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Comparative Study
, 42 (12), 3018-32

Localization and Expression of GABA Transporters in the Suprachiasmatic Nucleus

Affiliations
Comparative Study

Localization and Expression of GABA Transporters in the Suprachiasmatic Nucleus

Michael Moldavan et al. Eur J Neurosci.

Abstract

GABA is a principal neurotransmitter in the suprachiasmatic hypothalamic nucleus (SCN), the master circadian clock. Despite the importance of GABA and GABA uptake for functioning of the circadian pacemaker, the localization and expression of GABA transporters (GATs) in the SCN has not been investigated. The present studies used Western blot analysis, immunohistochemistry and electron microscopy to demonstrate the presence of GABA transporter 1 (GAT1) and GAT3 in the SCN. By using light microscopy, GAT1 and GAT3 were co-localized throughout the SCN, but were not expressed in the perikarya of arginine vasopressin- or vasoactive intestinal peptide-immunoreactive (-ir) neurons of adult rats, nor in the neuronal processes labelled with the neurofilament heavy chain. Using electron microscopy, GAT1- and GAT3-ir was found in glial processes surrounding unlabelled neuronal perikarya, axons, dendrites, and enveloped symmetric and asymmetric axo-dendritic synapses. Glial fibrillary acidic protein-ir astrocytes grown in cell culture were immunopositive for GAT1 and GAT3 and both GATs could be observed in the same glial cell. These data demonstrate that synapses in the SCN function as 'tripartite' synapses consisting of presynaptic axon terminals, postsynaptic membranes and astrocytes that contain GABA transporters. This model suggests that astrocytes expressing both GATs may regulate the extracellular GABA, and thereby modulate the activity of neuronal networks in the SCN.

Keywords: Western blot; circadian rhythm; electron microscopic imaging; hypothalamus; immunohistochemistry; suprachiasmatic nucleus.

