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Keywords:

  • GABA ;
  • glutamate;
  • metabolism;
  • MRI;
  • olfactory bulb;
  • striatum

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information
Thumbnail image of graphical abstract

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 Parkinson's 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 Parkinson's disease and acute l-DOPA therapy could temporarily recover the cerebral metabolism.

Cerebral atrophy, neurometabolite homeostasis, and neural energetics have been evaluated in an MPTP model of Parkinson's disease using MRI, in vivo 1H MRS and 1H-[13C]-NMR spectroscopy, respectively. MPTP treatment led to reduced paw grip strength and neuronal function. Acute Levodopa treatment was able to recover the diminished motor function and cerebral function. CMRGlc, Cerebral metabolic rate of glucose oxidation; MPTP, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridin.

Abbreviations used
1H-[13C]-NMR

Proton observed carbon-edited NMR spectroscopy

CMRGlc

Cerebral metabolic rate of glucose oxidation

DA

Dopamine

Gln

Glutamine

Glu

Glutamate

m-Ino

myo-Inositol

MPTP

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridin

NS

Normal saline

OB

Olfactory bulb

PD

Parkinson's disease

SNc

Substantia Nigra pars compacta

Parkinson's disease (PD) is a debilitating neurodegenerative disorder caused by selective degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). The loss of dopaminergic neurons in the SNc results in diminished level of dopamine (DA) in the putamen of the dorsolateral striatum which leads to dysfunction of cortico-basal ganglio-thalamo-cortical pathway in brain. The proper functioning of this pathway is critically important for the control of movement (Alexander 1994; Turner 2009). Clinical manifestations of PD include cardinal symptoms such as bradykinesia, resting tremors and muscular rigidity (Obeso et al. 2000; Gazewood et al. 2013). The diagnosis of PD is based on these motor symptoms which are evident only after the loss of 60–80% DA in the striatum.

Currently, there is no quantitative diagnosis and cure available for PD. Quantitative measurement of cerebral metabolism will provide assessment of brain function, which may yield biomarkers for the diagnosis of the disease and for monitoring the efficacy of therapeutic treatment. Several genetic, chemical, and toxin-induced models are employed to study the etiopathology of PD in rodents (Blandini and Armentero 2012; Blesa et al. 2012). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP) exposure in mice is a well established model of PD. MPTP, a neurotoxin, causes specific degeneration of nigrostriatal dopaminergic neurons which is accompanied by loss of DA in the striatum of non-human primates, rodents, and humans (Kohutnicka et al. 1998; Araki et al. 2001) leading to PD like syndrome. MPTP is highly lipophilic and freely crosses the blood–brain barrier thereby enters into glial cells. In glial cells, MPTP is converted into its active form 1-methyl-4-phenylpyridinium (MPP+) by the enzyme MAO-B (Markey et al. 1984). MPP+ is taken up specifically by the dopaminergic neurons through the dopamine transporters and accumulated in the mitochondria, where it interferes with complex I of the electron transport chain (Scotcher et al. 1990; Gluck et al. 1994). This causes depletion in the level of ATP in dopaminergic neurons leading to selective loss of these cells in the SNc and hence loss of DA in the striatum.

l-DOPA, a direct precursor of DA, has been used to ameliorate PD symptoms and for the management of PD (Hornykiewicz 2010). DA is synthesized from l-DOPA by the enzyme DOPA decarboxylase. l-DOPA can pass through the blood–brain barrier and provide symptomatic relief by replenishing the lost DA in the striatum which is required for the proper regulation of the cortico-basal ganglio-thalamo-cortical pathway of movement control.

Although there are many reports suggesting hypometabolism in cortical and subcortical regions in the PD brain (Lenzi et al. 1979; Otsuka et al. 1991; Borghammer et al. 2012b), the function of different cell types (glutamatergic, GABAergic, and astroglia) in regions of brain involved with movement control path way is still elusive. 13C NMR spectroscopy has provided important insights for the neural metabolism and interactions between neurons and astroglia commonly known as neurotransmitter cycling (Schousboe et al. 1997; de Graaf et al. 2003a). In this study, we have employed an approach of infusion of 13C labeled [1,6-13C2]glucose or [2-13C]acetate in conjunction with 1H-[13C]-NMR spectroscopy to understand the neuronal (glutamatergic, GABAergic) and astroglial metabolism in different regions of the brain in the MPTP model of PD. In addition, the effect of acute l-DOPA therapy for the reversal of impaired motor function as well as cerebral metabolism is also assessed. We hypothesized that because of loss of dopaminergic neurons, glutamatergic and GABAergic neuronal function would be altered in the areas involved in the cortico-basal ganglio-thalamo-cortical loop in PD which could be temporarily restored following supplementation of l-DOPA. Glucose is the major source of energy and is mostly oxidized by neurons (glutamatergic and GABAergic). Oxidative metabolism of pyruvate-C3 (PyrC3, the glycolytic end product of [1,6-13C2]glucose) via the TCA cycle in the glutamatergic and GABAergic neurons incorporates 13C label into gluatamte-C4 (GluC4) and GABAC2, respectively (Fig. 1a). Glutamine-C4 (GlnC4) is labeled by cycling of neurotransmitters, glutamate, and GABA into astrocytes. In contrast, [2-13C]acetate is specifically transported and metabolized in astroglia (Badar-Goffer et al. 1990; Waniewski and Martin 1998) and labels GlnC4 by the action of glutamine synthetase (Fig. 1b). GluC4 and GABAC2 are labeled via cycling of GlnC4 into neurons (Patel et al. 2005). Our findings indicate that excitatory and inhibitory neuronal metabolism is reduced in different regions of brain following subacute treatment of MPTP, which could be recovered by acute l-DOPA treatment. The preliminary findings of the study have been presented in the Annual Meeting of the International Society for Magnetic Resonance in Medicine, Utah, USA (Patel et al. 2013).

