Additional related information may be found at: |
Neuropsychopharmacology: The Fifth Generation of Progress |
Electron Microscopy of Central Dopamine
Systems
Virginia M. Pickel and Susan R. Sesack
The
most important ultrastructural features of dopaminergic neurons are those that
directly reflect dopaminergic transmission and functional interactions with
neurons that contain other identifiable transmitters. Dopaminergic transmission requires the synthesis, vesicular storage,
and release of dopamine to activate dopaminergic receptors that are present
on the surface of dopamine containing neurons and their targets. Electron microscopic immunolabeling of antisera
directed against tyrosine hydroxylase (TH), vesicular monoamine transporters,
and dopamine receptors have allowed us to precisely identify these functional
sites. Furthermore, the localization
of the plasmalemmal dopamine transporter (DAT) has shown the major sites involved
in the termination of dopamine transmission through reuptake into dopaminergic
neurons [see (77) ].
The known ultrastructure
of central dopaminergic neurons has been determined mainly from analysis of
midbrain dopaminergic cells of the A8 and A9 groups that comprise the substantia
nigra (SN) and the A10 group in the ventral tegmental area (VTA;15).
The nigral dopaminergic neurons are critical for motor functions involving
the release of dopamine from axon terminals in the dorsal striatum or caudate-putamen
nucleus (CPN) and core of the nucleus accumbens [see (65) for a more detailed discussion of these motor functions].
In contrast, the release of dopamine from terminals within mesolimbic
and mesocortical projections arising from the A10 group in the VTA play a more
dominant role in motivation, reward-related behaviors, and cognition [see Mesocorticolimbic
Dopaminergic Neurons: Functional and Regulatory Roles and (3,33)]. The ultrastructural
features of these and other dopaminergic systems that are described in this
chapter have been revealed mainly through methodological advances occuring since
publication of the review by Pickel and Milner in the last volume of Psychopharmacology:
The Third Generation of Progress (76). The described morphological features are based
primarily on studies of dopaminergic neurons in rat brain, which has been most
extensively studied by using electron microscopic immunocytochemistry.
A detailed species comparison is beyond the scope of the present chapter,
but can be found in the review by Lewis and Sesack (56). We do, however, include some of the more recent
studies from primates that directly indicate the significance of ultrastructural
observations for the understanding and treatment of clinical disorders involving
abnormalities in dopaminergic transmission.
Our
knowledge of the ultrastructure of dopaminergic neurons and their targets
is mainly attributed to the localization of antisera against dopamine-related
antigens by using immunoperoxidase and/or pre-embedding immunogold labeling
methods (71). Thus, advances in this field have
been dependent on the generation of antibodies specific to substances that
regulate dopamine neurotransmission. Furthermore,
detailed information regarding the cellular localization of these substances,
some of which may be low-abundance proteins (e.g., receptors), has been gained
by application of immunoperoxidase methods with enhanced sensitivity, such
as avidin-biotin or double bridged peroxidase techniques (71,77). Knowledge
regarding the subcellular distribution of dopamine-related antigens can be
acquired using immunoperoxidase methods (13a,62,65,77,86). However, interpretation
of this data is sometimes complicated by potential diffusion of the peroxidase
reaction product. In this regard, the pre-embedding immunogold technique permits
a fixed, albeit less sensitive, marker for subcellular antigen localization
(27,38,62,65,77,113). Finally, the combined use of immunocytochemistry and
tract-tracing methods has allowed advances in the identification of major
inputs to dopaminergic neurons or their targets (19,21,37,89,93). The following is a description of the antigens
that have proven to be most useful in ultrastructural analysis of central
dopaminergic neurons and their projections.
In brain regions
such as the SN, which contains no other known catecholaminergic somata, the
presence of the catecholamine-synthesizing enzyme, TH, is a reliable indicator
of dopaminergic neurons (77). This enzyme is expressed in high levels and
is readily detected in tissue by using fixation methods that are compatible
with the ultrastructural localization of most other antigens. Accordingly, TH is the most widely used marker
in studies of midbrain dopaminergic neurons. TH is, however, also required for the synthesis of noradrenaline
and adrenaline, making the immunolabeling for this enzyme less useful for
unequivocal identification of dopaminergic axons in brain regions that receive
input from other catecholaminergic afferents.
