From Signals to Subtypes: Mapping GABAergic Neuron Diversity and Migration in the Developing Ventral Midbrain

We investigate the development of GABAergic neurons (GABAn) in the ventral midbrain, which play a crucial role in providing inhibitory control within local circuits and also have close interactions with dopaminergic (mDA) neurons, which are central to motor and reward-related processes. Any disruption in the formation or positioning of GABAn can contribute to various
neurological disorders.
The diversity and migration of GABAn in the ventral midbrain have long been
understudied. Recent insights, partly obtained through new genetic techniques and single-cell RNA-sequencing (scRNA-seq), show that these GABAn are more
heterogeneous than previously assumed. The goal of this dissertation is to unravel the mechanisms underlying their diversity, migration, and subtype specification, with particular emphasis on the guidance molecule Netrin-1.

In Chapter 2, we explore how the axon guidance molecule Netrin-1, traditionally known for its role in axonal outgrowth, also regulates the migration of GABAn in
the ventral midbrain. We demonstrate that Netrin-1 is not only produced locally in the midbrain but is also supplied by axons coming from the forebrain. This axon-derived source of Netrin-1 acts as a long-range signal that guides GABAn to the substantia nigra pars reticulata (SNr). Accurate migration of GABAn is crucial for proper positioning of dopaminergic neurons in the substantia nigra pars compacta (SNc). We show that the absence or reduction of Netrin-1 disturbs this balance, leading to mislocalization of both GABAergic and dopaminergic cells. These findings establish a direct link between Netrin-1–guided GABAergic neuron migration.

Chapter 3 focuses on the molecular diversity of GABAergic neurons. To unravel this heterogeneity, we employ scRNA-seq of GABAn isolated during embryonic and early postnatal stages. We discover that GABAn fall into multiple subpopulations, each characterized by a specific expression profile of transcription factors and receptors. Some subgroups exhibit a combination of GABAergic and dopaminergic markers, suggesting a potential dual neurotransmitter function or an overlap in their developmental programs. Our experiments further reveal that different GABAergic subtypes utilize diverse sources of Netrin-1 and adopt distinct Netrin-1 receptors to migrate to their correct location. Through electrophysiological recordings, we confirm that these subpopulations vary not only in molecular terms but also in functional properties, underscoring their multifaceted role in both local and long-range signaling.

In Chapter 4, we describe the experimental and computational approach used to generate and analyze single-cell transcriptomics data. We address the isolation of GABAn via cell sorting, the protocol optimizations needed to preserve RNA integrity, and the various steps in library preparation for RNA sequencing.
We then discuss clustering methods. These methodological insights form the backbone for understanding and interpreting the findings presented in the previous chapters.

This dissertation reveals new mechanisms governing the migration and subtype
specification of GABAn in the ventral midbrain. The combination of genetic techniques, scRNAseq, and functional assays demonstrates that GABAn are subdivided not only anatomically but also molecularly and functionally into multiple subpopulations. This new knowledge provides a foundation for future research into neurological and psychiatric disorders where the balance between GABAergic and dopaminergic signaling may be disrupted.