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In the traditional model of the basal ganglia rmotor
circuit, the striatum is the primary site of dopamine release. In
the striatum, dopamine is thought to influence intrinsic basal ganglia
connections, which eventually results in reduced basal ganglia output
to the thalamus. This sequence of events may result in disinhibition
of thalamocortical connections and, ultimately, in the facilitation
of movement. In recent studies in rodents and cats, dopamine release
and dopamine receptors have also been described at extrastriatal
sites, most prominently the substantia nigra (SN). In rodents, dopamine
released in the SN appears to act predominately on D1-like receptors
(D1LRs, comprised of the D1- and D5 subtypes of dopamine receptors)
which are primarily located on terminals of the GABAergic striatonigral
projections. To investigate whether a similar arrangement is also
true in primates, we performed microdialysis studies in the SN of
a Rhesus monkey. Microdialysate samples were collected from the
SN before and during infusion with the D1LR agonist SKF 82958 (1
mM), the D1LR antagonist SCH 23390 (30 µM), or vehicle.
The GABA content was determined by high performance liquid chromatography
(HPLC). The available results show a 390% increase in GABA levels
in response to D1LR activation, while D1LR blockade decreased GABA
levels by 23% when compared to baseline levels. The data suggest
that activation of D1LRs may enhance GABA release in the primate
SN. Increased GABA levels may act to reduce the activity of nearby
basal ganglia output neurons in the substantia nigra pars reticulata
(SNr), resulting in downstream facilitation of cortical activities
involved in movement and other functions.
In Parkinson's Disease, the nigrostriatal projection
from dopamine-producing cells of the substantia nigra pars compacta
(SNc) degenerates. In most models of the pathophysiology of this
disorder, the resulting reduction of striatal dopamine triggers
a cascade of activity changes in the intrinsic basal ganglia circuitry,
and results in increased inhibitory output from the basal ganglia
to the thalamus, which reduces the facilitation of cortical areas
(Figure 1). Recent studies in rodents and cats describe that dopamine
release and dopamine receptors also takes place in the SN, and that
nigral dopamine loss may contribute to parkinsonism. Dopamine may
be released at this site from dendrites of SNc cells, and appears
to act on D1LRs that are primarily located on terminals of the GABAergic
striatonigral projections, as well as D2LR-autoreceptors (Figure
2). Our laboratory studies whether nigral dopamine serves similar
roles in primates as well, and whether loss of nigral dopamine also
contributes to primate parkinsonism. The experiments presented here
describe the effects of activation of D1LRs on GABA release in the
SN. Based on the aforementioned rodent studies we hypothesized that
activation of nigral D1LRs would increase GABA release in the SN,
and blockade of D1LRs would have the opposite effect.
We utilized microdialysis methods to sample the biochemical
composition of small areas of the brain in vivo. The microdialysis
approach can also be used to deliver drugs during the experiments
(so-called reverse microdialysis) (Figure 3). In this case, we measured
GABA concentrations in the primate SN before, during and after local
application of D1LR-active compounds. A Rhesus monkey was surgically
prepared for chronic access to the SN area. Under isoflurane gas
anesthesia, a metal recording cylinder was placed over a circular
craniectomy (18 mm diameter) at an angle of 25 degrees; from the
vertical in the parasagittal plane, stereotactically aimed at the
SN. The cylinder, as well as head holding screws were affixed to
the skull with dental acrylic. The preparation allowed us to record
repeatedly from the SN. The exact location of the SN area was identified
using standard electrophysiologic mapping procedures. In each microdialysis
experiment, a microdialysis probe was inserted into a new region
in SNr (Figure 4). The probe was continuously perfused with artificial
cerebrospinal fluid (aCSF, an electrolyte mixture resembling the
composition of CSF) at a rate of 2 microliters/min. After a two
hour equilibration period, we collected the microdialysate emanating
from the outlet tube of the system in 20-minute aliquots. After
collection of three baseline samples, the system was perfused for
an additional three samples with a solution of either the SKF 82958
(1mM) or SCH23390 (30 microM) in aCSF. The experiment was finished
after the sixth sample had been collected. The GABA content in the
samples were then analyzed by high performance liquid chromatography
(HPLC).
