THE JOURNAL OF COMPARATIVE NEUROLOGY 364931-253 (1996)

Synaptic Innervation of Midbrain

Dopaminergic Neurons
by Glutamate-Enriched Terminals
in the Squirrel Monkey
YOLAND SMITH, ALI CHARARA, A N D ANDRB PARENT
Centre de Recherche en Neurobiologie, H8pital de 1’Enfant-Jesus and Universite Laval,
Quebec, Canada G1J 124

ABSTRACT
The excitatory amino acid, glutamate, has long been thought to be a transmitter that plays
a major role in the control of the firing pattern of midbrain dopaminergic neurons. The present
study was aimed at elucidating the anatomical substrate that underlies the functional
interaction between glutamatergic afferents and midbrain dopaminergic neurons in the
squirrel monkey. To do this, we combined preembedding immunocytochemistry for tyrosine
hydroxylase and calbindin D-28k with postembedding immunostaining for glutamate.
On the basis of their ultrastructural features, three types (so-called types I, 11, and 111) of
glutamate-enriched terminals were found to form asymmetric synapses with dendrites and
perikarya of midbrain dopaminergic neurons. The type I terminals accounted for more than
70% of the total population of glutamate-enriched boutons in contact with dopaminergic cells in
the dorsal and ventral tiers of the substantia nigra pars compacta as well as in the ventral
tegmental area, whereas 5-20% of the glutamatergic synapses with dopaminergic neurons
involved the two other types of terminals. The major finding of our study is that the
glutamate-enriched boutons were involved in 70% of the axodendritic synapses in the ventral
tegmental area. In contrast, less than 40% of the boutons in contact with dopaminergic
dendrites were immunoreactive for glutamate in the dorsal and ventral tiers of the substantia
nigra pars compacta. Approximately 50% of the terminals in contact with the perikarya of the
different populations of midbrain dopaminergic neurons displayed glutamate immunoreactiv-
ity.
In conclusion, our findings provide the first evidence that glutamate-enriched terminals
form synapses with midbrain dopaminergic neurons in primates. The fact that the proportion
of glutamatergic boutons in contact with dopaminergic cells is higher in the ventral tegmental
area than in the substantia nigra pars compacta suggests that the different groups of midbrain
dopaminergic neurons are modulated differently by extrinsic glutamatergic afferents in
primates. I IWi Wile?.-Liss, Inc.

Indexing terms: substantia nigra, ventral tegmental area, GABA, postembedding

immunocgtochemistry, primate

In vivo, the dopaminergic neurons in the substantia nigra
pars compacta (SNc) and the ventral tegmental area (VTA)
exhibit two different firing patterns, namely irregular
single spiking or burst firing (Bunney et al., 1973; Grace
and Bunney, 1983, 1984a,b; Clark and Chiodo, 1988;
Overton and Clark, 1992; Smith and Grace, 1992; Niss-
brandt et al., 1994; Zhang et al., 1994). The exact mecha-
nism for the generation of burst firing in midbrain dopamin-
ergic neurons has not been elucidated. However, it is well
established that afferent input to these cells must play a
critical role, because burst firing is absent when dopamine
neurons are recorded in brain slices that are devoid of
afferents (Grace and Onn, 1989; Johnson and North, 1992;
Wang and French, 1993). Recent pharmacological and
electrophysiological data have demonstrated that glutama-
tergic afferents are likely to participate in the generation of
burst firing in midbrain dopaminergic neurons. For in-
Accepted July 10. 1995.
Address reprint requests to Dr. Yoland Smith, Centre de Recherche en
Neurobiologie, HBpital de I’Enfant-Jesus, 1401, 18ieme Rue, Quebec, QC,
CANADA, G I J 124.

( 1996 WILEY-LISS. INC.

232
stance, microiontophoretic application of glutamate stimu-
lates the burst activity in nigral dopamine neurons (Grace
and Bunney, 1984b). In keeping with these data, stimula-
tion of the cerebral cortex (Gariano and Groves, 1988) and
the subthalamic nucleus (Smith and Grace, 1992; Chergui
et al., 19941, which are known to be potential sources of
glutamatergic afferents to the SNc and VTA (Carter, 1982;
Kornhuber et al., 1984; Christie et al., 1985; Kita and Kitai,
1987; Groenewegen and Berendse, 1990; Robledo and
Feger, 1990; Smith et al., 1990; Sesack and Pickel, 1992a),
induce burst firing in midbrain dopaminergic neurons. On
the other hand, intraventricular infusion (Grenhoff et al.,
1988a) or local perfusion (Charlety et al., 1991) of the
broad-spectrum excitatory amino acid antagonist, kynure-
nate, reduce burst activity in midbrain dopaminergic cells.
Several studies support the notion that activation of
N-methyl-D-aspartate (NMDA) (Johnson and North, 1992;
Overton and Clark, 1992; Zhang et al., 1994) or non-NMDA
(Zhang et al., 1994) glutamatergic receptors produces burst
firing in nigral and VTA dopaminergic neurons. In line with
these findings, results obtained by means of ligand binding,
in situ hybridization and immunohistochemistry have re-
vealed the existence of different types of NMDA and
non-NMDA glutamatergic receptors associated with mid-
brain dopaminergic neurons in the rat (Albin et al., 1992;
Petralia and Wenthold, 1992; Martin et al., 1993; Sat0 et
al., 1993; Standaert et al., 1993, 1994; Laurie and Seeburg,
1994; Petralia et al., 1994).
On the basis of these data demonstrating that glutamate
plays a major role in controlling the activity of midbrain
dopaminergic neurons in the rat, it is reasonable to believe
that the SNc and VTA neurons are contacted by glutamater-
gic terminals. To our knowledge, there is no direct evidence
of synaptic contacts between glutamatergic terminals and
dopaminergic cells, except for a recent report demonstrat-
ing that cortical boutons, which probably contain gluta-
mate, form asymmetric synapses with tyrosine hydroxylase
(TH)-immunoreactive neurons in the VTA of the rat (Se-
sack and Pickel, 1992a). In the light of data suggesting that
not only the cerebral cortex but also the pedunculopontine
nucleus (Scarnati et al.,1986; Di Loreto et al., 1992; Lavoie
and Parent, 1994) and the subthalamic nucleus (Smith and
Grace, 1992; Chergui et al., 1994) provide glutamatergic
afferents to the midbrain dopaminergic neurons, we under-
took a series of experiments aimed at elucidating the
ultrastructural features and synaptic organization of the
glutamatergic boutons in contact with TH-immunoreactive
neurons in the SNc and the VTA of squirrel monkeys.
Taking into account that burst-firing neurons are more
frequent in the VTA than in SNc (Chiodo et al., 1984;
Grenhoff et al., 1988b), we paid particular attention to the
comparison of the proportion of glutamatergic terminals in
contact with these two groups of dopaminergic neurons. In
the light of recent colocalization studies in rat and monkey
showing that more than 90% of the calbindin D-28k-
positive neurons in the VTA display TH immunoreactivity
(Rogers, 1992; Gaspar et al., 1993) and that none of the
dopaminergic cells in the ventral tier of the SNc contain
calbindin (Rogers, 1992; Gaspar et al., 1993), we used the
immunoreactivity for calbindin D-28k as a specific marker
for dopaminergic neurons in the VTA of the squirrel
monkey. Therefore, we combined the postembedding immu-
nogold labelling for glutamate with the preembedding
immunocytochemistry for TH or calbindin D-28k. The
Y. SMITH ET AL.
results of this study have been presented in abstract form
(Smith et al., 1994a, 1995).
MATERIALS AND METHODS
Animals and preparation of tissue
Three adult (body weight 950-1,050 g) male squirrel
monkeys (Saimiri sciureus) were used in the present study.
They were deeply anesthetized with an overdose of pentobar-
bital and were perfused transcardially with 300 ml of cold
(4°C) oxygenated Ringer's solution containing 126 mM
NaCl, 26 mM NaHC03, 3 mM KCl, 1.2 mM KH2P04,1.3
mM MgS04, 2.4 mM CaC12, 5 mM HEPES, and 15 mM
dextrose. The brains were fixed with 1.2 liters of a solution
containing 2% paraformaldehyde and 1%glutaraldehyde in
phosphate buffer (PB; 0.1 M), pH 7.4, at room temperature.
The free aldehydes were then washed out with 1liter of PB
at 4°C. The brains were then removed from the skull, cut
into l0mm-thick blocks in the transverse plane, and placed
into cold phosphate-buffered saline (PBS; 0.01 M), pH 7.4,
until sectioning. The blocks were then sectioned at 60 km
in the transverse plane on a vibrating microtome, collected
in cold PBS, and treated for 20 minutes in sodium borohy-
dride (1%in PBS). After repeated washes in PBS, the
sections including the SNc and the VTA were processed for
TH or calbindin D-28k immunocytochemistry.
TH and calbindin D-28k immunocytochemistry
Light microscopy. Serial sections were first preincu-
bated in PBS (0.01 M), pH 7.4, containing 1%normal horse
serum (NHS), 1%bovine serum albumin (BSA, Sigma, St.
Louis, MO), and 0.3% Triton X-100 for 1 hour at room
temperature. They were then incubated overnight at room
temperature in the primary antisera solution containing
either mouse anti-TH antibodies (1:1,000; INCSTAR, Still-
water, MI) or mouse anti-calbindin D-28k antibodies (1:
2,500; Sigma). After three washes (10 minutes each) in
PBS, the sections were incubated for 1 hour at room
temperature in biotinylated horse anti-mouse immuno-
globulin (IgG; 1:200;Vector Labs, Burlingame, CA), washed
again in PBS, and incubated for 1 hour at room tempera-
ture in avidin-biotin-peroxidase complex (ABC; 1:100; Vec-
tor Labs) according to the method of Hsu et al. (1981). The
primary and secondary antibodies as well as the ABC were
diluted in the preincubation solution. The bound peroxi-
dase was then revealed by placing the sections in Tris buffer
(0.05M), pH 7.6, containing 3,3' diaminobenzidine tetrahy-
drochloride (DAB;0.025%;Sigma), 0.01 M imidazole (Fisher
Scientific, Nepean, Ontario, Canada), and 0.006% hydrogen
peroxide for 10 minutes. The reaction was stopped by
repeated washes in PBS. The sections were then mounted
on chrome ahmigelatin-coated slides, dehydrated, and a
coverslip was applied with Permount.
Electron microscopy. The sections were placed in a
cryoprotectant solution (PB, 0.05 M, pH 7.4, containing
25% sucrose and 10% glycerol) for 20-30 minutes. After
sinking, they were frozen at -80°C for 20 minutes. They
were then thawed and washed many times in PBS before
being processed to localize TH or calbindin D-28k according
to the protocol described for light microscopy except that
Triton X-100 was not included in the solutions, and the
incubation in the primary antisera was carried out at 4°C
for 48 hours. The bound peroxidase was localized with
benzidine dihydrochloride (BDHC; Sigma). The sections
GLUTAMATE INPUT TO MIDBRAIN DOPAMINERGIC CELLS 233

