Figure 1.
Schematic representation of the brain areas (black boxed regions) in which expression of mRNA and protein immunoreactivity as well as colocalization was examined.
Figures are adapted and modified from The Allen Mouse Brain Atlas (Reference Atlas Version 1, 2008). Nomenclature is obtained from Paxinos and Franklin [108]. Distance from bregma in the rostrocaudal planes is indicated. Abbreviations: aco, anterior commissure, olfactory limb; act, anterior commissure, temporal limb; Cg, cingulate cortex; LSD, dorsal part of lateral septal nucleus; LSI, intermediate part of lateral septal nucleus; LSV, ventral part of lateral septal nucleus; MPOA, medial preoptic area; VL, lateral ventricle.
Figure 2.
Schematic diagram depicting probe design for GAD65 and GAD67 in situ hybridization assay.
For each gene, three sets of oligonucleotide probes were generated to target three distinct sequences of each mRNA. Recognition of GAD65 and GAD67 mRNA sequences and length covered by each oligonucleotide probe are indicated. There was no overlap of sequences among all the probes and the probes designed for the two GADS had no homology to one another to ensure the specificity of the probe. The GAD67 probes were labeled with digoxigenin, while GAD65 probes were labeled with either biotin or digoxigenin. Note that probes for GAD65 target the mRNA 3’-non-coding sequences, whereas probes for GAD67 primarily recognize the mRNA 3’-coding sequences (blue box).
Figure 3.
Expression and colocalization of neurons expressing mRNAs for GAD65 and GAD67 in the LS, Cg and MPOA.
The number (A) and colocalization ratio (B) is presented. Note that mRNAs for both GAD65 and GAD67 are colocalized in the vast majority of neurons of the three brain regions. Each bar represents the mean+SEM obtained from six mice. Colocalization ratio of GAD67/GAD65 was calculated by dividing the number of neurons expressing both GAD65 and GAD67 mRNAs (colocalization) by total number of neurons expressing GAD65 mRNA, and multiplying by 100. A: a p < 0.001 versus LSr_D and LSr_I, b p < 0.05 versus LSc_D and LSc_I, c p < 0.001 versus LSc_D, d p < 0.001 versus LSr_D and LSr_I, e p < 0.05 versus LSc_D and LSc_I, f p < 0.001 versus LSc_D, g p < 0.001 versus GAD65 in LSr_V, h p < 0.001 versus GAD65 in LSc_V; B: a p < 0.05 versus LSc_D and LSc_I, b p < 0.05 versus GAD67/GAD65.
Figure 4.
Double fluorescence in situ hybridization labeling of GAD65 and GAD67 mRNA-expressing neurons in the LSD (A–D), LSI (E–H) and LSV (I–L).
Low magnification images of the white boxed regions (A, E and I) show neurons expressing mRNAs for GAD65 and GAD67 were counted for colocalization analysis. High magnification images show the colocalization of neurons containing both GAD65 and GAD67 mRNAs in the LSD (B–D), LSI (F–H) and LSV (J–L). Two typical examples of neurons coexpressing mRNAs for GAD65 and GAD67 in each subdivision are indicated in arrows. Note that in all three subdivisions of the LS, GAD65 and GAD67 mRNAs are highly colocalized in a single neuron. Scale bars = 150 µm in A, E and I; 50 µm in B–D, F–H, J-L.
Figure 5.
High magnification photomicrographs of z-series stacks displaying double fluorescence in situ hybridization labeling of GAD65 and GAD67 mRNA-expressing neurons in a single neuron of the LSI.
Images represent a series of four sequential photomicrographs (from B-E, G-J and L-O) that were captured at a distance of 0.5 micron apart through the entire thickness of the brain section, while images in A, F and K were projected images from four sequential photomicrographs correspondingly. Arrows indicate intensely labeled GAD65 mRNA at the periphery of cell bodies and axon initial segment, and arrowheads indicate robust and even labeling throughout the soma of the GAD67 mRNA-expressing cells. Scale bar = 250 µm.
