Hindawi Publishing Corporation
Volume 2012, Article ID 321406, 8 pages
L-DOPA Uptake inAstrocyticEndfeetEnwrapping
M. Y.Inyushin,1A. Huertas,1Y.V.Kucheryavykh,1L.Y.Kucheryavykh,1V.Tsydzik,1
1Department of Physiology, Universidad Central del Caribe, Bayam´ on, PR 00956, USA
2Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
Correspondence should be addressed to M. Y. Inyushin, firstname.lastname@example.org
Received 14 March 2012; Accepted 13 June 2012
Academic Editor: Heinz Reichmann
Copyright © 2012 M. Y. Inyushin et al.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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
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.
It is a widely held opinion that the transport systems at the
blood-brain barrier (BBB) are localized in the brain capillary
endothelial cells and control the exchange of various endoge-
nous and exogenous compounds between the circulating
blood and brain. Astrocytes, the glial cells that compose
the gliovascular interface , are an important part of
the brain vascular system. Astrocytic endfeet wrap blood
vessels, forming the second barrier around the endothelial
cells that is separated from endothelium by a space filled
with basal lamina. Basal lamina, a gel that consists of
laminin, fibronectin, tenascin, collagens, and proteoglycan
, separates the astrocytic endfeet and endothelium cell
layers but does not prevent passage of macromolecules. The
∼20nm gap between adjacent astrocytic endfeet, which is
diffusible by horseradish peroxidase , sometimes gives
rise to questions about the ability of astrocytes to physically
contribute to the BBB; hence, the barrier and transport
roles of astrocyteshave been underappreciated. Nevertheless,
astrocytes participate in the transport of substrates to the
brain  and possess a variety of transport systems that play
roles in the delivery of therapeutic drugs for Parkinson’s dis-
ease (PD). Tyrosine, a precursor of dopamine, and l-DOPA
are extracted from the circulating blood by the amino acid
transport system l, involving l-type amino acid transporter
1 (LAT1)/4F2 heavy chain (4F2hc) complex [5–9]. This
transporter has previously been identified and characterized
in cultured astrocytes [10, 11]. Additionally, variants of
the organic cation transporter 1, also previously identified
in cultured astrocytes [12, 13], may also be involved in
transport of l-DOPA [14, 15]. These observations led us to
ask whether astrocytes participate in l-DOPA transport in
the brain as well.
Early whole animal studies showed that after an injection
of l-DOPA to the circulation, l-DOPA or its products accu-
18]. Here, using rat brain slices and confocal microscopy,
2 Parkinson’s Disease
we report the accumulation of l-DOPA in astrocyte cell
bodies and in the endfeet surrounding blood vessels. Also,
the distribution pattern of MAO type B in pericytes and
astrocytic endfeet was similar to the l-DOPA accumulation
2.1. Histochemistry. A modified Falck-Hillarp method was
used to visualize l-DOPA uptake in slices . Briefly,
slices were incubated in freshly made glyoxylate-containing
solution at 4◦C for 2h, placed on a glass slide, dried
with air blower at room temperature for 1.5–2h, heated
in an oven at 85◦C for 9min, covered with paraffin oil,
coverslipped, and viewed using a Fluoview FV1000 fluo-
rescence confocal microscope (Olympus, Japan) with UV-
GFP filter set. The incubating solution contained (in mM)
500 sodium glyoxylate, 40 N-2-hydroxyethylpiperazine-N?-
2-ethanesulfonic acid (HEPES), and 100 sucrose, dissolved
in deionized water, pH 7.0. The final pH of the solution
was adjusted to 7.0 with either glyoxylic acid or sodium
To visualize MAO-B, we used diaminobenzidine as a
chromogen and tyramine as a substrate according to the
protocol of Ryder et al.  as modified by Willoughby et
al. . During this reaction, we blocked MAO-A using
2.2. Animals and Slice Preparation. All experimental pro-
cedures were performed in accordance with the US Public
Health Service Publication Guide for the Care and Use of
Laboratory Animals and were approved by the Animal Care
and Use Committee at the Universidad Central del Caribe.