were washed three to five times in PB (0.01 M), pH 6.8, nonimmune rabbit serum. Another series of control grids
before being preincubated in a solution of BDHC that was was incubated with GABA and glutamate antisera that had
prepared according to Levey et al. (1986).After a 10 minute undergone liquid-phase preadsorption with structurally
preincubation, the reaction was initiated by adding hydro- related amino acids conjugated to ethanolamine with glutar-
gen peroxide to a final concentration of 0.005% in fresh aldehyde (Dale et al., 1986). Each antiserum was pread-
BDHC solution. The reaction was terminated after 5-7 sorbed with taurine, GABA, glutamate, and glutamine
minutes by extensive washings in PB (0.01 M),pH 6.8, conjugates. After incubation, the tissue was almost com-
before processing for electron microscopy. For controls, a pletely devoid of gold particles in the cases where the GABA
few sections were incubated in a solution in which the and glutamate antisera were preadsorbed with GABA- and
primary antisera had been replaced by nonimmune serum. glutamate-glutaraldehyde-conjugates, respectively. In con-
trast, preadsorption of the antisera with other amino acids
Processing for electron microscopy conjugates had no effect on the intensity of staining.
The sections were washed in PB (0.01M), pH 6.8, before
being postfixed in osmium tetroxide (1%solution in the Analysis of the material
corresponding PB) for 20 minutes. They were then washed Selection o f the appropriate areas for electron micro-
in PB and dehydrated in a graded series of alcohol and scopic analysis. A total of 15 blocks were selected for
propylene oxide. Uranyl acetate was added to the 70% electron microscopic analysis of the synaptic relationships
ethanol (30 minutes) to improve the contrast in the electron between glutamate-enriched terminals and midbrain dopa-
microscope. The sections were then embedded in resin minergic neurons. All of these blocks were taken from the
(Durcupan ACM; Fluka) on microscope slides, placed in the same animal except for three that were selected from the
oven, and cured for 48 hours at 60°C. After examination by other cases. Although the chemical characteristics were a
light microscopy, regions of interest were drawn, and some good criterion for differentiating the dorsal from the ven-
were photographed, cut out from the slides, and glued on tral tiers of the SNc, such was not the case for the dorsal
the top of resin blocks with cyanoacrylate glue. Serial SNc or the VTA. The exact limit between these two regions
ultrathin sections were then cut on a Reichert-Jung Ultra- was difficult to trace on the basis of chemical characteris-
cut E ultramicrotome, collected on Pioloform-coated single- tics, because both neuronal groups displayed calbindin and
slot nickel grids, and processed for postembedding immuno- T H immunoreactivity. To make sure that we selected the
gold labelling for glutamate or y-aminobutyric acid (GABA). appropriate area for electron microscopic analysis, the
Postembedding immunocytochemistry blocks in the VTA were taken only from regions medial to
the third nerve, including the so-called parabrachial pig-
Serial sections from different sectors of SNc and VTA mented nucleus and the rostra1 linear nucleus (Halliday
were processed for postembedding immunocytochemistry and Tork, 1986). Therefore, five blocks were cut from the
for GABA or glutamate. The GABA immunocytochemistry ventral tier of the SNc (SNc-v),four blocks were taken from
was used as a control to identify terminals in which the dorsal tier of the SNc (SNc-d), and six blocks included
glutamate serves as a precursor for the synthesis of GABA. the VTA. All of the blocks in the SNc-v were taken from
The postembedding immunogold procedure was carried out TH-immunostained tissue (Fig. lA), whereas two blocks in
with a n antiserum raised in rabbit against GABA (Hodgson the SNc-d and three blocks in the VTA were cut from
et al., 1985) and with two different antisera raised in rabbit calbindin-immunostained sections (Fig. 1B).
against glutamate (Arne1 Products, New York, NY; Sigma). Measurement ofglutamate immunoreactivity. The level
The production, characterization, and specificity of these of glutamate immunoreactivity was measured over a sample
antisera have been described in detail elsewhere (Hodgson of type I, type 11, and type I11 terminals that were randomly
et al., 1985; Somogyi and Hodgson, 1985; Somogyi et al., selected in ultrathin sections of the VTA and the SNc-v.
1985; Hepler et al., 1988). The protocol used for immuno- The level of glutamate immunoreactivity was also mea-
staining was introduced by Hodgson et al. (1985) and sured over a randomly selected population of terminals that
included recent modifications (Phend et al., 1992). Briefly, a formed symmetric synapses and displayed GABA immuno-
series of adjacent ultrathin sections was preincubated for reactivity in adjacent sections. A terminal was considered
10 minutes in Tris-buffered saline (TBS; 0.05 M), pH 7.6, immunoreactive for GABA if the density of gold particles
containing 0.01% Triton X-100. This was followed by an associated with it was at least four times higher than that
overnight incubation at room temperature with the pri- overlying neighboring terminals that formed asymmetric
mary antisera (GABA 1:10,000 or glutamate 1:10,000) synapses and displayed strong glutamate immunoreactivity
diluted in TBS/O.Ol% Triton X-100. The sections were then in adjacent sections. For a control for the level of glutamate
washed three times (twice for 10 minutes and once for 30 involved in metabolic functions, the density of gold particles
minutes) in TBS/0.01% Triton X-100 followed by TBS (0.05 associated with glial cells was also measured in the gluta-
M),pH 8.2, for 10 minutes. They were then incubated with mate-immunostained sections. Although the pattern of
the gold-conjugated goat anti-rabbit IgG (BioCell; 1:25 in GABA and glutamate immunostaining was not signifi-
TBS 0.05 M), pH 8.2, for 90 minutes at room temperature, cantly different in the three animals used in the present
washed in distilled water, and stained with uranyl acetate study, the ultrastructural features and the intensity of
( 1%in distilled water) for 90 minutes. Finally, after wash- immunostaining were better in one of these cases. There-
ing in distilled water and staining with lead citrate (Rey- fore, we selected all of the terminals for the quantitative
nolds, 1963), they were examined with a Phillips EM 300 analysis from this animal. To determine the level of immu-
electron microscope. noreactivity, the cross-sectional area of the boutons was
measured with the aid of the Ultimage software (Graftek,
Control experiments France) and a Macintosh computer. All gold particles
The specificity of labelling was tested by incubation with (including those over mitochondria) were counted over each
solutions in which the primary antisera were replaced with profile. The results of these measurements are illustrated
234
in Figure 3. Student’s t test was used to ascertain the
significance or the differences between the mean values.
Relative distribution ofpostsynaptic targets. The rela-
tive distribution of the postsynaptic targets in contact with
glutamate-enriched terminals was determined by analysis
of the synaptic inputs to randomly chosen perikarya and
dendritic shafts in the SNc and the VTA. The ultrastruc-
tural features and the level of glutamate immunoreactivity
of the terminals that formed synapses with these elements
were determined, and were included in one of the three
categories of glutamate-enriched boutons described in our
study. To ensure that the pattern of synaptic innervation of
neuronal perikarya was similar throughout their full ex-
tent, they were examined in five to ten serial sections.
RESULTS
Identification of the different groups
of dopaminergic neurons at midbrain level
The midbrain dopaminergic neurons were subdivided
into three groups on the basis of their localization, morphol-
ogy, and immunoreactivity for calbindin D-28k. The tissue
examined in the electron microscope was taken from sec-
tions of the middle one-third of the substantia nigra, where
the three groups of dopaminergic cells were well defined
(Fig. 1).The anatomical and histochemical criteria used in
the present study for identifying these three groups of
neurons were as follows: The neurons in the SNc-v were
densely packed and displayed immunoreactivity for TH but
not for calbindin D-28k (Fig. 1);the neurons in the SNc-d
were more loosely distributed and displayed immunoreactiv-
ity for both TH and calbindin D-28k (Fig. 1);finally, the
neurons in the VTA also displayed immunoreactivity for
TH and calbindin D-28k but were localized more medially
than neurons in the SNc-d.
Overall labelling at the electron
microscopic level
In the material prepared for electron microscopy, the TH-
and calbindin-immunoreactive neurons were localized by
means of immunohistochemical techniques using BDHC as
chromogen. At the light microscopic level, the BDHC-
containing elements were recognized by the presence of
large, dark crystals that were randomly dispersed through-
out the labelled structures (Fig. 6A). At the electron
microscopic level, the BDHC deposits appeared as large,
electron-dense aggregates dispersed throughout the cyto-
plasm of immunoreactive elements (Figs. 2 , 5 , 6 , 10, 11).In
the TH-immunostained tissue, the BDHC deposits oc-
curred exclusively in perikarya and dendritic shafts, whereas
in the calbindin-immunostained material, axon terminals
were also labelled.
In sections that were processed for the localization of
glutamate or GABA immunoreactivity by the postembed-
ding immunogold method, the tissue was overlaid with gold
particles (Figs. 2, 5-8, 10, 11).Overall, the perikarya and
dendritic shafts containing the BDHC deposit were lightly
labelled except in the glutamate-immunostained material,
where the mitochondria sometimes contained a substantial
number of gold particles (Figs. 2D, 8E). The glial cells were
almost completely devoid of labelling in both glutamate-and
GABA-immunostained tissue (Fig. 7C,D). The axon termi-
nals were the neuronal elements that contained the highest
density of gold particles. However, the intensity of labelling
Y. SMITH ET AL.
was not homogeneous throughout the section, some termi-
nals were associated with a large density of gold particles,
whereas other boutons were almost completely devoid of
labelling. Therefore, we measured the density of gold
particles in order to determine the immunoreactivity or
nonimmunoreactivity of a particular element for glutamate
or GABA.
Quantitative measurements of glutamate
immunoreactivity
Glutamate is an essential component of intermediary
metabolism and is a precursor for the synthesis of GABA.
For this reason, all cells in the brain, including neuronal
elements that use GABA as a neurotransmitter, are ex-
pected to contain a certain level of glutamate. In sections of
midbrain reacted with antiserum to glutamate, gold par-
ticles were, in fact, associated with all neuronal and nonneu-
ronal structures. However, their surface density differed
significantly upon visual inspection. Quantitative measure-
ments were therefore carried out to substantiate these
differences. The density of gold particles associated with
putative glutamate-immunoreactive boutons, i.e., those
forming asymmetric synapses and overlaid by a high den-
sity of gold particles (Fig. 2A,C,D), was compared to the
density over glial cells and boutons that formed symmetric
synapses and displayed GABA immunoreactivity in adja-
cent sections. Statistical comparison of the immunoreactiv-
ity associated with different processes gave similar results
in the three animals used in the present study. The overall
pattern of immunostaining did not change from one animal
to the other. The possibility that the intensity of labelling
associated with a particular group of neuronal elements
differed from one postembedding reaction to the other was
tested. A comparative analysis of the density of gold
particles associated with different structures in sections
processed in six different postembedding reactions revealed
that the intensity of labelling over a particular group of
neuronal elements remained the same after each incuba-
tion. The total number of terminals measured in the
present study is given in Table 1. The measurements
obtained in sections taken from one incubation are shown
in Figure 3.
On the basis of their ultrastructural features, three
populations of glutamate-enriched boutons were found to
form asymmetric synapses with dopaminergic neurons in
the SNc and the VTA. The type I terminals were small to
medium-sized (maximum diameter ranging from 0.5 pm to
2.0 pm) and contained round synaptic vesicles and one or
two mitochondria (Figs. 2A, 5D, 7A, 8E, 1OC).The density
of gold particles associated with these boutons was signifi-
cantly higher than that associated with glial cells or bou-
tons forming symmetric synapses (Fig. 3A). A particular
feature concerning these terminals was the fact that the
intensity of labelling associated with them was significantly
higher in the VTA than in the SNc (Fig. 3A). This was not
due to a higher background staining in the VTA, because
the density of gold particles associated with glial cells or
boutons forming symmetric synapses in the VTA was not
significantly different from that in the SNc (Fig. 3A).
Like the type I terminals, the type I1 boutons were
packed with small round vesicles but also contained numer-
ous large dense-core vesicles (Figs. 2C, 10D, 11).Overall,
these boutons were larger than the type I terminals (maxi-
mum diameter ranging from 1.0 km to 3.0 pm). Com-
parative measurements of the density of gold particles
GLUTAMATE INPUT TO MIDBRAIN DOPAMINERGIC CELLS
Fig 1. Photomicrographs showing t h e three groups of midbrain
dopaminergic neurons at t h e level where t h e blocks were taken for
electron microscopic analysis. A: Section stained for tyrosine hydroxy-
lase ITHI. B: Adjacent Section stained for calbindin D-28k (CaBP).
Note that neurons in t h e ventral tier of t h e substantia nigra pars
associated with the type I1 terminals in SNc (n = 7 ) and
VTA ( n = 5 ) revealed that, in contrast to the type I
terminals, the intensity of labelling associated with the type
235
cornpacta (SNc-v) a r e immunoreactive for TH but not for CaBP,
whereas neurons in t h e dorsal tier of t h e SNc (SNc-d) and t h e ventral
tegmental area (VTA) display both TH and CaBP immunoreactivity.
Scale bar = 1 mm in A (also applies to B).
I1 boutons remained the same in both structures. There-
fore, we decided to pool the values obtained in SNc and VTA
to prepare the histogram presented in Figure 3B. The
 236 Y. SMITH ET AL.

