Epilepsy Res. 2015 Aug;114:32-46. doi: 10.1016/j.eplepsyres.2015.03.009.
Disinhibition reduces extracellular glutamine and elevates extracellular glutamate in rat hippocampus in vivo.
Huntington Medical Research Institutes, 660 South Fair Oaks Avenue, Pasadena, CA 91105, USA. Electronic address: firstname.lastname@example.org.
Disinhibition was induced in the hippocampal CA1/CA3 region of normal adult rats by unilateral perfusion of the GABA(A)R antagonist, 4-[6-imino-3-(4-methoxyphenyl)pyridazin-1-yl] butanoic acid hydrobromide (gabazine), or a GABA(B)R antagonist, p-(3-aminopropyl)-p-diethoxymethyl-phosphinic acid (CGP 35348), through a microdialysis probe. Effects of disinhibition on EEG recordings and the concentrations of extracellular glutamate (GLU(ECF)), the major excitatory neurotransmitter, and of extracellular glutamine (GLN(ECF)), its precursor, were examined bilaterally in freely behaving rats. Unilateral perfusion of 10 μM gabazine in artificial CSF of normal electrolyte composition for 34 min induced epileptiform discharges which represent synchronized glutamatergic population bursts, not only in the gabazine-perfused ipsilateral hippocampus, but also in the aCSF-perfused contralateral hippocampus. The concentration of GLU(ECF) remained unchanged, but the concentration of its precursor, GLN(ECF), decreased to 73 ± 4% (n = 5) of the baseline during frequent epileptiform discharges, not only in the ipsilateral, but also in the contralateral hippocampus, where the change can be attributed to recurrent epileptiform discharges per se, with recovery to 95% of baseline when epileptiform discharges diminished. The blockade of GABA(B)R, by CGP 35348 perfusion in the ipsilateral hippocampus for 30 min, induced bilateral Na(+) spikes in extracellular recording. These can reasonably be attributed to somatic and dendritic action potentials and are indicative of synchronized excitatory activity. This disinhibition induced, in both hippocampi, (a) transient 1.6-2.4-fold elevation of GLU(ECF) which correlated with the number of Na(+) spike cluster events and (b) concomitant reduction of GLN(ECF) to ∼ 70%. Intracellular GLN concentration was measured in the hippocampal CA1/CA3 region sampled by microdialysis in separate groups of rats by snap-freezing the brain after 25 min of gabazine perfusion or 20 min of CGP perfusion when extracellular GLN (GLN(ECF)) was 60-70% of the pre-perfusion level. These intracellular GLN concentrations in the disinhibited hippocampi showed no statistically significant difference from the untreated control. This result strongly suggests that the observed decrease of GLN(ECF) is not due to reduced glutamine synthesis or decrease in the rate of efflux of GLN to ECF. This strengthens the likelihood that reduced GLN(ECF) reflects increased GLN uptake into neurons to sustain enhanced GLU flux during excitatory population bursts in disinhibited hippocampus. The results are consistent with the emerging concept that neuronal uptake of GLN(ECF) plays a major role in sustaining epileptiform activities in the kainate-induced model of temporal-lobe epilepsy. Copyright © 2015 Elsevier B.V.
KEYWORDS: Disinhibition; Epileptiform discharge; Extracellular glutamate; Extracellular glutamine; GABA(A)/GABA(B) receptor antagonists; Rat hippocampus
Epilepsy, which affects 60 million worldwide, is a chronic neurological disorder characterized by spontaneous recurrent seizures. A seizure occurs when a large number of excitatory neurons fire synchronously, resulting in neuronal hyperactivity. The mechanisms underlying neuronal hyperactivity are not fully understood. Two well-supported hypotheses are glutamate excitotoxicity and reduced inhibitory control (disinhibition).
Fig. 1. The excitatory neurotransmitter glutamate (GLU) released from the presynaptic vesicles binds to the receptors of the postsynaptic neuron for neurotransmission. In the normal brain, GLUECF is rapidly taken up primarily into astrocytes. In an epileptic brain, either excessive release of GLUECF due to neuronal hyperactivity or its impaired uptake can cause an accumulation of GLUECF, resulting in excessive stimulation of the receptors and glutamate excitotoxicity.
