Metabolism of [1,6-¹³C]glucose in the cerebellum of 18-day old rats: Comparison with cerebral metabolism

Abstract

There is little information on metabolism in developing cerebellum despite the known importance of this region in cognition and motor tasks. Ex vivo ¹H- and ¹³C-NMR spectroscopy were used to determine metabolism during late postnatal development in cerebellum and cerebrum from 18-day old rat pups after intraperitoneal (i.p.) injection of [1,6-¹³C]glucose. The concentration of several metabolites in cerebellum was distinctly different than cerebrum; alanine, glutamine, creatine and myo-inositol were higher in cerebellum than cerebrum, the concentrations of lactate, GABA, aspartate and N-acetylaspartate (NAA) were lower in cerebellum than in cerebrum, and levels of glutamate, succinate, choline and taurine were similar in both brain regions. The incorporation of label from the metabolism of [1,6-¹³C]glucose into most isotopomers of glutamate (GLU), glutamine (GLN), GABA and aspartate was lower in cerebellum than in cerebrum. Incorporation of label into the C2 position of lactate via the pyruvate recycling pathway was found in both brain regions. The ratio of newly synthesized GLN/GLU was significantly higher in cerebellum than in cerebrum indicating relatively active metabolism via glutamine synthetase in cerebellar astrocytes at postnatal day 18. This is the first study to determine metabolism in the cerebellum and cerebrum of male and female rat brain.

Introduction

The cerebellum is indirectly connected to both cortical and subcortical regions of the cerebrum and plays an integrative role in brain function (Andreasen and Pierson, 2008). Cerebellar function was classically conceived as largely motor, based on tedious observational study of the effect of lesions on changes in motor function in animals (reviewed in (Peterson, et al., 2012). A role in cognition is now well accepted, as visualization of cerebellar activity and metabolic changes during language processing and problem solving confirmed involvement in non-motor tasks (Bloedel and Bracha, 1997, Kim, et al., 1994, Peterson, et al., 2012, Yang, et al., 2014).

The diverse population of neurons and astrocytes in the cerebellum converge at the level of the GABAergic Purkinje cells, which comprise the sole output of the cerebellar cortex and vermis (Altman, 1982, Altman and Das, 1966, Takayasu, et al., 2006, Takayasu, et al., 2009). Glutamatergic granule neurons are the most abundant cell type in the cerebellum, and a complex sequence of events between these principal neurons and astrocytes regulate presumably permanent patterns of cerebellar wiring through the first three weeks of postnatal development (Nakayama, et al., 2012, Takayasu, et al., 2009). Purkinje cells have high metabolic rates primarily to restore membrane potential in the many dendrites after tonic firing to inhibit motor nuclei in cerebellum (Howarth, et al., 2012, Howarth, et al., 2010, Connolly, et al., 2007, Welsh, et al., 2002). GABAergic inhibition of Purkinje cells by molecular layer interneurons regulates the elimination of surplus climbing fiber synapses between postnatal day 10 to 16 in the developing cerebellum (Nakayama, et al., 2012). The maturation of oxidative metabolism in brain, including the pyruvate dehydrogenase complex and increased incorporation of carbons from glucose into amino acids including glutamate and GABA, which occurs in concert with the development of parallel fiber synapses on Purkinje cells, has been proposed to have a role in the maturation of cerebellar function in rat brain (Butterworth and Giguere, 1984).

Metabolism provides the energy to support all brain functions, including production of precursors for neurotransmitter, lipid and protein synthesis, processes that are particularly high in developing brain (McKenna, et al., 2011). ¹H-NMR spectroscopy can be used to determine the concentration of many key metabolites in brain (Sanches, et al., 2019, Shi, et al., 2012, Xu, et al., 2015), but it does not provide insight into the dynamic trafficking of metabolites from neurons to astrocytes and astrocytes to neurons that is required for brain function. ¹³C-NMR spectroscopy is the only technique that can determine the metabolism via cell specific pathways in neurons and astrocytes in brain (Haberg, et al., 1998, McKenna, et al., 2011, Sonnewald and Kondziella, 2003). Studies of metabolism in developing brain have largely focused on the cerebral cortex (cerebrum), often to determine changes after injury or other pathological conditions, or metabolism via specific pathways (Chowdhury, et al., 2007, Morken, et al., 2014a, Morken, et al., 2014b, Scafidi, et al., 2009). Many groups have determined the concentration of key metabolites and neurotransmitters in specific brain regions (Kulak, et al., 2010, Sanches, et al., 2019, Shi, et al., 2012, Tkac, et al., 2003, Xu, et al., 2015). However, relatively few studies used ¹³C-NMR to determine metabolism in different brain regions using in vivo dynamic ¹³C-MRS (Cherix, et al., 2020, de Graaf, et al., 2004, Lizarbe, et al., 2018) or ex vivo ¹³C-NMR spectroscopy (Eloqayli, et al., 2003, Eyjolfsson, et al., 2011, Haberg, et al., 2009). Chowdhury et al. (Chowdhury, et al., 2007) infused [1,6-¹³C]glucose and [2-¹³C]acetate and found lower TCA cycle activity and glutamate-glutamine cycling in cortex from 10 day old (P10) rat brain compared to 30 day old (P30) brain. From P10 to P30 the neuronal TCA cycle flux increased ~ 3-fold and glutamatergic neurotransmitter cycling increased from 3.1-5 fold. GABAergic metabolism increased more steeply during the same time frame with ~ 5 fold increase in the TCA cycle and ~ 7 fold increase in glutamate-glutamine cycling (Chowdhury, et al., 2007). Their findings were consistent with an earlier report of active glutamine synthesis and astrocyte-neuron interactions in developing brain (Nehlig and Pereira de Vasconcelos, 1993).

It should be noted that in contrast to the study by Chowdhury et al. (Chowdhury, et al., 2007) that used in vivo dynamic ¹³C-MRS, in conjunction with data modeling to provide metabolic flux values, the ex vivo ¹³C-NMR used in the present study and studies by other groups (Eloqayli, et al., 2003, Eyjolfsson, et al., 2011, Haberg, et al., 2009, Scafidi, et al., 2009) used a bolus injection in unanesthetized rats with brain extraction early after injection to determine metabolism in the first phase of a labelling study well before a steady-state is reached. Each of these techniques, in vivo dynamic ¹³C-MRS and ex vivo ¹³C-NMR, has its advantages and limitations and both methods are frequently used to determine metabolism in brain.

Clinical positron emission tomography (PET) studies of ¹⁸F-deoxyglucose uptake show a high rate of glucose metabolism in cerebellum and subcortical areas of newborn infants compared to neocortex (Suhonen-Polvi, et al., 1993). Neuronal and astrocytic glutamate transporters change in a coordinated fashion during cerebellar development (Takayasu, et al., 2005, Takayasu, et al., 2006, Takayasu, et al., 2009) underscoring the importance of dynamic metabolic interactions between these brain cells. Despite the protracted postnatal development of this region (Nakayama, et al., 2012, Takayasu, et al., 2005) and the essential role of glia in proper wiring of connections onto Purkinje cells (Miyazaki, et al., 2017) there is no information about metabolism via neuronal and astrocyte specific pathways in developing cerebellum. Metabolite concentrations in cerebellum of 30-day old (Melo, et al., 2007) and 50-day old rats (Sanches, et al., 2019) have been reported; however, no ¹³C-NMR studies of cerebellar metabolism in developing brain have been reported.

We studied cerebrum and cerebellum from 18-day old rat brain because our group has a strong interest in the metabolism that supports brain maturation during postnatal development. Studies were done at postnatal day 18 which is a period of active synaptogenesis and rapid myelination in both regions studied and maturation of cerebellar connections including formation and remodeling of connections between climbing fibers and Purkinje cells in cerebellum of developing rat brain (Nakayama, et al., 2012, Takayasu, et al., 2009). Ex vivo ¹H-and ¹³C-NMR spectroscopy were used to compare metabolism in cerebellum and cerebrum from 18-day old male and female rat pups (n=16; 8 males and 8 females) at 30 minutes after intraperitoneal (i.p.) injection of [1,6-¹³C]glucose (Morken, et al., 2014b).