Fig. 2 . Electron micrographs showing the three types of glutamate-
enriched terminals visualized in the SNc-v of the squirrel monkey. A
and B illustrate adjacent sections of a TH-immunoreactive dendrite
( d l ) that have been processed using the postembedding immunogold
method for revealing glutamate (GLU; A) or y-aminobutyric acid
(GABA; B). Note that b l is immunoreactive for glutamate but not for
GABA. The density of gold particles associated with this terminal (bl)
in the GLU-immunostained section is significantly higher than that
overlying neighboring boutons (asterisks), whereas this is the reverse
in the GABA-immunostained material. This bouton displays the ultra-
structural features of the type I terminals and forms an asymmetric
synapse (arrowhead in B) with the dendrite. C illustrates a type I1
terminal (b2) in contact with a TH-positive dendrite (d2) in a section
that has been incubated with an antibody against GLU. Note the
occurrence of numerous dense-core vesicles (indicated by arrows) and
the high density of gold particles in b2. D shows a GLU-enriched type
I11 terminal that forms an asymmetric synapse (arrowheads) with a
TH-containing dendritic shaft (d3). This bouton contains a few dense-
core vesicles (arrows) and numerous mitochondria. Note the row of
postjunctional dense bodies associated with the synaptic contact. Scale
bars = 1 pm in A (also applies to B), 1 pm in C (also applies to D).

