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Glutamic acid decarboxylase 67 expression by a distinct population of mouse vestibular supporting cells.

Tavazzani E, Tritto S, Spaiardi P, Botta L, Manca M, Prigioni I, Masetto S, Russo G - Front Cell Neurosci (2014)

Bottom Line: While no evidence for GAD65 expression was found, GAD67 was detected in a distinct population of peripherally-located supporting cells, but not in hair cells or in centrally-located supporting cells.GABA, on the other hand, was found in all supporting cells.The present result indicate that only a discrete population of supporting cells use GAD67 to synthesize GABA.

View Article: PubMed Central - PubMed

Affiliation: Department of Brain and Behavioral Sciences, University of Pavia Pavia, Italy.

ABSTRACT
The function of the enzyme glutamate decarboxylase (GAD) is to convert glutamate in γ-aminobutyric acid (GABA). Glutamate decarboxylase exists as two major isoforms, termed GAD65 and GAD67, that are usually expressed in GABA-containing neurons in the central nervous system. GAD65 has been proposed to be associated with GABA exocytosis whereas GAD67 with GABA metabolism. In the present immunofluorescence study, we have investigated the presence of the two GAD isoforms in the semicircular canal cristae of wild type and GAD67-GFP knock-in mice. While no evidence for GAD65 expression was found, GAD67 was detected in a distinct population of peripherally-located supporting cells, but not in hair cells or in centrally-located supporting cells. GABA, on the other hand, was found in all supporting cells. The present result indicate that only a discrete population of supporting cells use GAD67 to synthesize GABA. This is the first report of a marker that allows to distinguish two populations of supporting cells in the vestibular epithelium. On the other hand, the lack of GABA and GAD enzymes in hair cells excludes its involvement in afferent transmission.

No MeSH data available.


Related in: MedlinePlus

Agarose gel image of PCR products for GAD67 and GAD67-GFP from wild-type and heterozygous GAD67-GFP knock-in mice. Representative PCR products for GAD67 (265 pb) and GAD67-GFP (564 pb) were observed in +/GFP lanes (n = 3) while only the PCR product for GAD67 was present in the wild type lanes (WT, n = 3). The control lane (blank) was negative as expected. The left lane (MW) shows DNA molecular weight markers.
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Figure 1: Agarose gel image of PCR products for GAD67 and GAD67-GFP from wild-type and heterozygous GAD67-GFP knock-in mice. Representative PCR products for GAD67 (265 pb) and GAD67-GFP (564 pb) were observed in +/GFP lanes (n = 3) while only the PCR product for GAD67 was present in the wild type lanes (WT, n = 3). The control lane (blank) was negative as expected. The left lane (MW) shows DNA molecular weight markers.

Mentions: Transgenic mice were obtained by crossing female wild-type C57BL/6 mice with male heterozygous GAD67-GFP mice. Transgenic mice were sorted by two ways: (1) by examining the heads of P1-2 mice under a fluorescence lamp. GAD67-GFP knock-in mice exhibit a striking green fluorescence in the brain that can be visualized through the skull at this age; (2) by extracting DNA from mouse tails and carrying out the PCR. In the latter case, genomic DNA was extracted from mouse tail biopsies with the PureLink® Genomic DNA Midi Kit (Invitrogen, Italy). The extracted DNA was kept frozen at −80°C until use. PCR was performed on 2 μg DNA with the GoTaq® Flexi DNA Polymerase (Promega, Italy) and with specific primers for GAD67-GFP tagged mice (TR-1b 5′-GGCACAGCTCTCCCTTCTGTTTGC-3′; TR-3 5′-GCTCTCCTTTCGCGTTCCGACAG-3′; TRGFP-8 5′-CTGCTTGTCGGCCATGATATAGACG-3′). An initial denaturation at 94°C for 3 min was followed by 20 s at 96°C, 30 s at 68°C and 30 s at 72°C for 30 cycles. A final extension at 72°C for 10 min was performed. The molecular weight of the PCR products was compared to the DNA molecular weight marker VIII (Roche Molecular Biochemicals, Italy). The bands acquired with the Image Master VDS (Amersham Bioscience Europe, Germany) were at the expected size of 265 bp for GAD67 in wild type mice and of 265 bp and 564 bp for heterozygous GAD67-GFP mice (Figure 1).