Methods:

Animals

Timed-pregnant female Sprague-Dawley rats (RRID:RGD_10395233) were obtained from Charles River (Frederick, MD, USA). This study was approved by the University of Maryland, Baltimore, Animal Care and Use Committee (IACUC Protocol #0518009). All care and handling of rats was in compliance with the National Institutes of Health guidelines. The day of birth was considered postnatal day 1 (PND). PND 18 male (8) and female (8) Sprague–Dawley rats were used in the studies. The sample size needed was determined from prior NMR studies by our group and others. We have previously determined that a sample size of 8/sex/group is sufficient for ¹³C-NMR studies (Scafidi, et al., 2009). Rats were arbitrarily selected from 3 different litters, with a maximum of 3 rat pups of each sex used from any of the litters. The study was not blinded as all rats were used and there were no treatment groups. However, the extraction of brain regions and preparation of samples for NMR was performed in a blinded fashion with regards to the sex of the rats. The study was not pregistered.

Biochemicals

The [1,6-¹³C]glucose (CLM-2717-0, 99% ¹³C enriched) and sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TMSP, DLM-48-1, 98%) were obtained from Cambridge Isotope Laboratories, Woburn, MA, USA. Dioxane (D111-500, 99.9%) was obtained from Thermo Fisher Scientific, Waltham, MA, USA. Perchloric acid (244252-500ML, 70%) was obtained from Sigma-Aldrich, St. Louis, MO.

Administration of labeled [1,6-¹³C]glucose

On PND 18 rats were weighed, given intraperitoneal injections of [1,6-¹³C]glucose (543 mg/kg) in early afternoon and euthanized by rapid decapitation 30 min after injection (Brekke, et al., 2012, Morken, et al., 2014b). It should be noted that our paradigm differed from Morken et al. (Morken, et al., 2014b) which used [1-¹³C]glucose and [1,2-¹³C]acetate as labeled substrates. At the time of the experiment pups were housed with dams and had free access to food and water. No anesthesia was used as it is known to decrease brain metabolism (Sibson, et al., 1998). Brains were rapidly removed and the cerebrum (cerebral cortex, including the subcortical structures, i.e., hippocampus, thalamus, hypothalamus, parietal and motor areas) and cerebellum were removed and snap-frozen in liquid nitrogen. The time from decapitation to freezing brain regions was less than 30 seconds, and the order of freezing cerebral cortex and cerebellum was alternated. All samples were stored at −80°C until extraction.

Tissue extraction

The frozen tissue samples, cerebellum and cerebrum samples from each animal, were homogenized in 2 mL ice-cold 7% perchloric acid (PCA) and extracted as described by Richards et al. (Richards, et al., 2007). Neutralized, lyophilized extracts were stored at −80°C.

NMR spectroscopy

Lyophilized samples were reconstituted in deuterium oxide (D₂O) containing 0.25% dioxane and 0.02% sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TMSP) as internal standards for quantification of ¹³C and ¹H spectra, respectively, and adjusted to pH 6.95-7.05. ¹H spectra and proton-decoupled ¹³C-NMR spectra were obtained on a Bruker AVANCE III 950 MHz NMR spectrometer at the NMR Center, University of Maryland Baltimore. Fully relaxed ¹H-NMR spectra (64 scans) were acquired with a 90° pulse angle, acquisition time of 2.7 sec, and relaxation delay of 20 sec. Chemical shifts for ¹H-spectra are reported relative to the TMSP at 0.0 ppm. ¹³C-spectra were acquired at 25°C using a 35° pulse angle, 25 KHz spectra width, and 64 K data points, with an acquisition time 0.99 sec, and 3 sec relaxation time. The number of scans was typically 2400 for cortex and 6400 for cerebellum. ¹³C-spectra were corrected for nuclear Overhauser effects (nOe) (Richards et al. 2007) and relaxation time (Melo, et al., 2007). Additional brain samples from rat pups injected with [1,6-¹³C]glucose were used to obtain data from spectra decoupled only during acquisition and run with a 25.7 sec relaxation time to obtain combined nOe and relaxation time correction factors for each isotopomer as described by Melo (Melo, et al., 2007). A line broadening of 5 Hz was used. Chemical shifts are reported relative to the dioxane peak at 67.4 ppm, and peak assignments were made by comparison to spectra from ¹³C standards and literature values. All peaks in ¹³C-NMR spectra were corrected for natural abundance and data reported as nmol ¹³C incorporated/mg protein.

Labeling from the metabolism of [1,6-¹³C]glucose in brain

Brain metabolites are rapidly labeled after intraperitoneal injection of [1,6-¹³C]glucose. The labeling pattern from glucose metabolism via glycolysis and the first turn of the TCA cycle is shown in Figure 1. [1,6-¹³C]Glucose is metabolized via the glycolytic pathway to form [3-¹³C]pyruvate, which can give rise to [3-¹³C]lactate and [3-¹³C]alanine. Pyruvate is primarily metabolized via pyruvate dehydrogenase (PDH) to form [2-¹³C]acetyl CoA which enters the TCA cycle and condenses with oxaloacetate to form citrate. Metabolism in the TCA cycle leads to labeling of [4-¹³C]α-ketoglutarate in the first turn, which can be converted to [4-¹³C]glutamate (GLU C4) in neurons; this labeling is typically stronger in neurons but also occurs in astrocytes (Sonnewald and Kondziella, 2003). After release by neurons, uptake of GLU C4 into astrocytes followed by metabolism via glutamine synthetase leads to formation of the corresponding [4-¹³C]glutamine (GLN C4) in astrocytes. GLU C3 is labeled from metabolism in the second turn of the TCA cycle and can be converted to GLN C3 in astrocytes. Due to randomization of the C3 label in the symmetrical molecule succinate equal labeling in GLU C2 is formed in the second turn and subsequently GLN C2 also occurs. The GLU C4 formed in neurons can be converted directly to [2-¹³C]GABA (GABA C2). Later in the first turn of the TCA cycle [3-¹³C]oxaloacetate is formed which can give rise to [3-¹³C]aspartate (ASP C3). In astrocytes, metabolism of glucose occurs via the pyruvate carboxylase (PC) pathway and also via PDH. Metabolism via PC leads to the formation of [2-¹³C]glutamate and incorporation of unlabeled carbon into GLU and GLN C3. This differential labeling in GLU/GLN C2 and C3 allows for estimation of the metabolism via the PC pathway. However, due to backflux of the TCA cycle some [3-¹³C]GLU is also formed from metabolism via PC, thus calculation of PC from the differential labeling in GLN C2/GLU C2 and GLN C3/GLU C3 provides a minimal value for labeling from metabolism via PC. Labeling in GLU C3 that occurs via PC can not be distinguished from the GLU C3 formed in the second turn of the neuronal TCA cycle. Glutamine C2 and C3 formed via PC is released by astrocytes and taken up by neurons where it can be converted to GLU C2 and GLU C3 by phosphate activated glutaminase. The GLU C2 formed from the precursors synthesized in astrocytes, or from labeling in the second turn of the neuronal TCA cycle, can subsequently be converted to GABA C4 in neurons. Labeling in GABA C3 is from metabolism of GLU C3 formed in neurons, and from the metabolism of the GLU C3 formed in astrocytes.

Figure 1. A simplified figure of labeling from metabolism of [1,6-¹³C]glucose in brain.

