Puneet Bagga

Dysfunctional Glutamatergic and γ-Aminobutyric Acidergic Activities in Prefrontal Cortex of Mice in Social Defeat Model of Depression

BACKGROUND Depression is a complex neuropsychiatric syndrome that is often very severe and life threatening. In spite of the remarkable progress in understanding the neural biology, the etiopathophysiology of depression is still elusive. In this study, we have investigated molecular mechanisms in the prefrontal cortex of mice showing depression-like phenotype induced by chronic defeat stress. METHODS Depression-like phenotype was induced in C57BL/6 mice by subjecting them to a 10-day social defeat paradigm. The metabolic activity of excitatory (glutamatergic) and inhibitory (γ-aminobutyric acid [GABA]ergic) neurons of the prefrontal cortex was measured by (1)H-[(13)C]-nuclear magnetic resonance spectroscopy together with infusion of [1,6-(13)C2]glucose. In addition, the expression level of genes associated with glutamatergic and GABAergic pathways was monitored by quantitative polymerase chain reaction. RESULTS Mice showing depression-like phenotype exhibit significant reduction in the levels of glutamate, glutamine, N-acetyl aspartate, and taurine in the prefrontal cortex. Most importantly, findings of reduced (13)C labeling of glutamate-C4, glutamate-C3, and GABA-C2 from [1,6-(13)C2]glucose indicate decreased glutamatergic and GABAergic neuronal metabolism and neurotransmitter cycling in the depressed mice. The reduced glutamine-C4 labeling suggests decreased neurotransmitter cycling in depression. Quantitative polymerase chain reaction analysis revealed reduced transcripts of Gad1 and Eaat2 genes, which code for enzymes involved in the synthesis of GABA and the clearance of glutamate from synapses, respectively. CONCLUSIONS These data indicate that the activities of glutamatergic and GABAergic neurons are reduced in mice showing a depression-like phenotype, which is supported by molecular data for the expression of genes involved in glutamate and GABA pathways.

In vivo NMR studies of regional cerebral energetics in MPTP model of Parkinson's disease: recovery of cerebral metabolism with acute levodopa treatment

In this study, we have evaluated cerebral atrophy, neurometabolite homeostasis, and neural energetics in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridin (MPTP) model of Parkinsons disease. In addition, the efficacy of acute l‐DOPA treatment for the reversal of altered metabolic functions was also evaluated. Cerebral atrophy and neurochemical profile were monitored in vivo using MRI and 1H MR Spectroscopy. Cerebral energetics was studied by 1H‐[13C]‐NMR spectroscopy in conjunction with infusion of 13C labeled [1,6‐13C2]glucose or [2‐13C]acetate. MPTP treatment led to reduction in paw grip strength and increased level of GABA and myo‐inositol in striatum and olfactory bulb. 13C Labeling of glutamate‐C4 (1.93 ± 0.24 vs. 1.48 ± 0.06 μmol/g), GABA‐C2 (0.24 ± 0.04 vs. 0.18 ± 0.02 μmol/g) and glutamaine‐C4 (0.26 ± 0.04 vs. 0.20 ± 0.04 μmol/g) from [1,6‐13C2]glucose was found to be decreased with MPTP exposure in striatum as well as in other brain regions. However, glutamine‐C4 labeling from [2‐13C]acetate was found to be increased in the striatum of the MPTP‐treated mice. Acute l‐DOPA treatment failed to normalize the increased ventricular size and level of metabolites but recovered the paw grip strength and 13C labeling of amino acids from [1,6‐13C2]glucose and [2‐13C]acetate in MPTP‐treated mice. These data indicate that brain energy metabolism is impaired in Parkinsons disease and acute l‐DOPA therapy could temporarily recover the cerebral metabolism.

Regional cerebral metabolism in mouse under chronic manganese exposure: implications for manganism.

Chronic manganese (Mn) exposure in rodents, non-human primates and humans has been linked to Parkinsons disease like condition known as Manganism. Mn being a cofactor for many enzymes in brain has been known to be accumulated in various regions differentially and thus exert toxic effect upon chronic overexposure. In present study, neuropathology of Manganism was investigated by evaluating regional neuronal and astroglial metabolism in mice under chronic Mn exposure. Male C57BL6 mice were treated with MnCl(2) (25 mg/kg, i.p.) for 21 days. Cerebral metabolism was studied by co-infusing [U-(13)C(6)]glucose and [2-(13)C]acetate, and monitoring (13)C labeling of amino acids in brain tissue extract using (1)H-[(13)C] and (13)C-[(1)H]-NMR spectroscopy. Glutamate, choline, N-acetyl aspartate and myo-inositol were found to be reduced in thalamus and hypothalamus indicating a loss in neuronal and astroglial cells due to Mn neurotoxicity. Reduced labeling of Glu(C4) from [U-(13)C(6)]glucose and [2-(13)C]acetate indicates an impairment of glucose oxidation by glutamatergic neurons and glutamate-glutamine neurotransmitter cycle in cortex, striatum, thalamus-hypothalamus and olfactory bulb with chronic Mn exposure. Additionally, reduced labeling of Gln(C4) from [2-(13)C]acetate indicates a decrease in acetate oxidation by astroglia in the same regions. However, GABAergic function was alleviated only in thalamus-hypothalamus. Our findings indicate that chronic Mn impairs excitatory (glutamatergic) function in the majority of regions of brain while inhibitory (GABAergic) activity is perturbed only in basal ganglia.