density of gold particles overlying this population of termi- TABLE 1. Total Number of Terminals and Glial Cells of Which the
nals was significantly higher than that associated with Glutamate ImmunoreactivitvWas Measured in SNc-V and VTA
nonimmunoreactive structures (Fig. 3B). Elements examined
The type I11 boutons were characterized by their high
Terminals with
number of mitochondria and by the occurrence of postjunc- Brain Glial symmetric Type I Type 11 Type I11
tional dense bodies at the synaptic junctions (Fig. 2D). structures cells synapses glu glu glu
These boutons contained numerous round synaptic vesicles
SNc-V 31 127 95 8 19
and a few dense-core vesicles (Fig. 2D). Like the type I1 VTA 22 79 135 25 8
terminals, no difference in the intensity of labelling associ- Total number of
elements examined 53 206 230 33 27
ated with these boutons was found between the SNc (n = 5)
 GLUTAMATE INPUT TO MIDBRAIN DOPAMINERGIC CELLS 237

% 701 VTA, Type I ( f SEM=103.9 f 3.9; n 4 0 )

60-
x
SNc-V, Type I ( _+ SEM=68.8 f 3.5; n=26)

Glial cell processes ( x fSEM=8.5f 0.2; n=17)

50 - VTA, symmetric synapses (Ef SEM=18.8 f 2.4; n=19)
SNc-V, symmetric synapses ( 57 f SEM=16.4 f 0.8; n=38)
40 -

30 -

20 - 3

10 -

0 -- I
1 0 . 2 0 . 30 ' 40 ' 50 ' 60 ' 70 ' 80 ' 90 '100'110'120'130'140'150'160'170'
A DENSITY OF GOLD PARTICLES / pn2

% 60 701 VTA/SNc-V, Type I1

VTNSNc-V, Type I11 (

(xf SEM=63.7 f 1.2; n=12)
f SEM=70.8 f 0.9; n=8)

10 ' 20 ' 30 ' 40 ' 50 ' 60 ' 70 ' 80 ' 90 ' 100' 110' 120' 130' 140' 150' 160' 170'

B DENSITY OF GOLD PARTICLES / pm2

Fig. 3. A,B: Histograms comparing the density of gold particles
associated with the three types of glutamate-enriched terminals de-
scribed in the present study to the density of gold particles overlying
glial cells and boutons that form symmetric synapses in the SNc-v and
in the VTA. The measurements were made from elements stained in
the same incubation for GLU or GABA. In B, the type I1 and type 111
terminals in the VTA and SNc-v were pooled because there was no
difference in the intensity of labelling between these two regions. Note
that the glia and boutons forming symmetric synapses display the
lowest GLU immunoreactivity. The density of gold particles associated
with these elements is significantly lower ( P < 0.0051 than that
associated with the three types of glutamate-enriched boutons in the
SNc and VTA. Note also that the density of gold particles associated
with the type I terminals in the SNc is significantly lower ( P < 0.0051
than that associated with the same type of terminals in the VTA.
238 Y. SMITH ET AL.

0 SNc, ventral tier

SNc, dorsal tier
VTA

70 4
3

F9
30 1

20 : 1
I
I I1 I11
TYPE OF GLUTAMATE-ENRICHED TERMINALS
Fig. 4. Histogram showing the relative abundance of the three types of GLU-enriched boutons in the
different groups of midbrain dopaminergic neurons. To prepare this histogram, a total of 238 type I, 24 type
11, and 38 type I11 terminals were randomly encountered in the different midbrain dopaminergic cell
groups