Labeling pattern from the metabolism of [1,6-¹³C]glucose via glycolysis, pyruvate dehydrogenase (PDH) and metabolism in the first turn of the TCA cycle and subsequent formation of glutamate is shown in black circles. Formation of glutamate C4 (GLU C4) from metabolism in the first turn of the TCA cycle, which is stronger in neurons but also occurs in astrocytes, and subsequent conversion to glutamine C4 (GLN C4) in astrocytes is indicated by black circles. Further metabolism of α-ketoglutarate C4 (α-KG) in the TCA cycle results in randomization of the label in the symmetrical molecule succinate and leads to formation of malate and oxaloacetate (OAA) equally labeled in the C2 and C3 positions (half-filled black circles). OAA can remain in the TCA cycle or be transaminated to form aspartate (ASP C2 and C3). Labeling of OAA from metabolism via the pyruvate carboxylase (PC) pathway in astrocytes leading is shown in red circles. Backflux of OAA C3 labeled via PC leads to partial labeling of OAA C2 (partially filled red circle). Metabolism of OAA labeled via PC leads to labeling of GLU and GLN in the C2 indicated by red circles. Some labeling in the C3 positions of GLU and GLN occurs from the backflux labeling in OAA. GABA C2 is formed from glutamate labeled via PDH in the first turn of the TCA cycle (black circles) and GABA C4 and some GABA C3 is labeled from precursors formed via PC (red circles). For simplicity labeling from the first turn of the TCA cycle is shown and labeling of GLU and GLN from the second turn is shown. It should be noted that GLU C3 and C2 is also labeled in the second turn of the TCA cycle and GABA C3 can be formed from GLU C3 formed in neurons. Additional details of the labeling and pathways are given in methods section. Abbreviations: AAT, aspartate aminotransferase; GABA, ɣ-aminobutyric acid; GAD, glutamic acid decarboxylase; GLU, glutamate; GLN, glutamine; GS, glutamine synthetase; OAA, oxaloacetate; PDH, pyruvate dehydrogenase complex; PC, pyruvate carboxylase.

Anaplerotic ratio, which is a measure of labeling from metabolism via the PC pathway in astrocytes was calculated from the labeled isotopomers as described by Rae et al. (Rae, et al., 2009). The equation for the anaplerotic ratio for aspartate was (ASP C3-ASP C2)/ ASP C2. The PC/PDH ratio was calculated for glutamine (GLN C2-GLN C3)/GLN C4 as described by Haberg et al. (Haberg et al., 2009).

Determination of protein and metabolite concentration

The protein content of the pellets from the PCA extracts of the brains was determined using the Pierce BCA microreagent assay (Smith, et al., 1985). Concentrations of amino acid and other metabolites were determined from the fully relaxed ¹H NMR spectra (Morken, et al., 2014a). In addition, amino acids were measured on a Biochrom 30 amino acid analyzer by ion exchange high-performance liquid chromatography with post-column ninhydrin detection (Duran, 2008, Shapira, et al., 1989). This additional analysis was done to correct for any ¹³C sideband contamination of peaks in the ¹H spectra. The amino acid analysis concentration values have been used rather than the ¹H spectra. However, some samples were not available for amino acid analysis. To correct for sideband contamination in these samples. correction factors determined for each of the amino acid peaks in the ¹H spectra from the dataset of samples with both amino acid analysis were determined. Since there was very little variability in the ¹H spectra data, the regional correction factors determined were applied to the ¹H values for samples that were not available for amino acid analysis.

Statistical analysis

Paired sampled t-tests were used to determine differences in the concentration of metabolites between the cerebellum and cerebrum after confirming the normality of variables using the Shapiro-Wilk test of normality (IBM® SPSS® Statistics Version 24.0, 2016). The cerebellum and cerebrum from each animal were coded as a pair. Paired sampled t-tests were also used to determine differences in compounds labeled from metabolism of [1,6-¹³C]glucose, and in percent enrichment between cerebellum and cerebrum. Variables that did not have normal distributions were evaluated using non-parametric Wilcoxon Signed Ranked tests. ANOVA was used to determine the presence of sex differences in normally distributed data. When no sex differences were detected, data were pooled across sex. Mann-Whitney U was used to test for comparisons within brain regions and Wilcoxon Signed Ranks were used for comparisons between brain regions. The choline concentration data contained two outliers (concentration > group mean ± 3*σ) in the cerebellum group; data from these outliers were removed. Two animals with negative ratios were not included in the analysis of cycling ratios. Results are presented as means ± SEM.

Results

Concentration of metabolites in cerebellum and cerebrum

Data for metabolite concentrations and incorporation of label into newly synthesized metabolites were analyzed separately to determine differences in male and female brain. High-resolution ¹H-NMR spectra of extracts of cerebellum and cerebrum from 18-day old male and female rats were obtained. Figure 2a shows representative spectra from these brain regions. The concentration of several metabolites in cerebellum was distinctly different than cerebrum as shown in Figure 3. The concentrations of alanine, glutamine, creatine and myo-inositol were higher in cerebellum than cerebrum, whereas the concentrations of lactate, GABA, aspartate and N-acetylaspartate (NAA) were lower in cerebellum than in cerebrum from 18-day old rat brain. Total levels of glutamate, succinate, choline and taurine were similar in both brain regions.

Figure 2.

Partial high resolution (a) ¹H-NMR and (b) ¹³C-NMR spectra of perchloric extracts of cerebellum (top) and cerebrum (bottom) from PND 18 male and female rat brain. A line broadening of 5 Hz was used. Chemical shifts for ¹H-NMR spectra are reported relative to the TMSP peak at 0.0 ppm; chemical shifts for ¹³C-NMR spectra are reported relative to the dioxane peak at 67.4 ppm. Abbreviations: For ¹H-spectra: Glu, glutamate; Gln, glutamine; Asp, aspartate; NAA, N-acetylaspartate; Cr, creatine; Cho, choline; Tau, taurine; Myo Inos; myoinositol. For ¹³C-spectra: Ala C3, alanine labeled in the C3 position; Lac C3; lactate labeled in the C3 position; GABA C3, C2, C4, GABA labeled in the C3, C2 and C4 positions, respectively; Gln C3, C4, C2, glutamine labeled in the C3, C4 and C2 positions, respectively; Glu C3, C4, C2, glutamate labeled in the C3, C4 and C2 positions, respectively; Succ 2/3, succinate labeled in C2 or C3 (indistinguishable as it is a symmetrical molecule); Asp C3, C2, aspartate labeled in the C3 and C2 position, respectively. Details for acquisition of ¹H-NMR and ¹³C-NMR spectra are in the methods section.

Figure 3.

Concentration of metabolites in cerebellum (white bars) and cerebrum (gray bars) from 18-day old rat brain. Values were obtained from ¹H spectra and concentrations calculated from the internal standard TMSP as described in the methods. Values for amino acids were determined using an amino acid analyzer or in some cases from ¹H spectra data corrected for ¹³C sideband contamination as described in the methods section. Individual data points in cerebellum are indicated by circles and in cerebrum by triangles. The concentrations of glutamine, creatine and myo-inositol were higher in cerebellum than cerebrum. In contrast, the concentrations of lactate, N-acetylaspartate (NAA) and GABA were lower in cerebellum than in cerebrum. Data from cerebellum and cerebrum from 16 rat pups were analyzed by paired Student’s t-test. *** p < 0.001, * p < 0.05

Male and Female Brain

There were no differences in the concentration of metabolites in cerebrum from 18-day old male and female rat brain. In contrast, the concentration of aspartate was lower in cerebellum from female rats compared to male rat brain (Figure 4). Values for aspartate were 22.9 ± 1.3 and 29.0 ± 1.3 nmol/mg protein, for female and male cerebellum, respectively; Z = −2.699, p = 0.007. The concentration of all other metabolites in cerebellum were comparable in male and female rat brain. There were no sex differences in the incorporation of label from metabolism of [1,6-¹³C]glucose into any metabolites in 18-day old male and female rat brain. Therefore, the data from male and female brain were combined for comparison of differences in cerebellum and cerebrum.

