10.1124/jpet.109.156034. [DOI] [PubMed] [Google Scholar]
  34. Malthankar-Phatak GH, de Lanerolle N, Eid T, Spencer DD, Behar KL, Spencer SS, et al. Differential glutamate dehydrogenase (GDH) activity profile in patients with temporal lobe epilepsy. Epilepsia. 2006;47:1292–1299. doi: 10.1111/j.1528-1167.2006.00543.x. [DOI] [PubMed] [Google Scholar]
  35. Dalsgaard MK, Madsen FF, Secher NH, Laursen H, Quistorff B. High glycogen levels in the hippocampus of patients with epilepsy. J Cereb Blood Flow Metab. 2007;27:1137–1141. doi: 10.1038/sj.jcbfm.9600426. [DOI] [PubMed] [Google Scholar]
  36. Seidel JL, Shuttleworth CW. Contribution of astrocyte glycogen stores to progression of spreading depression and related events in hippocampal slices. Neuroscience. 2011;192:295–303. doi: 10.1016/j.neuroscience.2011.05.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sickmann HM, Walls AB, Schousboe A, Bouman SD, Waagepetersen HS. Functional significance of brain glycogen in sustaining glutamatergic neurotransmission. J Neurochem. 2009;109 (Suppl 1:80–86. doi: 10.1111/j.1471-4159.2009.05915.x. [DOI] [PubMed] [Google Scholar]
  38. Xu J, Song D, Xue Z, Gu L, Hertz L, Peng L. Requirement of glycogenolysis for uptake of increased extracellular K+ in astrocytes: potential implications for K+ homeostasis and glycogen usage in brain. Neurochem Res. 2013;38:472–485. doi: 10.1007/s11064-012-0938-3. [DOI] [PubMed] [Google Scholar]
  39. Cataldo AM, Broadwell RD. Cytochemical staining of the endoplasmic reticulum and glycogen in mouse anterior pituitary cells. J Histochem Cytochem. 1984;32:1285–1294. doi: 10.1177/32.12.6094657. [DOI] [PubMed] [Google Scholar]
  40. Gibbs ME, Lloyd HG, Santa T, Hertz L. Glycogen is a preferred glutamate precursor during learning in 1-day-old chick: biochemical and behavioral evidence. J Neurosci Res. 2007;15:3326–3333. doi: 10.1002/jnr.21307. [DOI] [PubMed] [Google Scholar]

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