Lactate Chemical Exchange Saturation Transfer (LATEST) Imaging in vivo A Biomarker for LDH Activity.

Non-invasive imaging of lactate is of enormous significance in cancer and metabolic disorders where glycolysis dominates. Here, for the first time, we describe a chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) method (LATEST), based on the exchange between lactate hydroxyl proton and bulk water protons to image lactate with high spatial resolution. We demonstrate the feasibility of imaging lactate with LATEST in lactate phantoms under physiological conditions, in a mouse model of lymphoma tumors, and in skeletal muscle of healthy human subjects pre- and post-exercise. The method is validated by measuring LATEST changes in lymphoma tumors pre- and post-infusion of pyruvate and correlating them with lactate determined from multiple quantum filtered proton magnetic resonance spectroscopy (SEL-MQC 1H-MRS). Similarly, dynamic LATEST changes in exercising human skeletal muscle are correlated with lactate determined from SEL-MQC 1H-MRS. The LATEST method does not involve injection of radioactive isotopes or labeled metabolites. It has over two orders of magnitude higher sensitivity compared to conventional 1H-MRS. It is anticipated that this technique will have a wide range of applications including diagnosis and evaluation of therapeutic response of cancer, diabetes, cardiac, and musculoskeletal diseases. The advantages of LATEST over existing methods and its potential challenges are discussed.

Characterization of cerebral glutamine uptake from blood in the mouse brain: implications for metabolic modeling of 13C NMR data

13C Nuclear Magnetic Resonance (NMR) studies of rodent and human brain using [1-13C]/[1,6-13C2]glucose as labeled substrate have consistently found a lower enrichment (~25% to 30%) of glutamine-C4 compared with glutamate-C4 at isotopic steady state. The source of this isotope dilution has not been established experimentally but may potentially arise either from blood/brain exchange of glutamine or from metabolism of unlabeled substrates in astrocytes, where glutamine synthesis occurs. In this study, the contribution of the former was evaluated ex vivo using 1H-[13C]-NMR spectroscopy together with intravenous infusion of [U-13C5]glutamine for 3, 15, 30, and 60 minutes in mice. 13C labeling of brain glutamine was found to be saturated at plasma glutamine levels > 1.0 mmol/L. Fitting a blood–astrocyte–neuron metabolic model to the 13C enrichment time courses of glutamate and glutamine yielded the value of glutamine influx, VGln(in), 0.036 ± 0.002 μmol/g per minute for plasma glutamine of 1.8 mmol/L. For physiologic plasma glutamine level (~0.6 mmol/L), VGln(in) would be ~0.010 μmol/g per minute, which corresponds to ~6% of the glutamine synthesis rate and rises to ~11% for saturating blood glutamine concentrations. Thus, glutamine influx from blood contributes at most ~20% to the dilution of astroglial glutamine-C4 consistently seen in metabolic studies using [1-13C]glucose.

Neuroprotective effects of caffeine in MPTP model of Parkinson's disease: A (13)C NMR study.

Parkinsons disease (PD) is a neurodegenerative disorder characterized by degeneration of nigrostriatal dopaminergic neurons with an accompanying neuroinflammation leading to loss of dopamine in the basal ganglia. Caffeine, a well-known A2A receptor antagonist is reported to slow down the neuroinflammation caused by activated microglia and reduce the extracellular glutamate in the brain. In this study, we have evaluated the neuroprotective effect of caffeine in the MPTP model of PD by monitoring the region specific cerebral energy metabolism. Adult C57BL6 mice were treated with caffeine (30 mg/kg, i.p.) 30 min prior to MPTP (25 mg/kg, i.p.) administration for 8 days. The paw grip strength of mice was assessed in order to evaluate the motor function after various treatments. For metabolic studies, mice were infused with [1,6-(13)C2]glucose, and (13)C labeling of amino acids was monitored using ex vivo(1)H-[(13)C]-NMR spectroscopy. The paw grip strength was found to be reduced following the MPTP treatment. The caffeine pretreatment showed significant protection against the reduction of paw grip strength in MPTP treated mice. The levels of GABA and myo-inositol were found to be elevated in the striatum of MPTP treated mice. The (13)C labeling of GluC4, GABAC2 and GlnC4 from [1,6-(13)C2]glucose was decreased in the cerebral cortex, striatum, olfactory bulb, thalamus and cerebellum suggesting impaired glutamatergic and GABAergic neuronal activity and neurotransmission of the MPTP treated mice. Most interestingly, the pretreatment of caffeine maintained the (13)C labeling of amino acids to the control values in cortical, olfactory bulb and cerebellum regions while it partially retained in striatal and thalamic regions in MPTP treated mice. The pretreatment of caffeine provides a partial neuro-protection against severe striatal degeneration in the MPTP model of PD.

