L-DOPA Uptake in Astrocytic Endfeet Enwrapping Blood Vessels in Rat Brain

Abstract

Astrocyte endfeet surround brain blood vessels and can play a role in the delivery of therapeutic drugs for Parkinson's disease. However, there is no previous evidence of the presence of LAT transporter for L-DOPA in brain astrocytes except in culture. Using systemic L-DOPA administration and a combination of patch clamp, histochemistry and confocal microscopy we found that L-DOPA is accumulated mainly in astrocyte cell bodies, astrocytic endfeet surrounding blood vessels, and pericytes. In brain slices: (1) astrocytes were exposed to ASP(+), a fluorescent monoamine analog of MPP(+); (2) ASP(+) taken up by astrocytes was colocalized with L-DOPA fluorescence in (3) glial somata and in the endfeet attached to blood vessels; (4) these astrocytes have an electrogenic transporter current elicited by ASP(+), but intriguingly not by L-DOPA, suggesting a different pathway for monoamines and L-DOPA via astrocytic membrane. (5) The pattern of monoamine oxidase (MAO type B) allocation in pericytes and astrocytic endfeet was similar to that of L-DOPA accumulation. We conclude that astrocytes control L-DOPA uptake and metabolism and, therefore, may play a key role in regulating brain dopamine level during dopamine-associated diseases. These data also suggest that different transporter mechanisms may exist for monoamines and L-DOPA.

Figure 1

Uptake in rat hippocampus following an intravenous injection of l-DOPA (a) or ASP⁺ (b). Red arrows: pericytes, white arrows: presumable astrocytes, identified by morphology. Scale for (a) and (b): 20 μm.

Figure 2

Accumulation of ASP⁺, the fluorescent substrate for high-affinity monoamine transporters and organic cation transporters in astrocytes in hippocampal brain slices. (a) Astrocytes current response to voltage step protocol application reveals a linear IV-relationship. (b) Puff-application of ASP⁺ elicited transporter current in astrocytes, maintained previously at zero current. Arrow indicates the moment of application. (c) and (d) The same astrocyte with attached patch-pipette before (in (c), infrared DIC) and after (in (d), fluorescence) the application of 50 μM ASP⁺. Scale on (c) and (d) 20 μm.

Figure 3

Double staining of hippocampal brain slice with l-DOPA and ASP⁺ (fluorescent substrate for transporters present in astrocytes and some other cells). Uptake revealed with the Falck-Hillarp method (see. Section 2). Astrocytes take up both l-DOPA and ASP⁺ (indicated with arrows). Scale: 20 μm.

Figure 4

l-DOPA uptake by astrocytes and their endfeet on brain capillaries. (a) and (b) Uptake of l-DOPA as revealed by the Falck-Hillarp method is concentrated in astrocyte cell body, astrocyte processes that project to the vessels and in the endfeet touching the vessel wall. Note that the capillary vessel walls are completely enwrapped by the endfeet. (c) Current responses of an astrocyte during puff-application of l-DOPA (100 μM) or ASP⁺ (50 μM). Note that l-DOPA application did not elicit a current response, while ASP⁺ elicited an inward current. Scale: 20 μm.

Figure 5

The pattern of MAO-B distribution in capillaries and astrocytes ((a) and (b)). (b) General pattern of MAO-B distribution in blood vessels and nearby astrocytes. Arrow indicates the insert. (a) Insert from (b), revealing at larger magnification a part of a capillary with a pericyte and an astrocyte cell body (both shown by arrows) with an endfoot process extending toward the vessel. Scale: 10 μm in (a), 100 μm, in (b).