image

Figure 1. Schematic of 13C labeling of cerebral metabolites from (a) [1,6-13C2]glucose and (b) [2-13C]acetate. (a) Metabolism of [1,6-13C2]glucose via glutamatergic and GABAergic TCA cycles labels α-ketoglutarate-C4 (α-KG4) thereby labeling of Glu4 and GABA2 in glutamatergic and GABAergic neurons, respectively, during the first turn of the TCA cycle.13C Labeling of Gln4 occurs from Glu4 and GABA2 via glutamate-glutamine and GABA-glutamine cycle. Metabolism of amino acids in the subsequent turns of TCA cycle incorporates label into Glu2/3, GABA3/4, Gln2/3, and Asp2/3 (not shown). (b) [2-13C]Acetate is selectively transported in astroglia and metabolized thereby labels Gln4 by combined action of astroglial TCA cycle and glutamine synthetase. Neurotransmitters, Glu4 and GABA2, are labeled from Gln4 via glutamate-glutamine and GABA-glutamine substrates cycling between astroglia and neurons, respectively. Glu2/3, GABA3/4, Gln2/3 and Asp2/3 are labeled by the metabolism in the subsequent turns of TCA cycle (not shown). AcCoA2, acetylCoA-C2; GABA2, γ-aminobutyric acid-C2; Gln4, glutamine-C4; Glu4, glutamate-C4; α-KG4, α-ketoglutarate-C4; Pyr3, Pyruvate-C3.

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Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

MPTP administration

All the animal experiments were approved by the Institutional Animal Ethics Committee of CCMB, Hyderabad. ARRIVE guidelines have been followed in the preparation of the manuscript. C57BL6 mice were procured from the Jackson Laboratory, Bar Harbor, ME, USA and bred at the CCMB Animal House. Mice were housed in a humidity controlled room ~ 24°C under a 12 h light/dark cycle with ad libitum access to food and water. Sixty male mice (2 month old) were divided into four groups: Group (i) Control + Normal Saline (NS) (n = 15), Group (ii) Control + l-DOPA (n = 15), Group (iii) MPTP + NS (n = 15), Group (iv) MPTP + l-DOPA (n = 15). MPTP (Sigma/Aldrich, St. Louis, MO, USA) solution (2.5 mg/mL) was made in normal saline. Mice in Group (iii) and (iv) received MPTP (25 mg/kg, i.p.) once in a day for 7 days, while the control mice [Group (i) and (ii)] received the same volume of NS (Fig. 2a) for the same period.

image

Figure 2. (a) Schematic representation of timeline of various treatments and measurements. The paw grip strength (PG) test was carried out on day 0, 8, 14, and 16 to monitor motor function in mice undergoing various treatments. l-DOPA was administered on days 15 and 21. In vivo MRI/MRS studies were performed on day 16. Metabolic measurements were carried out on 22nd day. (b) Paw grip strength of the mice during different treatments. (c) Representative T1 weighted MRI images of the mice brain during different treatments. MRI images were recorded using FLASH method. Increased size of ventricles (marked by arrow) following MPTP administration in mice could be seen in mice treated with MPTP. ***p < 0.001 when compared with control.

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Levodopa administration

l-DOPA (Sigma-Aldrich Chemical Co.) solution (3.25 mg/mL) was prepared in normal saline. After the assessment of the motor function on day 14, mice in Group (ii) and (iv) were administered l-DOPA (35 mg/kg, i.p.) four times (accumulative dose 140 mg/kg) on day 15 at an interval of 2 h while those in Group (i) and (iii) received NS. Behavioral analysis and MRI/MRS was carried out on day 16. Mice were re-administered l-DOPA on day 21 and the metabolic analysis was carried out on day 22 (Fig. 2a).

Assessment of motor function

The grip strength of the forepaws of each animal was measured on day 0, 8, 14, and 16 (before and after every treatment) using a grip strength meter (MK-380M; Muromachi Kikai Co. Ltd, Tokyo, Japan) to evaluate motor function. The paw grip strength meter contains a digital force gauge attached to a grip strength platform having 3 mm × 3 mm size mesh. Mice were allowed to hold the platform with their forelimbs and pulled backwards with the tail in a horizontal plane. The maximum force applied to the platform immediately before the release of the paw grip was recorded in the meter.

MRI of brain and 1H-MRS of striatum

In vivo MRI/MRS measurements were carried out on day 16 (12 h after the last dose of l-DOPA/NS). Mice anesthetized with isoflurane (1.0–1.5%) in 70% air and 30% O2 were placed in an MR probe and a transceiver surface coil (i.d. 15 mm) was positioned on the head of the mice. The probe was positioned into a vertical wide bore (89 mm) 14.1 T magnet, equipped with 60 mm actively shielded gradient, interfaced with Avance II Microimager (Bruker Biospin, Karlsruhe, Germany). Body temperature of the animals was maintained at ~ 37°C by circulating warm water through the imaging gradient and respiration was maintained at 50–70 breaths per minute by adjusting the level of isoflurane.

T1 Weighted MR images were acquired using FLASH method to monitor cerebral atrophy as well as for positioning of the spectroscopic voxel in the striatum. The typical parameters used for MRI acquisition were: Repetition time (TR) = 250 ms, Echo time (TE) = 5 ms, Flip angle = 40°, Number of slices = 16, Slice thickness = 0.25 mm, Field of View = 16 mm × 16 mm, Matrix size = 256 × 256 and Number of averages = 8. FASTMAP method was used for the adjustment of the first and second order shims in the spectroscopic voxel positioned in striatum yielding water line width ~ 20 Hz. Localized in vivo 1H NMR spectroscopy was carried out using STEAM method in conjunction with outer volume suppression from a voxel positioned in the striatum (2 mm × 2 mm × 2 mm) using TE/TR = 3.5/3000 ms (Frahm et al. 1989). Water resonance was suppressed using VAPOR method (Tkac et al. 1999). 1H NMR spectra were processed in Topspin 1.5. FID's were zero filled to 16384, multiplied by Gaussian window function, Fourier transformed and phase corrected.