In these regions, antisera against glutaraldehyde conjugates of dopamine
have proven to be more useful for light and electron microscopic identification
of dopaminergic neurons (96) The usefulness is, however, somewhat diminished
by the requirement of fixation conditions that are not always compatible with
other antigens. Furthermore, a number
of studies have demonstrated that TH antisera primarily label dopaminergic
versus noradrenergic axons in forebrain targets (reviewed in 5,56). Thus,
antibodies directed against TH continue to be used in many investigations
as putative markers of dopaminergic profiles.
Molecular cloning techniques have been used to identify the
vesicular monoamine transporters (VMAT1 and VMAT2), which are two membrane‑bound
markers that identify the sites of intracellular monoamine uptake [reviewed
in (64)]. These transporters
use the proton electrochemical gradient to mediate the uptake of monoamines
into vesicles or other acidic intracellular organelles [reviewed in (84)]. When expressed
in vitro, VMAT1 and VMAT2 confer
reserpine-sensitivity and other known properties of the vesicular monoamine
carrier. Only VMAT2 has been detected
in the central and peripheral nervous systems in a distribution paralleling
that of monoaminergic neurons (68). Polyclonal antisera directed against a synthetic
peptide sequence at the C-terminus of VMAT2 have been generated, and shown
to be localized to vesicular membranes in dopaminergic neurons (62).
A single gene encoding a sodium- and chloride-dependent DAT has been identified and cloned ( see The
Dopamine Transporter: Potential Involvement in Neuropsychiatric Disorders, this volume for
references). The cloned DAT has all
of the known characteristics of the dopamine uptake carrier, including sensitivity
to psychostimulants (52). Moreover, in situ hybridization techniques have shown that DAT mRNA is abundant
in midbrain dopaminergic neurons. The
sequencing of the gene encoding DAT has led to the development of specific
polyclonal antisera directed against peptide sequences that are unique to
this transporter. These antisera provide
new markers that allow unequivocal identification of dopaminergic neurons
and the functional sites for dopamine reuptake [for further discussion and
references on central functions of DAT see (64)].
Dopamine Receptors
Genes encoding five subtypes of dopaminergic receptors have
been cloned and sequenced. Each of
these subtypes has homology with either dopamine D1 or D2
receptor families that were predicted by differences in affinities for specific
ligands (for references see Molecular
Biology of the Dopamine Receptor Subtypes and Dopamine
Receptor Expression in the Central Nervous System for review and references).
All subtypes have seven membrane-spanning domains that are typical
of G-protein coupled receptors. They differ, however, in that activation of
D1-like receptors (D1 and D5) stimulates,
but D2-like receptors (D2, D3, and D4)
inhibits adenylate cyclase. The D2-like
receptors also are associated with potassium channels and other signal transduction
systems. The cloning of dopaminergic
receptors has permitted the production of mRNA probes and antipeptide antibodies
specific for particular subtypes. These have been used to precisely map brain
sites of synthesis and functional activation by using in situ hybridization and immunocytochemistry (see Dopamine
Receptor Expression in the Central Nervous System for more details and references).
MIDBRAIN
DOPAMINERGIC SOMATA AND DENDRITES
Substantia Nigra
and Ventral Tegmental Area
Midbrain dopaminergic neurons are sparsely spiny, multipolar
cells having a mean diameter of approximately 10 μm in rat brain (26). These neurons are distinguished from others
mainly by their content of dopamine and proteins
required for dopamine synthesis, vesicular storage and release. Plasma membranes of the dopaminergic neurons
also are endowed with proteins that are actively involved in dopamine uptake
or physiological responses to dopamine or other neurotransmitters released
from afferent axon terminals. Dopamine
neurons may also have functional interactions with neighboring glia (discussed
below), as suggested by the close association of portions of the plasma membrane
with astrocytic processes.
Somata and proximal dendrites of midbrain dopaminergic neurons
contain mitochondria, rough and smooth endoplasmic reticulum and other typical
neuronal cytoplasmic organelles (69). TH-immunogold labeling is seen free within
the cytoplasm of neuronal perikarya or in contact with membranes of smooth
or rough endoplasmic reticulum (Fig. 1).
The TH-immunogold particles are usually not associated with vesicular
cytoplasmic organelles such as dense core vesicles (DCVs), whose membranes
are enriched in VMAT2 (62). Electron microscopic dual labeling has shown that DCVs within VTA
dopaminergic neurons often contain neurotensin (11),
and many of these vesicles may also contain cholecystokinin (see Colocalization
in Dopamine Neurons).
