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Differential Anatomical Expression of Ganglioside GM1 Species Containing d18:1 or d20:1 Sphingosine Detected by MALDI Imaging Mass Spectrometry in Mature Rat Brain.

Weishaupt N, Caughlin S, Yeung KK, Whitehead SN - Front Neuroanat (2015)

Bottom Line: Across layers of the sensory cortex, opposing expression gradients were found for GM1d18:1 and GM1d20:1.By far the highest GM1d18:1/d20:1 ratio was found in the amygdala.Differential expression of GM1 with d18:1- or d20:1-sphingosine bases in the adult rat brain suggests tight regulation of expression and points toward a distinct functional relevance for each of these GM1 species in neuronal processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London ON, Canada.

ABSTRACT
GM1 ganglioside plays a role in essential neuronal processes, including differentiation, survival, and signaling. Yet, little is known about GM1 species with different sphingosine bases, such as the most abundant species containing 18 carbon atoms in the sphingosine chain (GM1d18:1), and the less abundant containing 20 carbon atoms (GM1d20:1). While absent in the early fetal brain, GM1d20:1 continues to increase throughout pre- and postnatal development and into old age, raising questions about the functional relevance of the GM1d18:1 to GM1d20:1 ratio. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a novel technology that allows differentiation between these two GM1 species and quantification of their expression within an anatomical context. Using this technology, we find GM1d18:1/d20:1 expression ratios are highly specific to defined anatomical brain regions in adult rats. Thus, the ratio was significantly different among different thalamic nuclei and between the corpus callosum and internal capsule. Differential GM1d18:1/GM1d20:1 ratios measured in hippocampal subregions in rat brain complement previous studies conducted in mice. Across layers of the sensory cortex, opposing expression gradients were found for GM1d18:1 and GM1d20:1. Superficial layers demonstrated lower GM1d18:1 and higher GM1d20:1 signal than other layers, while in deep layers GM1d18:1 expression was relatively high and GM1d20:1 expression low. By far the highest GM1d18:1/d20:1 ratio was found in the amygdala. Differential expression of GM1 with d18:1- or d20:1-sphingosine bases in the adult rat brain suggests tight regulation of expression and points toward a distinct functional relevance for each of these GM1 species in neuronal processes.

No MeSH data available.


Cortical sampling areas are depicted in a histological section stained with thionine (A). Mass spectra were generated from a superficial, an intermediate and a deep region of interest within the somatosensory cortex (B, modified from Paxinos and Watson, 1998), and corresponding regions were also sampled in two more posterior sections per animal. GM1d18:1/GM1d20:1 ratios were significantly different (Tukey’s multiple comparisons, ∗∗∗∗p < 0.0001) among cortical layers (C). Molecular images show the anatomical expression of GM1d18:1 (green) and GM1d20:1 (red) in the cortex (D). Scale bar = 3 mm. Dashed box marks cortical region magnified in bottom row (E). Dashed lines in composite image mark the borders between sampling areas for different cortical layers. Scale bar = 3 mm. All images are optimized for visualization and do not represent absolute concentrations (GM1d18:1 is the predominant species even in the superficial layers of the cortex).
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Figure 2: Cortical sampling areas are depicted in a histological section stained with thionine (A). Mass spectra were generated from a superficial, an intermediate and a deep region of interest within the somatosensory cortex (B, modified from Paxinos and Watson, 1998), and corresponding regions were also sampled in two more posterior sections per animal. GM1d18:1/GM1d20:1 ratios were significantly different (Tukey’s multiple comparisons, ∗∗∗∗p < 0.0001) among cortical layers (C). Molecular images show the anatomical expression of GM1d18:1 (green) and GM1d20:1 (red) in the cortex (D). Scale bar = 3 mm. Dashed box marks cortical region magnified in bottom row (E). Dashed lines in composite image mark the borders between sampling areas for different cortical layers. Scale bar = 3 mm. All images are optimized for visualization and do not represent absolute concentrations (GM1d18:1 is the predominant species even in the superficial layers of the cortex).