Quantification of in vivo 1H NMR spectra

Signal intensity in the in vivo 1H NMR spectra was quantified using the LCModel software (Provencher 1993). The following metabolites were included in the basis set: alanine, aspartate (Asp), creatine, GABA, glucose, Glu, Gln, glycerophosphorylcholine, phosphorylcholine, m-Ino, lactate, N-acetylaspartate, N-acetylaspartylglutamate, phosphocreatine, scyllo-inositol, and taurine. In addition, pre-simulated 9 macromolecule (MM) FIDs were incorporated in the basis set. For analysis of in vivo 1H MRS, a spectral range of 0–4.2 ppm was used. The error in the quantification of various metabolites was estimated as Cramer Rao Bound (CRB) and metabolites having a CRB < 20% were considered for further analysis (Srinivasan et al. 2004).

Infusion of 13C labeled substrates for the metabolic measurements

Overnight (12–14 h) fasted mice were anesthetized with urethane (1.5 g/kg i.p.) and the lateral tail vein catheterized for infusion of 13C labeled substrates. Body temperature was maintained ~ 37°C using a heating pad connected to a temperature-regulated re-circulating water bath. The respiration rate was monitored using Biopack device. [1,6-13C2]Glucose (99 atom%; Cambridge Isotope Laboratory, Andover, MA, USA) dissolved in de-ionized water (0.225 mol/L) was administered intravenously in mice via lateral tail vein cannula for 10 min using bolus variable infusion rate (Fitzpatrick et al. 1990). Mice received a bolus administration of 135 μL of [1,6-13C2]glucose (per 30 g body wt) in 15 s, following which the rate of the infusion was decreased exponentially every 30 s until 8.25 min. Thereafter, the infusion rate was set to a constant value (6.8 μL/min). In another set of animals, [2-13C]Acetate (99 atom%; Cambridge Isotope Laboratory) (0.8 mol/L) dissolved in de-ionized water, pH adjusted to 7.0, was infused (i.v.) into mice (per 30 g body wt) for 10 min using an infusion protocol similar to Patel et al. (2010). In brief, [2-13C]acetate was infused using a bolus-variable rate infusion of 117.2 μL/min (0 to 15 s), 23.4 μL/min (15 s to 4 min), 11.7 μL/min (4 to 8 min) and 4.7μL/min (8 min onward) until conclusion of the experiment. Blood was collected from retro-orbital sinus artery for the measurement of plasma glucose and acetate enrichment just before the conclusion of infusion experiment. The mouse head was frozen in situ in liquid nitrogen and stored at −80°C until further processing.

Extraction of metabolites from brain tissue

Different brain regions, namely cerebral cortex, cerebellum, striatum, thalamus-hypothalamus, and olfactory bulb (OB) were dissected in a cryostat maintained at −20°C. Metabolites were extracted from the frozen tissue using a protocol described previously (Patel et al. 2001). Briefly, weighed tissue was powdered with 0.1 N HCl/methanol in a dry ice-ethanol bath and [2-13C]glycine was added for concentration reference. Powdered tissue was homogenized with ice-cold 60% ethanol and the homogenate was centrifuged at 14 000 g for 45 min. The supernatant was lyophilized and the powder was dissolved in 550 μL of D2O containing 0.25 mM sodium 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionate for NMR analysis.

Analysis of plasma glucose and acetate labeling

Plasma (30–50 μL) was mixed with D2O (500 μL) containing sodium formate (0.5 mmol/L) and passed through a centrifugal filter (10-kDa cut-off) to remove macromolecules. The percentage 13C enrichment of plasma glucose-C1 and acetate-C2 was determined from the 1H NMR spectrum obtained at 600 MHz NMR spectrometer. 13C Enrichment of glucose-C1α and acetate-C2 was calculated by dividing the intensity of the two 13C satellites by the total (12C + 13C) intensity at 5.23 and 1.91 ppm, respectively.

NMR spectroscopy of brain tissue extracts

1H-[13C]-NMR spectra of brain tissue extracts were acquired using 600 MHz NMR spectrometer with the following parameters: repetition time = 5.5 s, echo time = 8 ms, number of points in FID = 16384, spectral width = 7212 kHz, number of averages ranging from 64 (cerebral cortex) to 1024 (OB) (de Graaf et al. 2003b; Bagga and Patel 2012). In this experiment, two sets of spin-echo 1H NMR spectra were acquired with ON/OFF 13C inversion pulse. FIDs were zero filled to 128 K data points, apodized (0.8 Hz Lorentzian line broadening), Fourier transformed, and phase corrected. C-13 edited NMR spectrum [1H(2x13C)] was obtained by subtracting a subspectrum obtained with 13C inversion pulse ON [1H(12C-13C)] from that acquired under OFF condition [1H(12C+13C)]. The peak intensity of different metabolites was measured from the spectrum acquired under OFF condition using inbuilt integration tool of the Topspin 2.1. The intensity of the labeled metabolites was obtained from the C-13 edited spectrum. The total concentration of metabolites was calculated from the peak intensity in the spectrum obtained without 13C pulse (OFF condition) relative to [2-13C]glycine (obtained from the edited spectrum). The isotopic 13C enrichment of amino acids at different carbon positions was calculated from the ratio of intensity in the difference spectrum and the non-edited spectrum.

Glucose oxidation by glutamatergic and GABAergic neurons

Glutamatergic and GABAergic neuronal glucose oxidation rate was calculated from the initial rate of labeling from [1,6-13C2]glucose as described earlier by Patel et al. (2005). The cerebral metabolic rate of glucose oxidation by glutamatergic neurons is given by the following:

  • display math(1)

where Glui and Aspi are percent 13C labeling of glutamate and aspartate, respectively, at carbon ‘i’ from [1,6-13C2]glucose during infusion time ‘t’, and [Glu] and [Asp] are the respective concentrations of glutamate and aspartate in the concerned region of the brain.

Similarly, the cerebral metabolic rate of glucose oxidation by GABAergic neurons could be obtained from the following equation:

  • display math(2)

where GABAi is the labeling of GABA at carbon ‘i’ from [1,6-13C2]glucose during ‘t, and [GABA] is the concentration of GABA.