Figure 4.

Concentration of aspartate in cerebellum and cerebrum from 18-day old female (white) and male (grey) rat brain. Values represent means ± SEM of data from cerebrum and cerebellum from 8 male and 8 female brains. Data from cerebellum and cerebrum were analyzed by paired Wilcoxin signed rank test: Female cerebellum vs. female cerebrum, Z= −2.366, p=0.018; Male cerebellum vs. male cerebrum, Z= −2.666, p= 0.008. Sex differences within regions were analyzed by Mann-Whitney U test: Female vs. Male cerebellum, Z=−2.699, p= 0.007; Female vs. Male, Cerebrum, Z= −1.217, p=0.223

Incorporation of label from the metabolism of [1,6-¹³C]glucose into glutamate, glutamine and GABA in cerebellum and cerebrum

Representative ¹³C-NMR spectra obtained from cerebellum and cerebrum are shown in Figure 2b. A 30 minute time point for labelling with [1,6-¹³C]glucose was used for this study as oxidative metabolism of glucose is lower in developing brain (Morken, et al., 2014b), and the time for conversion of TCA cycle intermediates to glutamate, glutamine and GABA is longer in developing brain than in adult brain (Chowdhury, et al., 2007, Morken, et al., 2014b). The amount of unmetabolized [1,6-¹³C]glucose was significantly higher in cerebellum than in cerebrum of 18-day old rat brain. α-Glucose C1 was 0.671 ± 0.048 and 0.439 ± 0.044 nmol/mg protein in cerebellum and cerebrum, respectively (t(15)= 3.818, p = 0.002). However, there was no difference in the percent enrichment of 41.71 ± 3.59 and 36.76 ± 3.08 percent in cerebellum and cerebrum, respectively, (Z = −1.138, p = 0.255).

In the 18-day old rat brain label from the metabolism of [1,6-¹³C]glucose was detected in isotope isomers (isotopomers) of glutamate, glutamine, GABA, aspartate, lactate, alanine and succinate. Incorporation of label into C3 position of lactate (LAC C3), from the pyruvate formed via glycolysis, was significantly lower in cerebellum than in cerebrum. LAC C3 was 10.47 ± 0.47 and 16.20 ± 0.91 nmol ¹³C incorporated/mg protein in cerebellum and cerebrum, respectively; t(15)= −9.896, p<0.001 (Figure 5a) The formation of [3-¹³C]alanine at 30 minutes after labeled glucose injection was lower in cerebellum than in cerebrum. ALA C3 was 1.36 ± 0.06 and 1.82 ± 0.08 in ¹³C incorporated/mg protein in cerebellum and cerebrum, respectively; Z(15) = −3.516, p<0.001); Figure 5b. However, the ratio of newly synthesized ALA C3/LAC C3 was slightly but significantly higher in cerebellum than in cerebrum, t(15)=−2.339, p=0.034.

Figure 5a & b.

Incorporation of label from metabolism of [1,6-¹³C]glucose via glycolysis into newly synthesized lactate (LAC) and alanine (ALA C3) in cerebellum (white bars) and cerebrum (grey bars) from 18-day old rat brain. Values are mean ± SEM nmol ¹³C incorporated/mg protein from n =16 rat pups. Data from cerebellum and cerebrum were analyzed by paired Student’s t-test and by Wilcoxin Signed Ranked test. *** p < 0.001, ** p < 0.01

The incorporation of label into the C4, C3 and C2 carbons of glutamate was lower in cerebellum than in cerebrum (Figure 6a); GLU C4: t(15) = −6.120, p< 0.001; GLU C3: t(15) = −7.157, p< 0.001 and GLU C2: Z = −3.516, p<0.001. Incorporation of label into the C4, C3 and C2 positions of glutamine was also lower in cerebellum than in cerebrum (Figure 6b); GLN C4: t(15)= −4.180, p= 0.001; GLN C3: t(15)= −2.399, p=0.030; GLN C2: t(15)= −3.101, p= 0.007.

Figure 6a & b.

Incorporation of label from metabolism of [1,6-¹³C]glucose into newly synthesized isotopomers of glutamate (a) and glutamine (b) in cerebellum (white bars) and cerebrum (grey bars) from 18-day old rat brain. Note the difference in scale of the y-axis for glutamine compared to glutamate. Values are mean ± SEM nmol ¹³C incorporated/mg protein from n =16 rat pups. Data from cerebellum and cerebrum were analyzed by paired Student’s t-test and by Wilcoxin Signed Ranked test. *** p < 0.001, ** p < 0.01, * p < 0.05

Ratios of newly synthesized compounds in brain can provide additional insight into differences in regional metabolism. In 18-day old rat brain the overall synthesis of glutamate and glutamine was lower in cerebellum than cerebrum. However, the higher ratio of total newly synthesized GLN to GLU, t(15)= 6.594, p<0.001, in cerebellum than in cerebrum (Figure 7), demonstrates that there is particularly active metabolism via glutamine synthetase in cerebellar astrocytes at postnatal day 18. The higher ratio of newly synthesized GLN/GLU was evident in all isotopomers, as shown in Figure 7. GLN C4/GLU C4, Z = −2.275, p < 0.023; GLN C3/ GLU C3, Z = −2.689, p=0.007; GLN C2/ GLU C2, t(15) = 6.798, p< 0.001.

Figure 7.

Ratio of newly synthesized glutamine to glutamate in cerebellum (white bars) and cerebrum (gray bars) from 18-day old rat brain (n = 16). Values are mean ± SEM. Data comparing cerebellum and cerebrum were analyzed by Wilcoxin Signed Ranked test. *** p < 0.001, ** p < 0.01, * p = 0.023

The incorporation of label from the metabolism of [1,6-¹³C]glucose into the C2, C3 and C4 positions of GABA was lower in cerebellum than in cerebrum (Figure 8); GABA C2: Z = −3.309, p<0.001, GABA C3: Z = −2.430, p=0.015, GABA C4 t(15)=−2.304, p= 0.036. Although the labeling of newly synthesized GLU and GABA were significantly lower in cerebellum than in cerebrum, the ratio of total newly synthesized GABA/GLU was not different in these regions, Z(15)= −1.138, p=0.255; and was 0.15 ± 0.01 and 0.13 ± 0.01 in cerebellum and cerebrum, respectively. In the short timeframe of this ex vivo study the ratio of GABA C2/GLU C4 likely reflects direct formation of GABA from neuronally synthesized glutamate, which was similar in both regions; Z = −1.344 p=0.179 (data not shown).

Figure 8.

Incorporation of label from metabolism of [1,6-¹³C]glucose into newly synthesized isotopomers of GABA in cerebellum (white bars) and cerebrum (grey bars) from 18-day old rat brain. Values are mean ± SEM nmol/¹³C incorporated/mg protein. Data from cerebellum and cerebrum were analyzed by paired Student’s t-test and and by Wilcoxin Signed Ranked test. *** p < 0.001, ** p = 0.006, * p < 0.036.

Incorporation of label from the metabolism of [1,6-¹³C]glucose into succinate and aspartate

The incorporation of label from the metabolism of [1,6-¹³C]glucose into the indistinguishable C2/3 position of the symmetrical TCA cycle intermediate succinate was lower in cerebellum than in cerebrum (Figure 9); Succ C2/3: t(15)=−3.564, p=0.003. The incorporation of label from the metabolism of [1,6-¹³C]glucose into the C2 and C3 positions of aspartate was lower in cerebellum than in cerebrum (Figure 9); ASP C2: t(15)=−9.472, p<.001, ASP C3: t(15)=−9.583, p<.001. Asp C3 was 2.54 ± 0.15 and 5.46 ± 0.35 nmol ¹³C incorp/mg protein in cerebellum and cerebrum, respectively; t(15)= −9.583, p<.001. Asp C2 was 2.23 ± 0.15 and 5.03 ± 0.313 nmol ¹³C incorp/mg protein in cerebellum and cerebrum, respectively; t(15)= −9.472, p<.001.

