Abstract
The pedunculopontine tegmental nucleus (PPN) and laterodorsal tegmental nucleus (LDT) are functionally associated brainstem structures implicated in behavioral state control and sensorimotor integration. The PPN is also involved in gait and posture, while the LDT plays a role in reward. Both nuclei comprise characteristic cholinergic neurons intermingled with glutamatergic and GABAergic cells whose absolute numbers in the rat have been only partly established. Here we sought to determine the complete phenotypical profile of each nucleus to investigate potential differences between them. Counts were obtained using stereological methods after the simultaneous visualization of cholinergic and either glutamatergic or GABAergic cells. The two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67, were separately analyzed. Dual in situ hybridization revealed coexpression of GAD65 and GAD67 mRNAs in ∼90% of GAD-positive cells in both nuclei; thus, the estimated mean numbers of (1) cholinergic, (2) glutamatergic, and (3) GABAergic cells in PPN and LDT, respectively, were (1) 3,360 and 3,650; (2) 5,910 and 5,190; and (3) 4,439 and 7,599. These data reveal significant differences between PPN and LDT in their relative phenotypical composition, which may underlie some of the functional differences observed between them. The estimation of glutamatergic cells was significantly higher in the caudal PPN, supporting the reported functional rostrocaudal segregation in this nucleus. Finally, a small subset of cholinergic neurons (8% in PPN and 5% in LDT) also expressed the glutamatergic marker Vglut2, providing anatomical evidence for a potential corelease of transmitters at specific target areas.
Highlights
The pedunculopontine tegmental nucleus (PPN) and the laterodorsal tegmental nucleus (LDT) are two closely associated brainstem structures that jointly participate in a variety of functions such as behavioral state control, sensorimotor integration and reinforcement, and learning (Winn, 2006; Garcia-Rill et al, 2016; Mena-Segovia, 2016)
The specificity of the biotinylated riboprobes used for the detection of vesicular glutamate transporter 2 (Vglut2), GAD65, and GAD67 transcripts was assessed in control experiments using the sense and antisense riboprobes
Control sections were dually immunoreacted against choline acetyl transferase (ChAT) using an antibody reported to colocalize with all ChAT mRNA-containing cells in the PPN and LDT (Wang and Morales, 2009), and the labeling of ChAT-positive cells and fibers exhibited the characteristic distribution of cholinergic cell groups and terminal fields in the diencephalon and brainstem (Figures 1B,B’,C,C’; Mesulam et al, 1983; Rye et al, 1987)
Summary
The PPN and the LDT are two closely associated brainstem structures that jointly participate in a variety of functions such as behavioral state control, sensorimotor integration and reinforcement, and learning (Winn, 2006; Garcia-Rill et al, 2016; Mena-Segovia, 2016). Segregation of their respective output projections and/or regional complementarity at their target structures has been reported (Woolf and Butcher, 1986; Hallanger et al, 1987; Hallanger and Wainer, 1988; Semba and Fibiger, 1992; Oakman et al, 1995; Kita and Kita, 2011). A dual colorimetric method was used that enabled to distinctly visualize either cholinergic and glutamatergic cells, or cholinergic and either GAD67- or GAD65-positive cells, directly in a single section
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