Mapping the alterations in glutamate with GluCEST MRI in a mouse model of dopamine deficiency

Glutamate chemical exchange saturation transfer (GluCEST) MRI was used to measure metabolic changes in mice treated with 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) by mapping regional cerebral glutamate. The GluCEST contrast following MPTP treatment was correlated with 1H‐MR spectroscopy, motor function, and immunohistochemical measures. The GluCEST contrast was found to be significantly higher in the striatum and motor cortex of mice treated with MPTP than in controls (p < 0.001), which was confirmed by localized 1H‐MR spectroscopy. Elevated striatal GluCEST was positively associated with local astrogliosis measured by immunohistochemistry for glial fibrillary acidic protein. Additionally, a negative correlation was found between motor function, measured by the four‐limb grip strength test, and GluCEST of the striatum (R = −0.705, p < 0.001) and the motor cortex (R = −0.617, p < 0.01), suggesting a role of elevated glutamate in the abnormal cerebral motor function regulation. The GluCEST contrast and glial fibrillary acidic protein immunostaining were unaltered in the thalamus indicating glutamate elevation was localized to the striatum and the motor cortex. These findings suggest that in addition to measuring spatial changes in glutamate, GluCEST may serve as an in vivo biomarker of metabolic and functional changes that may be applied to the assessment of a broad range of neuropathologies.

High quality three-dimensional gagCEST imaging of in vivo human knee cartilage at 7 Tesla

To develop a new faster and higher quality three‐dimensional (3D) gagCEST MRI technique for reliable quantification of glycosaminoglycan (GAG) present in the human knee cartilages.

In vivo GluCEST MRI: Reproducibility, background contribution and source of glutamate changes in the MPTP model of Parkinson’s disease

Glutamate Chemical Exchange Saturation Transfer (GluCEST) MRI is a recently developed technique to image glutamate. In the present study, we evaluated the reproducibility and background contamination to the GluCEST and source of the GluCEST changes in a mouse model of Parkinson’s disease. Repeated measurements in five mice demonstrated an intra-animal coefficient of variation (CV) of GluCEST signal to be 2.3 ± 1.3% and inter-animal CV of GluCEST to be 3.3 ± 0.3%. Mice were treated with MPTP to create a localized striatal elevation of glutamate. We found an elevation in the GluCEST contrast of the striatum following MPTP treatment (Control: 23.3 ± 0.8%, n = 16; MPTP: 26.2 ± 0.8%, n = 19; p ≤ 0.001). Additionally, the positive association between glutamate concentration measured via 1H MRS and GluCEST signal was used to estimate background contribution to the measured GluCEST. The contribution of signal from non-glutamate sources was found to be ~28% of the total GluCEST. Immunohistochemical analysis of the brain showed co-localization of glutamate with GFAP in the striatum. This suggests that the elevated glutamate present in the striatum in this mouse model reflects astroglial proliferation or reactivity due to the action of MPTP. The potential of GluCEST as a biomarker for imaging inflammation mediated gliosis is discussed.

Pretreatment of caffeine leads to partial neuroprotection in MPTP model of Parkinson's disease

Parkinsons disease (PD) is a common neurodegenerative disorder affecting more than 1% people above 60 years of age worldwide, manifesting as the impaired motor function such as tremors, rigidity, akinesia/bradykinesia and postural inefficiency with a reduced life expectancy (Dorsey et al., 2007). PD is believed to be the end result of the progressive death of dopaminergic neurons in the substantia nigra pars compacta (SNc). This loss of dopaminergic neurons results in deficit in neurotransmitter dopamine in the basal ganglia that leads to impairment of motor control. The pathologic changes are reported to precede by a decade or more before the manifestation of clinical symptoms. Analysis in postmortem human brain tissue and various animal studies have reported activation of inflammatory microglial cells causing the oxidative stress. The reactive oxygen species (ROS) may cause severe damage to DNA, proteins and lipids in cells (Jenner, 2003).