The major excitatory neurotransmitter in the brain is glutamate (GLU). As shown in Fig. 1, GLU is released from the vesicles of the presynaptic neuron into the extracellular fluid (ECF) and binds to the receptor of the postsynaptic neuron for neurotransmission. In a normal brain, extracellular glutamate (GLUECF) is rapidly cleared from the synaptic fluid by glutamate transporters primarily into astrocytes where it is metabolized to glutamine. In a pathological brain, GLUECF accumulates in the synaptic fluid as a result of either an excessive GLU release due to neuronal hyperactivity or an impaired uptake into astrocytes. This accumulation of GLUECF leads to an overstimulation of glutamate receptors occasionally resulting in neuronal death, a phenomenon known as glutamate excitotoxicity. Glutamate excitotoxicity has been implicated in a number of neurological diseases including epilepsy. The pioneering study by During and Spencer reported an elevation of GLUECF before and during spontaneous seizures in the epileptogenic hippocampus of temporal-lobe epilepsy patients . Subsequently, chronic elevation of GLUECF has been reported in the brain of numerous awake epileptic patients who are resistant to anti-epileptic drugs ( and references cited therein). Thus, a clearer understanding of the underlying mechanisms of glutamate excitotoxicity is an important goal.
In the brain, the CA3 region of the hippocampus shown in Fig. 2 (top) is particularly susceptible to synchronized excitatory population bursts because the region is highly populated with pyramidal glutamatergic neurons with recurrent networks, enabling a chain reaction of excitation. The CA3 glutamatergic neurons innervate the dendrites of the CA1 glutamatergic neurons.
Disinhibition. GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the brain. Normally, excitation of the glutamatergic neurons is under control of GABAergic inhibitory interneurons. As shown in Fig. 2 (center), the axon terminals of GABAergic neurons target and inhibit the somatodendritic region and the axon initial segment of the glutamatergic neurons. Fig. 2 (bottom) shows how GABA, acting on the ionotropic GABAA receptor on glutamatergic neurons, mediates fast inhibitory post-synaptic potentials via Cl– influx, which results in hyperpolarization of the postsynaptic glutamatergic neurons and an increase in the threshold for firing. As a result, glutamatergic neurons are subject to two antagonistic polarizations: dendritic excitation from glutamatergic neurons and somatic/perisomatic inhibition from GABAergic interneurons. When the inhibitory control is weakened or lost (disinhibition), massive depolarization of the target glutamatergic neurons occurs, resulting in epileptiform activity (reviewed by ).
Fig. 2. An image of the rat hippocampus showing CA3, CA1 and dentate gyrus (DG). Glutamatergic neurons are controlled by two antagonistic polarizations: dendritic excitation from glutamatergic neurons and somatic inhibition from GABAergic neurons. When inhibitory control is weakened or lost (disinhibition), the target glutamatergic neurons undergo massive depolarization, resulting in epileptiform activity.
GLNECF decreases in response to neuronal hyperactivity – a novel finding
Fig. 3 shows schematically the major metabolic and transport pathways of glutamate (GLU) and glutamine (GLN) in the brain compartments of the neuron, the astrocyte and the extracellular fluid (ECF). GLUECF is taken up into the astrocyte, primarily by the excitatory amino acid transporter type 2, and is metabolized by the astrocyte-specific enzyme, glutamine synthetase, to GLN. GLN is later transported to the extracellular fluid. GLNECF is taken up into neurons by the sodium-coupled neutral amino acid transporters subtypes 1 and 2. In the neuron, GLN is hydrolyzed by glutaminase (GLNase) to provide the metabolic and neurotransmitter pools of GLU. This completes the glutamate-glutamine cycle.