and the VTA (n = 3 ) . The density of gold particles that
overlaid these terminals was significantly higher than that
overlying nonimmunoreactive structures (Fig. 3 ) .
Relative distribution of the three types
of glutamate-enrichedboutons
Among the three types of glutamate-enriched terminals
described above, the type I boutons formed the largest
population (Fig. 4).Examination of 161glutamate-enriched
boutons in randomly chosen ultrathin sections revealed
that the type I boutons accounted for 73-88% of the total
number of glutamate-enriched terminals in contact with
dopaminergic neurons in the two tiers of SNc and VTA (Fig.
4).In contrast, the type I1 terminals represented less than
10% of the glutamate-enriched boutons in contact with the
different populations of midbrain dopaminergic neurons
(Fig. 4).The type I11 terminals were slightly more abundant
(20%) in the SNc-v than in other midbrain dopaminergic
cell groups ( 5 1 0 % ) (Fig. 4).
Pattern of synaptic innervation
of dopaminergic neurons by
glutamate-enrichedboutons
The pattern of synaptic innervation of dopaminergic
neurons was determined by examination of boutons in
contact with randomly chosen perikarya and dendritic
shafts in the ventral and dorsal tiers of the SNc as well as in
the VTA. These elements were examined on adjacent
sections immunostained for glutamate or GABA. The num-
ber of terminals in contact with each dopaminergic element
was determined. Among these boutons, the percentage of
those that displayed a high level vs. a low level of glutamate
immunoreactivity was established (see Fig. 9). On the basis
of the quantitative data presented in Figure 3, we consid-
ered that terminals with a “high” level of glutamate
immunoreactivity were those associated with more than 50
gold particles/ pm2. In addition, these terminals had to be
nonimmunoreactive for GABA in adjacent sections (Fig. 9).
The typical ultrastructural features of the terminals form-
ing synapses with the different populations of dopaminergic
neurons are depicted in Figures 2 , 5 , 7 , 8 , 10, and 11.
SNc-u. A total of 41 proximal dendrites (diameter larger
than 2.0 pm), 28 distal dendrites (diameter smaller than 2
pm), and 7 perikarya were examined in the SNc-v. Typical
examples of dendritic shafts receiving inputs from gluta-
mate-enriched terminals in this region are illustrated in
Figures 2 and 5 . The vast majority (more than 70%) of
terminals in contact with dendritic shafts displayed a low
level of immunoreactivity for glutamate but were strongly
labelled for GABA (Fig. 2A,B). Most of the glutamate-
enriched terminals in contact with distal dendrites dis-
played the ultrastructural features of type I boutons (Fig.
2A,B), whereas both type I and type I11 terminals formed
synapses with proximal dendrites (Fig. 2D). A few type I1
terminals encountered in this region were in contact with
 GLUTAMATE INPUT TO MIDBRAIN DOPAMINERGIC CELLS
Fig. 5. Electron micrographs showing various features of boutons
forming synapses with TH-immunoreactive dendrites in the SNc-v.
A-C: Adjacent sections of the same dendrite ( d l )contacted by different
types of terminals (indicated by b l , b2, b3, and asterisks). One of these
houtons ( b l ) forms an asymmetric synapse (A,B, arrowheads) but
displays a low immunoreactivity in sections immunostained with two
different antisera for GLU (A,B). In contrast, this terminal is strongly
labelled for GABA (C). In the GABA-immunostained section (C), the
density of gold particles associated with b l is not different from that
overlying neighboring terminals, which, in some cases (h2), form
239
symmetric synapses (A-C, arrows). D-F Three adjacent sections of a
type I GLU-enriched terminal (h4)in asymmetric contact (arrowheads)
with a small TH-positive dendrite (d2). Note that the density of gold
particles overlying b4 is much higher than t h a t associated with a
neighboring terminal (b5) in the sections immunostained with two
different glutamate antisera (D,E),whereas this is the reverse in the
section processed for GABA (F).The asterisks in D and E indicate other
boutons that display a low immunoreactivity for GLU. Scale bar = 1 km
in A (also applies to B-F).
240
distal and proximal dendrites. These glutamate-enriched
terminals were interspersed among a large number of
GABA-positive boutons, which for the most part, were
packed with ovoid vesicles and formed symmetric synapses
(Figs. 2A,B, 5A-C). Three terminals that had a morphology
similar to the GABA-immunoreactive terminals but that
formed asymmetric synapses were also seen in contact with
TH-immunoreactive dendrites (see Fig. 5A-C, bouton bl).
To ensure that these boutons displayed a low level of
immunoreactivity for glutamate, three adjacent sections
containing one of these terminals were immunostained
with two different glutamate antisera and the GABA anti-
body (Fig. 5A-c). This was compared t o the immunostain-
ing of a bouton in the same section that formed an
asymmetric synapse with a distal dendrite (Fig. 5D-F). In
the two cases, the glutamate antisera and the GABA
antibody gave opposite patterns of immunostaining. No
significant difference was noted between the patterns of
immunostaining revealed with either glutamate antisera,
except that the overall intensity of labelling was slightly
lower with the Sigma antibody (GLU-S; compare Fig. 5D to
Fig. 5E).
The pattern of innervation of perikarya in the SNc-v was
relatively similar to that in the SNc-d and the VTA, except
that the percentage of glutamate-enriched boutons was
slightly higher in the VTA than in the SNc (Figs. 6, 7, 9). A
typical example is shown in Figures 6 and 7. The perikarya
of dopaminergic neurons were pyramidal to fusiform in
shape and had a maximum diameter that ranged from 20
km to 35 IJ-m. They contained an invaginated nucleus
surrounded by cytoplasm that was rich in organelles includ-
ing endoplasmic reticulum, Golgi apparatus, mitochondria,
and numerous lysosomes. Overall, the density of boutons
forming synapses with perikarya was lower than the num-
ber of terminals in contact with dendritic shafts. Figure 7
shows that most of the glutamate-enriched terminals in
contact with dopaminergic somata displayed the ultrastruc-
tural features of type I terminals (Fig. 7A,B). These termi-
nals were interspersed among a larger number of boutons
that were immunoreactive for GABA (Fig. 7C-G). Most of
these GABA-positive terminals displayed ultrastructural
features that resembled those of boutons in contact with
dendritic shafts (Fig. 2A,B) except for a population of
large-sized terminals, which contained pleomorphic vesicles
and numerous mitochondria (Fig. 7E-G). These boutons
formed short symmetric synapses. Frequently, puncta ad-
herentia were associated with these terminals (Fig. 7E-G).
A particular chemical feature of these boutons was the
display of strong immunoreactivity for glutamate and
GABA (Fig. 7E-G). Overall, this population of terminals
represented 1 5 2 0 % of the boutons in contact with peri-
karya in the different groups of dopaminergic neurons
examined. None of these boutons was seen in contact with
dendritic shafts.
SNc-d. A total of 24 proximal dendrites, 28 distal den-
drites, and 5 perikarya were examined in this region. An
example of a calbindin-positive dendrite receiving synaptic
inputs from glutamate-enriched boutons is shown in Figure
8. The pattern of synaptic innervation of dopaminergic
neurons in this region resembled that described in the
SNc-v, except that the percentage of glutamate-enriched
terminals in contact with dopaminergic cells was slightly
higher in this region than in the ventral tier of SNc (Fig.
9A,B).Another difference between the two parts of SNc was
the higher percentage of type I11 terminals in contact with
Y. SMITH ET AL.
dopaminergic elements in the SNc-v than in the SNc-d (Fig.
4). The innervation of perikarya, as mentioned above, was
quite similar in the two tiers of the SNc (Figs. 6,7).
VTA. A total of 39 proximal dendrites, 51 distal den-
drites, and 6 perikarya were examined in the VTA. The
glutamatergic innervation of dopaminergic elements in this
region is depicted in Figures 10 and 11. The major differ-
ence between the VTA and the SNc was the higher percent-
age of glutamate-enriched terminals in contact with den-
drites in VTA than in the two tiers of SNc (Fig. 9). This
difference was particularly obvious for the distal dendrites
that received more than 70% of their innervation from
glutamate-enriched boutons in the VTA vs. 30-40% in the
SNc (Fig. 9). However, the percentage of glutamate-
enriched terminals in contact with perikarya was quite
similar in the VTA and the SNc (Fig. 9). Like the SNc, most
(88%) of the glutamate-enriched terminals visualized in the
VTA displayed the ultrastructural features of type I termi-
nals (Fig. lOA,C). The other glutamate-enriched boutons in
contact with VTA neurons were of the type I1 category.
Although they were much less abundant than the type I
terminals, some of the glutamate-positive type I1 boutons
were seen in contact with dendritic shafts and also with
perikarya of VTA neurons (Figs. 10B,D, 11).The type 111
terminals accounted for less than 5% of the glutamate-
enriched boutons in contact with dopaminergic neurons in
the VTA (Fig. 4).
DISCUSSION
The present study provides the first description of the
synaptic innervation of midbrain dopaminergic neurons by
glutamate-enriched terminals in primates. The major find-
ing of our investigation is that the proportion of glutamate-
enriched terminals in contact with dopaminergic neurons is
much higher in the VTA than in the SNc of squirrel monkey
(Fig. 12). These data serve as evidence of a potential
anatomical substrate underlying the powerful control ex-
erted by excitatory amino acids on the activity of midbrain
dopaminergic neurons.
Overall synaptic innervation of midbrain
dopaminergic neurons
Although the synaptic organization of the substantia
nigra was the topic of many early studies (Rinvik and
Grofova, 1970; Gulley and Wood, 1971; Schwyn and Fox,
19741, the fact that the dopaminergic neurons were not
identified makes the comparison difficult between these
data and those obtained in the present study. However, the
introduction of sophisticated immunohistochemical tech-
niques at the electron microscopic level allowed the chemi-
cal characterization of different populations of terminals in
contact with dopaminergic neurons in the rodent SNc. It is
now well established that boutons immunoreactive for
GABA (Van den Pol et al., 1985; Bolam and Smith, 19901,
substance P (Mahalik, 1988; Bolam and Smith, 1990;
Mendez et al., 19921, enkephalin (Bolam and Smith, 19911,
serotonin (Nedergaard et al., 1988; Corvaja et al., 19931,
and choline acetyltransferase (Bolam et al., 1991) form
synapses with SNc dopaminergic neurons in the rat. Our
results provide the first evidence of direct synaptic contact
between glutamate-enriched boutons and SNc dopaminer-
gic neurons in primates. The fact that the ultrastructural
features displayed by the peptidergic, serotonergic, and
cholinergic terminals correspond, in many cases, to those of
GLUTAMATE INPUT TO MIDBRAIN DOPAMINERGIC CELLS 241

Fig. 6. Correlated light (A) and electron (B) micrographs of a micrograph has been taken in a section immunostained for GLU. The
CaBP-immunoreactive perikaryon in the SNc-d. The arrows in B area in the rectangle and boutons b5 and h6 are shown at higher
indicate crystals of henzidine dihydrochloride (BDHC). The electron magnification in Figure 7. Scale bars = 10 Fm in A, 2 bm in B.
Figure 7
 GLUTAMATE INPUT TO MIDBRAIN DOPAMINERGIC CELLS 243