The total glucose oxidation by neurons and astrocytes was estimated by the following:

  • display math(3)

Statistics

Statistical analysis was carried out using the Data Analysis Tool package of Microsoft Excel 2007. Single factor anova was performed to determine the global differences in the paw grip strength, concentration, labeling of amino acids, and glucose oxidation among different groups. Further, differences at individual amino acid level were evaluated by Student's t-test. All results are presented as mean ± SD.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Motor function with different treatments

Treatment with MPTP and l-DOPA did not cause any significant alteration in the weight of mice. Subacute treatment of MPTP to mice led to significant reduction [F(1,58) = 25.31, = 5 × 10−6] in the paw grip strength of forelimb (0.55 ± 0.01 N, = 30 vs. 0.68 ± 0.02 N, = 30) (Fig. 2b). The acute l-DOPA therapy improved the paw grip strength significantly [F(1,26) = 7.57, = 0.011] in mice treated with MPTP (before 0.58 ± 0.02 N, = 14; after 0.68 ± 0.03 N, = 14). l-DOPA treatment did not affect the paw grip strength in control mice (before 0.68 ± 0.03 N; after 0.69 ± 0.02 N, = 0.736).

Cerebral atrophy with MPTP treatment

The MPTP induced brain atrophy was evaluated by in vivo MRI. T1 Weighted MR images of coronal sections of mice brain were acquired with slice thickness 250 μm and in plane resolution 62.5 μm × 62.5 μm. Subacute exposure of MPTP to mice led to enhanced ventricular size (Fig. 2c). Acute l-DOPA treatment did not change the ventricular size in MPTP-treated mice.

Regional neurochemical profile with different treatments

Typical in vivo 1H NMR spectra obtained from striatum of mice in different groups are presented in Fig. 3. Measurement of metabolites in the extract indicated that level of creatine was unchanged with different treatments. Hence, creatine was scaled to the same amplitude in all the groups. The NMR spectra suggested MPTP treatment (Fig. 3b) led to an increased level of Glu, GABA, glutamine and m-Ino while the level of N-acetylaspartate was unaltered. Indeed, the LCModel analysis of in vivo NMR spectra indicated that the ratio of GABA (Control + NS 0.28 ± 0.02; MPTP + NS 0.37 ± 0.07, = 0.017), Glu (Control + NS 1.15 ± 0.04, MPTP + NS 1.46 ± 0.05, = 6 × 10−7), Gln (Control + NS 0.39 ± 0.01, MPTP + NS 0.43 ± 0.05, = 0.046), m-Ino (Control + NS 0.53 ± 0.03, MPTP + NS 0.70 ± 0.06, = 0.00007) with total creatine (tCr) was increased significantly in the striatum of the MPTP-treated mice (Fig. 3e).

image

Figure 3. Representative localized in vivo 1H MR spectra from striatum of (a) Control, (b) MPTP treated, (c) Control + l-DOPA and (d) MPTP + l-DOPA mice. The inset images represent the coronal image of mouse brain depicting the positioning of MR voxel. In vivo 1H MR spectroscopy was carried out using STEAM method in conjunction with outer volume suppression from a voxel (2 mm × 2 mm × 2 mm). (e) Ratio of concentration of metabolite with total creatine. Signal intensity of various metabolites in the 1H MR spectra was quantified using LCModel software. Values represent mean ± SD. *< 0.05, **< 0.01, ***< 0.001 when compared with respective control, #p < 0.05, ##p < 0.01, ###p < 0.001 when compared with Control + L-DOPA group.

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In vivo findings were validated by the measurement of metabolites concentration in 1H NMR spectra of tissue extracts (Fig. 4). In accordance with findings from in vivo measurements, the levels of Glu (Control + NS 12.0 ± 0.8 μmol/g; MPTP + NS 13.0 ± 0.6 μmol/g, = 0.0004), GABA (Control + NS 4.8 ± 0.4 μmol/g; MPTP + NS 6.0 ± 0.7μmol/g, = 6 × 10−6), Gln (Control + NS 5.3 ±0.5 μmol/g; MPTP + NS 5.8 ± 0.7 μmol/g, = 0.028), Asp (Control + NS 1.7 ± 0.2 μmol/g; MPTP + NS 2.3 ± 0.4 μmol/g, = 0.0001), and m-Ino (Control + NS 6.6 ± 0.8 μmol/g; MPTP + NS 8.0 ± 1.0 μmol/g, = 0.0003) were elevated in the striatum of mice treated with MPTP. In addition, levels of GABA (Control + NS 6.2 ± 0.6 μmol/g; MPTP + NS 7.4 ± 1.0 μmol/g, = 0.0003) and m-Ino (Control + NS 6.2 ± 0.6 μmol/g; MPTP + NS 7.3 ± 0.08 μmol/g, = 0.0002) were also found to be elevated significantly in OB with subacute MPTP treatment (Table 1). Acute treatment of l-DOPA did not alter (> 0.11) level of neurometabolites in control as well as in the MPTP-treated mice.

Table 1. Concentration (μmol/g) of cerebral metabolites in different brain regions following subacute MPTP and acute l-DOPA treatment
Brain regionTreatmentGluGABAGlnNAAChom-Ino Cre
  1. Concentration of metabolites was measured from non-edited 1H-[13C]-NMR spectrum using [2-13C]glycine as standard. Values are represented as mean ± SD. *< 0.05, **< 0.01 when compared with the respective controls.

