Stereological estimations and neurochemical characterization of neurons expressing GABAA and GABAB receptors in the rat pedunculopontine and laterodorsal tegmental nuclei

Esther Luquin,Carmen Santomá,Inés Zugasti,Beatriz Paternain,Elisa Mengual

doi:10.1007/s00429-021-02375-9

Esther Luquin, Carmen Santomá + Show 3 more

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https://doi.org/10.1007/s00429-021-02375-9

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Abstract

To better understand GABAergic transmission at two targets of basal ganglia downstream projections, the pedunculopontine (PPN) and laterodorsal (LDT) tegmental nuclei, the anatomical localization of GABAA and GABAB receptors was investigated in both nuclei. Specifically, the total number of neurons expressing the GABAA receptor γ2 subunit (GABAAR γ2) and the GABAB receptor R2 subunit (GABAB R2) in PPN and LDT was estimated using stereological methods, and the neurochemical phenotype of cells expressing each subunit was also determined. The mean number of non-cholinergic cells expressing GABAAR γ2 was 9850 ± 1856 in the PPN and 8285 ± 962 in the LDT, whereas those expressing GABAB R2 were 7310 ± 1970 and 9170 ± 1900 in the PPN and LDT, respectively. In addition, all cholinergic neurons in both nuclei co-expressed GABAAR γ2 and 95–98% of them co-expressed GABAB R2. Triple labeling using in situ hybridization revealed that 77% of GAD67 mRNA-positive cells in the PPT and 49% in the LDT expressed GABAAR γ2, while 90% (PPN) and 65% (LDT) of Vglut2 mRNA-positive cells also expressed GABAAR γ2. In contrast, a similar proportion (~2/3) of glutamatergic and GABAergic cells co-expressed GABAB R2 in both nuclei. The heterogeneous distribution of GABAAR and GABABR among non-cholinergic cells in PPN and LDT may give rise to physiological differences within each neurochemical subpopulation. In addition, the dissimilar proportion of GABAAR γ2-expressing glutamatergic and GABAergic neurons in the PPN and LDT may contribute to some of the functional differences found between the two nuclei.

Highlights

The pedunculopontine (PPN) and the laterodorsal (LDT) tegmental nuclei are two complex brainstem structures characteristically containing cholinergic cells (Mesulam et al 1983), which form an anatomical continuum in the mesopontine tegmentum and are considered as a functional unit working in an integrated fashion (Mena-Segovia 2016)
Coronal brainstem sections were first stained for NADPHd, a histochemical marker of PPN and LDT cholinergic neurons (Vincent et al 1983) used to identify the cholinergic subpopulation and delineate PPT and LDT territories, using previously described criteria (Luquin et al 2017; 2018); secondly, they were immunoreacted against GABAA receptors (GABAARs) α1, GABAAR γ2 and GABAB R2
No statistically significant differences were found in the rostrocaudal distribution of VGlut2/GABAB R2-positive cells either in the PPN (Wilcoxon signed-rank test p = 0.345) or LDT (Wilcoxon signed-rank test p = 0.500). These results indicate that, glutamatergic cells are preferentially located in the caudal portion of the PPN (Luquin et al 2018), there are no regional rostrocaudal differences in the relative distribution of glutamatergic cells co-expressing either GABAAR γ2 or GABAB R2 in this nucleus, and the same stands for the LDT

Summary

Introduction

The pedunculopontine (PPN) and the laterodorsal (LDT) tegmental nuclei are two complex brainstem structures characteristically containing cholinergic cells (Mesulam et al 1983), which form an anatomical continuum in the mesopontine tegmentum and are considered as a functional unit working in an integrated fashion (Mena-Segovia 2016). Some of their functions, like locomotion and motor adaptive control, action selection and reward, are related to their reciprocal connections with basal ganglia structures (MenaSegovia et al 2004; Roseberry et al 2016; Takakusaki et al 2016; Gut and Winn 2016). As a first step to better understand the interactions between the downstream projections of basal ganglia output nuclei and the PPN and LDT at the cellular level and the neural circuitry within these nuclei, we aimed to anatomically confirm the presence of GABA receptors within the PPN and LDT, and to quantitatively determine their relative distribution among the three cell phenotypes in the two nuclei

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