the type I and type I1 terminals described in our study dopaminergic dendrites in the VTA formed asymmetric
suggests the possibility that other neuromediators coexist synapses and displayed strong glutamate immunoreactivity
with glutamate in subpopulations of glutamate-enriched in the squirrel monkey. The most likely explanation for this
terminals in contact with SNc neurons in primates. In discrepancy is a difference between the sampling method
keeping with previous data (Bolam and Smith, 1990), the used in these studies. In our case, the percentage of
vast majority of boutons in contact with SNc neurons were glutamate-enriched boutons in contact with dopaminergc
lightly labelled for glutamate but displayed strong GABA dendrites was obtained by counting the number of immuno-
immunoreactivity. We estimate that about 30% of the reactive and nonimmunoreactive terminals in contact with
terminals in contact with dopaminergic dendrites in the randomly selected dopaminergic dendrites in the VTA. In
SNc of squirrel monkey displayed glutamate immunoreac- the rat, the authors purposely selected areas that were
tivity. Most of the remaining terminals were immunoreac- enriched in GABA-positive boutons (Bayer and Pickel,
tive for GABA. These data are in keeping with those 1991) because of the problems relating to the penetration of
suggesting that 70% of the terminals that form synapses antibodies in tissue stained with preembedding methods. It
with SNc neurons displayed GABA immunoreactivity in the is therefore likely that the overall proportion of GABA-
rat (Bolam and Smith, 1990). Therefore, in both primate containing boutons in contact with dopaminergic neurons
and non-primate species, the vast majority of terminals in in the rat VTA is lower than that proposed in this study
contact with dendrites of dopaminergic neurons in the SNc (Bayer and Pickel, 1991). Of course, differences between
display GABA immunoreactivity. primate and nonprimate species must also be considered.
Similar to the dendrites, a minority of terminals in A particular feature that stood out in our material was
contact with perikarya of SNc neurons displayed strong the fact that the average density of gold particles associated
glutamate immunoreactivity. We estimated that about 40% with the glutamate-enriched boutons in the VTA was
of the axosomatic synapses in SNc involved glutamate- significantly higher than that in the SNc. This was not due
enriched boutons. A particular population of terminals that to a particular technical problem concerning the VTA,
was found in contact with perikarya, but not with dendritic because the intensity of labelling associated with nonimmu-
shafts of SNc neurons, displayed immunoreactivity for both noreactive structures was similar in the VTA and the SNc.
glutamate and GABA. The ultrastructural features of these Previous quantitative studies have established a positive
terminals corresponded to those arising from the external correlation between the density of gold particles and the
pallidum (or the globus pallidus in nonprimates). Direct absolute quantity of glutamate in brain tissue (Ottersen,
synaptic contacts between pallidal boutons and dopaminer- 1989). If this is the case in the VTA and the substantia
gic neurons were previously found in the rat (Smith and nigra of monkeys, then our data suggest that the level of
Bolam, 1990). Moreover, we recently showed that the glutamate in terminals that form synapses with VTA
terminals from the external pallidum in contact with neurons is higher than those in contact with SNc cells. The
neurons in the internal pallidum displayed strong immuno- other possibility is that a particular population of boutons
reactivity for both glutamate and GABA (Shink and Smith, richer in glutamate occurred in the VTA but not in the SNc
1994,1995).The possible functional significance of coenrich- of squirrel monkey. Future studies, like the one presented
ment of glutamate and GABA at the terminal level has been in the companion paper (Charara et al., 1996) in which the
discussed in detail in a previous study (Shink and Smith, anterograde tract-tracing method was combined with
1995). postembedding glutamate immunostaining, should help to
According to previous electron microscopic analysis of the resolve this issue.
VTA in the rat, the majority of terminals in contact with the It is likely that glutamate coexists with peptides or with
dopaminergic neurons in this region formed symmetric serotonin in terminals forming asymmetric synapses with
synapses and displayed GABA immunoreactivity (Bayer VTA neurons, as was the case in the SNc, because boutons
and Pickel, 1990, 1991). In contrast to these findings, our with ultrastructural features that resembled those of the
results show that 60-70% of the terminals in contact with type I and type I1 terminals described in our study displayed
immunoreactivity for enkephalin (Sesackand Pickel, 1992b),
dynorphin (Pickel et al., 19931, or serotonin (Herve et al.,
1987; Van Bockstaele et al., 1994) in the rat VTA.
Fig. 7. Electron micrographs showing ultrastructural features and Possible sources of glutamate-enriched
neurotransmitter content of houtons in contact with t h e CaBP-
immunoreactive perikaryon illustrated in Figure 6. A,B: Adjacent terminals in contact with midbrain
sections of t h e area in the rectangle in Figure 6. T h e section in A was dopaminergic neurons
processed for GLU immunostaining, whereas t h a t in B was immuno-
stained for GABA. Note t h a t b l , b2, and b 3 display a high level of GLU There are at least four possible sources of glutamate-
immunoreactivity but a r e nonimmunoreactive for GABA. In contrast, enriched terminals in contact with midbrain dopaminergic
b4 is lightly Iahelled for GLU but is strongly immunostained for GABA. neurons: the cerebral cortex, the subthalamic nucleus, the
C,D: Bouton h6 shown in adjacent sections immunostained for GLU pedunculopontine nucleus, and the raphe nuclei.
tCi o r GABA (D). This bouton and a neighboring terminal (D, open Cerebral cortex. The existence of a direct connection
arrow) a r c lightly labelled for GLU b u t are strongly immunoreactive for
GABA. Bouton b6 forms a symmetric synapse (D, paired solid arrows), from the cerebral cortex to the substantia nigra and the
whereas the neighboring terminal establishes a n asymmetric synaptic VTA was first suggested on the basis of results obtained in
contact ID, arrowhead) with t h e perikaryon. Note t h e low GLU degeneration studies (Afifi et al., 1974; Usunoff et al., 1982;
immunoreactivity associated with t h e glial cell in t h e same field. E-G Nitsch et al., 1984). These data were further confirmed
Bouton h5 shown in adjacent sections stained with two different GLU with retrograde (Bunney and Aghajanian, 1976; Gerfen et
antisera IE,FIand with t h e GABA antibody (GI. This bouton is strongly
immunoreactive for both GLU and GABA and forms a symmetric
al., 1982) and anterograde (Beckstead, 1979; Sakai, 1988;
synapse I G , arrows) with the perikaryon. A punctae adherentia ( E , F , Shook et al., 1991; Sesack and Pickel, 1992a; Naito and
arrows) is associated with this terminal. Scale bars = 1 k m in A (also E t a , 1994) tracing techniques. Although there is still some
applies to BI, 1 pm in C (also applies to D-G). controversy regarding the exact origin of the corticonigral
244
Fig. 8. Electron micrographs showing two boutons in contact with a
CaBP-immunoreactivedendrite in the SNc-d. A: Low-power view of the
dendrite in a GABA-immunostained section. Two boutons in contact
with this dendrite ( b l and b2) are shown at higher magnification in
B-E. B,C: Bouton b l shown in adjacent sections stained for GABA (B)
or for GLU (C). This bouton, which is immunoreactive for GABA but
projection, anterograde tract-tracing studies revealed that