Figure 9.

Incorporation of label from metabolism of [1,6-¹³C]glucose into newly synthesized isotopomers of aspartate (ASP), succinate (Succ 2/3), and Lactate C2 (Lac C2) in cerebellum (white bars) and cerebrum (grey bars) from 18-day old rat brain. Labeling in the C2 and C3 positions of succinate are indistinguishable as succinate is a symmetrical molecule. Values are mean ± SEM nmol/¹³C incorporated/mg protein. Data from cerebellum and cerebrum from n = 16 rat pups were analyzed by paired Student’s t-test. *** p < 0.001, ** p < 0.003

Anaplerosis and metabolism via the pyruvate recycling pathway

Anaplerotic ratios are a measure of labeling from metabolism via the pyruvate carboxylase pathway in astrocytes (Rae, et al., 2009). The anaplerotic ratios calculated from labeling in aspartate (ASP C3-ASP C2)/ ASP C2 of 0.16 ± 0.06 and 0.08 ± 0.02 in cerebellum and cerebrum, respectively, were not different Z = −1.475, p=0.140 (Figure 10). When [1,6-¹³C]glucose is used as a precursor metabolism via the pyruvate carboxylase pathway leads to ¹³C labeling in the C2 position of GLU, it also incorporates the same amount of unlabeled ¹²C into the GLU C3 pool which leads to an apparent “dilution” of ¹³C GLU C3 pool and the subsequent GLN formed in astrocytes. Thus the difference in C2 and C3 labeling in GLN can be used to calculate anaplerosis which makes it possible to assess the contribution of metabolism via pyruvate carboxylase (Henry, et al., 2006, Rae, et al., 2009). The relative amount of metabolism via PC/PDH for glutamine (GLN C2-GLN C3)/GLN C4 (Haberg et al., 2009) was 0.23 ± 0.07 and 0.24 ± 0.04 in cerebellum and cerebrum suggesting relatively active metabolism via the pyruvate carboxylase pathway at the less mature cerebellum at this age.

Figure 10.

Anaplerotic ratio of metabolism via pyruvate carboxylation in aspartate. in cerebellum (white bars) and cerebrum (gray bars) from 18-day old rat brain. Ratios were calculated using the following equation for ASP (ASP C3-ASP C2)/ASP C2 (Rae, et al., 2009). Values are mean ratio ± SEM for n=14 for ASP. Data comparing the ratios cerebellum and cerebrum were analyzed by Wilcoxin Signed Ranked test. *** p < 0.001, ** p < 0.01

Evidence of metabolism via the neuroprotective pyruvate recycling pathway (labeling in LAC C2) was present in both cerebellum and cerebrum of 18-day old rat brain (Figure 9). Lac C2, which can only be labeled from [1,6-¹³C]glucose by metabolism via the pyruvate recycling pathway, was 0.39 ± 0.04 and 0.63 ± 0.04 nmol ¹³C incorp/mg protein in cerebellum and cerebrum, respectively; t(15)= −5.446, p<0.001.

Cycling ratios

Calculation of cycling ratios of labeling from the second turn of the TCA cycle, relative to labeling from the first turn of the cycle revealed an interesting difference between cerebellum and cerebrum at 18 days of age (Supplemental Figure 1). In ex vivo bolus injection studies with short delay before brain extraction, these ¹³C position ratios are considered to be good approximations of upstream fluxes (Qu, et al., 2003, Sonnewald and Kondziella, 2003). Although GLU C3 and C4 are also labeled from astrocyte metabolism particularly in longer infusion studies, in the short timeframe of this ex vivo study the synthesis of GLU C4 and GLU C3 is primarily labeled from the first turn of the TCA cycle in neurons (Sonnewald and Kondziella, 2003). The cycling ratio for glutamate (GLU C3/GLU C4) was lower in cerebellum than in cerebrum, t(15)= −3.949, p=0.001, demonstrating relatively slower TCA cycle metabolism in glutamatergic neurons in cerebellum than in cerebrum.

The cycling ratio GLN C3/GLN C4 labeled from the metabolism of glucose has been used by many groups (Eloqayli, et al., 2003, Qu, et al., 2003, Richards, et al., 2007, Scafidi, et al., 2009) as an accepted measure of relative metabolism in astrocytes. In contrast to the ratio of GLU, the cycling ratios for glutamine (GLN C3/GLN C4) and for GABA (GABA C3/GABA C2) were not significantly different in cerebellum and cerebrum; Z = −0.155, p= 0.877 and t(15)= 1.496, p=0.162, for glutamine and GABA respectively. The lack of a regional difference in the cycling ratios for glutamine demonstrates that, in contrast to the lower labeling of glutamine in cerebellum, the rate of metabolism in cerebellar astrocytes is comparable to that in cerebrum at 18 days of age.

Percent enrichment

The percent enrichment reveals how much of a given isotopomer (isotope isomer; e.g. Glutamate C4) is labeled relative to the entire pool of that metabolite (e.g., total glutamate pool). The percent enrichment for all isotopomers of glutamate, glutamine, lactate, alanine, aspartate and succinate was lower in cerebellum than in cerebrum (Table 1), reflecting the overall higher metabolic rate in cerebrum at 18 days of age. The enrichment in C3 alanine was considerably lower in cerebellum than in cerebrum. The lower enrichment in [3-¹³C]alanine is not directly related to the difference in lactate C3 enrichment, although both reflect lower glycolysis in cerebellum. The lower enrichment reflects the combination of lower labeling of ALA C3 from glycolysis and subsequent transamination in cerebellum and the higher concentration of alanine in cerebellum compared to cerebrum, which would lead to a much lower percent enrichment. The lower percent enrichment in all isotopomers of glutamine in cerebellum compared to cerebrum also reflects the combination of lower incorporation of label and the significantly larger size of the glutamine pool in cerebellum compared to cerebrum. In contrast, the lower percent enrichment in glutamate in cerebellum is due solely to lower oxidative metabolism in this region in 18 day old rat brain as the size of the glutamate pool was comparable in both regions at this age. The lower percent enrichment of lactate in cerebellum is due to lower metabolism via glycolysis in this region as is evident from the higher concentration of lactate in cerebrum in 18 day old rat brain.

Table 1.

Percent enrichment of newly synthesized metabolites in cerebellum and cerebrum from 18-day old rat brain

Metabolite	Cerebellum % enrichment	Cerebrum % enrichment	Significance*
Glucose	41.71 ± 3.59	36.76 ± 3.08	Z= −1.138, p= 0.255
Lactate C3	18.74 ± 0.90	23.01 ± 1.26	t= −4.279, p<0.001
Alanine C3	20.29 ± 1.12	31.89 ± 1.61	t= −6.305, p<0.001
Glutamate C4	16.08 ± 0.56	21.42 1 ± 1.18	t= −5.348, p <.001
Glutamate C3	6.32 ± 0.29	9.39 ± 0.54	t= −5.698, p <.001
Glutamate C2	5.51 ± 0.35	9.60 ± 0.56	Z= −3.516, p <.001
Glutamine C4	7.05 ± 0.36	11.37 ± 0.71	t= −6.647, p <0.001
Glutamine C3	3.92 ± 0.26	5.78 ± 0.43	t= −4.496, p <0.001
Glutamine C2	5.40 ± 0.42	8.60 ± 0.61	t= −4.766, p <0.001
GABA C2	12.40 ± 0.63	12.74 ± 0.72	Z=−0.724, p=0.469
GABA C3	5.50 ± 0.41	5.28 ± 0.36	Z= −0.310, p=0.756
GABA C4	6.56 ± 0.32	5.24 ± 0.34	Z= −2.844, p=0.004
Lactate C2	0.70 ± 0.08	0.90 ± 0.05	t= −3.190, p<0.001
Aspartate C2	8.56 ± 0.63	14.11 ± 0.94	Z=−3.361, p=0.001
Aspartate C3	9.79 ± 0.71	15.30 ± 1.01	t=−4.929, p <0.001
Succinate C2/3	10.46 ± 0.91	14.11 ± 1.00	Z= −2.379, p <0.017
GABA/GLU	0.88 ± 0.03	0.58 ± 0.02	Z=−3.516, p<0.001
GLN/GLU	0.59 ± 0.02	0.63 ± 0.02	t=−1.965, p=0.068

^*Normally distributed data were analyzed by paired Students t-test to determine differences in enrichment in cerebellum and cerebrum. Data that were not normally distributed were analyzed by the Wilcoxon Signed Ranked test. n = 16 for cerebellum and 16 for cerebrum obtained from the same rat pups.