Cerebral cortexControl + NS13.8 ± 0.62.5 ± 0.25.5 ± 0.48.3 ± 0.41.9 ± 0.16.0 ± 0.510.7 ± 0.3
Control + l-DOPA13.9 ± 0.72.6 ± 0.25.2 ± 0.88.1 ± 0.61.8 ± 0.15.8 ± 1.311.0 ± 0.3
MPTP + NS13.2 ± 0.82.5 ± 0.35.4 ± 0.67.9 ± 0.61.8 ± 0.16.3 ± 0.410.8 ± 0.5
MPTP + l-DOPA14.0 ± 0.42.5 ± 0.25.5 ± 0.88.2 ± 1.01.8 ± 0.16.3 ± 0.411.0 ± 0.4
CerebellumControl + NS11.9 ± 0.72.3 ± 0.35.9 ± 0.47.7 ± 0.41.9 ± 0.37.6 ± 0.715.5 ± 1.0
Control + l-DOPA12.2 ± 1.12.4 ± 0.35.9 ± 0.77.4 ± 0.62.0 ± 0.28.0 ± 0.716.0 ± 0.9
MPTP + NS11.4 ± 0.72.3 ± 0.35.9 ± 0.77.3 ± 0.41.9 ± 0.27.9 ± 0.816.0 ± 0.8
MPTP + l-DOPA11.8 ± 0.72.1 ± 0.35.9 ± 0.77.3 ± 0.51.9 ± 0.27.6 ± 0.815.2 ± 1.6
StriatumControl + NS12.0 ± 0.84.8 ± 0.55.3 ± 0.56.7 ± 0.62.3 ± 0.26.6 ± 0.810.4 ± 0.8
Control + l-DOPA12.3 ± 0.84.9 ± 0.55.3 ± 0.66.7 ± 1.02.2 ± 0.37.0 ± 0.610.6 ± 0.6
MPTP + NS13.0 ± 0.6*6.0 ± 0.7**5.8 ± 0.77.0 ± 0.62.4 ± 0.28.0 ± 1.0**10.8 ± 1.1
MPTP + l-DOPA12.7 ± 0.45.5 ± 0.5**5.3 ± 0.77.0 ± 0.62.3 ± 0.27.6 ± 0.8*11.0 ± 0.6
Thalamus-hypothalamusControl + NS12.5 ± 0.94.4 ± 0.45.0 ± 0.57.7 ± 0.72.1 ± 0.27.0 ± 0.810.8 ± 0.8
Control + l-DOPA12.4 ± 0.74.3 ± 0.44.8 ± 0.67.6 ± 0.62.1 ± 0.37.3 ± 0.710.8 ± 0.6
MPTP + NS12.5 ± 0.94.6 ± 0.55.1 ± 0.77.9 ± 0.42.1 ± 0.27.5 ± 0.511.3 ± 0.4
MPTP + l-DOPA12.4 ± 0.64.2 ± 0.54.9 ± 0.67.6 ± 0.52.1 ± 0.27.2 ± 1.110.6 ± 0.5
Olfactory bulbControl + NS12.5 ± 0.96.2 ± 0.65.9 ± 0.89.1 ± 0.82.1 ± 0.26.2 ± 0.68.8 ± 0.6
Control + l-DOPA12.4 ± 0.76.4 ± 0.75.6 ± 0.89.5 ± 0.92.0 ± 0.36.4 ± 0.88.9 ± 0.8
MPTP + NS12.5 ± 0.97.4 ± 1.0**6.4 ± 0.69.5 ± 0.82.1 ± 0.27.3 ± 0.8**9.3 ± 0.7
MPTP + l-DOPA12.4 ± 0.66.9 ± 0.5*6.1 ± 1.09.7 ± 0.82.2 ± 0.26.9 ± 0.6*9.2 ± 0.8
image

Figure 4. Representative 1H-[13C]-NMR spectra of striatal tissue extract. Mice were infused with [1,6-13C2]glucose for 10 min and metabolites were extracted from frozen striatal tissue and lyophilized. 1H-[13C]-NMR spectroscopy was carried out in powdered extract dissolved in D2O. The top spectra represent the total concentration of metabolites (1H{12C + 13C}) in (a) Control and (b) MPTP-treated mice, while the rest depict concentration of 13C labeled metabolites from [1,6-13C2]glucose in (c) Control + NS, (d) MPTP + NS, (e) Control + l-DOPA, and (f) MPTP + l-DOPA. Peak labels are: Asp3, Asparate-C3; Creatine, Cre; Cho, Choline; GABA2, GABA-C2; Gln4, Glutamine-C4; Glu3, Glutamate-C3; Glu4, Glutamate-C4; m-Ino, myo-Inositol; NAA, N-acetyl aspartate; Tau, Taurine.

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Enrichment of plasma glucose and acetate

Acute treatment with l-DOPA did not change the percent 13C labeling of plasma glucose-C1 in control (Control + NS 24.9 ± 5.6%, = 5; Control + l-DOPA 25.5 ± 11.0%, = 5, = 0.68) and MPTP-treated (MPTP + NS 28.7 ± 3.2%, = 5; MPTP + l-DOPA 26.4 ± 7.7%, = 5, = 0.53) mice following 10 min of [1,6-13C2]glucose infusion. Similarly, plasma acetate-C2 enrichment was not significantly (> 0.06) different among different groups (Control + NS 85.5 ± 2.8%; Control + l-DOPA 85.5  ± 2.4%; MPTP + NS 79.9 ± 7.9% and MPTP + l-DOPA 83.4 ± 4.7%) suggesting that MPTP and l-DOPA treatment did not alter the homeostasis of glucose and acetate in blood.