the prefrontal cortex contributes more significantly to this
pathway than other cortical areas (Kiinzle, 1978; Sesack et
Y. SMITH ET AL.
not for GLU, forms a symmetric synapse (B, arrows) with the dendritic
shaft. D,E: Illustrations of b2 in adjacent sections immunostained for
GABA (D) or for GLU (El. This bouton is strongly immunoreactive for
GLU but is nonimmunoreactive for GABA. It forms a synapse (arrow-
heads) with the dendrite. Note the crystal of BDHC at the bottom of E,
which indicates immunoreactivity for CaBP.
al., 1989; Shook et al., 1991; Sesack and Pickel, 1992a;
Naito and Kita, 1994). A common feature that stood out
from these anatomical studies is that the cerebral cortex is
GLUTAMATE INPUT TO MIDBRAIN DOPAMINERGIC CELLS
the source of a relatively sparse input to midbrain dopamin-
e r g c neurons.
Early analysis at the electron microscopic level revealed
that the cortical terminals are packed with round electron-
lucent vesicles and form mainly asymmetric synapses with
SNc neurons (Malinov et al., 1982; Usunoff et al., 1982;
Nitsch et al., 1984). More recently, the use of double
immunocytochemical techniques at the electron micro-
scopic level has allowed researchers to demonstrate that
some of the VTA neurons that receive cortical inputs
displayed TH immunoreactivity (Sesack and Pickel, 1992a).
To our knowledge, the existence of direct synaptic contacts
between cortical boutons and dopaminergic neurons in the
SNc still remains to be demonstrated. The ultrastructural
features of the anterogradely labelled terminals in contact
with dopaminergic neurons in the rat VTA (Sesack and
Pickel, 1992a) largely correspond to those of the type I
terminals described in our study. These data combined with
data suggesting the glutamatergic nature of the cortical
input to midbrain dopaminergic neurons (Carter, 1982;
Kornhuber et al., 1984; Christie et al., 1985; Gariano and
Groves, 1988) indicate that the cerebral cortex is likely to be
the source of some of the glutamate-enriched boutons that
form synapses with dopaminergic neurons in the SNc and
the VTA of squirrel monkeys. However, because of the
scarcity of labelled terminals at the midbrain level after
cortical injections of anterograde tracers (Kiinzle, 1978;
Jurgens, 1984; Huerta et al., 1986; Leichnetz, 1986; Stan-
ton et al., 1988; Shook et al., 1991), the cerebral cortex
should not be considered to be the sole source of glutamater-
gic boutons in contact with midbrain dopaminergic neurons
in primates. This is particularly true for VTA neurons,
because our data demonstrate that more than 60% of the
boutons in contact with these cells display glutamate
immunoreactivity.
Subthalamic nucleus. The existence of a direct connec-
tion from the subthalamic nucleus to the substantia nigra
has been demonstrated in various species by means of
anterograde (Nauta and Cole, 1978; Carpenter et al., 1981;
Kita and Kitai, 1987; Groenewegen and Berendse, 1990;
Smith et al., 1990; Rinvik and Ottersen, 1993; Bevan et al.,
1994) and retrograde (Kanazawa et al., 1976; Parent and
Smith, 1987) tract-tracing techniques. In primates, this
projection arises from a group of neurons that is confined to
the ventromedial region of the subthalamic nucleus (Parent
and Smith, 1987). Electrophysiological data has shown that
the subthalamic nucleus is a powerful excitatory driver of
nigral neurons (Hammond et al., 1978; Nakanishi et al.,
1987; Robledo and Feger, 1990). These data combined with
immunohistochemical findings showing that the peri-
karyon (Smith and Parent, 1988; Albin et al., 1989) and
terminals (Rinvik and Ottersen, 1993; Shink and Smith,
1995)of subthalamic neurons display glutamate immunore-
activity indicate that the subthalamic nucleus is likely to be
the source of some of the glutamate-enriched boutons
described in our study. Although direct synaptic contacts
between subthalamic terminals and dopaminergic neurons
have never been shown, labelled varicosities occurred in the
SNc after injections of anterograde tracers in the subtha-
lamic nucleus of rat (Kita and Kitai, 1987; Groenewegen
and Berendse, 1990) and monkey (Smith et al., 1990).
Moreover, stimulation of the subthalamic nucleus en-
hanced dendritic release of dopamine in the substantia
nigra (Mintz et al., 1986; Rosales et al., 1994). On the other
hand, lesions of the subthalamic nucleus reduced signifi-
215
cantly the level of burst firing in nigral dopaminergic cells
in the rat (Smith and Grace, 1992). Therefore, the subtha-
lamic nucleus appears to contribute directly to the excita-
tion of dopaminergic neurons in the rat SNc. In keeping
with these data, our findings demonstrate that a population
of glutamate-enriched boutons, the so-called type I11 termi-
nals, display the ultrastructural features of subthalamic
boutons (Kita and Kitai, 1987; Rinvik and Ottersen, 1993;
Bevan et al., 1994; Smith et al., 199413; Shink and Smith,
1995) and form asymmetric synapses with dopaminergic
neurons in the SNc and VTA of squirrel monkey. However,
the type I11 terminals represent a minority of glutamatergic
boutons in contact with midbrain dopaminergic cells in the
squirrel monkey.
Pedunculopontine nucleus. The possibility that the
pedunculopontine nucleus is the source of glutamate-
enriched boutons in contact with midbrain dopaminergic
neurons was investigated and is discussed in detail in the
companion paper (Charara et al., 1996).
Kaphk nuclei. The neurons in the dorsal and median
raphe nuclei are well known as the sources of serotonin in
the substantia nigra and the VTA (Bobillier et al., 1976;
Van der Kooy and Hattori, 1980; Parent et al., 1981;
Steinbusch, 1984; Imai et al., 1986; Lavoie and Parent,
1990). On the basis of electron microscopic data, it appears
that the ultrastructural features of the serotonin-immuno-
reactive boutons that form asymmetric synapses with
midbrain dopaminergic neurons in the rat (Herve et al.,
1987; Nedergaard et al., 1988; Corvaja et al., 1993; Van
Bockstaele et al., 1994) resemble those of the type I
terminals described in our study. In light of the findings
suggesting the coexistence of glutamate and serotonin in
single raphe neurons (Fung et al., 1992; Nicholas et al.,
1992; Johnson, 1994), it is likely that serotonin and gluta-
mate coexist at the level of single terminals that form
synapses with dopaminergic neurons in the SNc and the
VTA of the squirrel monkey. On the other hand, it has been
shown that a substantial proportion of raphe neurons that
project to the striatum in the rat are nonserotonergic
(Steinbusch et al., 1980). These data combined with data
demonstrating that the raphe-nigral and raphe-striatal
projections largely arise from the same neurons (Van der
Kooy and Hattori, 1980) suggest the possibility that some of
the raphe neurons that project to the SNc and VTA in
monkeys use only glutamate as a neurotransmitter. In
keeping with this hypothesis, stimulation of the dorsal
raphe nucleus induces a nonserotonergic, fast, excitatory
effect in midbrain dopaminergic neurons in the rat (Gervais
and Rouillard, 1993). The chemical content of the raphe
terminals in contact with midbrain dopaminergic neurons
in primates is currently under investigation in our labora-
tory.
Other minor sources. The central nucleus of the amyg-
dala (Price and Amaral, 1981; Gerfen et al., 1982; Gonzales
and Chesselet, 1990), the intralaminar thalamic nuclei
(Gerfen et al., 1982; Sadikot et al., 1992), and the lateral
hypothalamus (Gerfen et al., 1982) are additional sources
that may contribute to the glutamatergic innervation of
midbrain dopaminergic neurons. However, the ultrastruc-
tural features and the exact proportion of the terminals
arising from these regions, which form asymmetric syn-
apses with dopaminergic neurons, still remain to be estab-
lished.
 W

