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The re-uptake of GABA in the CNS is mediated by two
main transporters named GAT-1 and GAT-3. Since little is known about
their distribution and respective roles in the functional circuitry
of the basal ganglia (BG), light microscopy (LM) double immunofluorescence
and electron microscopy (EM) immunoperoxidase techniques were used
to compare the cellular and subcellular localization of GAT-1 and
GAT-3 in two nuclei of the BG, the striatum (STr) and the external
segment of the globus pallidus (GPe). At the LM level, immunoreactivity
(IR) for GAT-1 was observed in both axon- and glial-like processes,
whereas labeling for GAT-3 was found mostly in glial-like elements.
Overall, the strongest level of IR was found for GAT-3 in GPe, while
the levels of immunostaining for GAT-1 in both STr/GPe and GAT-3
in the STr were significantly lower. At the EM level, both GAT-1
and GAT-3 IR was found predominantly in glial cell processes, but
a significant proportion of GAT-1 labeling was also expressed in
small, unmyelinated axons in STr and GPe. Surprisingly, very few
terminals were labeled for either transporter subtype, suggesting
that GABA re-uptake in STr and GPe is mainly regulated by pre-terminal
axons and glial cell processes. Interestingly, the pattern of glial
labeling for the two GATs resembled that of the major glutamate
transporter, GLT-1, suggesting that both GABA and glutamate re-uptake
may be regulated by the same glial cells in these brain regions.
In conclusion, our findings provide strong evidence for functional
glial and axonal GABA re-uptake in the monkey STr and GPe. These
data pave the way for further analyses of the differential roles
these two transporters may play in regulating GABA neurotransmission
in normal and pathological BG functions.
Cellular communication in the brain occurs through
a highly dynamic process known as chemical neurotransmission. Neurotransmitters
that do not bind to receptors in the plasma membrane of postsynaptic
neurons are then inactivated by degradation or re-uptake. A group
of proteins called transporters are responsible for the re-uptake
of neurotransmitters into the presynaptic terminal or glial cells.
These transporters are essential to maintain homeostasis of neurotransmission
in the CNS. GABA is the main inhibitory neurotransmitter in the
brain. The neuronal activity of a group of brain nuclei named basal
ganglia largely relies upon normal GABAergic transmission. An abnormal
regulation of the release and/or re-uptake of GABA in these brain
regions may result in various movement disorders such as Parkinson's
disease and Huntington's chorea. The localization of the two main
GABA transporters, GAT-1 and GAT-3, (Na+/Cl- dependent), in the
BG remains unknown. A better understanding of the localization and
functions of GABA transporters may provide novel targets for therapeutic
strategies in BG diseases. AIM: The purpose of this study is to
use EM immunocytochemical procedures to determine and compare the
cellular and subcellular localization of GAT-1 and GAT-3 within
two major nuclei of the monkey basal ganglia, the striatum and the
globus pallidus.
Animals:
Four adult Rhesus macaque monkeys, perfused with 4% paraformaldehyde
and 0.1% glutaraldehyde.
Pre-embedding Immunoperoxidase (Avidin biotin peroxidase method-ABC)
Primary Antibodies:
Rabbit anti-GAT-1 (Chemicon) used at a dilution of 1:500. Rabbit
anti-GAT-3 (Chemicon) used at a dilution of 1:1000
Secondary Antibody:
Goat anti-rabbit coupled to a biotin molecule (Vector) used at a
dilution of 1:200. Avidin biotin complex (ABC) used at a dilution
of 1:100
Immunofluorescence Primary Antibodies:
Rabbit anti-GAT-1 and anti-GAT-3 (Chemicon) at a dilution of 1:200
and 1:500, respectively. Guinea pig anti-GLT-1 (Chemicon) used at
a dilution of 1:5000.
Secondary Antibodies:
Goat anti-rabbit conjugated with a fluorescent molecule (FITC) at
a dilution of 1:100. Donkey anti-guinea pig conjugated with a fluorescent
molecule (Rhodamine) at a dilution of 1:100.
Both GAT-1 and GAT-3 are expressed in STr and GPe. Overall, the
GPe displays a stronger level of immunoreactivity for GAT-1 and
GAT-3 than the STr. GAT-3 is mainly expressed in glial processes,
while GAT-1 is found in glial cells and pre-terminal axons in both
STr and GPe. Very few immunoreactive terminals for either transporter
subtypes were found in STr and GPe. GABA re-uptake in STr and GPe
is mainly regulated by pre-terminal axons and glial cells. The pattern
of glial labeling for the GATs resemble that of the glutamate transporter
GLT-1, suggesting that glutamate and GABA re-uptake may be mediated
by the same glial cells in STr and GPe. The localization of GABA
transporters is consistent with extra-synaptic diffusion of GABA
in the STr and GPe.
Future Directions:
Study the respective roles of GAT-1 and GAT-3 in regulating GABAergic
transmission in STr and GPe using selective GABA transporter antagonists
in brain slices. Look at potential changes in the expression and
localization of GAT-1 and GAT-3 in animal models of Parkinson's
disease.
Special thanks to Mr. Dinesh Raju, Mr. Jeff Pare, and all members
of Dr. Yoland Smith's laboratory for assistance throughout the project.
Cathy Quinones and Pat Marstellar for organization of the SURE program.
This material is based upon work supported by the Howard Hughes
Medical Institute under Grant No 52003727. This research was also
supported by NIH grants to Dr. Yoland Smith.
Cellular communication in the brain occurs through a process known
as chemical neurotransmission. Neurotransmitters are released from
presynaptic nerve terminals into the synaptic cleft between the
pre- and postsynaptic neurons. The chemicals that do not bind to
receptors in the plasma membrane of postsynaptic neurons are then
inactivated by degradation or re-uptake into the presynaptic terminal
or glial cells. Various disorders can disrupt the homeostasis found
in normal brains and lead to pathological states through alterations
in transporter functions. GABA is the main inhibitory chemical neurotransmitter
in the brain. It is expected that GABA transporters would be located
close to GABA receptors, though their localization is unknown in
certain areas of the brain. More specifically, their localization
within the basal ganglia, one of the main brain regions responsible
for motor control, remains poorly characterized. It is important
to determine where the transporters are located in relation to the
sites of GABA release to further understand their role in regulating
GABAergic transmission in the basal ganglia. Furthermore, knowing
that abnormal inhibitory transmission underlies the symptoms of
various basal ganglia diseases such as Parkinson's Disease and Huntington's
Disease, a better understanding of the localization and functions
of GABA transporters may provide ideas on how to treat these diseases.
Immunoperoxidase, Immunogold, Immunofluorescence Light, Microscopy,
Electron Microscopy.
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