The higher ratio of GABA/GLU enrichment in cerebellum supports the concept of high metabolism in GABAergic neurons, relative to glutamatergic neurons in cerebellum at this age (Table 1). The percent enrichment in GABA C2 and C3 was comparable in cerebrum and cerebellum reflecting active metabolism in GABAergic neurons in both regions at this age. However, the higher enrichment of GABA C4 in cerebellum than in cerebrum likely reflects active metabolism in a specific pool of GABA formed primarily, but not exclusively, from glial precursors in the short timeframe of this ex vivo study.

The higher percent enrichment in ASP C3 and C2 and in succinate C2/3 in cerebrum than in cerebellum is due to much higher metabolism in cerebrum as the pool size of these metabolites is also larger in cerebrum. This also reflects the well established delay in maturation of cerebellum compared to cerebrum.

Discussion

Oxidative metabolism of glucose has a key role in neurotransmitter synthesis and brain maturation

Much of cerebellar development occurs perinatally in human infants and at the corresponding postnatal time points in rat brain (Hashimoto and Kano, 2012). The maturation of oxidative metabolism in brain, which is required for synthesis of the main excitatory neurotransmitter glutamate and subsequent formation of the main inhibitory transmitter GABA, has a key role in brain development (Butterworth and Giguere, 1984). We determined that synthesis of several key compounds including the neurotransmitters glutamate (GLU) and GABA, glutamine, aspartate and lactate from the metabolism of [1,6-¹³C]glucose are different in cerebellum than in the cerebrum of 18-day old rats. In particular, the synthesis of glutamate C4, which during the time frame of this ex vivo NMR study reflects primarily neuronal metabolism, was lower in cerebellum. This may reflect the delayed development of this region compared to cerebrum in 18-day old rat brain. Transport of glucose across the blood-brain barrier is not likely to be limiting at this age as the percent enrichment and amount of glucose detected in the present study were comparable to published values (Morken, et al., 2014b). The percent enrichment of glucose was not different in cerebellum and cerebrum; however, there was more unmetabolized ¹³C glucose in cerebellum as has been reported in this region by other groups (Eloqayli et al., 2003). The higher cycling ratio for GLU metabolism in cerebrum underscores the very active glutamatergic activity in this region in developing brain. The lower incorporation of label into glutamate, glutamine and GABA in cerebellum reflects the lower oxidative metabolism in this region compared to cerebrum in 18 day old rat brain. It is not related to the lower labeling in LAC C3 which is formed from the pyruvate from glycolysis.

Despite the protracted postnatal development of this region (Nakayama, et al., 2012, Takayasu, et al., 2005) and the essential role of glia in proper wiring of connections onto Purkinje cells (Miyazaki, et al., 2017), there are no reports about metabolism via neuron and astrocyte specific pathways in developing cerebellum. Glutamatergic cerebellar granule cells are the most abundant type of neuron in cerebellum and in the brain (Lee, et al., 2005). Indeed, most of the reports on metabolism in cerebellum are from in vitro studies using cerebellar granule cells, which determines metabolism in only one cell type and does not reflect metabolism in the GABAergic Purkinje cells, cerebellar astrocytes, neuron-astrocyte metabolic interactions and developmental changes in this brain region (Bak, et al., 2006, Bak, et al., 2005, Bak, et al., 2009, Brekke, et al., 2012, Waagepetersen, et al., 2001a, Waagepetersen, et al., 2005, Waagepetersen, et al., 2001b).

Both glutamatergic and GABAergic input have regulatory roles in proper cerebellar wiring and function (Nakayama, et al., 2012, Takayasu, et al., 2009). During the second postnatal week in the rat, a period analogous to birth in humans, excess climbing fiber input is eliminated so that the rapidly expanding dendritic tree of each GABAergic Purkinje cell is enervated by a single glutamatergic climbing fiber (Hashimoto and Kano, 2012). Glutamate transporters on neurons and Bergmann glial cells change in a coordinated fashion during cerebellar development to tightly regulate the extracellular concentration of glutamate (Takayasu, et al., 2005, Takayasu, et al., 2006, Takayasu, et al., 2009). The radial fibers of Bergman glial cells associate closely with developing and mature Purkinje cells, to remove excess glutamate and provide metabolic support (Takayasu, et al., 2009, Yamada and Watanabe, 2002). Bergmann glial cells have an active and essential role in cerebellar development (Inage, et al., 1998). Developing granule cells migrate adjacent to the processes of these specialized astrocytes (Yamada, et al., 2000).

During normal brain development, there is a transition from using both ketone bodies and glucose for energy in immature brain to almost complete reliance upon glucose in the adult brain (Vannucci and Simpson, 2003). Use of ketones peaks around postnatal day 14 in the rat, and is relatively uniform throughout brain, supporting energy metabolism and synthesis of lipid and amino acid precursors needed for brain growth (Cotter, et al., 2011, Nehlig, 1996, Nehlig, 2004, Vannucci and Simpson, 2003). In contrast, the uptake and use of glucose varies considerably in different brain regions during development and increases as neuronal pathways mature (Bondy, et al., 1992, Nehlig, 2004, Vannucci, et al., 1998, Vannucci and Simpson, 2003). The level of neuronal glucose transporter GLUT3 increases several fold in cerebellum and cerebrum between postnatal days 7-28 in rat brain, as neurons mature and increased neuronal activity requires higher glucose utilization (Vannucci, et al., 1998). Oxidative metabolism of glucose via pyruvate dehydrogenase (PDH) and the TCA cycle, which occurs in concert with the development of parallel fiber synapses on Purkinje cells, is instrumental in facilitating cerebellar development (Butterworth and Giguere, 1984). The high metabolic rate of Purkinje cells is primarily due to the energy required to restore membrane potential in the massive number of dendrites that inhibit motor nuclei in cerebellum (Connolly, et al., 2007, Howarth, et al., 2012, Howarth, et al., 2010, Welsh, et al., 2002). In the current study the ratio of total labeling in GABA/GLU of 0.15 in cerebellum and 0.13 in cerebrum, reflects metabolism primarily in the neuronal populations in these regions. Our findings are consistent with other studies of developing and mature brain that reported a ratio of ~85% to 15% overall metabolism in glutamatergic to GABAergic neurons in cortex, consistent with the high density of glutamatergic neurons in cortex (Chowdhury, et al., 2007). It is well established that dynamic metabolic interactions between neurons and astrocytes are essential for maintaining both glutamatergic and GABAergic homeostasis in brain (McKenna, et al., 2011, Schousboe, et al., 2013). The data obtained in the present study reflect the neuronal and astrocytic contributions to metabolism in glutamatergic and GABAergic neurotransmitter formation in cerebellum and cerebrum during late postnatal development. The identification of a functional pyruvate recycling pathway at this stage of brain development is important as it was not detected in P7 rat brain (Morken, et al., 2014b).

