13C NMRS of animal models of schizophrenia  

Altered brain metabolism is implicated in several brain disorders such as schizophrenia. Insights into underlying mechanisms and how they are altered could help find new treatment strategies. Animal models serve as tools to mimic human diseases. However, recreating the “normal” course of human disease in animals is difficult. Nevertheless, animal models have shown to be useful in providing knowledge about pathological processes. In this thesis several animal models of brain disorders were used. We used both pharmacological interventions mimicking one feature of schizophrenia, and a gene knock out model aimed at elucidating the role of GAD65 in disease. GABA metabolism is believed to be altered in several brain disorders. Two different protocols for studying the effect of MK-801 were used to mimic schizophrenia, repeated low dose (0.1 mg/kg) MK-801 and repeated high dose injections (0.5 mg/kg) MK-801 respectively. These models were used to gain knowledge about how altered neurotransmitter homeostasis possibly can lead to psychiatric disease. In paper I, repeated low dose MK-801 injections caused hypermetabolism of glucose and increased glutamatergic activity in the temporal areas only. Thus, it appears that this model does not show the same pattern as seen in patients with schizophrenia but rather mimics the toxic effects of MK-801 possibly caused by increased glutamate release into the synaptic cleft. Repeated injections of high doses of MK-801 (paper II) led to hypometabolism of glucose. It was further shown that perturbation of NMDA receptor function in the model of repeated injections of MK-801 caused changes not only in the glutamatergic and GABAergic systems, but also in that of dopamine. Changes were most pronounced in the frontal cortex (FCX) in analogy with the human condition. In paper II, repeated injection of high doses of MK-801 resulted in increased amounts of glutamate. However, reduced 13C labelling was observed in the same study, which might indicate a transition to reduced glutamate metabolism, and glutamate amounts seen in patients with chronic schizophrenia. In paper III, also using injection of repeated high doses of MK-801, we found similar results as in paper II, with reduced 13C labelling in glutamate and glutamine. Further we found reduced 13C labelling in GABA, lactate and NAA implying neuronal hypometabolism In paper IV studying GAD65 knockout mice, labelling from glucose was dramatically decreased in lactate and alanine reflecting attenuated glycolysis. In concurrence with this TCA cycle activity, was decreased in the GAD65 knockout animals. Consequently, decreased 13C labelling in GABA was observed, implying neuronal hypometabolism. Patients with schizophrenia constitute a heterogeneous group with a large variety of symptoms and it is likely that the underlying causes of psychosis are not always induced by the same mechanisms. Still, it is of great interest that blocking of the NMDA receptor using repeated injections of high doses of MK-801 caused neuronal hypometabolism as found in the GAD65 knockout model. Hypometabolism in FCX is a common finding in patients with schizophrenia

13C NMRS of animal models of schizophrenia

Altered brain metabolism is implicated in several brain disorders such as schizophrenia. Insights into underlying mechanisms and how they are altered could help find new treatment strategies. Animal models serve as tools to mimic human diseases. However, recreating the “normal” course of human disease in animals is difficult. Nevertheless, animal models have shown to be useful in providing knowledge about pathological processes. In this thesis several animal models of brain disorders were used. We used both pharmacological interventions mimicking one feature of schizophrenia, and a gene knock out model aimed at elucidating the role of GAD65 in disease. GABA metabolism is believed to be altered in several brain disorders. Two different protocols for studying the effect of MK-801 were used to mimic schizophrenia, repeated low dose (0.1 mg/kg) MK-801 and repeated high dose injections (0.5 mg/kg) MK-801 respectively. These models were used to gain knowledge about how altered neurotransmitter homeostasis possibly can lead to psychiatric disease. In paper I, repeated low dose MK-801 injections caused hypermetabolism of glucose and increased glutamatergic activity in the temporal areas only. Thus, it appears that this model does not show the same pattern as seen in patients with schizophrenia but rather mimics the toxic effects of MK-801 possibly caused by increased glutamate release into the synaptic cleft. Repeated injections of high doses of MK-801 (paper II) led to hypometabolism of glucose. It was further shown that perturbation of NMDA receptor function in the model of repeated injections of MK-801 caused changes not only in the glutamatergic and GABAergic systems, but also in that of dopamine. Changes were most pronounced in the frontal cortex (FCX) in analogy with the human condition. In paper II, repeated injection of high doses of MK-801 resulted in increased amounts of glutamate. However, reduced 13C labelling was observed in the same study, which might indicate a transition to reduced glutamate metabolism, and glutamate amounts seen in patients with chronic schizophrenia. In paper III, also using injection of repeated high doses of MK-801, we found similar results as in paper II, with reduced 13C labelling in glutamate and glutamine. Further we found reduced 13C labelling in GABA, lactate and NAA implying neuronal hypometabolism In paper IV studying GAD65 knockout mice, labelling from glucose was dramatically decreased in lactate and alanine reflecting attenuated glycolysis. In concurrence with this TCA cycle activity, was decreased in the GAD65 knockout animals. Consequently, decreased 13C labelling in GABA was observed, implying neuronal hypometabolism. Patients with schizophrenia constitute a heterogeneous group with a large variety of symptoms and it is likely that the underlying causes of psychosis are not always induced by the same mechanisms. Still, it is of great interest that blocking of the NMDA receptor using repeated injections of high doses of MK-801 caused neuronal hypometabolism as found in the GAD65 knockout model. Hypometabolism in FCX is a common finding in patients with schizophrenia