13C labeling of cerebral amino acids from [1,6-13C2]glucose and [2-13C]acetate

The percent labeling of amino acids from [1,6-13C2]glucose and [2-13C]acetate was measured in tissue extract using 1H-[13C]-NMR spectra. Representative 1H-[13C]-NMR spectra obtained from striatal region in [1,6-13C2]glucose experiment among different groups are depicted in Fig. 4. The 13C labeling of amino acids in the striatum was reduced in MPTP-treated mouse (Fig. 4d) as compared to control (Fig. 4c) and MPTP + l-DOPA-treated mouse (Fig. 4f). The measured percent 13C enrichment was normalized with plasma glucose enrichment and multiplied with the total concentration to obtain the amount of 13C label accumulated in different amino acids. The concentration of 13C labeled GluC4 (Control + NS 1.93 ± 0.24 μmol/g, = 6; MPTP + NS 1.48 ± 0.06 μmol/g, = 5, = 0.0028), GlnC4 (Control + NS 0.26 ± 0.04 μmol/g, = 6; MPTP  + NS 0.20 ± 0.04 μmol/g, = 5, = 0.015), and GABAC2 (Control + NS 0.24 ± 0.04 μmol/g, = 6; MPTP + NS 0.18 ± 0.02 μmol/g, = 5, = 0.013) from [1,6-13C2]glucose was found to be reduced in the striatum of MPTP-treated mice (Fig. 5). In addition, significant reduction in 13C labeling of amino acids was observed in the cerebral cortex (< 0.007), cerebellum (< 0.035), thalamus-hypothalamus (< 0.024), and OB (< 0.016) of MPTP-treated mice. Acute treatment with l-DOPA in MPTP-treated mice recovered the labeling of striatal GluC4 (Control + NS 1.93 ± 0.24 μmol/g, = 6; MPTP + l-DOPA 1.98 ± 0.22 μmol/g, = 5, = 0.367), GlnC4 (Control + NS 0.26 ± 0.04 μmol/g, = 6; MPTP + l-DOPA 0.29 ±  0.05 μmol/g, = 5, = 0.216), and GABAC2 (Control + NS 0.24 ± 0.04 μmol/g, = 5; MPTP + l-DOPA 0.28 ±  0.07 μmol/g, = 5, = 0.761) to the control value. There was no significant (> 0.86) change in the labeling of amino acids with l-DOPA treatment in control mice. Like striatum, other brain regions also exhibited recovery of 13C labeling of amino acids from [1,6-13C2]glucose following acute l-DOPA treatment in mice exposed to MPTP (Fig. 5).

image

Figure 5. Concentration of 13C labeled amino acids from [1,6-13C2]glucose. Mice were infused with [1,6-13C2]glucose for 10 min and 13C labeling of amino acids were monitored using 1H-[13C]-NMR spectroscopy in tissue extract. GABAC2, γ-aminobutyric acid-C2; GlnC4, glutamine-C4; GluC4, glutamate-C4. Values represent mean ± SD. *< 0.05, **< 0.01, ***p < 0.001 when compared with control; #< 0.05, ##< 0.01, ###< 0.001 when compared with MPTP + NS group.

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GlnC4 labeling from [2-13C]acetate was found to be increased in the striatum (Control + NS 0.46 ± 0.05μmol/g; MPTP + NS 0.54 ± 0.04 μmol/g, = 0.012) of MPTP-exposed mice (Fig. 6). There was no significant (> 0.8) change in the labeling of GlnC4 from [2-13C]acetate in other brain regions. The increased labeling of GlnC4 in the MPTP-treated mice (0.54 ± 0.04 μmol/g) was normalized to control value (0.46 ± 0.05 μmol/g) following acute l-DOPA treatment (0.46 ± 0.05 μmol/g, = 0.025). Acute l-DOPA treatment in control mice did not change (> 0.83) the labeling of brain amino acids from [2-13C]acetate (Fig. 6).

image

Figure 6. Concentration of 13C labeled amino acids from [2-13C]acetate in the striatum. Mice were infused with [2-13C]acetate for 10 min and 13C labeling of GluC4, GABAC2 and GlnC4 was measured in striatal tissue extract. Abbreviations used are same as described in Fig. 5. Values represent as mean ± SD. *= 0.01.

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Glucose oxidation by glutamatergic and GABAergic neurons with different treatment

Glucose oxidation associated with glutamatergic and GABAergic neurons was calculated based on the 13C label accumulated into amino acids from [1,6-13C2]glucose using eqns (1) and (2), respectively. Subacute exposure of mice to MPTP reduced the glucose oxidation by glutamatergic (Control + NS 0.24 ± 0.03 μmol/g/min, = 6; MPTP +NS 0.16 ± 0.02 μmol/g/min, = 5, = 0.0011) and GABAergic neurons (Control + NS 0.063 ± 0.01 μmol/g/min, = 6; MPTP + NS 0.050 ± 0.003 μmol/g/min, = 5, = 0.021) in the striatum. Total striatal glucose oxidation (Control + NS 0.33 ± 0.04 μmol/g/min, = 6; MPTP + NS 0.23 ± 0.02 μmol/g/min, = 5, = 0.0002) was also found to be reduced after MPTP treatment (Fig. 7). The effect of MPTP on the glucose oxidation was also evident in the cerebral cortex, cerebellum, OB, and thalamus–hypothalamus (Figure S1). Acute l-DOPA treatment in MPTP-treated mice recovered the glucose oxidation by glutamatergic (Control + NS 0.24 ± 0.03 μmol/g/min; MPTP + l-DOPA 0.25 ± 0.04 μmol/g/min, = 0.6488) and GABAergic neurons (Control + NS 0.063 ±0.01 μmol/g/min; MPTP + l-DOPA 0.074 ± 0.012 μmol/g/min, = 0.125) to the control value in the striatum (Fig. 7). Recovery of glucose oxidation by glutamatergic and GABAergic neurons with acute l-DOPA treatment was also observed in other brain regions (Figure S1). l-DOPA did not alter the glucose oxidation pertaining to different cell types across the brain in control mice.

image

Figure 7. Rate of glucose oxidation in the striatum. Mice were infused with [1,6-13C2]glucose for 10 min and 13C labeling of amino acids were monitored using 1H-[13C]-NMR spectroscopy in striatal extract. Rate of glucose oxidation associated with glutamatergic neurons, GABAergic neurons, neuronal (glutamatergic + GABAergic), and total glucose oxidation was calculated using eqns (1)-(3) as described in the Methods section. Values are represented as mean ± SD. *= 0.01, **= 0.001

