5-HT(2C) receptors localize to dopamine and GABA neurons in the rat mesoaccumbens pathway

Abstract

The serotonin 5-HT(2C) receptor (5-HT(2C)R) is localized to the limbic-corticostriatal circuit, which plays an integral role in mediating attention, motivation, cognition, and reward processes. The 5-HT(2C)R is linked to modulation of mesoaccumbens dopamine neurotransmission via an activation of γ-aminobutyric acid (GABA) neurons in the ventral tegmental area (VTA). However, we recently demonstrated the expression of the 5-HT(2C)R within dopamine VTA neurons suggesting the possibility of a direct influence of the 5-HT(2C)R upon mesoaccumbens dopamine output. Here, we employed double-label fluorescence immunochemistry with the synthetic enzymes for dopamine (tyrosine hydroxylase; TH) and GABA (glutamic acid decarboxylase isoform 67; GAD-67) and retrograde tract tracing with FluoroGold (FG) to uncover whether dopamine and GABA VTA neurons that possess 5-HT(2C)R innervate the nucleus accumbens (NAc). The highest numbers of FG-labeled cells were detected in the middle versus rostral and caudal levels of the VTA, and included a subset of TH- and GAD-67 immunoreactive cells, of which >50% also contained 5-HT(2C)R immunoreactivity. Thus, we demonstrate for the first time that the 5-HT(2C)R colocalizes in DA and GABA VTA neurons which project to the NAc, describe in detail the distribution of NAc-projecting GABA VTA neurons, and identify the colocalization of TH and GAD-67 in the same NAc-projecting VTA neurons. These data suggest that the 5-HT(2C)R may exert direct influence upon both dopamine and GABA VTA output to the NAc. Further, the indication that a proportion of NAc-projecting VTA neurons synthesize and potentially release both dopamine and GABA adds intriguing complexity to the framework of the VTA and its postulated neuroanatomical roles.

Figure 1. FluoroGold staining at injection site in the NAc.

Schematic diagrams depicting coronal sections of the NAc shell and surrounding brain areas at bregma +2.16, +1.80 and +1.56 mm 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.

Figure 2. FluoroGold-labeled cells in the VTA following NAc FG infusion.

Schematic diagrams depicting coronal sections of the rostral [bregma −5.16 mm; A], middle [bregma −5.64 mm; B], and caudal [bregma −6.12 mm; C,] levels of the VTA and surrounding brain areas overlaid on top of representative composite photomicrographs from Rat #3 (see Fig. 1) displaying FG (blue) labeling in the VTA one week following infusion of FG into the NAc shell. [D, E, F] Higher power magnification of yellow boxed regions in A, B, and C, respectively. Scale bars  = 30 µm.

Figure 3. Colocalization of TH and 5-HT_2CR immuonoreactivity with FG-labeled cells in the VTA.

[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 . 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 () indicate cells triple-labeled for FG+TH+5-HT_2CR, while open arrows () 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.

Figure 4. Colocalization of FG, TH and 5-HT_2CR in the VTA.

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.

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

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 . 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)] . 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.

Figure 6. Colocalization of GAD-67 and 5-HT_2CR immuonoreactivity with FG-labeled cells in the VTA.

[A] 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 Fig. 3 for abbreviations) at bregma -5.64 mm . 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 () indicate cells triple-labeled for FG+GAD-67+5-HT_2CR cells, while the open arrows () 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.

Figure 7. Colocalization of FG, GAD-67 and 5-HT_2CR in the VTA.

Photomicrographs display FG- [blue, A], GAD-67- [red, B] and 5-HT_2CR-labeling [green, C] in series of five sequential images (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 GAD-67- and 5-HT_2CR-IR in a FG-labeled cell in the VTA. Scale bars  = 10 µm.

Figure 8. Distribution of FG- GAD-67- and 5-HT_2CR-labeled cells in the VTA.

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 . Insets display schematic diagrams depicting the location of VTA (shaded) relative to surrounding brain areas (see Fig. 5 for abbreviations) . 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.

Figure 9. Colocalization of TH and GAD-67 immuonoreactivity with FG-labeled cells in the VTA.

[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 Fig. 3 for abbreviations] at bregma -5.64 mm. . 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 () indicate a cell triple-labeled for FG+TH+GAD-67, open arrows () 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.

Figure 10. Distribution of FG- TH- and GAD-67-labeled cells in the VTA.

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 . Insets display schematic diagrams depicting the location of VTA (shaded) relative to surrounding brain areas (see Fig. 5 for abbreviations) . 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.