In midbrain dopaminergic neurons, VMAT2 is frequently observed
along the membrane of tubulovesicules, intracellular organelles that are larger
than synaptic vesicles and most likely represent segments of smooth endoplasmic
reticulum (reviewed in 62,69). Tubulovesicular organelles expressing VMAT2
are often seen in aggregates near dendritic plasma membranes, suggesting that
they represent the major reserpine-sensitive pool for dendritic release of
dopamine [see (64) for additional review and
references]. Furthermore, dendrites
in the VTA contain significantly higher numbers of VMAT2-immunogold particles per unit area than
seen in the adjacent SN, providing evidence that dendrites in the VTA have
potentially greater ability to store and release dopamine (62). This is
consistent with the known prevalence in this region of appositional contacts
between two dendrites, both or only one of which contain TH (10). Apposing
TH-immunoreactive dendrites show equally spaced plasma membranes, with clusters
of VMAT-labeled tubulovesicles near dendro-dendritic appositions (62). Together, these observations suggest that these
appositions are functional sites for dopamine release.
Somata and dendrites of midbrain dopaminergic neurons also
show labeling for dopamine D2 receptors (113) and DAT (65). In somata and larger dendrites, immunolabeling
for these proteins are localized to membranes of tubulovesicles and multivesicular
bodies. The latter organelle is a structure for cytoplasmic transport of plasma
membrane products and may be confluent with the smooth endoplasmic reticulum
(69). In smaller dendrites and dendritic
spines, dopamine D2 receptors and DAT are more frequently identified
along plasma membranes consistent with their known respective roles in (i)
the autoregulation of dopaminergic neurons as discussed in Dopamine
Autoreceptor Signal Transduction and Regulation and (ii) the termination of dopaminergic transmission through
plasmalemmal reuptake as reviewed in The
Dopamine Transporter: Potential Involvement in Neuropsychiatric Disorders and Biochemical
Pharmacology of Midbrain Dopamine Neurons.
Glutamate and acetylcholine: Cortical glutamatergic afferents to VTA dopaminergic neurons
play an important role in determining subcortical dopamine release (101). Afferents
to the VTA from the prefrontal cortex form asymmetric synapses that are typical
of those containing glutamate (89). This input from the prefrontal cortex may largely
account for the greater prevalence of glutamate-containing terminals that
form asymmetric synapses with TH-immunoreactive dendrites in the VTA, as compared
with the SN that has been reported in primates (94). Anterograde tracing and immunocytochemistry
also have show that glutamatergic afferents from the pedunculopontine tegmental
nucleus (PPT) form asymmetric, excitatory-type synapses with dopaminergic
dendrites in the SN and VTA of primates (21). The PPT also contains many cholinergic neurons,
which together with the laterodorsal tegmental nucleus, provide the major
cholinergic input to the SN (83,102). In the SN, cholinergic terminals form mainly asymmetric synapses, suggesting
that acetylcholine also produces excitation of midbrain dopaminergic neurons
(16)
In addition to receiving glutamatergic inputs, some lines of
evidence suggest that midbrain dopamine neurons may actually colocalize glutamate
and release this excitatory transmitter (98b). However, most of this evidence derives from
studies of cultured dopamine neurons, raising the issue of possible phenotypic
changes associated with the culturing process. Furthermore, the localization
of glutamate immunoreactivity to dopamine neurons in situ does not provide definitive evidence for a transmitter function,
because much of this is likely to reside in mitochondria (94). The suggestion
that nigral dopamine neurons may release glutamate from nerve terminals in
the striatum is inconsistent with their exclusively symmetric synaptic morphology,
as described in numerous studies (see below). Finally, tract-tracing studies that have described
nigrostriatal axon terminals forming asymmetric synapses have utilized approaches
that do not rule out transport within non-dopamine neurons (e.g., retrograde
labeling of the striatal axon collaterals of other afferents). Additional
studies are therefore required before this controversial issue can be resolved.
GABA and co-stored peptides: Many afferents to midbrain dopaminergic neurons are derived
from GABAergic neurons in the striatum and globus pallidus (93), and most likely also from local GABAergic neurons,
as shown by combined intracellular recording and electron microscopic immunocytochemistry
(95). In the VTA, we have shown that neurons containing
lower levels of TH receive proportionally more GABAergic synapses and less
input from terminals forming excitatory-type junctions (10). These ultrastructural
observations support recent evidence showing that GABAergic input may influence
not only the physiological activity, but long-term TH expression in midbrain
dopaminergic neurons (97).