Sprague-Dawley rats of either sex between 20 and 30 days
of age were decapitated. Hippocampal slices (200µM) were
prepared using a vibratome (VT1000S, Leica Microsystems
GmbH, Wetzlar, Germany) in artificial cerebrospinal fluid
(ACSF) containing (in mM) 127 NaCl, 2.5 KCl, 1.25
NaH2PO4, 25 NaHCO3, 2 CaCl2, 1 MgCl2, and 25 d-glucose,
ice-cold, saturated with a 95% O2-5% CO2gas mixture at
pH = 7.4. Slices were perfused with the same ACSF at room
temperature. BaCl2 (100µM) was added to ACSF for the
For systemic injections we used 60-day-old Sprague-
Dawley rats. Intravenous injections of l-DOPA (2mM) or
ASP+(0.5mM) in a 0.5mL volume were made via the lateral
2.3. Whole Cell Recordings. Membrane currents were mea-
sured with the single-electrode whole-cell patch-clamp tech-
nique. Cells were visualized using an Olympus infrared
microscope equipped with DIC (BX51WI Olympus, Japan).
Two piezoelectric micromanipulators (MX7500 with MC-
1000 drive, Siskiyou, Inc., Grants Pass, OR) were used
for voltage-clamp and current-clamp recording and for
positioning a micropipette with a 30–50µm tip diameter
for application of test solutions. A MultiClamp 700A patch-
clamp amplifier with a DigiData 1322A interface (Molecular
Devices, Inc., Sunnyvale, CA) was used for recording and
stimulation. The pClamp 10 software package (Molecular
Devices, Inc., CA) was used for online data acquisition
and analysis. Borosilicate glass pipettes (O.D. 1.5mm, I.D.
1.0mm; World Precision Instruments, Sarasota, FL) were
pulled to a final resistance of 8–10MΩ for astrocyte record-
ings in four steps using a P-97 puller (Sutter Instrument Co.,
Novato, CA). Electrodes were filled with the following solu-
tion (in mM): 130K-gluconate, 10 Na-gluconate, 4 NaCl, 4
phosphocreatine, 0.3 GTP-Na2, 4 Mg-ATP, and 10 HEPES,
recordings were considered only if membrane potential was
negative to –75mV and if cells had linear current voltage
relation (variably rectifying astrocytesaccording to ) and
low input resistance (less than 20 MΩ). Pipette potential was
2.4. Materials. 4-[4-(dimethylamino)-styryl)-N-methylpyri-
22), sodium glyoxylate, N-2-hydroxyethylpiperazine-N?-2-
ethanesulfonic acid (HEPES), glyoxylic acid and other
chemicals were purchased from Sigma-Aldrich Corp. (St.
3.1. Uptake of l-DOPA and ASP+after Intravenous Injection.
l-DOPA is extracted from the circulating blood by LAT1
transport system, which is found in astrocytes [10, 11]. It
was shown previously that l-DOPA can pass through the
blood-brain barrier and for a short time (15–60sec) is accu-
mulated in blood vessel walls and pericytes, partially being
decarboxylated to dopamine [16–18]. Since the astrocytic
endfeet processes wrap blood vessels, we expected astrocyte
involvement as well after systemic injection of l-DOPA.
After a 2mM intravenous injection of l-DOPA we waited
30sec, then we decapitated the animal, removed the brain,
and dissected the hippocampal area out. This tissue was
fixed for 30sec in 4% paraformaldehyde, and then 200µm
Hillarp method .
Similar experiments were conducted after intravenous
injections of 0.5mM ASP+. ASP+is a known fluorescent
substrate for high-affinity monoamine transporters and
organic cation transporters [23–25].
After either l-DOPA or ASP+injections, fluorescence
was clearly visible from cells identified by appearance as
astrocytes, sending their processes to the walls of blood
vessels (Figures 1(a) and 1(b), presumable astrocytes marked
with white arrows). The intensive uptake of l-DOPA by
pericytes has been described previously [16, 17], and they
were also visible (Figure 1, pericytes marked by red arrows).