Figures

Figure 1
Figure 1. Relative expression levels of GAT1 and GAT3 in other brain regions compared to the SCN
A and B: Western blot and quantitative analysis of GAT1 and GAT3 expression, respectively. Upper: immunoblot, GAT bands (top line), and the loading control: GAPDH (bottom line). Molecular mass markers (kDa) are indicated. Lower: optical density (O.D.) was measured and quantified with ImageJ software, then normalized to the loading control (shown in ratio units). Each bar represents the ratio of GAT expression in each brain region to the SCN level. Error bars represent the SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 when compared with SCN. Each histogram represents O.D. measurements from 5 gels (mean ± SE). A. In insert: Blocking peptide (Bl. pept., Alomone Labs Ltd.) completely blocked GAT1 detection in the cerebral cortex sample (positive control). Anti-GAT1 antibody alone and anti-GAT1 antibody + blocking peptide (Bl. pept., Alomone Labs) are shown in duplicates. A detailed description of antibodies is in Table 1 and in Methods. These data demonstrate that GAT1 is more widely expressed in the brain than GAT3. In contrast to GAT1, the GAT3 expression is more prominent in the thalamus and hypothalamus including the SCN.
Figure 2
Figure 2. Diurnal expression of GAT3 and GAT1 in the SCN of rats on a 12:12h LD cycle
A. Western blot for GAT1, each band represents one of five zeitgeber times (ZT)s: ZT 4 - 6, ZT 8 - 10; ZT 14 - 16; ZT 18 - 20; ZT 22 - 24. Positive control is the cerebral cortex. Loading control (LC): GAPDH. B. Western blot for GAT3. Positive control is the thalamus. C. Graph shows GAT1 and GAT3 expression at different ZTs. The quantification of the optical density of the bands was performed, than data were normalized to the loading control. GAT1: 4 gels in duplicate i.e. 8 bands for each ZT. GAT3: 5 gels in duplicate i.e.10 bands for each ZT. The light and dark phases of the cycle are shown on the horizontal bar under the graph. Neither GAT1 nor GAT3 showed a diurnal rhythm of expression. Data shown as mean ± SEM. A detailed description of the antibodies is in Table 1 and in Methods. The rest of the notations are the same, as those in Fig.1.
Figure 3
Figure 3. GAT1-immunoreactivity in the SCN
A, B. Coronal section of the hypothalamus including the SCN, demonstrating high levels of GAT1 (red) expression in the region of the periventricular hypothalamic nuclei and the region between the lobes of the SCN. 10×, scale bar 200 μm. The brain was extracted and the tissue fixed at ZT 4 - 5 (A, B) and at ZT 18 - 20 (C – I). C. Higher magnification image showing GAT1-ir punctas surrounding cell bodies in the SCN (arrowheads), 60×, scale bar 10 μm. B and C. Sections were counterstained with DAPI (blue) to show the location of cellular nuclei and to outline the SCN. D - F. GAT1 (D) and NFH (green, E) expression. A merged GAT1 and NFH image is shown in F. Note the lack of overlap between the GAT1 and NFH expression. G. Low magnification image (20×) showing GAT1 and GFAP (green) expression in the SCN, scale bar 100 μm. H. Higher magnification showing GFAP (green) expression. I. Merged image of GAT1 and GFAP staining. There was only occasional overlap between the two stains. D - F, H, I, 60×, scale bar 50 μm. Rabbit anti-GAT1 antibody (ab72448, Abcam) was used for all images, except for A and B (rabbit anti-GAT1 antibody, AB1570, Millipore). SCN – hypothalamic suprachiasmatic nucleus, 3V – third ventricle, Och - optic chiasm. D – I. Asterisks – blood vessels. Description of antibodies is shown in Table 1.
Figure 4
Figure 4. GAT3-immunoreactivity in SCN
A, B. Coronal section of the hypothalamus including the SCN, demonstrating GAT3 (red) expression in hypothalamus and SCN with high level of expression around 3-rd ventricles and in SCNs. 10×, scale bar 200 μm. The brain was extracted and the tissue fixed at ZT 4 - 5 (A, B) and at ZT 18 - 20 (C – H). B. Section counterstained with DAPI (blue) to outline the SCN, 10×, scale bar 200 μm. C. Low magnification (20×) image showing the expression of GAT3 and NFH (green). D. Higher magnification image showing NFH expression. E. Merged image showing GAT3 and NFH expression. Note that the GAT3- and NFH-positive processes are not co-localized. F. Low magnification image demonstrating GAT3 and GFAP expression (green) in the SCN, 10× G. Higher magnification image of GFAP expression. H. Merged image showing GAT3 and GFAP expression. C, E, F, H, I. Confocal images, 60×. Scale bars 50 μm. D, G. Scale bar 100 μm. Description of antibodies is shown in Table 1. The rest of the notations are the same, as those in Fig. 3.
Figure 5
Figure 5. GAT1 and GAT3 were not expressed in neurons immunoreactive for arginine vasopressin (AVP) or vasoactive intestinal peptide (VIP)
A - I. Double-labeling with antibodies to GAT1 or GAT3 and AVP or VIP. A – C. Application of AVP (, green, (A)) and GAT3 (, red (B), merged images (C). D - F. Application of AVP (D) and GAT1 (, red, (E)), merged images (F). G – I. VIP (green, G) and GAT3 (H), merged images (I). Arrows – neurons immunoreactive to AVP or VIP. Note: neurons labeled for AVP or VIP did not show GAT1 or GAT3 expression. All images were taken at 60×, scale bar 10 μm. Rats were perfused for IHC at ZT 18 - 20.
Figure 6
Figure 6. GAT1 and GAT3 are co-localized in the SCN
A - F. Brain sections of adult rats stained for GAT1 (red) and GAT3 (green) showing significant overlap in the expression pattern of these transporters C, F. Merged images. A - C. Low magnification, 10×, scale bar: 100 μm. D - F. Higher magnification, 60×, scale bar: 50 μm. Rats were perfused for IHC at ZT 18 - 20.
Figure 7
Figure 7. GAT1- and GAT3-immunogold labeling (−IL) in glial processes surrounding perikarya in the SCN
A, B. GAT1- IL, C, D. GAT3-IL. A. An unlabeled cell body is surrounded by glial processes containing IL for GAT1 (arrow, GAT1-gl). B. A high magnification image shows immunogold particles (arrow) associated with glial processes near an unlabeled perikaryon (up). C. IL for GAT3 (arrow, GAT3-gl) is associated with glial processes near an unlabeled cell body. The cell displays a large nucleus (N) and limited cytoplasm. D. A high magnification image shows GAT3-ir glial processes between an unlabeled perikaryon (up) and groups of unmyelinated axons (ua) nearby. The immungold particles (arrow) are closely associated with glial cell membranes. Scale bars 500 nm.
Figure 8
Figure 8. GAT1- and GAT3-ir glial cells are closely associated with synaptic complexes
A. GAT1-immunogold labeled glial processes (arrow, GAT1-gl) surround a synaptic complex that consists of an unlabeled axon terminal (ut) and unlabeled dendrite (ud). B. Three unlabeled axon terminals (ut1 – ut3) form contacts with unlabeled dendrites (ud). One axon terminal (ut1) forms an asymmetric synapse (arrow head) with a dendrite, while nearby another axon terminal (ut3) forms a symmetric synapse (arrow head) with a dendrite. The third terminal (ut2) is apposed to an unlabeled dendrite without a clear synaptic junction. All three dendritic complexes are ensheathed in GAT3-ir glial processes (arrows, GAT3-gl) indicated by immungold particles. C. A GAT3-ir glial process (arrow) is apposed to an axon terminal that contains both small clear vesicles, as well as dense core vesicles (dcv). An unlabeled dendrite (ud) is also partially surrounded by GAT3-ir glial processes. Scale bars: 500 nm.
Figure 9
Figure 9. GAT1- and GAT3-ir glial processes are closely associated with unmyelinated axons
A. GAT1-ir glial processes surround axon bundles that include unmyelinated axons and an axonal process that contains small clear vesicles and dense core vesicles (dcv). Arrows – immunogold labeling. B. Immunogold particles (arrows) are closely associated with the membranes of GAT3-ir glial cells surrounding unmyelinated axons (ua). Scale bars 500 nm.
Figure 10
Figure 10. GAT1 and GAT3 are expressed in GFAP-immunopositive astrocytes
A - I. Glial cells in SCN cell cultures were stained for the presence of GAT1, GAT3, and GFAP. The images were taken from one-week old cultures. A - C. GFAP-positive (green) astrocytes were immunoreactive for GAT1 (red), 100×. C. The images shown in A and B were merged. D - F. GFAP-positive astrocytes were also immunoreactive for GAT3 (red), 60×. F. The GFAP and GAT3 images shown in D and E were merged. A – F. scale bar 25 μm. G – I. Co-expression of GAT1 and GAT3 in glial cells (presumably astrocytes), scale bar 10 μm. The GAT1 and GAT3 images were merged.
Figure 11
Figure 11. Schematic model of the location of GATs in SCN
This model schematically proposes that GAT1 and GAT3 are expressed in the distal processes of GFAP-positive astrocytes, and also by GFAP-negative glial cells. The model shows that GATs-ir processes of glial cells surround neuronal cell bodies and axo-dendritic synapses. The location of the GATs suggests they can restrict GABA diffusion from the synapses and regulate extracellular GABA concentration around the neuronal cell bodies.

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