Colocalization of GAD-67 and 5-HT_2CR immuonoreactivity with FG-labeled cells in the VTA.

<p>[<b>A</b>] Representative composite photomicrograph of the middle level of the VTA displaying the overlay of FG (blue), GAD-67-IR (red) and 5-HT_2CR-IR (green). Inset displays the schematic diagram of the middle VTA (shaded area) and surrounding brain areas (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone-0020508-g003" target="_blank">Fig. 3</a> for abbreviations) at bregma -5.64 mm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone.0020508-Paxinos1" target="_blank">[17]</a>. High magnification images of the boxed region in panel A depict FG labeling [blue; B], GAD-67-IR [red, C], and 5-HT_2CR-IR [green, D], as well as the overlay of images in B, C, and D to demonstrate colocalization [E]. Filled arrows (<b></b>) indicate cells triple-labeled for FG+GAD-67+5-HT_2CR cells, while the open arrows (<b></b>) indicate a cell double-labeled for FG+5-HT_2CR; as noted in the text, cells labeled for FG+GAD-67 alone were not often detected in the area represented by the boxed region. Scale bars  = 20 µm. Note: Portions of IP nucleus present in the composite photomicrograph in panel A were removed from the image prior to incorporation into the figure.</p

FluoroGold staining at injection site in the NAc.

<p>Schematic diagrams depicting coronal sections of the NAc shell and surrounding brain areas at bregma +2.16, +1.80 and +1.56 mm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone.0020508-Paxinos1" target="_blank">[17]</a> overlaid on top of a representative composite photomicrographs depicting the FG injection sites (100 nL; 1–2% FG) for the three animals with injections correctly placed in the NAc shell.</p

Colocalization of FG, TH and 5-HT_2CR in the VTA.

<p>Images display FG- [blue, A], TH- [red, B] and 5-HT_2CR-labeling [green, C] in series of five sequential photomicrographs (from left to right) captured using a confocal microscope in the VTA of a rat injected with FG in the NAc shell. Photomicrographs represent images captured at a distance of 1.0 µm apart through the thickness of the brain section. [D] Overlay of images in A–C shows colocalization of TH and 5-HT_2CR in a FG-labeled cell in the VTA. Scale bar  = 10 µm.</p

Distribution of FG- TH- and GAD-67-labeled cells in the VTA.

<p>Schematic representation of the location of cells labeled for FG alone (black squares), FG+TH (blue circles), FG+GAD-67 (green triangles) and FG+TH+GAD-67-labeled cells (red stars) in the [A] rostral (∼bregma −5.14 mm), [B] middle (∼bregma −5.67 mm), and [C] caudal (∼bregma −6.30 mm) levels of the VTA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone.0020508-Paxinos1" target="_blank">[17]</a>. Insets display schematic diagrams depicting the location of VTA (shaded) relative to surrounding brain areas (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone-0020508-g005" target="_blank">Fig. 5</a> for abbreviations) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone.0020508-Paxinos1" target="_blank">[17]</a>. Data represent the number and distribution of cells counted in one rostral, middle or caudal section from a animal injected with FG in the NAc shell.</p

Distribution of FG- TH- and 5-HT_2CR-labeled cells in the VTA.

<p>Schematic representation of the location of cells labeled for FG alone (black squares), FG+TH (blue circles), FG+5-HT_2CR (green triangles) and FG+TH+5-HT_2CR-labeled cells (red stars) in the [A] rostral (∼bregma −5.12 mm), [B] middle (∼bregma −5.67 mm), and [C] caudal (∼bregma −6.30 mm) levels of the VTA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone.0020508-Paxinos1" target="_blank">[17]</a>. Insets display schematic diagrams depicting the location of VTA (shaded) relative to surrounding brain areas [interpeduncular nucleus (IP); interpeduncular fossa (IPF); medial laminiscus (ml); mammillary peduncle (mp); mammillothalamic tract (MT) substantia nigra pars compacta, dorsal tier (SNCD); substantia nigra pars compacta, medial tier (SNCM); substantial nigra reticulata (SNR)] <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone.0020508-Paxinos1" target="_blank">[17]</a>. Data represent the number and distribution of cells counted in one rostral, middle or caudal section from an animal injected with FG in the NAc shell.</p

Primary antibodies employed in the experiments.