Some reduced fluorescence was also visible in capillary
endothelium, corresponding to Wade and Katzman’s 
results showing endothelium l-DOPA fluorescence that
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): 20µm.
peaked at 15sec after injection and was significantly reduced
3.2. Electrophysiological Characterization of Astrocytes and
ASP+Uptake in Slices. In order to confirm that the cells that
we identified after the fluorescent staining as astrocytes by
their shape and the size were really astrocytes, we used brain
slices for electrophysiological characterization and ASP+
uptake studies. In these experiments we were interested to
determine if ASP+was taken up by astrocytes in brain slices.
For this purpose, astrocytes in stratum radiatum area of
hippocampus were first electrophysiologically characterized
using whole-cell voltage-clamp; to be sure we are working
with passive (linear) astrocytes. We studied astrocytes that
were 30–50µm deep in the slice, cells (with cell body
∼10µm) were identified first under infrared DIC optics
(Figure 2(c)). After a standard patching procedure, a voltage
step protocol was applied, as described by . Only data
from cells showing a linear I–V curve (Figure 2(a)) were
considered for consistency. The mean membrane potential
of astrocytes was –85.2 ± 3.1mV (n = 126), and the mean
input resistance was 16.3 ±2.1M Ω (n = 126).
Ba++(100µM) was added to the solution after the
membrane potential measurements to reduce the potassium
current, which can interfere with the current from the
channels were not suitable for these experiments because
they are either substrates (Cs+) or nontransportable blockers
(quinine) of organic cation transporters . Organic cation
transporters are among the possible candidates for l-DOPA
and ASP+transport, as they were found in astrocytes 
and have been shown to transport both of these substrates
[14, 24, 25].
Astrocytes were voltage-clamped at their membrane
potential to get zero current. 50µM ASP+was applied to the
astrocyte from a distance of 100µM, using a puff-electrode
with a 4µM tip and a 0.5sec pressure injection (Figure 2(b),
the moment of the application marked with the arrow). An
ASP+-elicited inward current was recorded for each clamped
astrocyte, and the mean current value at maximum was
47 ± 14 pA (n = 126). The current varied from 120 pA to
15pA in individual astrocytes in different animals. Transport
other monoamine transporters is known to be electrogenic
, and thus the current we had recorded in the presence of
potassium channel blocker probably represents a transporter
current. After the puff-application of ASP+, fluorescence was
visualized using a CY3 filter set. Figure 2(d) shows that ASP+
was taken up by the majority of astrocytes in the slice,
including the astrocyte that was connected to the electrode.
In separate experiments we showed that 2µM decynium-
22, a well-known nonneuronal monoamine transporter
blocker [12, 25], reduced the ASP+-elicited current in astro-
cytes by 56.3 ± 14% (n = 12), if applied 30sec prior to the
were clearly identified by electrophysiological methods as
passive astrocytes, have effective mechanisms to take up
ASP+. The relatively large variation of transporter current
amplitudes from astrocytes in the same brain area suggests a
wide dispersion of transporter quantity in different animals,
and possibly a wide range of physiological regulation of the
3.3. l-DOPA and ASP+Uptake in Astrocytes in Slices
3.3.1. Double Staining for l-DOPA and ASP+Uptake. Brain
slices were incubated in oxygenated ACSF containing 10µM
l-DOPA and 1µM ASP+(pH 7.4) for 30min at room
temperature. Slices were then dried and processed accord-
ing to the modified Falck-Hillarp method (see Section 2).
Using both filter sets for l-DOPA and ASP+on the same
preparation, we found a cooccurrence of ASP+and l-
DOPA accumulation in the same cells, including astrocytes
4 Parkinson’s Disease
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 50µM ASP+. Scale on (c) and (d) 20µm.
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: 20µm.
and their endfeet on blood vessels (Figure 3). These data
confirmed that both substrates were taken up by astrocytes
in hippocampal slice preparations.
3.3.2. l-DOPA Uptake by Astrocytes. We used the Falck-
Hillarp method to determine that l-DOPA uptake alone
was more effective than in combination with ASP+, because
ASP+, especially at concentrations greater than 1µM, inter-
fered with l-DOPA uptake. Fluorescent astrocytic endfeet
surround the hippocampal capillaries, and some astrocyte
cell bodies and the processes that extend toward the
capillaries were also clearly fluorescent (Figure 4, also see
stacks in 3D supplementary material available online at
doi:10.1155/2012/321406). Confocal images revealed that
astrocytic endfeet cover the entire capillary wall without
visible gaps (Figures 4(a) and 4(b)). These data suggest that
astrocytes and their endfeet surrounding brain capillaries, as
well as the process or processes that extend toward the vessels
participate in l-DOPA uptake.
