Ognen A. C. Petroff MD, FAAN, FANA, FACNS
Associate Professor of Neurology
Epilepsy & seizure disorders; Cerebral metabolism; Brain glutamate, GABA & glutamine metabolism; Antiepileptic drug actions & brain metabolism; CNS physiology & neurochemistry; Neuroprotection
Epilepsy is a common neurological disorder affecting two to three million Americans. We have focused our research on brain glutamate, glutamine and GABA metabolism in epilepsy. Glutamate is the primary excitatory and GABA the primary inhibitory neurotransmitters. Alterations in both glutamatergic and GABAergic systems have been strongly implicated as the final common pathway in many epileptic syndromes. During neurotransmission, glutamate and GABA are released by the neurons into the synaptic cleft. Once taken up by the astroglia, glutamate and GABA are converted to glutamine. Astrocytes supply glutamine to neuron that convert glutamine to glutamate in glutamatergic neurons and GABA in GABAergic neurons. By using 13C-labeled precursors the rates of glutamate and GABA neurotransmission and astrocyte uptake and conversion to glutamine (recycling) may be measured through analysis of the time courses of glutamate, glutamine, and GABA isotopic labeling.
Extensive Research Description
The significance of understanding
the relationship between GABA levels and cortical excitability.
cortical excitability is a hallmark of several human epileptic syndromes,
facilitating the spread of the seizure in the localization related epilepsies
and as a final common pathway for some of the primary generalized and myoclonic
epilepsies. Photosensitive epilepsy is the most common of the
stimulus-triggered (reflex) epilepsies, and there is considerable evidence,
developed primarily in animal models, that it is associated with impairments in
GABAergic inhibition, possibly secondary to alterations in GABA metabolism.
GABA is the major inhibitory neurotransmitter and has a crucial role in
regulating cortical excitability. The concentration of GABA in both the
vesicular and cytosolic pools may have an important influence on GABAergic
inhibition. Our development of non-invasive magnetic resonance spectroscopy
(MRS) based measurements of cellular GABA in human cerebral cortex allowed us
to study GABA metabolism in human epilepsy. Our studies found profound
decreases in the level of cortical GABA in many patients with complex partial
seizures (CPS) and juvenile myoclonic epilepsy (JME). Our preliminary studies
suggest that GABA synthesis rates are decreased in patients with low GABA in
functional occipital cortex remote from the ictal onset zone. We found that, light-deprivation,
a condition that enhances excitability in the visual cortex, decreases GABA
levels in non-epileptic subjects in parallel with increased excitability measured
with paired-pulse stimulation using transcranial magnetic stimulation. These
results support an important role for cortical GABA level on cortical
excitability thereby facilitating the spread of seizure discharges from the
ictal onset zone to involve more normal functional brain. The primary goal is
to better understand the mechanisms by which GABA levels influence cortical
excitability, and the role of altered GABA levels in the etiology and
pathogenesis of epilepsy. Our primary general hypothesis is that reduced
cellular GABA levels result in an increase in cortical excitability through
decreased GABA release.
The significance of the relationship between GABA metabolism and cortical excitability. There is a renewed awareness of the interaction between metabolism and excitability as the relationships between different aspects of neuronal-glial neurotransmitter cycling are established. In order to fully understand the physiologic consequences on GABAergic inhibition of altering neurotransmitter cycling, our approach was to start at the final step and work backwards. We used a variety of physiologic and analytic methods to examine the consequences of glutamic acid decarboxylase (GAD) inhibition on network excitability in the rat hippocampus. We used field and intracellular recordings from the CA1 region of rat hippocampal slices to determine the physiological effects of blocking GABA synthesis with the convulsant, 3-mercaptoproprionic acid (MPA). We measured the rate of synthesis of GABA and glutamate in slices using 2-13C-glucose as a label source and liquid chromatography-tandem mass spectrometry. The primary goal was to establish whether GAD inhibition alone was sufficient to induce network hyperexcitability in an isolated preparation comparable to that seen with GABA receptor inhibition. The key findings were that inhibition of GABA synthesis via GAD only produces hyperexcitability following repetitive stimulation, demonstrating a pronounced use dependence when studied both intra and extracellularly. This is consistent with the data showing that the regulation of IPSC amplitudes and of GABA release can be regulated by glutamine availability in use-dependent fashion. In addition, our data with NO-711, which blocks neuronal GABA uptake, indicated that under baseline conditions, neuronal GABA uptake does not provide a significant degree of GABA that is available for release under resting conditions, but does once frank hyperexcitability has been produced by repetitive stimulation. Despite this clear disinhibition, we were unable to observe seizure-like activity comparable to that seen with GABAA receptor blockade until basal excitability was increased with elevated extracellular potassium concentrations. Finally, our data demonstrated that there is no significant change in the size of the GABA pool, even under conditions where synchronized activity is present. However, as expected, GAD inhibition with MPA significantly decreased the amount of newly formed GABA. These biochemical data demonstrated that full blown network excitability is seen only with a combination of >70% decrease in newly synthesized GABA with a concomitant moderate elevation in neuronal excitability. Thus, these data are comparable to those in circuit modeling studies showing that GABAergic inhibition is highly context-dependent.