The available data are from five experiments, two
agonist experiments and two antagonist experiments (Table 1 shows
a sample agonist experiment). The two groups of experiments were
analyzed separately. Given the small sample size, a meaningful statistical
comparison could not be carried out. In figure 5, baseline data
are presented as the mean of the third baseline samples, while the
drug exposure results represent the mean of the samples that gave
the maximum effect in response to the drugs.
Figure 1. Traditional model of basal ganglia-thalamocortical
circuitry. The basal ganglia (blue box) include the striatum, the
external and internal segments of the globus pallidus (GPe and GPi,
resp.), the subthalamic nucleus (STN), SNr and SNc. Red arrows indicate
excitatory projections, and black arrows indicate inhibitory projections.
Figure 2. The nigral dopamine system. Dopamine (DA)
molecules are released from SNc dendrites. Subsequent activation
of D1-receptors on striatonigral terminals may increase GABA release,
which in turn, may reduce the inhibitory output from the SNr cell
to the thalamus.
Figure 3. The membrane of a microdialysis probe. The
beveled silica tubing in the middle is connected to the inlet of
the probe, through which aCSF and drugs are injected. Drugs get
into the surrounding tissue by diffusing across the clear membrane.
By the same token, GABA diffuses into the membrane and is carried
up though the outlet of the probe.
Figure 4. Schematic of probe penetrations. This figure
shows the parasagittal slice of lateral 7 plane. The penetrations
are 25 degrees; posterior to the vertical, limiting tissue damage
to the basal ganglia. The SNr is about 2-3 mm in thickness. This
ensures that the 2 mm-long microdialysis membrane is completely
in the SNr.
Figure 5. Effects of activation or blockade of D1LRs
on the GABA concentrations in microdialysates from the primate SN.
The data show a 390% increase in GABA levels in response to D1LR
activation with SKF 82958 (Left Diagram) and a 23% decrease in GABA
levels in response to D1LR blockade with SCH 23390 (Right Diagram).
Table 1. Results of one of the agonist experiments.
First three samples were collected when only aCSF was infused, and
samples 4, 5, and 6 were collected when the D1LR agonist (SKF 82958)
was infused.
These preliminary results suggest that activation of D1LRs enhances
GABA release in the primate SN, likely originating from striatonigral
terminals. Increased GABA levels may act to reduce the activity
of the nearby basal ganglia output neurons in SNr, resulting in
downstream facilitation of cortical activity involved in movement
and other functions.
Significance:
If confirmed, these results will help to explain some of the effects
and side-effects of current anti-parkinsonian drugs, and may help
to direct the development of new drugs which are more specifically
aimed at reducing basal ganglia output at the nigral level. The
studies may also have relevance for the interpretation of ongoing
attempts to treat parkinsonism with nigral transplantation of dopaminergic
tissue or injections of dopaminergic growth factors at this site.
Future Studies:
1. Repetition of these microdialysis experiments in other animals
to increase our sample size so that statistical comparisons can
be done.
2. Measurements of dopamine in addition to GABA.
3. Assessment of the D1LR-mediated effects on GABA and dopamine
release under parkinsonian conditions.
4. Study of the electrophysiologic changes of single SNr cells in
response to local microinjections of D1LR-active drugs.
5. Assessment of the behavioral consequences of D1LR activation
in parkinsonism.
This project is supported by Howard Hughes Medical Institute Grant
No. 52003727, and by NIH grant R01 NS040432 through Dr. Thomas Wichmann's
laboratory. GABA levels were determined in Dr. Nigel Maidment's
laboratory (UCLA) through a fee-for-service consultant arrangement.
Parkinson's Disease results from loss of a brain chemical, dopamine.
Dopamine is crucial for modulation movement and other brain functions.
For years, it was believed that dopamine is released primarily in
a structure of the brain called striatum. Recently, in studies of
rodents and cats, dopamine has also been found in another structure
of the brain called substantia nigra pars reticulata (SNr). Our
experiments are conducted in a monkey to investigate the presence
of dopamine and its role in the SNr.
In vivo microdialysis, Electrophysiologic Mapping.
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