Figure 6.
Double fluorescence in situ hybridization labeling of GAD65 and GAD67 mRNA-expressing neurons in the Cg (A–F) and MPOA (G–L).
Low magnification images of the white boxed regions (A–C, G–I) show the GAD65 and GAD67 mRNA-expressing neurons were counted for colocalization analysis. High magnification images show the colocalization of GAD65 and GAD67 mRNA-expressing neurons in the Cg (D–F) and MPOA (J–L). Two typical examples of double-labeled neurons, indicating the colocalization of GAD65 and GAD67 mRNA in the Cg and MPOA are indicated in arrows. Note that mRNAs for GAD65 and GAD67 are highly coexpressed in a single cell. Scale bars = 150 µm in A-C, G-I; 50 µm in D–F, J-L.
Figure 7.
Expression and colocalization of cells expressing mRNAs for GAD65/GAD67 (GAD) and NeuN-immunoreactivity in the LS, Cg and MPOA.
The number (A) and colocalization ratio (B) was calculated. Note that the vast majority of cells coexpressed GAD and NeuN within the three brain regions. Each bar represents the mean+SEM obtained from six mice. A: a p < 0.001 versus LSr_D and LSr_I, b p < 0.05 versus LSc_D and LSc_I, c p < 0.001 versus LSc_D, d p < 0.001 versus LSr_D and LSr_I, e p < 0.05 versus LSc_D and LSc_I, f p < 0.001 versus LSc_D; B: a p < 0.05 versus LSr_D, b p < 0.05 versus LSc_D, c p < 0.01 versus LSc_D and LSc_I, d p < 0.05 versus GAD/NeuN.
Figure 8.
Double fluorescence in situ hybridization and immunohistochemistry labeling of neurons expressing mRNAs for GAD65/GAD67 (GAD) and NeuN-immunoreactivity in the LSD (A–D), LSI (E–H) and LSV (I–L).
Low magnification images of the white boxed regions (A, E and I) show the GAD- and NeuN-expressing neurons were counted for colocalization analysis. High magnification images show the colocalization of GAD and NeuN in the LSD (B–D), LSI (F–H) and LSV (J–L). Two typical examples of double-labeled neurons, indicating the colocalization of GAD and NeuN in each subdivision are indicated in arrows. Note that in all three subdivisions of the lateral septum, GAD and NeuN are highly coexpressed in a single cell. Scale bars = 150 µm in A, E and I; 50 µm in B–D, F–H, J-L.
Figure 9.
Double fluorescence in situ hybridization and immunohistochemistry labeling of neurons expressing mRNAs for GAD65/GAD67 (GAD) and NeuN-immunoreactivity in the Cg (A–F) and MPOA (G–L).
Low magnification images of the white boxed regions (A–C, G–I) show the GAD- and NeuN-expressing neurons were counted for colocalization analysis. High magnification images show the colocalization of GAD- and NeuN-expressing neurons in the Cg (D–F) and MPOA (J–L). Two typical examples of double-labeled neurons in the Cg and MPOA are indicated in arrows. Note that GAD and NeuN are highly coexpressed in a single cell. Scale bars = 150 µm in A-C, G-I; 50 µm in D–F, J-L.
Figure 10.
Expression and colocalization of neurons expressing mRNAs for GAD65/GAD67 (GAD) and immunoreactivity for calcium binding proteins in the LS, Cg and MPOA.