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

In the recent past, the proneurotoxin MPTP model has become a widely used approach to induce PD like symptoms in animals for understanding the pathology of PD (Schmidt and Ferger 2001). The MPTP model has been used to study the pathophysiology of PD in rodents and non-human primates because of the selective and irreversible loss of dopaminergic neurons, similar to PD patients, under subacute and chronic exposure to MPTP (Sedelis et al. 2001). Though MPTP is widely studied model, the perturbation in cerebral metabolism associated with different cell types in this model is poorly understood. In this study, we have used a novel approach of infusion of [1,6-13C2]glucose or [2-13C]acetate in conjunction with 1H-[13C]-NMR spectroscopy to assess the glutamatergic, GABAergic and astroglial function in different regions of the brain of mice exposed to MPTP. We have also evaluated the efficacy of acute l-DOPA treatment for the recovery of cerebral function in the MPTP model. Our findings indicate that GABA level is increased in the striatum and OB following subacute MPTP treatment in mice. Most importantly, we discovered that the energy metabolism of excitatory and inhibitory pathways is impaired with MPTP neurotoxicity in brain regions that are involved in regulating movement and also in the OB and cerebellum. In addition, we found that the perturbation in energy metabolism could be recovered temporarily following acute l-DOPA therapy in MPTP exposed mice. As per our knowledge, this is the first study demonstrating increased level of GABA in OB in MPTP model of PD and temporary recovery of neural function with acute l-DOPA treatment.

The findings of current study indicate increased ventricular size in the brain of mice treated with MPTP. Larger ventricular size is one of the indicators of cognitive impairment in late Parkinson's subjects (Lewis et al. 2009; Dalaker et al. 2011; Beyer et al. 2013). The finding of increased ventricular size with MPTP exposure suggests progression of PD in mice in line with the sporadic PD. The acute treatment with l-DOPA was unable to reduce the enlarged ventricular size in the brain of mice pre-treated with MPTP implying that l-DOPA therapy would not recover the cerebral atrophy in PD patients.

Maintenance of neurochemical homeostasis is critical for the proper functioning of the brain. The GABAergic system has been known to be involved in PD. Studies as early as in 1976 (Achar et al. 1976; Spokes 1979) reported that the PD patients have detectable amounts of GABA in the CSF which was lacking in healthy individuals and patients with other neurological disorders. The increased level of striatal GABA in PD has been shown in MR studies of human PD patients (Emir et al. 2012) as well as in rat (Coune et al. 2013) and mouse (Chassain et al. 2010) models of PD. In agreement with these reports, we found increased level of GABA in striatum following subacute treatment with MPTP. In addition, we discovered increased level of Glu in the striatum of MPTP-treated mice, which corroborates with the previous findings of higher levels of Glu in the mouse model of PD (Chassain et al. 2008, 2010). Moreover, we discovered elevated level of GABA in the OB following subacute MPTP exposure. Hence, in addition to striatum, GABA homeostasis is also perturbed in OB region of PD brain. In addition, m-inositol level was also found to be increased in the striatum and OB. m-Inositol is a marker for astroglial cells (Galanaud et al. 2003). The finding of increased m-inositol points toward microglial activation and inflammation caused by neurotoxicity induced dopaminergic cell death in the striatum and SNc following exposure to MPTP. The observations of disturbed neurometabolite homeostasis in OB points toward involvement of OB in PD and is in accordance with findings of loss of olfaction several years prior to the manifestation of motor symptoms in PD patients (Brodoehl et al. 2012; Doty 2012; Pouclet et al. 2012). The finding of enhanced level of striatal GABA and m-inositol in subacute MPTP model suggests that GABA and m-inositol could be used as biomarker for early diagnosis of PD. The failure in restoration of the altered neurometabolite homeostasis by acute l-DOPA treatment in MPTP model suggests that the temporary replenishment of DA is not able to bring assertive effects in the PD brain.

Neurons and astroglia work in a concerted manner to bring about normal functioning of the brain. Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the matured brain whose energetics is supported by oxidative glucose metabolism. Imbalance in flux through these pathways is believed to be the cause of many neurological/neurodegenerative disorders. Several studies have pointed toward impaired cerebral energy metabolism in PD patients (Lenzi et al. 1979; Otsuka et al. 1991; Borghammer et al. 2012a, b). However, the implication of loss of DA neurons in SNc on the regional brain activity, especially cortico-basal ganglio-thalamocortical pathway activity at cellular level is poorly understood. 13C NMR spectroscopy together with infusion of [1,6-13C2]glucose, and [2-13C]acetate offers a unique approach for monitoring the neuronal and astroglial activities under different pathological conditions (Henry et al. 2002; Patel et al. 2004; Alexander et al. 2005; Navarro et al. 2005; Bagga and Patel 2012) and during interventions (Bagga et al. 2013). The oxidative metabolism of [1,6-13C2]glucose in glutamatergic and GABAergic neurons labels GluC4 and GABAC2, respectively, which provides a functional measure of glutamatergic and GABAergic neurons, simultaneously. The finding of reduced 13C labeling of GluC4 and GABAC2 from [1,6-13C2]glucose indicates diminished activity of glutamatergic and GABAergic neurons in all the brain regions following MPTP neurotoxicity. This reduced activity in brain regions involved in direct and indirect movement pathways could be because of malfunctioning of basal ganglia owing to loss of dopaminergic neurons. The glutamine synthesis rate is the combined flux of glutamate-glutamine cycle, GABA-Gln cycle and pyruvate carboxylase pathway. The labeling of GlnC4 from [1,6-13C2]glucose represents the total flux of glutamate-glutamine cycle and GABA-Gln cycle. The finding of decreased GlnC4 labeling from [1,6-13C2]glucose is suggestive of reduced neurotransmission in the MPTP exposed mice.