Mentions: Molecular images were analyzed using Tissue View Software (Sciex). All images were optimized for visualization of expression, therefore color intensities do not reflect the same absolute signal intensity across different images. In each section, similar regions of interests (ROIs) were drawn for each anatomical region in the left and right hemisphere. As we cannot identify anatomically defined regions based on their cytoarchitecture in molecular images, we selected ROIs with reference to the Rat Brain Atlas by Paxinos and Watson (1998). The sampled cortical regions include putative primary and secondary somatosensory cortex (Figure 2B). The average mass spectral data for each ROI was exported, and the baseline noise was removed for each peak. GraphPad Prism software version 6 (GraphPad Software Inc., La Jolla, CA, USA) was used to measure the area under the curve (AUC) to quantify the highest peak for GM1d18:1 at a predicted m/z of 1544.87, and for GM1d20:1 at a predicted m/z of 1572.9, respectively (Figure 1B). Subsequently, the ratio of AUC of the highest GM1d18:1 peak to that of the highest GM1d20:1 peak was calculated for each ROI. Based on previous studies (Whitehead et al., 2011; Caughlin et al., 2015) measuring the AUC for the first (largest) peak is indicative of total signal for all isotope peaks of the species. Four measurements were taken for each animal (both hemispheres in two sections), and the average per animal was presented in graphs and used for statistical analyses. The corpus callosum and cortical layers were sampled in eight ROIs (in two anterior and two posterior sections, in both hemispheres). In cases where image resolution was insufficient for detailed analysis, the average value was taken from less than four sample measurements. Individual animal’s ROIs with less than two measurements were excluded. Each ROI is represented by average values from 16 to 20 brains. All calculated GM1d18:1/GM1d20:1 ratios are available in the MetaboLights database (MTBLS271).


Differential Anatomical Expression of Ganglioside GM1 Species Containing d18:1 or d20:1 Sphingosine Detected by MALDI Imaging Mass Spectrometry in Mature Rat Brain.

Weishaupt N, Caughlin S, Yeung KK, Whitehead SN - Front Neuroanat (2015)

Cortical sampling areas are depicted in a histological section stained with thionine (A). Mass spectra were generated from a superficial, an intermediate and a deep region of interest within the somatosensory cortex (B, modified from Paxinos and Watson, 1998), and corresponding regions were also sampled in two more posterior sections per animal. GM1d18:1/GM1d20:1 ratios were significantly different (Tukey’s multiple comparisons, ∗∗∗∗p < 0.0001) among cortical layers (C). Molecular images show the anatomical expression of GM1d18:1 (green) and GM1d20:1 (red) in the cortex (D). Scale bar = 3 mm. Dashed box marks cortical region magnified in bottom row (E). Dashed lines in composite image mark the borders between sampling areas for different cortical layers. Scale bar = 3 mm. All images are optimized for visualization and do not represent absolute concentrations (GM1d18:1 is the predominant species even in the superficial layers of the cortex).
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Related In: Results  -  Collection