Each bar represents the mean+SEM obtained from four mice. A: number of calbindin-immunoreactive cells: a p < 0.001 versus LSr_D and LSr_I, b p < 0.05 versus LSc_D and LSc_I, c p < 0.001 versus LSc_D, d p < 0.05 versus LSr_I and LSr_V, e p < 0.001 versus LSr_I and LSc_D, f p < 0.01 versus LSr_V, LSc_D and LSc_I; B: ratio of colocalization of GAD and calbindin. a p < 0.001 versus LSc_I, b p < 0.001 versus LSc_I and LSc_V, c p < 0.001 versus GAD/Calbindin; C: number of calretinin-immunoreactive cells, a p < 0.001 versus LSr_D and LSr_I, b p < 0.05 versus LSc_D and LSc_I, c p < 0.001 versus LSc_D, d p < 0.001 versus LSr_I and LSc_D, e p < 0.001 versus LSr_V and LSc_D; D, ratio of colocalization of GAD and calretinin, a p < 0.05 versus LSr_D and LSr_V, b p < 0.001 versus LSc_I and LSc_V, c p < 0.001 versus LSc_V, d p < 0.001 versus LSc_V, e p < 0.001 versus GAD/Calretinin.
Figure 11.
Double fluorescence in situ hybridization and immunohistochemistry labeling of neurons expressing mRNAs for GAD65/GAD67 (GAD) and calbindin-immunoreactivity in the LSD (A–C), LSI (D–F) and LSV (G–I).
High magnification images show the colocalization of neurons expressing GAD and CB. Two typical examples of double-labeled (arrows) or single-labeled (arrowheads) neurons in each subdivision are indicated. Note that in all three subdivisions of the lateral septum, GAD and CB are highly coexpressed in a single cell, and most CB-immunoreactive cells express GAD mRNA (GABAergic). Scale bar = 50 µm.
Figure 12.
Double fluorescence in situ hybridization and immunohistochemistry labeling of neurons expressing mRNAs for GAD65/GAD67 (GAD) and calbindin-immunoreactivity in the Cg (A–C) and MPOA (D–F).
High magnification images show the colocalization of neurons expressing GAD and CB. Two typical examples of double-labeled (arrows) or single-labeled (arrowheads) neurons are indicated. Note that GAD and CB are highly coexpressed in a single cell, and most CB-immunoreactive cells express GAD mRNA (GABAergic). Scale bar = 50 µm.
Figure 13.
Double fluorescence in situ hybridization and immunohistochemistry labeling of neurons expressing mRNAs for GAD65/GAD67 (GAD) and calretinin-immunoreactivity in the LSD (A–C), LSI (D–F) and LSV (G–I).
High magnification images show the colocalization of neurons expressing GAD and CR.
Typical examples of double-labeled (arrows) or single-labeled (arrowheads) neurons in each subdivision are indicated. Note that in all three subdivisions of the lateral septum, GAD and CR are highly coexpressed in a single cell, and most CR-immunoreactive cells express GAD mRNA (GABAergic). Scale bar = 50 µm.
Figure 14.
Double fluorescence in situ hybridization and immunohistochemistry labeling of neurons expressing mRNAs for GAD65/GAD67 and calretinin-immunoreactivity in the Cg (A–C) and MPOA (D–F).
High magnification images show the colocalization of neurons expressing GAD and CR. Two typical examples of double-labeled (arrows) or single-labeled (arrowheads) neurons are indicated. Note that GAD and CR are highly coexpressed in a single cell, and most CR-immunoreactive cells express GAD mRNA (GABAergic). Scale bar = 50 µm.
Figure 15.
Double fluorescence in situ hybridization and immunohistochemistry labeling of neurons expressing mRNAs for GAD65/GAD67 (GAD) and parvalbumin-immunoreactivity in the Cg (A–F).
High magnification images show the colocalization of neurons expressing GAD and PV. Typical examples of double-labeled (arrows) or single-labeled (arrowheads) neurons are indicated. Widely spreading dendrites (D, open arrows) and cell bodies (D, arrows) of PV-immunoreactive neurons are clearly stained. Note that GAD and PV are highly coexpressed in a single cell, and most PV-immunoreactive cells express GAD mRNA (GABAergic). Scale bars = 50 µm in A-C; 50 µm in D–F.