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. 2007 Jan;25(1):106-18.
doi: 10.1111/j.1460-9568.2006.05263.x.

Glutamatergic Neurons Are Present in the Rat Ventral Tegmental Area

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Free PMC article

Glutamatergic Neurons Are Present in the Rat Ventral Tegmental Area

Tsuyoshi Yamaguchi et al. Eur J Neurosci. .
Free PMC article

Abstract

The ventral tegmental area (VTA) is thought to play an important role in reward function. Two populations of neurons, containing either dopamine (DA) or gamma-amino butyric acid (GABA), have been extensively characterized in this area. However, recent electrophysiological studies are consistent with the notion that neurons that utilize neurotransmitters other than DA or GABA are likely to be present in the VTA. Given the pronounced phenotypic diversity of neurons in this region, we have proposed that additional cell types, such as those that express the neurotransmitter glutamate may also be present in this area. Thus, by using in situ hybridization histochemistry we investigated whether transcripts encoded by genes for the two vesicular glutamate transporters, VGluT1 or VGluT2, were expressed in the VTA. We found that VGluT2 mRNA but not VGluT1 mRNA is expressed in the VTA. Neurons expressing VGluT2 mRNA were differentially distributed throughout the rostro-caudal and medio-lateral aspects of the VTA, with the highest concentration detected in rostro-medial areas. Phenotypic characterization with double in situ hybridization of these neurons indicated that they rarely co-expressed mRNAs for tyrosine hydroxylase (TH, marker for DAergic neurons) or glutamic acid decarboxylase (GAD, marker for GABAergic neurons). Based on the results described here, we concluded that the VTA contains glutamatergic neurons that in their vast majority are clearly non-DAergic and non-GABAergic.

