Pedunculopontine and laterodorsal tegmental nuclei contain distinct populations of cholinergic, glutamatergic and GABAergic neurons in the rat

Abstract

The pedunculopontine tegmental nucleus (PPTg) and laterodorsal tegmental nucleus (LDTg) provide cholinergic afferents to several brain areas. This cholinergic complex has been suggested to play a role in sleep, waking, motor function, learning and reward. To have a better understanding of the neurochemical organization of the PPTg/LDTg we characterized the phenotype of PPTg/LDTg neurons by determining in these cells the expression of transcripts encoding choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or the vesicular glutamate transporters (vGluT1, vGluT2 and vGluT3). Within the PPTg/LDTg complex we found neurons expressing ChAT, vGluT2 or GAD transcripts, these neuronal phenotypes were intermingled, but not homogeneously distributed within the PPTg or LDTg. Previous studies suggested the presence of either glutamate or gamma-aminobutyric acid (GABA) immunolabeling in a large number of PPTg/LDTg cholinergic neurons, leading to the widespread notion that PPTg/LDTg cholinergic neurons co-release acetylcholine together with either glutamate or GABA. To assess the glutamatergic or GABAergic nature of the PPTg/LDTg cholinergic neurons, we combined in situ hybridization (to detect vGluT2 or GAD transcripts) and immunohistochemistry (to detect ChAT), and found that over 95% of all PPTg/LDTg cholinergic neurons lack transcripts encoding either vGluT2 mRNA or GAD mRNA. As the vast majority of PPTg/LDTg cholinergic neurons lack transcripts encoding essential proteins for the vesicular transport of glutamate or for the synthesis of GABA, co-release of acetylcholine with either glutamate or GABA is unlikely to be a major factor in the interactions between acetylcholine, glutamate and GABA at the postsynaptic site.

Figure 1. Cellular co-expression of ChAT immunoreactivity and ChAT mRNA in the PPTg and the LDTg (sagittal sections)

Frames A and B: Bright field microscopy showing neurons with ChAT immunoreactivity (DAB: brown cells; ChAT-IR, arrows) or GAD mRNA (purple cells; arrow heads). Frames A' and B': Epiluminescence microscopy showing cellular expression of ChAT mRNA (silver grains seen as white grain aggregates). Note examples of neurons expressing ChAT mRNA (arrows) shown in frames A and B as ChAT immunoreactive. Frames A” and B”. Cellular co-expression of ChAT immunoreactivity and ChAT mRNA under bright field and epiluminescence microscopy. Note detection of ChAT mRNA (silver grains seen as green grain aggregates) in all ChAT immunoreactive neurons (DAB: brown cells). ChAT, choline acetyltransferase; PPTg, pedunculopontine tegmental nucleus; LDTg, laterodorsal tegmental nucleus. Scale bar is 50 μm.

Figure 2. Cellular expression of GAD mRNA in the PPTg and the LDTg (sagittal sections)

Frames A and B: Bright field microscopy showing ChAT immunoreactive (DAB: brown cells; ChAT-IR, arrows) neurons in the PPTg (A) and the LDTg (B). Frames A' and B': Same sections under epiluminescence microscopy show cellular expression of GAD mRNA (silver grains seen as white grain aggregates, arrow heads). Frames A” and B”: Note under bright field and epiluminescence microscopy cells expressing GAD mRNA (silver grains seen as green grain aggregates) intermingle with ChAT immunoreactive neurons (DAB: brown cells). Scale bar is 135 μm.

Figure 3. Lack of expression of vGluT1mRNA or vGluT3 mRNA in the LDTg (coronal sections)

Frame A: Bright field microscopy showing ChAT immunoreactive cells (DAB: brown). Rectangle in frame A delimitates low magnification area shown at higher magnification in 1A (bright field microscopy) and 1A' (epiluminescence microscopy). Cellular expression of vGluT1 mRNA is present within the mesencephalic trigeminal nucleus, Me5 (arrow heads), note lack of vGluT1 mRNA within the LDTg. Frame B: Epiluminescence microscopy, vGluT3 mRNA is clearly seen within the Dorsal Raphe (DR), note lack of vGluT3 mRNA in the dorsal tegmental nucleus (DTg) and in the LDTg. Frame B': Bright field microscopy of same section shown in frame B, ChAT immunoreactive neurons (DAB: brown cells) are prominent in the LDTg while those with GAD mRNA (purple cells) appear to be concentrated in the DTg. Scale bar is 610 μm for A, 360 μm for 1A and 1A', and 265 μm for B and B'.