Cerebral cortex, specifically, motor cortices are the regions involved in planning and execution of movement. The basal ganglia interacts with cerebral cortex via the direct and indirect pathways of movement that involve cortico-basal ganglia-thalamo-cortical loop. Therefore, neuronal damage in the striatum/SNc will perturb the activity in cortical and subcortical regions. Reduced metabolism of excitatory and inhibitory neurons in the cerebral cortex following MPTP treatment points toward impaired functioning of the areas which are involved in planning and execution of movement. Reduction in excitatory and inhibitory neuronal function was also found in the major input and output nuclei of basal ganglia, that is, striatum and thalamus, indicating malfunctioning of basal ganglia leading to reduced control of movement in PD. In addition, we also observed reduced GluC4 and GABAC2 labeling in OB suggesting impaired neuronal activity for both glutamatergic and GABAergic neurons with MPTP exposure in this region. The observed hypometabolism of glutamatergic and GABAergic neurons in OB provides the first direct experimental evidence for a dysfunctional OB system and a quantitative measure for the loss of olfaction in PD. Glu, GABA, and DA are the major neurotransmitters in OB (Halasz et al. 1977; Aroniadou-Anderjaska et al. 2000). The reduced excitatory and inhibitory activity together with increased GABA level in MPTP induced Parkinsonism could explain the effect of loss of DA innervations in the OB and hence the reduced DA in the region. To the best of our knowledge, the dysfunction in glutamatergic and GABAergic neuronal activities in OB in the MPTP model of PD is reported for the first time. Another interesting observation of the study is the recovery of labeling of GluC4 and GABAC2 from [1,6-13C2]glucose to control value following l-DOPA therapy in MPTP pre-exposed mice. These data suggest that the supplementation of l-DOPA recovered the function of glutamatergic (excitatory) and GABAergic (inhibitory) neurons in every brain region including striatum.

Astrocytes play an important role in the normal functioning of various circuitries in the brain. In addition to their involvement in neurotransmission, microglia also act as immunocompetent and phagocytic cells (Kim and de Vellis 2005). During neuronal damage, microglia are activated (Block and Hong 2005) and produce various potentially anti-inflammatory compounds for the containment of inflammation (Block and Hong 2007). Increased number of glial cells in the striatum has been shown recently in 6-hydroxydopamine induced lesion (Aponso et al. 2008) and MPTP induced SNc dopaminergic lesion (Tristao et al. 2013). Microglial activation was seen from 1st to 14th day in the SNc and in the striatum following MPTP insult in mice (Czlonkowska et al. 1996; Kohutnicka et al. 1998). Our finding of increased m-inositol level in MPTP-treated mice points toward increased number of microglial cells in the striatum and OB. However, acute treatment of l-DOPA was not able to normalize the elevated m-inositol level suggesting that astroglial population is unaffected with DA supplementation.

Astroglial function could be evaluated using [2-13C]acetate, a glial specific substrate (Deelchand et al. 2009; Patel et al. 2010). [2-13C]Acetate is selectively transported into astrocytes and oxidized therein to label GlnC4. Therefore, the labeling of GlnC4 from [2-13C]acetate provides a quantitative estimate of astroglial activity. The finding of increased labeling of GlnC4 from [2-13C]acetate with MPTP exposure suggests enhanced activity of astrocytes and microglia in the striatum caused by inflammation because of MPTP neurotoxicity. We further found that though acute l-DOPA treatment could normalize the enhanced astroglial activity (Fig. 6), it failed to normalize the increased level of m-inositol under MPTP neurotoxicity. These data suggest that l-DOPA could temporarily subside the microglial activity but does not normalize their number in MPTP model of PD.

DA replacement therapy by l-DOPA treatment has been shown to be quite efficient in managing the symptoms of PD patients in early stages of the disease. We have evaluated the effect of acute l-DOPA on recovery of motor function in MPTP model of PD. Our finding of a rapid and strong response to l-DOPA for the recovery of paw grip strength is quite similar to that observed earlier in animal models of PD (Contin and Martinelli 2010). It is noteworthy that the amount of acute l-DOPA administered to stimulate motor activity in MPTP-treated mice did not have adverse effects in control animals over a short duration. However, l-DOPA-induced dyskinesia has been reported after repeated administration of l-DOPA for a longer period (Calon et al. 2000; Alam and Schmidt 2004; Lundblad et al. 2004).

It has been well established that the rate of neurotransmitter cycling is stoichiometrically coupled to the neuronal glucose oxidation over the long range of brain activity indicating that neuronal energetics is supported by oxidative glucose metabolism. The 13C NMR spectroscopy in combination with the infusion of 13C labeled substrates could identify subtle perturbations in the cerebral function in different neurological diseases (Meisingset et al. 2010; Eyjolfsson et al. 2011; Tiwari and Patel 2012). One of the major advantages of the 1H-[13C]-NMR spectroscopy is that it provides simultaneous measurement of the level of neurometabolites as well as the neural function. Moreover, because of enhanced sensitivity, it also provides higher spatio-temporal resolution in in vivo measurements. The application of 1H-[13C]-NMR spectroscopy in neurodegenerative disorders like Alzheimer's, Huntington's, Amyotophic Lateral Sclerosis will provide better insights about pathophysiology of disease which may yield biomarkers that will aid in the preclinical diagnosis of these disorders.

Conclusion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Loss of dopaminergic neurons because of MPTP neurotoxicity in SNc led to reduction in the paw grip strength, increased ventricular size and perturbation in the homeostasis of neurometabolites in striatum and OB. Furthermore, loss of DA in striatum resulted in compromised activity of excitatory and inhibitory neurons across the brain including in the striatum, while astroglial activity was enhanced only in the striatum. Acute l-DOPA therapy in the MPTP model of PD restored the paw grip strength, and neural function across the brain but failed to normalize the ventricle size and altered neurochemical homeostasis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

We thank Dr. Robin A. de Graaf, Yale University for providing the 1H-[13C]-NMR sequence, Mr. Dwarkanath for breeding and providing mice, Mr. Bhargidhar Babu for assistance in metabolic study, Ms. Rajeeva Voleti for help with the extraction of metabolite from brain tissue and Ms. Judith M. Noronha for critical reading of the manuscript. All NMR experiments were performed at NMR Microimaging and Spectroscopy Facility, CSIR-CCMB, Hyderabad, India. PB gratefully acknowledges the Senior Research Fellowship from CSIR. The authors declare no conflict of interest. This study was supported by funding from DST SR/SO/BB-63/2008 and CSIR network project BSC0115.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information
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jnc12407-sup-0001-FigS1.pdfapplication/PDF162KFigure S1. Rate of glucose oxidation in different brain regions following different treatments.

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