Figures

FIG. 1
FIG. 1
Expression of VGluT2 mRNA in the VTA. (A and A′) Frames A (bright field microscopy) and A′ (epiluminescence microscopy) of a section hybridized with the antisense radioactive VGluT2 riboprobe. At low magnification in frame A′ notice positive signal for the detection of VGluT2 mRNA in the ventral tegmental area (VTA), medial terminal nucleus of the accessory optic tract (MT), medial lemniscus (ml) and red nucleus (R). Signal for VGluT2 mRNA is not seen in the substatia nigra compacta (SNC), substantia nigra reticulata (SNR) or fasciculus retroflexus (fr). (B and B′) Frames B (bright field microscopy) and B′ (epiluminescence microscopy) of a section hybridized with sense radioactive VGluT2 riboprobe showing lack of signal. (C and D′) Frames C, C′, D and D′ are high magnification micrographs of the VTA shown at low magnification in A, A′, B and B′. Arrows in C′ indicate cells expressing mRNA within the VTA. Bregma −5.16 mm. Scale bar shown in D′ represents 230 µm for A, A′, B and B′; 42 µm for C, C′, D and D′.
FIG. 2
FIG. 2
Expression of VGluT2 mRNA (white grains in A′, B′, and C′), TH immunoreactivity (dark brown label in A) and TH mRNA (purple label in B and C) in the rostral VTA. Frames A and B are adjacent sections at low power in bright field. The blue outline delimits the VTA (parabrachial pigmental area, PBP) and substantia nigra compacta (SNC) dopamine groups as defined by the extent of their cell bodies (purple cells in B and C). The magenta outline delimits the ventral tegmental area rostral (VTAR), which is rich in TH immunoreactive dendrites (dark brown label in A) but contains very few neurons expressing TH mRNA (B and C). Neurons expressing VGluT2 mRNA in the PBP and VTAR appear as aggregates of white grains in frames A′, B′ and C′. Note the high concentration of neurons expressing VGluT2 mRNA in the VTAR, an area having only occasional neurons expressing TH mRNA (compare B with B′), but with high levels of TH immunoreactive dendrites (compare A with B). Arrows in C′ indicate VGluT2 mRNA expressing neurons in the PBP. Frames C (bright field microscopy) and C′ (epiluminescence microscopy) are enlargement of the area delimited by rectangles in B and B′. Bregma −4.80 mm. Scale bar shown in C represents 480 µm for A, A′, B and B′ and 125 µm for C and C′.
FIG. 3
FIG. 3
Expression of VGluT2 mRNA (white grains in A′ and C′), TH mRNA (purple label in A and C) and TH immunoreactivity (dark brown label in B) in the medial VTA. Frames A and B are adjacent sections at low power in bright field. In frame A′ the cells expressing VGluT2 mRNA are seen in the VTA, red nucleus (R), caudal linear nucleus of the raphe (CLi), interfascicular nucleus (IF) and interpeduncular nucleus (IP). Note high levels of VGluT2 mRNA expression in red nucleus clearly seen as black dots under bright field (A and B) and white grains under epiluminescence microscopy (A′). Squares in A and A′ delimitate low magnification areas shown at higher magnifications in C and C′. (C′) Note higher concentration of cells expressing VGluT2 mRNA (arrows) in the paranigral nucleus (PN) than in the parabrachial pigmented nucleus (PBP). Bregma −5.40 mm. Scale bar shown in C represents 800 µm for A, A′ and B, and 80 µm for C and C′.
FIG. 4
FIG. 4
(A–C) Summary diagram of rostro-caudal distribution of cells expressing VGluT2 (closed circles) or TH (open circles) mRNAs in the VTA. Note that cells expressing TH mRNA are most densely aggregated in the caudal VTA, where cells expressing VGluT2 mRNA are sparse. Also, in the rostral and medial VTATH neurons are more densely aggregated in the PBP than in the PN or VTAR, where the converse is true for VGluT2 cells. Cells expressing VGluT2 mRNA or TH mRNA in neighbouring areas of the VTA are not included in the diagram. MT, medial terminal nucleus of the accessory optic tract; ml, medial lemniscus. Subregions of the VTA are named according to Paxinos & Watson (2005). Bregma −5.04 mm for A, −5.28 mm for B and −5.52 mm for C.
FIG. 5
FIG. 5
Expression of VGluT2 mRNA in the VTA. (A and A′) Low magnification of a section simultaneously hybridized with Dig-TH (A) and radioactive VGluT2 (A′) antisense riboprobes. Rectangle in A delimitates low magnification area shown at higher magnification in C and C. Rectangle in A′ delimitates low magnification area shown at higher magnification in B′. (B) Bright field microscopy of TH expressing cells seen before developing silver grains. (B′) Epiluminescence microscopy of the same section showing cells expressing VGluT2 mRNA. (C) Bright field microscopy of the same section after developing silver grains, note cells expressing either TH mRNA (purple cells) or VGluT2 mRNA (black grains). Bregma −5.40 mm. Scale bar shown in C represents 3.8 mm for A and A′ and 700 µm for B, B′ and C.
FIG. 6
FIG. 6
VGluT2 mRNA is mainly expressed in cells lacking expression of TH mRNA. (A and A′) Low magnification of a section simultaneously hybridized with Dig-TH (A) and radioactive VGluT2 (A′) antisense riboprobes. Rectangle in A delimitates low magnification area shown at higher magnification in B and B′. (B and B′) Pair of micrographs showing TH mRNA expressing cells in purple lacking VGluT2 mRNA (small arrows), and cells expressing VGluT2 mRNA lacking TH mRNA (large arrows). One cell appears to coexpress VGluT2 and TH transcripts (arrow-head). Bregma −5.40 mm. Scale bar shown in B′ represents 1 mm for A and A′, and 130 µm for B and B′.
FIG. 7
FIG. 7
Expression of VGluT2 mRNA in the VTA. (A and A′) Low magnification of a section simultaneously hybridized with Dig-GAD (A) and radioactive VGluT2 (A′) antisense riboprobes. Rectangle in A delimitates low magnification area shown at higher magnification in C and C. Rectangle in A′ delimitates low magnification area shown at higher magnification in B′. (B) Bright field microscopy of GAD expressing cells seen before developing silver grains. (B′) Epiluminescence microscopy of the same section showing cells expressing VGluT2 mRNA. (C) Bright field microscopy of the same section after developing silver grains, note cells expressing either GAD mRNA (purple cells) or VGluT2 mRNA (black grains). Bregma −5.40 mm. Scale bar shown in C represents 3.8 mm for A and A′ and 700 µm for B, B′ and C.
FIG. 8
FIG. 8
VGluT2 mRNA is mainly expressed in cells lacking expression of GAD mRNA. (A and A′) Low magnification of a section simultaneously hybridized with Dig-GAD (A) and radioactive VGluT2 (A′) antisense riboprobes. Rectangle in A delimitates low magnification area shown at higher magnification in B and B′. (B and B′) Pair of micrographs showing GAD mRNA expressing cells in purple lacking VGluT2 mRNA (small arrows), and cells expressing VGluT2 mRNA lacking GAD mRNA (large arrows). One cell appears to coexpress VGluT2 and GAD transcripts (arrow-head). Bregma −5.40 mm. Scale bar shown in B′ represents 1 mm for A and A′, and 130 µm for B and B′.

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