Fig. 4. Expression of vGluT2 mRNA in the PPTg (coronal sections)

Frame A and B: Low (A) and high magnification (B) of a Nissl stained section. Frame C and C': Adjacent section to the one showed in frame B, section was hybridized with vGluT2 antisense riboprobe. Note in frame C expression of ChAT immunoreactive neurons (DAB: brown cells, arrows) and vGluT2 mRNA (silver grains seen as green aggregates, arrow-heads) seen under bright field and epiluminescence microscopy. Cellular expression of vGluT2 mRNA is seen as white aggregates under epiluminescence microscopy in frame C'. Frames D and D': Adjacent section to the one showed in frames C and C', section was hybridized with a sense radioactive vGluT2 riboprobe, note lack of green aggregates in frame D and white aggregates in frame D'. Scale is 250 μm for A and 180 μm for B, C, C', D and D'.

Fig. 5. Expression of vGluT2 mRNA in the LDTg (coronal sections)

Frame A and B: Low (A) and high magnification (B) of a Nissl stained section. Frame C and C': Adjacent section to the one showed in frame B, section was hybridized with vGluT2 antisense riboprobe. Note in frame C expression of ChAT immunoreactive neurons (DAB: brown cells, arrows) and vGluT2 mRNA (silver grains seen as green aggregates, arrow-heads) seen under bright field and epiluminescence microscopy. Cellular expression of vGluT2 mRNA is seen as white aggregates under epiluminescence microscopy in frame C'. Frames D and D': Adjacent section to the one showed in frames C and C', section was hybridized with a sense radioactive vGluT2 riboprobe, note lack of green aggregates in frame D and white aggregates in frame D'. Scale bar is 325 μm for A and 165 μm for B, C, C', D and D'.

Figure 6. ChAT immunoreactive neurons of the PPTg lack either vGluT2 mRNA or GAD mRNA (sagittal sections)

Frames A and B correspond to a low magnification view of the same section showing ChAT immunoreactivity under bright field microscopy (A) or vGluT2 mRNA under epiluminescence microscopy (B). Rectangles in frames A and B delimitate low magnification areas shown at higher magnification in 1A and 1B. In frames 1A and 1B, the blue outlines demarcate the profile of ChAT (+) neurons; note in frame 1B lack of concentricity of white aggregates (vGluT2 mRNA) within most of the blue outlines, concentricity is apparent in two cells (arrows). An adjacent section to the one shown in frames A and B is displayed in frames C and D. Frames C and D correspond to a low magnification view of the same section showing ChAT immunoreactivity (C) or GAD mRNA (D). Rectangles in frames C and D delimitate low magnification areas shown at higher magnification in frames 2C and 2D. In frames 2C and 2D, the blue outlines demarcate the profile of ChAT (+) neurons; note in frame 2D lack of concentricity of white aggregates (GAD mRNA) within the blue outlines. Scale bar is 60 μm for 1A, 1B, 2C and 2D, and 150 μm for A, B, C and D.

Figure 7. ChAT immunoreactive neurons of the LDTg lack either vGluT2 mRNA or GAD mRNA (sagittal sections)

Frames A and B correspond to a low magnification view of the same section showing ChAT immunoreactivity (A) or vGluT2 mRNA (B). Rectangles in frames A and B delimitate low magnification areas shown at higher magnification in frames 1A and 1B. In frames 1A and 1B, the blue outlines demarcate the profile of ChAT (+) neurons; note in frame 1B the lack of concentricity of white aggregates (vGluT2 mRNA) within the blue outlines. An adjacent section to the one shown in frames A and B is displayed in frames C and D. Frames C and D correspond to low magnification view of the same section showing ChAT immunoreactivity (C) or GAD mRNA (D). Rectangles in C and D delimitate a low magnification areas shown at higher magnification in frames 2C and 2D. In frames 2C and 2D, the blue outlines demarcate the profile of ChAT (+) neurons; note in 2D lack of concentricity of white aggregates (GAD mRNA) within the blue outlines, concentricity is clearly seen in one cell (arrow). Scale bar is 63 μm for 1A, 1B, 2C and 2D, and 164 μm for A, B, C and D.