<p>Primary antibodies employed in the experiments.</p

Total FG-labeled cells containing immunoreactivity for TH, GAD-67, and 5-HT_2CR.

a<p>Bregma locations according to the brain atlas of Paxinos and Watson (1998).</p>b<p>Average (± SEM) per section of 12 rostral and middle sections, 9 caudal sections.</p>c<p>Average (± SEM) number of total FG+5-HT_2CR-labeled cells per section in 24 rostral and middle, 18 caudal sections.</p>d<p>Average (± SEM) of FG+TH-labeled cells/total FG cells in 12 rostral and middle, 9 caudal sections.</p>e<p>Average (± SEM) of FG+TH+5-HT_2CR-labeled cells/total FG+TH-labeled cells in 12 rostral and middle, 9 caudal sections.</p>f<p>Average (± SEM) of FG+GAD-67-labeled cells/total FG cells in 12 rostral and middle, 9 caudal sections.</p>g<p>Average (± SEM) of FG+GAD-67+5-HT_2CR-labeled cells/total FG/+GAD-67-labeled cells in 12 rostral and mid, 9 caudal sections.</p><p>**<i>p</i><0.001 vs. rostral level;</p><p>*<i>p</i><0.01 vs. rostral level;</p><p>∧<i>p</i><0.001 vs. middle level.</p

Colocalization of TH and GAD-67 immuonoreactivity with FG-labeled cells in the VTA.

<p>[A] Representative composite photomicrograph of the middle level of the VTA displaying the overlay of FG (blue), TH-IR (green) and GAD-67-IR (red). Inset displays the schematic diagram of the middle VTA (shaded area) and surrounding brain areas (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone-0020508-g003" target="_blank">Fig. 3</a> for abbreviations] at bregma -5.64 mm. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone.0020508-Paxinos1" target="_blank">[17]</a>. High magnification images of the boxed region in panel A depict FG labeling [blue, B], TH-IR [green, C], and GAD-67-IR [red, D], as well as the overlay of images in B, C, and D to demonstrate colocalization [E]. Filled arrows (<b></b>) indicate a cell triple-labeled for FG+TH+GAD-67, open arrows (<b></b>) indicate a cell double-labeled for FG+TH, solid arrows () indicate a cell double-labeled for FG+GAD-67, and the arrowheads () point to a cell double-labeled for TH+GAD-67 in the absence of FG; Scale bars  = 20 µm. Note: Portions of IP nucleus present in the composite photomicrograph in panel A were removed from the image prior to incorporation into the figure.</p

Distribution of FG- GAD-67- and 5-HT_2CR-labeled cells in the VTA.

<p>Schematic representation of the location of cells labeled for FG alone (black squares), FG+GAD-67 (blue circles), FG+5-HT_2CR (green triangles) and FG+GAD-67+5-HT_2CR-labeled cells (red stars) in the [A] rostral (∼bregma −5.10 mm), [B] middle (∼bregma −5.69 mm), and [C] caudal (∼bregma −6.26 mm) levels of the VTA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone.0020508-Paxinos1" target="_blank">[17]</a>. Insets display schematic diagrams depicting the location of VTA (shaded) relative to surrounding brain areas (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone-0020508-g005" target="_blank">Fig. 5</a> for abbreviations) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone.0020508-Paxinos1" target="_blank">[17]</a>. Data represent the number and distribution of cells counted in one rostral, middle or caudal section from an animal injected with FG in the NAc shell.</p

Colocalization of TH and 5-HT_2CR immuonoreactivity with FG-labeled cells in the VTA.

<p>[A] Representative composite photomicrograph of the middle level of the VTA displaying the overlay of FG (blue), TH-IR (red) and 5-HT_2CR-IR (green). Inset displays the schematic diagram of the middle VTA (shaded area) and surrounding brain areas [interpeduncular nucleus (IP); medial laminiscus (ml); mammillary peduncle (mp); substantia nigra pars compacta, dorsal tier (SNCD); substantial nigra reticulata (SNR)] at bregma −5.64 mm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020508#pone.0020508-Paxinos1" target="_blank">[17]</a>. High magnification images of the boxed region in panel A depict FG labeling [blue; B], TH-IR [red, C], and 5-HT_2CR-IR [green, D], as well as the overlay of images in B, C and D to demonstrate colocalization [E]. Filled arrows (<b></b>) indicate cells triple-labeled for FG+TH+5-HT_2CR, while open arrows (<b></b>) indicate a cell double-labeled for FG+TH; Scale bars  = 20 µm. Note: Portions of IP nucleus present in the composite photomicrograph in panel A were removed from the image prior to incorporation into the figure.</p