Glutamic acid decarboxylase 67 expression by a distinct population of mouse vestibular supporting cells.

Tavazzani E, Tritto S, Spaiardi P, Botta L, Manca M, Prigioni I, Masetto S, Russo G - Front Cell Neurosci (2014)

Agarose gel image of PCR products for GAD67 and GAD67-GFP from wild-type and heterozygous GAD67-GFP knock-in mice. Representative PCR products for GAD67 (265 pb) and GAD67-GFP (564 pb) were observed in +/GFP lanes (n = 3) while only the PCR product for GAD67 was present in the wild type lanes (WT, n = 3). The control lane (blank) was negative as expected. The left lane (MW) shows DNA molecular weight markers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4269132&req=5

Figure 1: Agarose gel image of PCR products for GAD67 and GAD67-GFP from wild-type and heterozygous GAD67-GFP knock-in mice. Representative PCR products for GAD67 (265 pb) and GAD67-GFP (564 pb) were observed in +/GFP lanes (n = 3) while only the PCR product for GAD67 was present in the wild type lanes (WT, n = 3). The control lane (blank) was negative as expected. The left lane (MW) shows DNA molecular weight markers.
Mentions: Transgenic mice were obtained by crossing female wild-type C57BL/6 mice with male heterozygous GAD67-GFP mice. Transgenic mice were sorted by two ways: (1) by examining the heads of P1-2 mice under a fluorescence lamp. GAD67-GFP knock-in mice exhibit a striking green fluorescence in the brain that can be visualized through the skull at this age; (2) by extracting DNA from mouse tails and carrying out the PCR. In the latter case, genomic DNA was extracted from mouse tail biopsies with the PureLink® Genomic DNA Midi Kit (Invitrogen, Italy). The extracted DNA was kept frozen at −80°C until use. PCR was performed on 2 μg DNA with the GoTaq® Flexi DNA Polymerase (Promega, Italy) and with specific primers for GAD67-GFP tagged mice (TR-1b 5′-GGCACAGCTCTCCCTTCTGTTTGC-3′; TR-3 5′-GCTCTCCTTTCGCGTTCCGACAG-3′; TRGFP-8 5′-CTGCTTGTCGGCCATGATATAGACG-3′). An initial denaturation at 94°C for 3 min was followed by 20 s at 96°C, 30 s at 68°C and 30 s at 72°C for 30 cycles. A final extension at 72°C for 10 min was performed. The molecular weight of the PCR products was compared to the DNA molecular weight marker VIII (Roche Molecular Biochemicals, Italy). The bands acquired with the Image Master VDS (Amersham Bioscience Europe, Germany) were at the expected size of 265 bp for GAD67 in wild type mice and of 265 bp and 564 bp for heterozygous GAD67-GFP mice (Figure 1).

Bottom Line: While no evidence for GAD65 expression was found, GAD67 was detected in a distinct population of peripherally-located supporting cells, but not in hair cells or in centrally-located supporting cells.GABA, on the other hand, was found in all supporting cells.The present result indicate that only a discrete population of supporting cells use GAD67 to synthesize GABA.

View Article: PubMed Central - PubMed

Affiliation: Department of Brain and Behavioral Sciences, University of Pavia Pavia, Italy.

ABSTRACT
The function of the enzyme glutamate decarboxylase (GAD) is to convert glutamate in γ-aminobutyric acid (GABA). Glutamate decarboxylase exists as two major isoforms, termed GAD65 and GAD67, that are usually expressed in GABA-containing neurons in the central nervous system. GAD65 has been proposed to be associated with GABA exocytosis whereas GAD67 with GABA metabolism. In the present immunofluorescence study, we have investigated the presence of the two GAD isoforms in the semicircular canal cristae of wild type and GAD67-GFP knock-in mice. While no evidence for GAD65 expression was found, GAD67 was detected in a distinct population of peripherally-located supporting cells, but not in hair cells or in centrally-located supporting cells. GABA, on the other hand, was found in all supporting cells. The present result indicate that only a discrete population of supporting cells use GAD67 to synthesize GABA. This is the first report of a marker that allows to distinguish two populations of supporting cells in the vestibular epithelium. On the other hand, the lack of GABA and GAD enzymes in hair cells excludes its involvement in afferent transmission.

No MeSH data available.


Related in: MedlinePlus