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

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-(13)C]acetate and [1-(13)C]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-(13)C]glutamine and an increase in the calculated astrocytic TCA cycle activity. In contrast, the convulsive dose led to decrements in the cortical content and (13)C 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

Altered 13C glucose metabolism in the cortico-striato-thalamo-cortical loop in the MK-801 rat model of schizophrenia

Using a modified MK-801 (dizocilpine) N-methyl--aspartic acid (NMDA) receptor hypofunction model for schizophrenia, we analyzed glycolysis, as well as glutamatergic, GABAergic, and monoaminergic neurotransmitter synthesis and degradation. Rats received an injection of MK-801 daily for 6 days and on day 6, they also received an injection of [1-(13)C]glucose. Extracts of frontal cortex (FCX), parietal and temporal cortex (PTCX), thalamus, striatum, nucleus accumbens (NAc), and hippocampus were analyzed using (13)C nuclear magnetic resonance spectroscopy, high-performance liquid chromatography, and gas chromatography–mass spectrometry. A pronounced reduction in glycolysis was found only in PTCX, in which (13)C labeling of glucose, lactate, and alanine was decreased. (13)C enrichment in lactate, however, was reduced in all areas investigated. The largest reductions in glutamate labeling were detected in FCX and PTCX, whereas in hippocampus, striatum, and Nac, (13)C labeling of glutamate was only slightly but significantly reduced. The thalamus was the only region with unaffected glutamate labeling. γ-Aminobutyric acid (GABA) labeling was reduced in all areas, but most significantly in FCX. Glutamine and aspartate labeling was unchanged. Mitochondrial metabolites were also affected. Fumarate labeling was reduced in FCX and thalamus, whereas malate labeling was reduced in FCX, PTCX, striatum, and NAc. Dopamine turnover was decreased in FCX and thalamus, whereas that of serotonin was unchanged in all regions. In conclusion, neurotransmitter metabolism in the cortico–striato–thalamo–cortical loop is severely impaired in the MK-801 (dizocilpine) NMDA receptor hypofunction animal model for schizophrenia

Knockout of GAD65 has major impact on synaptic GABA synthesized from astrocyte-derived glutamine

γ-Aminobutyric acid (GABA) synthesis from glutamate is catalyzed by glutamate decarboxylase (GAD) of which two isoforms, GAD65 and GAD67, have been identified. The GAD65 has repeatedly been shown to be important during intensified synaptic activity. To specifically elucidate the significance of GAD65 for maintenance of the highly compartmentalized intracellular and intercellular GABA homeostasis, GAD65 knockout and corresponding wild-type mice were injected with [1-13C]glucose and the astrocyte-specific substrate [1,2-13C]acetate. Synthesis of GABA from glutamine in the GABAergic synapses was further investigated in GAD65 knockout and wild-type mice using [1,2-13C]acetate and in some cases γ-vinylGABA (GVG, Vigabatrin), an inhibitor of GABA degradation. A detailed metabolic mapping was obtained by nuclear magnetic resonance (NMR) spectroscopic analysis of tissue extracts of cerebral cortex and hippocampus. The GABA content in both brain regions was reduced by ∼20%. Moreover, it was revealed that GAD65 is crucial for maintenance of biosynthesis of synaptic GABA particularly by direct synthesis from astrocytic glutamine via glutamate. The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism. Furthermore, a severe neuronal hypometabolism, involving glycolysis and tricarboxylic acid (TCA) cycle activity, was observed in cerebral cortex of GAD65 knockout mice

Loss or mislocalization of aquaporin-4 affects diffusion properties and intermediary metabolism in gray matter of mice

The first aim of this study was to determine how complete or perivascular loss of aquaporin-4 (AQP4) water channels affects membrane permeability for water in the mouse brain grey matter in the steady state. Time-dependent diffusion magnetic resonance imaging was performed on global Aqp4 knock out (KO) and α-syntrophin (α-syn) KO mice, in the latter perivascular AQP4 are mislocalized, but still functioning. Control animals were corresponding wild type (WT) mice. By combining in vivo diffusion measurements with the effective medium theory and previously measured extra-cellular volume fractions, the effects of membrane permeability and extracellular volume fraction were uncoupled for Aqp4 and α-syn KO. The second aim was to assess the effect of α-syn KO on cortical intermediary metabolism combining in vivo [1-13C]glucose and [1,2-13C]acetate injection with ex vivo 13C MR spectroscopy. Aqp4 KO increased the effective diffusion coefficient at long diffusion times by 5%, and a 14% decrease in membrane water permeability was estimated for Aqp4 KO compared with WT mice. α-syn KO did not affect the measured diffusion parameters. In the metabolic analyses, significantly lower amounts of [4-13C]glutamate and [4-13C]glutamine, and percent enrichment in [4-13C]glutamate were detected in the α-syn KO mice. [1,2-13C]acetate metabolism was unaffected in α-syn KO, but the contribution of astrocyte derived metabolites to GABA synthesis was significantly increased. Taken together, α-syn KO mice appeared to have decreased neuronal glucose metabolism, partly compensated for by utilization of astrocyte derived metabolites