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Figure 2: Cortical sampling areas are depicted in a histological section stained with thionine (A). Mass spectra were generated from a superficial, an intermediate and a deep region of interest within the somatosensory cortex (B, modified from Paxinos and Watson, 1998), and corresponding regions were also sampled in two more posterior sections per animal. GM1d18:1/GM1d20:1 ratios were significantly different (Tukey’s multiple comparisons, ∗∗∗∗p < 0.0001) among cortical layers (C). Molecular images show the anatomical expression of GM1d18:1 (green) and GM1d20:1 (red) in the cortex (D). Scale bar = 3 mm. Dashed box marks cortical region magnified in bottom row (E). Dashed lines in composite image mark the borders between sampling areas for different cortical layers. Scale bar = 3 mm. All images are optimized for visualization and do not represent absolute concentrations (GM1d18:1 is the predominant species even in the superficial layers of the cortex).
Mentions: Molecular images were analyzed using Tissue View Software (Sciex). All images were optimized for visualization of expression, therefore color intensities do not reflect the same absolute signal intensity across different images. In each section, similar regions of interests (ROIs) were drawn for each anatomical region in the left and right hemisphere. As we cannot identify anatomically defined regions based on their cytoarchitecture in molecular images, we selected ROIs with reference to the Rat Brain Atlas by Paxinos and Watson (1998). The sampled cortical regions include putative primary and secondary somatosensory cortex (Figure 2B). The average mass spectral data for each ROI was exported, and the baseline noise was removed for each peak. GraphPad Prism software version 6 (GraphPad Software Inc., La Jolla, CA, USA) was used to measure the area under the curve (AUC) to quantify the highest peak for GM1d18:1 at a predicted m/z of 1544.87, and for GM1d20:1 at a predicted m/z of 1572.9, respectively (Figure 1B). Subsequently, the ratio of AUC of the highest GM1d18:1 peak to that of the highest GM1d20:1 peak was calculated for each ROI. Based on previous studies (Whitehead et al., 2011; Caughlin et al., 2015) measuring the AUC for the first (largest) peak is indicative of total signal for all isotope peaks of the species. Four measurements were taken for each animal (both hemispheres in two sections), and the average per animal was presented in graphs and used for statistical analyses. The corpus callosum and cortical layers were sampled in eight ROIs (in two anterior and two posterior sections, in both hemispheres). In cases where image resolution was insufficient for detailed analysis, the average value was taken from less than four sample measurements. Individual animal’s ROIs with less than two measurements were excluded. Each ROI is represented by average values from 16 to 20 brains. All calculated GM1d18:1/GM1d20:1 ratios are available in the MetaboLights database (MTBLS271).

Bottom Line: Across layers of the sensory cortex, opposing expression gradients were found for GM1d18:1 and GM1d20:1.By far the highest GM1d18:1/d20:1 ratio was found in the amygdala.Differential expression of GM1 with d18:1- or d20:1-sphingosine bases in the adult rat brain suggests tight regulation of expression and points toward a distinct functional relevance for each of these GM1 species in neuronal processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London ON, Canada.

ABSTRACT
GM1 ganglioside plays a role in essential neuronal processes, including differentiation, survival, and signaling. Yet, little is known about GM1 species with different sphingosine bases, such as the most abundant species containing 18 carbon atoms in the sphingosine chain (GM1d18:1), and the less abundant containing 20 carbon atoms (GM1d20:1). While absent in the early fetal brain, GM1d20:1 continues to increase throughout pre- and postnatal development and into old age, raising questions about the functional relevance of the GM1d18:1 to GM1d20:1 ratio. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a novel technology that allows differentiation between these two GM1 species and quantification of their expression within an anatomical context. Using this technology, we find GM1d18:1/d20:1 expression ratios are highly specific to defined anatomical brain regions in adult rats. Thus, the ratio was significantly different among different thalamic nuclei and between the corpus callosum and internal capsule. Differential GM1d18:1/GM1d20:1 ratios measured in hippocampal subregions in rat brain complement previous studies conducted in mice. Across layers of the sensory cortex, opposing expression gradients were found for GM1d18:1 and GM1d20:1. Superficial layers demonstrated lower GM1d18:1 and higher GM1d20:1 signal than other layers, while in deep layers GM1d18:1 expression was relatively high and GM1d20:1 expression low. By far the highest GM1d18:1/d20:1 ratio was found in the amygdala. Differential expression of GM1 with d18:1- or d20:1-sphingosine bases in the adult rat brain suggests tight regulation of expression and points toward a distinct functional relevance for each of these GM1 species in neuronal processes.

No MeSH data available.