Regional differences in concentration of metabolites

Data obtained from ¹H spectra demonstrate that the concentration of metabolites in cerebellum from 18-day old rat brain was distinctly different than cerebrum from the same rat pups. The higher concentration of total creatine in cerebellum than in cerebrum is consistent with the high activity and labeling of specific creatine kinase isoforms in Purkinje cells and Bergmann glia in developing cerebellum (Holtzman, et al., 1993, Kaldis, et al., 1996). The high creatine kinase in these cells and in the granular layer may provide a PCr circuit to support the high energy requirement for restoring K⁺ gradients as well as for neurotransmitter release and uptake (Kaldis, et al., 1996, Wallimann and Hemmer, 1994, Wallimann, et al., 1992) in the developing cerebellum at this age. These findings are consistent with the positron emission tomography (PET) studies showing high uptake of ¹⁸F-deoxyglucose in cerebellum and subcortical areas of infant brain compared to neocortex (Suhonen-Polvi, et al., 1993). In the present study the amount of unmetabolized [1,6-¹³C]glucose was significantly higher in cerebellum than in cerebrum of 18-day old rat brain. This finding may reflect regional developmental differences and decreased labeling from glucose metabolism in GLU and GABA in cerebellum in the current study. Our results are in line with the report of considerably higher level of unmetabolized glucose in cerebellum, compared to cortex and subcortex, in adult male Wistar rats, which suggests possible intrinsic differences in glucose uptake and/or utilization between cerebellum and cerebrum (Eloqayli, et al., 2003). Vannucci et al. (Vannucci, et al., 1998) reported that expression and protein level of the neuronal glucose transporter GLUT3 is lower in cerebellum when compared to cerebrum (cortex and hippocampus) at this age; however, it should be noted that the GLUT1 on the blood-brain barrier transports glucose into brain (Vannucci, et al., 1998).

Although the concentration of lactate was lower in cerebellum than in cerebrum, the concentration of alanine, which is formed by transamination of pyruvate was higher in cerebellum than in cerebrum. The lower enrichment in [3-¹³C]alanine in cerebellum than in cerebrum may not be directly related to the difference in lactate C3 enrichment, although both reflect lower glycolysis in cerebellum. Instead, it reflects the combination of lower labeling of ALA C3 from glycolysis and subsequent transamination in cerebellum and the higher concentration of alanine in cerebellum compared to cerebrum, which would lead to a much lower percent enrichment.

Our data showing higher concentrations of glutamine and the glial-specific osmolyte myo-inositol and higher GLN C4/GLU C4 labeling suggest active metabolism in astrocytes in cerebellum at this age. The glutamine pool size is larger in cerebellum than in cerebrum; whereas, the glutamate pool size is comparable in both regions (Figure 3). Thus, the higher ratio of labeling in GLN/GLU may reflect pool size; however, the fact that the concentration of glutamine is higher in cerebellum (McKenna, 2007) does support relatively high glutamine formation in this region compared to cerebrum or slower turnover of glutamine. The ratio may also reflect lower loss of glutamate to other pathways (e.g. glutathione formation, etc.) in the developing cerebellum compared to cerebrum. In steady-state infusion experiments there is no direct link between the influxes and outfluxes and the total pool size; however, the situation may be different in ex vivo experiments like the current study that determine metabolism in the first phase of a labelling study well before a steady-state is reached. In the study by Lizarbe et al. (Lizarbe, et al., 2018) steady-state labeling in GLU C4 and GLN C4 was not reached until 130 min, and in GABA C3 at 150 minutes after the beginning of labeled glucose infusion.

The findings in the present study are consistent with earlier reports that glutamine levels in adult cerebellum were comparable to or slightly higher than cortex (Eloqayli, et al., 2003, Haberg, et al., 2009). The higher concentration of myo-inositol in cerebellum is consistent with the role phosphoinositide 3-kinase (PI3K) signaling plays in differentiation and subsequent cell growth in developing brain (Peltier, et al., 2007, Sanchez, et al., 2004). Our study found a lower aspartate concentration in developing cerebellum than in cerebrum, which is consistent with the data reported for 30 day old control rats (Melo, et al., 2007). The lower concentration of GABA in cerebellum than in cerebrum of 18-day old rats in the present study is likely due to the nature of the cerebrum sample used which contained both cortical and subcortical regions. Although the GABA concentration in cortex is comparable to cerebellum, the concentration of GABA in subcortex is considerably higher than either cortex or cerebellum (Eloqayli, et al., 2003, Haberg, et al., 2009, Melo, et al., 2007). NAA is a marker of neuronal mitochondrial function (Moffett, et al., 2007); the higher levels in cerebrum reflect greater mitochondrial activity in cerebrum which is also reflected in the higher amount of newly synthesized glutamate and GABA (Figs. 6 and 8) in this region. Indeed, the lower concentrations of aspartate and N-acetylaspartate in cerebellum reflects the relative immaturity of this region and lower TCA cycle activity in neuronal mitochondria compared to the cerebrum in 18-day old rat brain, which is consistent with the lower labeling in glutamate and GABA from glucose metabolism in cerebellum at this age. The only sex difference found was a 14% lower concentration of aspartate in the cerebellum of female rats compared to males. It is not known if this difference in female brain has physiological or functional effects. Interestingly, there was no sex difference in the concentration of N-acetylaspartate (Fig. 3) which is formed directly from aspartate synthesized in neuronal mitochondria (Moffett, et al., 2007, Rae, 2014).

Metabolism in astrocytes is relatively high in developing cerebellum

There was only slightly less labeling of glutamine in cerebellum than in cerebrum, which underscores the importance of astrocyte metabolism in this region of brain. The higher ratio of newly synthesized GLN C4/GLU C4 and total labeling of glutamine/glutamate in cerebellum indicates particularly active metabolism in cerebellar astrocytes in 18-day old rat brain (Figure 7). Active metabolism via glutamine synthetase in cerebellum was apparent in the higher ratio of labeled GLN/GLU in all isotopomers determined in this region compared to cerebrum (Figure 7). In support of this concept, the ratio of labeling in GLN/GLU calculated from the percent of total glucose incorporation (Supplemental Table 1) was higher in cerebellum than in cerebrum (t=−6.707, p<0.001). In contrast, the ratio of percent enrichment in GLN/GLU, which may be more meaningful in steady state experiments than in bolus injection initial metabolism studies like the current study was not different in cerebrum than in cerebellum. However, the higher ratio of metabolites would suggest that the higher ratio of labeling in GLN/GLU in this short bolus ex vivo experiment is a distinct increase of the reaction rates involved in GLN synthesis.

Data in the literature report that between 14-21 days of age GS activity in brain reaches ~ 75-100% of the postnatal day (P) P28 activity (Brekke, et al., 2015). Whereas PC activity increases more slowly between 14-21 days of age, reaching ~ 45-60% of P28 activity (Brekke, et al., 2015). The finding that the PC/PDH ratio for glutamine [(GLN C2 – GLN C3)/GLN C4] was comparable in both regions indicates that metabolism via pyruvate carboxylase in cerebellum is comparable to cerebrum in 18 day old rat brain. This activity likely increases rapidly between P18 and adulthood as Haberg et al. (Haberg, et al., 2009) reported significantly higher pyruvate carboxylation in cerebellum compared to mainly cortical and subcortical regions in adult rat brain.