Using live brain slices perfused with ACSF, we also
patch-clamped astrocytes in voltage clamp mode, under
similar conditions as used for studying ASP+uptake
(Figure 4(c)). The holding potential was maintained to
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 (100µM) or ASP+(50µM). Note that l-DOPA application did not elicit a current response, while ASP+elicited an inward current.
keep zero current, and 100µM l-DOPA was puff-applied
to the astrocyte, followed 100s later with 50µM ASP+
similarly applied for comparison (applications marked by
arrows). There was an obvious difference between the
effects of ASP+and l-DOPA on astrocyte currents: there
was no elicited inward transporter current in the case
of l-DOPA application, suggesting l-DOPA may enter
the astrocyte via a different pathway or that it is trans-
ported by the same transporter, but in a different man-
3.4. The Distribution of Monoamino Oxidase Type B (MAO-
B) in Astrocytes and Capillaries. The presence of MAO-B
in astrocytes [27–29] and in blood vessels with pericytes
 is well established. Blood vessels with dark staining
pericytes can be easily identified (Figure 5(b)) in our slices
after visualization of MAO-B. In addition, stained astrocytes
and their processes projecting towards the vessel can also
be observed (Figure 5(a)). In these experiments, we were
interested in determining if the pattern of MAO distribution
resembles that of l-DOPA uptake. Generally, MAO-B was
located in the pericytes and in the astrocyte cell bodies, the
astrocyte processes projecting to the vessel and in the astro-
cytic endfeet. This was similar to the l-DOPA accumulation
pattern suggesting that l-DOPA may be destroyed in these
cells after uptake, at least partially.
Our experiments have confirmed that astrocytes, charac-
terized as electrically passive astrocytes by patch-clamp
methods, readily take up l-DOPA, as well as the fluorescent
monoamine analog ASP+in hippocampal slices. l-DOPA
accumulation in astrocyte endfeet, in the astrocyte processes
that project to the capillaries and in cell bodies suggests
that the gliovascular interface may play an important role in
dopamine precursor uptake, previously overlooked. Passive
astrocytes are known to form an astrocytic network [1, 22,
30], in which astrocytes are interconnected with each other
via gap junctions, and substances such as l-DOPA may be
taken up near the blood vessels and may spread through this
Participation of astroglia in l-DOPA uptake is very
intriguing because astrocytes have definitely been shown to
contribute to l-DOPA-to-dopamine conversion. Dopamine
was detected in both rat and mouse cultured astrocytes after
a 30min incubation with l-DOPA, indicating the existence
of aromatic l-amino acid decarboxylase [10, 31]. Also,
aromatic l-amino acid decarboxylase mRNA was detected
6 Parkinson’s Disease
in primary cultures of astrocytes, and Western immunoblots
demonstrated aromatic l-amino acid decarboxylase expres-
sion in astrocytes . Interestingly, just 15 seconds after
carotid injection of 3mM/mL l-DOPA half of it was already
decarboxylated to dopamine . This occurred relatively
uniformly and similarly in different parts of the brain and
only about 10% more effectively in brain regions with
pronounced dopaminergic and serotoninergic innervation
. The authors credited this decarboxylation to the
endothelium cells, probably because previously aromatic
l-amino acid decarboxylase activity was demonstrated in
blood vessels and was attributed to the endothelium .
The close relationship between astrocytic endfeet and brain
vessels was not well appreciated at that time. Even a
very conservative estimate of the brain capillary-astrocyte
contribution to decarboxylation of l-DOPA can account for
at least 12% of all the aromatic l-amino acid decarboxylase
activity after a double dopaminergic and serotoninergic
chemical lesion .