Figure 8. ChAT immunoreactive neurons in the PPTg are intermingled with neurons expressing either vGluT2 mRNA or GAD mRNA (triple labeling)

Frame A: Bright field microscopy of a sagittal section showing ChAT immunoreactive neurons (DAB: brown cells) and cells expressing GAD mRNA (purple neurons). Frame A': Epiluminescence microscopy of the section showed in frame A, note cellular expression of vGluT2 mRNA (silver grains seen as white aggregates). Frames B, B', C and C' correspond to higher magnification areas delimited by rectangles in frames A and A' of the pars compacta (B and B') and the pars dissipata (C and C') of the PPTg. scp, superior cerebellar peduncle. Scale bar is 346 μm for A and A', and 95 μm for B, B', C and C'.

Figure 9. Distribution of neurons expressing transcripts encoding either vGluT2 or GAD within the PPTg (double labeling)

Frames A and B: Bright field microscopy of a sagittal section showing ChAT immunoreactive neurons (DAB: brown cells) in the pars compacta (A) and pars dissipata (B) of the PPTg. Frame A' and B': Epiluminescence microscopy of the section showed in frame A and B, note cellular expression of vGluT2 mRNA (silver grains seen as white aggregates) within both sub-divisions of the PPTg. Frames C and D: Bright field microscopy of a sagittal section, adjacent to the one in frames A and B, showing ChAT immunoreactive neurons (DAB: brown cells) in the pars compacta (C) and pars dissipata (D) of the PPTg. Frame C' and D': Epiluminescence microscopy of the section showed in frame C and D, note cellular expression of GAD mRNA (silver grains seen as white aggregates) within both subdivisions of the PPTg. Scale bar is 84 μm.

Figure 10. Summary diagram of the distribution of cells containing ChAT immunoreactivity (circles in frames A and D), vGluT2 mRNA (stars in frames B and E) or GAD mRNA (triangles in frames C and F) in the PPTg

Very few ChAT (+) cells co-express either vGluT2 mRNA (solid squares in frames B and E) or GAD mRNA (solid circles in frames C). PPTg (c), pars compacta; PPTg (d), pars dissipata.

Figure 11. ChAT (+) neurons in the LDTg are intermingled with neurons expressing either vGluT2 mRNA or GAD mRNA (triple labeling)

Frame A: Bright field microscopy of a sagittal section showing ChAT immunoreactive neurons (DAB: brown cells) and cells expressing GAD mRNA (purple neurons). Frame A': Epiluminescence microscopy of the section showed in frame A, note cellular expression of vGluT2 mRNA (silver grains seen as white aggregates). Frames B and B', correspond to higher magnification areas delimited by rectangles in frames A and A'. Scale bar is 225 μm for A and A', and 88 μm for B and B'.

Figure 12. Distribution of neurons expressing either vGluT2 mRNA or GAD mRNA within the LDTg (double labeling)

Frames A and B: Bright field microscopy of two consecutive sections showing ChAT immunoreactive neurons (brown cells) within the LDTg (sagittal sections). Note the highest concentration of ChAT (+) cells in the medial portion of the LDTg. Frame A': Epiluminescence microscopy of section showed in frame A. Note an apparent descending rostral-caudal gradient of expression of vGluT2 mRNA (silver grains seen as white aggregates). Frame B': Epiluminescence microscopy of section showed in frame B. Note an apparent ascending rostral-caudal gradient of expression of GAD mRNA (silver grains seen as white aggregates). LDTg, dorsal LDTg; LDTgV, ventral LDTg. Scale bar is 285 μm.

Figure 13. Summary diagram of the distribution of cells containing ChAT immunoreactivity (circles in frames A and D), vGluT2 mRNA (stars in frames B and E) or GAD mRNA (triangles in frames C and F) in the LDTg

Very few ChAT (+) cells co-express either vGluT2 mRNA (solid squares in frames B and E) or GAD mRNA (solid circles in frames C and F).