% OF TERMINALS % OF TERMINALS % OF TERMINALS

2
~ r u W P c n ( n d c o c o 0
00000000000

W
-1
1
0
 GLUTAMATE INPUT TO MIDBRAIN DOPAMINERGlC CELLS
What does glutamate do to midbrain
dopaminergic neurons?
The dopaminergic neurons in the SNc and the VTA, as
mentioned at the beginning of this paper, exhibit two
different types of firing patterns: irregular single spiking or
burst firing (see above). The pattern of discharge of mid-
brain dopaminergic cells is quite important, because the
burst activity in these neurons is positively correlated with
the release of transmitter in their targets (Gonon and Buda,
1985; Suaud-Chagny et al., 1992; Nissbrandt et al., 1994).
It has been shown that the shift from the irregular single-
spike mode to the burst-firing mode is regulated by extrin-
sic glutamatergic afferents (see above). If this is also the
case in primates (Schultz, 1986; Romo and Schultz, 19901,
then our data provide evidence of a potential anatomical
substrate that underlies the control of the firing pattern of
midbrain dopaminergc neurons in monkeys. However, it is
important to remember that glutamaterac afferents alone
cannot induce burst firing in midbrain dopaminergic neu-
rons. For instance, in vitro recording from rat midbrain
slices revealed that superfusion with excitatory amino acid
agonists increased the firing rate but never induced burst
firing in SNc and VTA neurons (Wang and French, 1993;
Wu et al., 1994). Therefore, other modulatory afferents
must be intact in order to allow glutamatergic inputs to
change the firing pattern of midbrain dopaminergic cells. It
is difficult to speculate on this issue, because the neuronal
mechanisms that underlie the generation of burst firing in
midbrain dopaminergic cells are still poorly understood. In
light of recent data showing that burst firing can be induced
in vitro if the superfusion solution contains a blocker of
Caz+-activatedK+-conductance (Johnson and North, 1992),
one possibility could be that afferents that reduce the
transmembrane potassium current facilitate the induction
of burst firing in midbrain dopaminergic cells by excitatory
amino acids.
An important finding in our study is that the proportion
of glutamate-enriched terminals in contact with dopaminer-
gic neurons is significantly higher in the VTA than in the
SNc. In light of the data discussed above, one might expect
that VTA neurons may have a pattern of discharge different
from SNc cells. In keeping with this suggestion, it has been
demonstrated that VTA dopaminergic neurons display a
much higher degree of burst firing than SNc neurons in
anaesthetized rats (Chiodo et al., 1984; Grenhoff et al.,
1988b). These findings strengthen the role of glutamatergic
afferents in the generation of burst firing in midbrain
dopaminergic cells.
Our data have revealed the existence of three major
categories of glutamate-enriched boutons in contact with
dopaminergic cells in the SNc and the VTA of squirrel
monkeys. Various brain structures, as discussed above,
may be the source of these terminals. On the basis of
Fig. 9 Histograms comparing the relative abundance of GLU-
enriched terminals (open bars; > 50 gold particles/FmZ)and boutons
with a low level of GLU immunoreactivity (solid bars; <50 gold
Z )contact with different parts of dopaminergc neurons
p a r t i c l e s ~ ~ n iin
in the ventral tA1 and dorsal (B)tiers of SNc as well as in the VTA iC).
Note that the percentage of GLU-enriched terminals in contact with
deiidritic shafts is higher in the VTA than i n the ventral and dorsal tiers
of the SNc. In contrast, the proportion of GLU-enriched boutons in
contact with perikarya remained the same in the three different regions
of midbrain dopaminergc neurons.
247
electrophysiological data, it is likely that terminals arising
from the cerebral cortex and the subthalamic nucleus
participate in the generation of burst firing in midbrain
dopaminergic cells. For instance, stimulation of these brain
structures led to a higher degree of burst firing in subpopu-
lations of midbrain dopaminergic neurons in the rat (Gari-
ano and Groves, 1988; Smith and Grace, 1992; Chergui et
al., 1994). On the other hand, lesions of the cerebral cortex
or the subthalamic nucleus led to opposite effects, i.e., a
decrease of burst firing (Svensson and Tung, 1989; Smith
and Grace, 1992). However, it is worth noting that a large
proportion of recorded dopaminergic neurons were not
affected by changes of activity at cortical or subthalamic
level (Svensson and Tung, 1989; Smith and Grace, 1992).
Therefore, it is important to keep in mind that other
glutamatergic afferents must be involved in the generation
of burst firing in dopaminergc cells. The potential role of
the glutamate-enriched terminals arising from the peduncu-
lopontine nucleus is discussed in the companion paper
(Charara et al., 1996). The raphe nuclei, which contain
glutamatergic neurons, may also exert an important con-
trol on the activity of SNc and VTA neurons, as mentioned
above.
The recent introduction of specific oligonucleotide probes
and antibodies has revealed the existence of different types
of ionotropic and metabotropic glutamate receptors associ-
ated with midbrain dopaminergic neurons in the rat. The
NMDARl (Laurie and Seeburg, 1994; Petralia et al., 1994;
Standaert et al., 1994) as well as the subunits GLURl and
GLUR213 of the a-amino-3-hydroxy-5-methyl-4-isoxa-
zoleproprionate (AMPA) ionotropic receptors (Petralia and
Wenthold, 1992; Martin et al., 1993) abound at midbrain
level. On the other hand, mGLURl is the only subunit of
metabotropic receptors expressed by midbrain dopaminer-
gic cells (Testa et al., 1994). Taken together, these data
indicate that glutamate may generate excitatory influences
in midbrain dopaminergic neurons through the activation
of various types of glutamate receptors. An issue that
remains controversial is the possibility that AMPA recep-
tors are involved in the induction of burst firing. For
instance, in vivo extracellular recordings combined with
microiontophoretic application of excitatory amino acid
agonists and antagonists revealed, on one hand, that ago-
nists of NMDA receptors, (but not those ofAMPAreceptors,
generate bursts in SNc and VTA neurons (Chergui et al.,
1993): On the other hand, another group showed that
AMPA, NMDA, and kainate agonists induce burst firing in
midbrain dopaminergic cells in the rat (Zhanget al., 1994).
Moreover, the latter study showed that blockade of NMDA
receptors had no effect on the induction of bursts by AMPA
or kainate agonists (Zhang et al., 1994). On the basis of
recent electrophysiological and pharmacological studies, it
appears that NMDA receptors mediate the excitatory ef-
fects generated by subthalamic afferents (Chergui et al.,
1994), whereas glutamatergic inputs from the pedunculo-
pontine nucleus act preferentially on non-NMDA receptors
located on SNc dopaminergic neurons (Di Loreto et al.,
1992).It is worth noting that stimulation of the pedunculo-
pontine nucleus does not induce burst firing in dopaminer-
gic cells (Scarnati et al., 1986; Di Loreto et al., 1992),
whereas activation of the subthalamic nucleus does so.
These observations support the hypothesis that activation
of NMDA is essential for generating bursts in midbrain
dopaminergic cells (Chergui et al., 1994). Recent evidence
has suggested a close interaction between NMDA and
248
Fig. 10. Electron micrographs depicting CaBP-immunoreactive den-
drites ( d l and d2) that are contacted by GLU-enriched terminals in the
VTA. A,B: Low-power views of the two dendrites in sections immuno-
stained for GLU (A) or for GABA (B). Three boutons (bl,b3, b5) in
contact with d l and the bouton b4 in contact with d2 display a high level
of immunoreactivity for GLU. In contrast, b2 is lightly labelled for
GLU. In the GABA-immunostained section, b l , b3, and b4 are unla-
non-NMDA receptors to generate excitatory postsynaptic
currents in SNc neurons (Mereu et al., 1991).Although the
controversies regarding these data are difficult to explain,
Y. SMITH ET AL.
belled, whereas b5 is immunoreactive. C,D: High-power views of these
terminals in GLU-immunostained sections. Boutons b l and b3 display
the ultrastructural features of type I terminals and form asymmetric
synapses (arrowheads) with d l (C). Bouton b4 displays the ultrastruc-
tural features of a type I1 terminal and forms an asymmetric synapse
(arrowhead) with d2 (D). Scale bars = 1 pm in A (also applies to B), 1
pm in C (also applies to D).
they all indicate that the type of glutamate receptors
activated by a particular population of glutamatergic affer-
ents is a key feature in the global understanding of the
GLUTAMATE INPUT TO MIDBRAIN DOPAMINERGIC CELLS
Fig. 11. A,B: Electron micrographs showing a bouton t h a t forms a n
asymmetric synapse iB, arrowheads) with a TH-immunoreactive peri-
karyon t PER) in the VTA. In the GLU-immunostained section (A). this
SNc (ventral tier) SNc (dorsal tier)
w HIGH GLU-IR
[7 LOW GLU-IR
Fig. 12. Schematic drawing summarizing the pattern of distrihu-
tion of boutons enriched (solid terminals; > 50 gold particlesikm') or
poor topen terminals; < 50 gold particles/pm2) in GLU a t the level of
single dopaminergic cells in t h e two tiers of the substantia nigra
compacta iSNc-v, SNc-d) and t h e VTA. All of the GLU-enriched
houtons form asymmetric synapses, whereas the terminals with low
synaptic mechanisms involved in the control of the firing
pattern of midbrain dopaminergic neurons. Future experi-
ments designed to elucidate the subcellular localization of
the different subtypes of glutamate receptors at the level of
single midbrain dopaminergic cells would significantly con-
tribute to resolving this issue.
249
bouton is strongly immunoreactive, whereas it is unlabelled in the
section processed for GABA immunostaining (B). The arrows indicate
dense-core vesicles. Scale bar = 1 km in A (also applies to B).
VTA
HIGH GLU-IR HIGH GLU-IR
c]LOW GLU-IR LOW GLU-IR
levels of GLU immunoreactivity are involved in symmetric synaptic
contacts. Note that the proportion of GLU-enriched boutons in contact
with dendritic shafts is higher in the VTA than in the SNc-v and the
SNc-d. The innervation of perikarya is relatively similar in the three
groups of midbrain dopaminergic neurons.
Potential clinical relevance
Although various hypotheses have been suggested for the
etiology of Parkinson's disease, none of them is substanti-
ated well enough to explain the different features of the
disease. An important source of toxicity for the midbrain
 250
dopaminergic cells could be a rise in the intracellular
concentration of calcium, which, by activating enzymes and
inducing mitochondria1 dysfunction, can lead to cell death
(Rothman and Olney, 1987). An interesting aspect of this
hypothesis is the fact that a subpopulation of midbrain
dopaminergic neurons that contain the calcium-binding
protein calbindin D-28k is selectively preserved in Parkin-
son’s disease (Yamada et al., 1990; Lavoie and Parent,
1991; Iacopino et al., 1992; Ito et al., 1992). Although the
exact role of calbindin in the midbrain dopaminergic neu-
rons still remains to be determined, it is tempting to
speculate that it exerts a neuroprotective function in
Parkinson’s disease by preventing the intracellular calcium
to reach a lethal concentration. If this is the case, then it
implies that the calcium homeostasis is altered in mesence-
phalic dopaminergic neurons of patients who suffer from
Parkinson’s disease. A major source of changes in the
intracellular concentration of calcium is the stimulation of
NMDA glutamatergic receptors (Michaels and Rothman,
1990; Nakanishi, 1992). For instance, it has been shown
that, in vitro, glutamate neurotoxicity is calcium dependent
(for reviews, see Beal, 1992; Choi, 1988, 1992). Therefore, a
dysfunction in glutamatergic neurotransmission that af-
fects dopaminergic neurons may result in an increased
concentration of calcium that will ultimately lead to the
death of neurons that do not contain calcium-binding
proteins. Future studies aimed a t elucidating the sources of
the glutamatergic afferents in contact with dopaminergic
cells combined with electron microscopic analysis of the
subcellular localization of glutamate receptors associated
with these afferents should help to understand better the
mechanism by which glutamatergic terminals may become
cytotoxic in midbrain dopaminergic neurons in Parkinson’s
disease.
ACKNOWLEDGMENTS
The authors thank Jean-Frangois Pare, Carole Harvey,
and Lisette Bertrand for technical assistance as well as
Isabelle Deaudelin and Louise Bertrand for the art work.
We also thank Dr. Peter Somogyi for the generous gift of
the GABA antiserum. This research was supported by
grants from the Medical Research Council of Canada (A.P.)
and the Parkinson Foundation of Canada (Y.S.).A.C. holds
a studentship from the FCAR.
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