Differences in Glutamate, Glutamine, GABA and Aspartate formation

The labeling of newly synthesized GABA was lower in cerebellum than cerebrum; however, the difference was not as pronounced as the difference observed with glutamate. The size of the glutamate pool is comparable in cerebellum and cerebrum. In contrast, the pool size for GABA is smaller in cerebellum than in cerebrum. Thus, the comparable ratio of labeling in GABA/GLU seen in the present study is not just due to the difference in pool size. It is also a reflection of the finding that although the incorporation of label into GLU and GABA was lower in cerebellum than in cerebrum, the magnitude of difference in labeling was more pronounced for glutamate than for GABA. The lower labeling in the isotopomers of GABA in cerebellum is also not just an effect of pool size. The enrichment of GABA C2 and GABA C3 are comparable in both regions; however, enrichment in GABA C4 is higher in cerebellum than in cerebrum which would not be the case if the differences were due only to pool size. The finding that the ratio of GABA/GLU enrichment in cerebellum 0.88 ± 0.03 is higher than in cerebrum 0.58 ± 0.02 (Z = −3.516, p<0.001) supports this concept. Indeed, the higher enrichment ratio and enrichment in GABA C4 in cerebellum would suggest that the higher ratio of labeling in GABA C4/GLU C2 (Z = −2.999, p=0.003) in the short timeframe of this short bolus injection ex vivo experiment is a distinct increase of the reaction rates involved in a specific pool of GABA synthesis formed primarily, but not exclusively, from glial precursors. However, regional differences in the subsequent metabolism of labeled GABA and GLU may also contribute to this ratio.

This finding suggests that the relative proportion of inhibitory (GABAergic) to excitatory (glutamatergic) neurotransmission is higher in cerebellum in developing brain. It may also be a reflection of the differentiation of Purkinje cells and the relative immaturity of synapse formation in cells formed during the second wave of cerebellar granule cell expansion that takes place from birth to P15 in rat brain (Takayasu, et al., 2005, Takayasu, et al., 2006, Takayasu, et al., 2009). Astrocytes provide critical metabolic support to GABAergic Purkinje neurons in this brain region. Neuronal and glial glutamate transporters change in coordination during development of cerebellum (Takayasu, et al., 2005, Takayasu, et al., 2006, Takayasu, et al., 2009).

Few studies have determined metabolism in cerebellum in adult brain and the published studies to date have focused on regional changes in metabolism in different injury models (Eloqayli, et al., 2003, Haberg, et al., 2009, Melo, et al., 2007). Values reported for metabolism in the control rats in these studies are useful for comparison; however, differences between cerebellum and other regions were not compared. After i.p. injection of [1-¹³C]glucose the labeling of glutamate and glutamine in cerebellum was only slightly lower than in cortex from 30 day old non-epileptic control rats; whereas, labeling of GABA in cerebellum was comparable to cortex (Melo, et al., 2007). In adult Wistar rats (240-280g; ~ 7-8 weeks old) given subcutaneous injections of [1-¹³C]glucose labeling in glutamate C4 was only slightly higher in cortex than in cerebellum (305 ± 23 nmol ¹³C incorp/g tissue vs 249 ± 18) and comparable levels of labeling in glutamine and GABA were found (75 ± 8 vs 68 ± 7 nmol ¹³C incorp/g tissue for GLN C4; 43 ± 4 vs 37 ± 5 nmol ¹³C incorp/g tissue for GABA C2) in cortex and cerebellum, respectively (Eloqayli, et al., 2003). These earlier studies underscore the active metabolism in astrocytes and GABAergic neurons in cerebellum in developing and mature brain. The study in adult brain found labeling of ASP C2 to be higher in cortex than cerebellum, 61 ± 4 and 45 ± 5 nmol ¹³C incorp/g tissue, respectively (Eloqayli, et al., 2003). Data from the present study are comparable to the values reported for the 30 day old control rats (Melo, et al., 2007) and somewhat lower than the 7-8 week old control rats in the study by Eloqayli (Eloqayli, et al., 2003). Thus, our data do suggest that cerebellum in 18-day old rat brain has not yet reached metabolism comparable to adult brain (Eloqayli, et al., 2003).

The lower incorporation of label into aspartate C2 and C3 in cerebellum reflects the relative immaturity of cerebellum compared to cerebrum and the lower TCA cycle activity in cerebellum in 18 day old rat brain. The pool size is also lower in cerebellum as evidenced by the lower concentration of aspartate compared to cerebrum. It may also reflect an overall lower rate of transamination of OAA to aspartate in mitochondria in cerebellum. However, the anaplerotic ratio calculated from labeling in aspartate (ASP C3-ASP C2)/ ASP C2 that reflects metabolism via the pyruvate carboxylase pathway (Rae, et al., 2009) in cerebellum and cerebrum was not different suggesting active metabolism via the pyruvate carboxylase pathway in cerebellum at this age.

Haberg et al. (Haberg, et al., 2009) determined metabolism in subcortical (“encompassing anteriorly the lateral caudoputamen and more posterior the lateral thalamus and lateral parts of hippocampus, and lower temporo-parietal cortex”), pure cortical samples (“obtained from the lateral parts of the frontal cortex plus the upper parietal cortex”) and cerebellum in sham rats and rats that underwent the middle cerebral artery ligation stroke procedure (MCAO). They infused [1-¹³C]glucose over 2 minutes via femoral vein catheters in the sham adult male Wistar rats (320-340g; ~10 weeks old) 15 minutes prior to tissue collection and determined metabolism using ex vivo ¹³C-NMR spectroscopy. Their study (which did not do statistical comparisons of the regional data for sham rats) showed slightly higher enrichment of label from glucose metabolism in glutamate and aspartate in cerebellum compared to cortex, and comparable enrichment of glutamine and GABA (Haberg, et al., 2009). They also reported that the relative contribution to labeling of GABA from metabolism via the pyruvate carboxylase pathway in cerebellar astrocytes, compared to labeling from metabolism via pyruvate dehydrogenase (PC/PDH), was several fold higher in cerebellum than in subcortical or pure cortical regions of sham rat brain (Haberg, et al., 2009). The high labeling from metabolism via pyruvate carboxylation in cerebellum observed by Haberg et al. (Haberg, et al., 2009) is consistent with an early study by Griffin et al. (Griffin, et al., 1999) reporting a substantial portion of the labeling of glutamate from glucose metabolism occurred via the pyruvate carboxylase pathway in cerebellum of awake guinea pig brain.

Conclusions

In cerebellum from developing rat brain the overall labeling of newly synthesized compounds from metabolism of [1,6-¹³C]glucose was lower than in cerebrum. However, the higher ratio of newly synthesized glutamine/glutamate in cerebellum compared to cerebrum indicates particularly active metabolism via glutamine synthetase in cerebellar astrocytes in 18 day old rat brain. The present study found distinct differences in the concentration of metabolites in cerebellum compared to cerebrum in 18 day old brain. This is the first study to determine metabolism in the cerebellum and cerebrum of male and female rat brain. The only sex difference found was a 14% lower concentration of aspartate in the cerebellum of female rats compared to males. It is not known whether this difference is present in mature brain. Our data provide new information about metabolism and neurotransmitter synthesis during a critical period of development in cerebellum.

Abbreviations:

ALA C3

alanine C3

ASP C2 and C3

aspartate labeled in the C2 and C3 carbon, respectively

AAT

aspartate aminotransferase

D₂O

deuterium oxide

GABA

ɣ-aminobutyric acid

GAD

glutamic acid decarboxylase

GLU C2, C3 and C4

glutamate labeled in the C2, C3 and C4 carbon, respectively

GLN C2, C3 and C4

glutamine labeled in the C2, C3 and C4 carbon, respectively

GS

glutamine synthetase

NAA

N-acetylaspartate

nOe

nuclear Overhauser effects

OAA

oxaloacetate

PCA

perchloric

PC

pyruvate carboxylase

PDH

pyruvate dehydrogenase

PET

positron emission tomography

Succ 2/3

succinate labeled in the C2 and C3 positions

TCA cycle

tricarboxylic acid cycle

TMSP

3-(trimethylsilyl)propionate-2,2,3,3-d4