Uptake of ASP+by astrocytes is very interesting because
the well-known nonfluorescent analog and neurotoxic agent,
1-methyl-4-phenylpyridinium (MPP+), is probably trans-
ported into astrocytes by the same transporter. Astro-
cytes produce MPP+from its precursor, MPTP , and
MPP+reverse transport has been shown to contribute to
MPP+-related dopamine neuronal death . It was also
shown that organic cation transporters on the astrocytes
adjacent to dopamine neurons contribute to damage by
bidirectionally regulating the local bioavailability of the
MPP+and other similar toxic species . ASP+, being
a monoamine analog, is partly transported by intracel-
lular transport to mitochondria where MAO is situated
and concentrated . It is known that MPP+pro-
duces significant damage to mitochondria, what about
In some atypical forms of Parkinsonism, the glial cells
have been shown to malfunction due to other disease
processes. This highlights the involvement of astrocytes in l-
DOPA uptake and metabolism. One of clinical implications
of glial l-DOPA processing may be the Parkinsonism mani-
is compromised [38, 39]. To what extent astrocytes are
involved in MSA is unknown, but l-DOPA fails to improve
the parkinsonian symptoms of most MSA patients. Poor
in the differential diagnosis of MSA from Parkinson’s disease
cells can also contribute to pulsatile changes of dopamine
levels in cerebrospinal fluid after oral l-DOPA pill con-
sumption, as well as oxidation of excess monoamines or
methylation of l-DOPA to its main metabolites (ex. 3-O-
methyldopa). These concentration spikes are associated with
the development of motor complications and even the onset
of psychosis .
It is important to note that MAO-B is considered to
be the main pathway for the dopamine degradation in
astrocytes [28, 41]. For this reason we used MAO-B staining
tovisualizeglia. Butolderdata about theMAO-Aabundance
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:
10µm in (a), 100µm, in (b).
in glia  and newer data about the therapeutic potential of
mixed nonselective MAOA/B monoamine oxidase inhibitors
 raise the possibility that glial MAO-A may also partici-
levels are in excess.
Glial turnover of the l-DOPA metabolite of dopamine
via MAO-B is a further source of increased synthesis of free
radicals , producing gliosis and compensatory changes
in protein expression in astrocytes. For example, production
of endothelial growth factor (EGF) is upregulated by l-
DOPA in the Parkinsonian brain. EGF is expressed mainly
by astrocyte processes and astrocyte endfeet on blood vessels
and overexpression can lead to abnormal vessel density and
ultimately to the development of dyskinesia . Earlier it
was proposed that endothelial proliferation after exposure
to high concentrations of l-DOPA can lead to breach in
the blood-brain barrier (BBB) that itself can be the cause
of dyskinesia . This finding is controversial because
a later study using in vivo neuroimaging demonstrated
that the BBB is intact after l-DOPA-induced dyskinesias
in parkinsonian animals . Also, M¨ uller and coauthors
 showed that the level of 3-O-methyldopa does not
affect significantly l-DOPA pharmacokinetics and motor
responses in patients and they concluded that the BBB
was not affected.
Our aim was to demonstrate that astrocytes, as well
as pericytes and endothelial cells, are able to take up l-
DOPA, convert it to dopamine, and also have all necessary
oxidative machinery to metabolize dopamine. They can
release dopamine using different mechanisms; for example,
by reverse transport. MAO-B blockers may be able to avert
a lot of open questions. It was shown that astrocytes release
dopamine [10, 31], but it is not known which transporter is
releasing dopamine from astrocytes. Could it be the organic
cation transporter? How important is the glial participation
in l-DOPA uptake and conversion? Does glial uptake of l-
DOPA change during the progression of Parkinson’s disease?
Finally, we can conclude that (i) astrocytes participate
in l-DOPA uptake and metabolism via the gliovascular
interface and, therefore, may play a key role in regulating
brain dopamine, (ii) the role of astroglia in l-DOPA uptake
and processing must be reconsidered specifically in cases
of Parkinson’s disease, and (iii) our data also suggest that
different transporter mechanisms may exist for monoamines
and l-DOPA uptake by astrocytes.
This project was supported by Grants from the National
Center for Research Resources (2 G12 RR03035-26) and the
(8G12MD007583-27) from the National Institutes of Health.
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