A New Method for the Visualization of Living Dopaminergic Neurons and Prospects for Using It to Develop Targeted Drug Delivery to These Cells

Abstract

This is the first study aiming to develop a method for the long-term visualization of living nigrostriatal dopaminergic neurons using 1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)piperazine-BODIPY (GBR-BP), the original fluorescent substance, which is a derivative of GBR-12909, a dopamine uptake inhibitor. This method is based on the authors’ hypothesis about the possibility of specifically internalizing into dopaminergic neurons substances with a high affinity for the dopamine transporter (DAT). Using a culture of mouse embryonic mesencephalic and LUHMES cells (human embryonic mesencephalic cells), as well as slices of the substantia nigra of adult mice, we have obtained evidence that GBR-BP is internalized specifically into dopaminergic neurons in association with DAT via a clathrin-dependent mechanism. Moreover, GBR-BP has been proven to be nontoxic. As we have shown in a primary culture of mouse metencephalon, GBR-BP is also specifically internalized into some noradrenergic and serotonergic neurons, but is not delivered to nonmonoaminergic neurons. Our data hold great promise for visualization of dopaminergic neurons in a mixed cell population to study their functioning, and can also be considered a new approach for the development of targeted drug delivery to dopaminergic neurons in pathology, including Parkinson’s disease.

Isolation of living dopaminergic neurons labeled with a fluorescent ligand of the dopamine transporter from mouse substantia nigra as a new tool for basic and applied research

“…One dye, DRAQ5, stains the nuclei of both living and fixed cells (Smith et al, 2004). Another dye, GBR-BODIPY (GBR-BP), specifically stains DNs by binding to DAT and subsequent internalization into the neurons (Blokhin et al, 2022). These dyes have non-overlapping emission spectra: DRAQ5 with a maximum at 697 nm; GBR-BP with a maximum at 511 nm.…”

“…The cells were then precipitated by centrifugation at 300× g for 5 min at 4°C, resuspended in 50 μl HBSS and stored in the dark at 4°C until fluorescent microscopy. This control was used in a previous study to assess the specificity of GBR-BP staining of DNs derived from primary culture of mouse embryonic mesencephalon and LUHMES cells (Blokhin et al, 2022).…”

“…After the dissociation of SN, which is heterogeneous in cellular composition, it was necessary to identify DNs in the resulting cell suspension. For this, along with DRAQ5, which stains cell nuclei, GBR-BP, a recently manufactured specific fluorescent dye for monoaminergic neurons, was used (Lavrova et al, 2019;Blokhin et al, 2022). Indeed, GBR-BP has been shown to specifically stain DNs in a primary culture of mouse embryonic mesencephalon cells and LUHMES cells (Blokhin et al, 2022).…”

“…At the first stage of developing the optimal protocol for specifically staining DNs in the SN cell suspension, GBR-BP was used at a concentration of 5 nM, which was previously used to stain DNs in a primary culture of mouse mesencephalon (Blokhin et al, 2022).…”

“…It follows that methods for isolating nigrostriatal DNs from wild-type animals are in great demand, since they can be used for transcriptomic analysis, avoiding some problems associated with the use of transgenic animals. In this context, the synthesis of a fluorescent dopamine transporter ligand that specifically stains DNs has provided new opportunities for isolating these neurons (Blokhin et al, 2022).…”