Our previous studies on the metabolic and pathophysiological bases of glutamate excitotoxicity showed, for the first time, that GLNECF, which upon uptake into neurons, serves as the precursor of the metabolic and neurotransmitter pools of GLU (Fig. 3), is significantly reduced in response to spontaneous recurrent seizures in a kainate-induced rat model of epilepsy . This model closely resembles the EEG, biochemical and morphological abnormalities of the human temporal lobe epilepsy. A subsequent study in our laboratory  showed that the spontaneous recurrent seizures are significantly reduced when neuronal uptake of GLNECF is inhibited by perfusion of 2-(methylamino)isobutyrate (MeAIB), a non-toxic, non-metabolizable and competitive inhibitor of this uptake in vivo (Fig. 3). These novel findings raised an intriguing possibility that neuronal uptake of GLNECF is accelerated during epileptic seizures to replenish the neurotransmitter pool of glutamate. To examine this hypothesis further, the paper featured at this site  investigated the effects of glutamatergic population bursts, induced by disinhibition in normal awake rats, on (a) seizure activity recorded by EEG and (b) the concentrations of GLUECF and GLNECF collected by microdialysis in awake, freely behaving rats. In the first experiment, disinhibition was induced by unilateral intrahippocampal perfusion of the antagonist gabazine, which inhibits GABA binding to the ionotropic GABAA receptor (Fig. 2). This experiment was combined with EEG recordings and the collection of GLNECF by microdialysis in both hippocampi. Although gabazine was perfused in only one hippocampus (ipsilateral) and the contralateral hippocampus was perfused with an artificial cerebrospinal fluid (aCSF) of normal electrolyte composition, epileptic discharges were observed synchronously in both hippocampi (Fig. 1 ) This finding is observed because the CA3 glutamatergic neurons target the dendrites of CA1 glutamatergic cells, not only of the same hippocampus, but also of the opposite hippocampus through the commissural fibers. As a result, the glutamatergic population bursts are transmitted to, and appear as epileptiform discharges in, the opposite hippocampus as well. This finding enables us to examine the effects of epileptiform discharges per se on GLNECF concentration without possible additional effects from the perfused gabazine. As shown in Fig. 2A of , GLNECF decreased significantly, to 73 ± 4% (n = 5) of the pre-infusion concentration, in response to the frequent occurrence of epileptiform discharges that represent synchronized bursts of glutamatergic neuron firing. Additionally, GLNECF was restored to 95% of the basal level when the epileptiform discharges became rare upon washout of the antagonist.
In the second experiment, disinhibition was induced by perfusion of the metabotropic GABAB receptor antagonist, CGP 35348, in the ipsilateral hippocampus as described in . This induced bilateral Na+ spikes (Fig. 3 of ) which can reasonably be attributed to somatic and dendritic action potentials and are indicative of synchronized excitatory activity. This excitatory activity also resulted in a reduction of GLNECF to 68 ± 4% (n = 5) in the aCSF-perfused contralateral hippocampus (Fig. 5B of ).
Intracellular GLN concentration in the hippocampal region sampled by microdialysis was unchanged in the treated rats compared to the control rats in both disinhibition paradigms (Fig. 6 of ). This result suggests that neither GLN synthesis nor the rate of efflux of GLN to ECF (Fig. 3) was altered by disinhibition. Taken together, the results strongly suggest that the observed decrease of GLNECF in vivo reflects enhanced uptake into neurons when its flux is accelerated by glutamatergic population bursts induced by disinhibition. This result is consistent with our previous findings [4, 5] regarding the role of GLNECF in sustaining epileptiform activities in the kainate-induced rat model of temporal lobe epilepsy.
Fig. 3. Major metabolic and transport pathways of glutamate (GLU) and glutamine (GLN) in the neuron, the astrocyte and the extracellular fluid (ECF) according to the concept of glutamate/glutamine cycle. GLNase, glutaminase; MeAIB, 2-(methylamino)isobutyrate (an inhibitor of neuronal uptake of GLNECF in vivo).
Importance of the study
Collectively, our recent studies ( [5, 6]) strongly suggest that neuronal uptake of GLNECF (Fig. 3) can be one of the key regulatory sites at which excitatory and inhibitory neurotransmissions are modulated by fluctuations in the supply of the precursor. In clinical studies, brain microdialysis in epilepsy patients undergoing presurgical intracranial EEG evaluation has provided a unique opportunity to examine GLUECF, GLNECF and GABAECF concentrations in epileptogenic vs non-epileptogenic brain regions ([2, 7] and references cited therein). These examinations were performed when the patients, who were on anti-epileptic drugs, were quietly resting at least 6 hours from any seizure activity [2, 7]. Interestingly, the mean GLNECF concentration was lower in the epileptogenic compared to the non-epileptogenic hippocampus  although the difference was not statistically significant. It is possible that progressive decrease in GLNECF in response to seizures, such as that reported in our studies [4, 5], is undetectable in epileptic patients during seizure-free resting periods. Animal models of temporal-lobe epilepsy, such as the kainate model, permit the examination of the correlation of epileptiform activity with changes in the concentrations of GLUECF and GLNECF during seizure activity in the absence of anti-seizure treatment [4, 5]. Thus animal studies can make a unique contribution to the examination of neurochemical changes associated with seizure activity and thereby to clarification of the mechanisms underlying neuronal hyperactivity. Our hope is that our novel findings will contribute to a better understanding of the role of glutamine in sustaining epileptiform activity in vivo, and open the way to therapeutic interventions for suppressing chronic recurrent seizures.
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Acknowledgements: 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.
Keiko Kanamori, Ph.D.
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