Understanding the role of homocarnosine in modulating human cerebral excitability. One aspect of GABA biochemistry that has not received significant attention is the role of GABA-containing compounds. One such compound is homocarnosine, a dipeptide formed from histidine and GABA. The role of homocarnosine in the human brain is poorly understood. Homocarnosine has been proposed as an inhibitory neuromodulator, which is hydrolyzed into GABA and histidine in the extracellular fluid (ECF), thereby increasing GABAergic activity. Consistent with this proposal, studies of refractory human epilepsy using vigabatrin show that CSF homocarnosine concentrations are significantly higher in patients whose seizure control improved than in those who failed to benefit. Free GABA concentrations were the same. Our own observational studies of human epilepsy suggest that increased cortical levels of intracellular homocarnosine appear to be associated with decreased cortical excitability. Low intracellular homocarnosine and GABA levels, measured in the occipital lobe using magnetic resonance spectroscopy (MRS), are associated with frequent complex partial seizures in patients treated with valproate or lamotrigine. Patients with juvenile myoclonic epilepsy with excellent seizure control and treated with the same drugs usually have high homocarnosine levels, but very low intracellular GABA levels. What is unknown is whether higher levels of intracellular homocarnosine are a characteristic of patients with primary epilepsies, perhaps compensating for the low intracellular GABA levels and contributing to better seizure control by decreasing cortical excitability.
Topiramate, gabapentin and levetiracetam are three new drugs that increase cortical homocarnosine concentrations. The mechanisms through which these drugs increase human homocarnosine levels are unknown. Unlike vigabatrin, none of these three drugs alter intracellular GABA concentrations in rodent models Topiramate and gabapentin increase human cortical GABA levels within two hours of the first dose and homocarnosine levels rise after one day (with topiramate) to one week (with gabapentin) of daily use. Levetiracetam was studied only after two weeks of treatment. Our studies using three antiepileptic drugs show that patients with refractory complex partial seizures with better seizure control had higher homocarnosine levels than those with poor seizure control. Cortical intracellular GABA levels were the same in patients, who responded, compared with those, who failed to benefit. The findings suggest, but do not prove, that homocarnosine may decrease cortical excitability. The alternative explanation would be frequent seizures either decreases the synthesis of homocarnosine or enhances its catabolism. Taken as a whole these findings suggest, but do not prove, that homocarnosine may decrease cortical excitability.
Homocarnosine appears to directly decrease neuronal excitability of the human hippocampus. Mechanistic studies have been carried out in whole animal and cell culture models, which may not apply to humans because of the very low endogenous levels of homocarnosine present in non-primate models. However, under conditions when GABA is elevated, abundant homocarnosine is found in the rodent brain and CSF, demonstrating that it can be synthesized de novo in the rodent. Moreover, biochemical studies showed that homocarnosine blocks GABA uptake into synaptosomes, suggesting that homocarnosine has the potential to be an endogenous modulator of GABAergic function. Our preliminary data using hippocampal slices incubated in ACSF indicate that homocarnosine decreases neuronal excitability in both rat and human hippocampus. We studied the effects of bath-applied homocarnosine using electrophysiological recording techniques in the CA1 region of rat hippocampal slices. We found that both homocarnosine and GABA shifted the input-output relationship for evoked synaptic responses rightward and thus was inhibitory. GABA and homocarnosine did not have additive effects, suggesting a common mechanism of action. However, in intracellular recordings, homocarnosine had no significant effects on the membrane potential and slightly decreased the input resistance of the cells. Therefore, the dipeptide is not a direct GABAA agonist.
An excess of extracellular glutamate in the sclerotic hippocampus may be one of the key molecular causes of seizures and brain damage in human mesial temporal lobe epilepsy. Research by the Yale Epilepsy Clinical Research group revealed that several important characteristics of the epileptogenic human hippocampus include: above normal interictal extracellular glutamate levels and enhanced glutamate release during spontaneous seizures with abnormally slow post-ictal glutamate clearance. Paradoxically, interictal extracellular glutamate concentrations were considerably higher in patients with hippocampal sclerosis (MTLE) than in patients without this pathology (non-MTLE), despite the 60–80% neuronal loss and doubling of glial density in the sclerotic hippocampus. Surprisingly, a considerable (35–40%) loss of glutamine synthetase protein and activity was demonstrated in astrocytes of the epileptogenic hippocampus resected from patients with MTLE. Isotopic tracer (13C-glucose) studies during epilepsy surgery suggested that the accumulation and impaired clearance of glutamate in MTLE is due to a slowing of the glutamate–glutamine cycle metabolism in the sclerotic hippocampus compared with the non-gliotic epileptogenic hippocampus. Based on these data we hypothesize that elevated extracellular glutamate is a consequence of impaired glial function due to both decreased rates of glial uptake and/or metabolism. The isotopic enrichment of microdialysis (extracellular) glutamine was higher for probes with nearly normal glutamate after infusion of labeled substrates, which suggests the rate of glutamine synthesis, thus glutamate detoxification, is lower in those areas of the brain with above normal extracellular glutamate. The data obtained with an infusion of 13C-glucose or 13C-acetate on different days in the same subjects were the same, which increased our confidence in the findings. The variation among multiple probes in the same patient reflected regional variation in glutamine synthesis and extracellular glutamate. Glutamine synthesis is limited to glia; therefore, our data suggested glial dysfunction in regions with above normal extracellular glutamate concentrations.