Many of the GABAergic terminals that form synapses with midbrain
dopaminergic dendrites also contain substance P and are presumably derived
from striatal neurons (17). These results are consistent with studies showing
(i) the presence of substance P receptor mRNA in nigral dopaminergic neurons
(29), and
(ii) a hypothesized neuroprotective influence of substance P on dopaminergic
neurons in this brain region (61). In addition, we have shown that dynorphin,
which is co-expressed with substance P in certain striatal neurons (105) is present in axons and axon terminals in contact
with midbrain TH-immunoreactive neurons [Fig.
1; (73)]. In contrast, enkephalin, another endogenous opioid peptide, is prominently
distributed in striatal efferents to the globus pallidus (54a), but is also found in many axon terminals within
the VTA that (i) contain GABA (90), or (ii)
form synapses with dopaminergic dendrites (88). In axon terminals, opioids and other neuropeptides
are localized within large DCVs that are often distributed around the perimeter
of the plasma membrane at a distance from synaptic junctions, suggesting that
they are released by exocytosis to activate receptors on neighboring neurons
or glia [see (73) for further discussion and
references on peptide exocytosis].
Midbrain dopaminergic neurons are often apposed by thin glial
processes (11). Although astrocytes can take up dopamine, the uptake appears to
occur largely by diffusion and is not DAT mediated (41). In vivo
support for this conclusion is provided by the lack of DAT labeling in glial
profiles apposed to dopaminergic dendrites showing plamalemmal distributions
of DAT in the SN and VTA (65). In contrast, astrocytic processes apposed to
midbrain dopaminergic dendrites that express D2 receptors show
light peroxidase labeling for this receptor (86).
While the presence of mRNA for D2 receptors has not yet been demonstrated
in midbrain glial cells, D2 mRNA has been localized to striatal
astrocytes (9). Thus, these observations suggest that dendritically released
dopamine may modulate the activity of neighboring glial cells through D2
receptor activation. These glia may secrete growth factors that are required
for survival of dopaminergic neurons and protect against L-DOPA induced toxicity
(58). Through
uptake and/or release of glutamate, the apposed glial cells may also be important
for glutamate signaling and cytotoxicity in midbrain dopaminergic neurons
(60). Interestingly, in the retina, D2
receptors on glial cells have been reported to close inwardly rectifying K+
channels, suggesting that dopamine may modulate local cerebral blood flow
by regulating astrocytic K+ clearance (13b).
Ultrastructure
The ultrastructural features of dopaminergic terminals have
been most extensively examined in the CPN and core of the nucleus accumbens,
two striatal regions prominently involved in motor function [see (56, 63, 115) for pertinent references and comparison
with the limbic shell region, which is the major site of action for psychostimulants
(3)]. These dopaminergic terminals are usually small (0.1-0.5 μm in diameter) and show thin and symmetric, or non-detectable,
synaptic membrane specializations [see (77)
for further discussion]. The dopaminergic
terminals that are identified by the presence of TH and DAT commonly contact
the necks of dendritic spines that receive asymmetric excitatory-type junctions
(Fig. 2A).
These junctions are typical of corticostriatal afferents (37), which also often are apposed to dopaminergic terminals
(Fig. 2A).
When dopaminergic terminals form synapses, either with the dendritic
spines or dendrites, the junctions show symmetric distributions of electron
dense material on either side of the synaptic cleft (Fig.
2B). Such junctions are typical
of terminals containing inhibitory neurotransmitters such as GABA (1). Dopamine is, however, also known to produce excitation, most likely
through modulatory interactions with glutamate (55;
but, see 98b).
Dopaminergic striatal terminals contain densely packed small
synaptic vesicles (SSVs). The membranes
of these vesicles, as well as the occasionally observed DCV in dopaminergic
terminals, are selectively identified by labeling for VMAT2 (64). These vesicles
are usually not aggregated near synaptic junctions. Furthermore, in striatal dopaminergic terminals,
DAT is rarely seen within presynaptic membrane specializations, but is prominently
displayed along non-synaptic portions of the plasma membrane [Fig.
2 (38,63)]. These observations are consistent with the idea that dopamine reuptake
and consequently the "spatial
buffering" of dopamine is most likely not restricted to the region of
the synaptic cleft (77). These results clearly support the growing evidence
that, in addition to classic synaptic transmission, dopaminergic striatal
terminals participate in volume transmission, affecting all neighboring neurons
or glia that express dopamine receptors (24,116).
Dopaminergic transmission involves the release of dopamine
mainly from axon terminals, followed by the activation of specific receptors
on target cells [see (77) for review]. In the striatum, many of the postsynaptic dopamine
receptors belong to the D1 subtype (37). D1 receptor labeling is, however,
not limited to regions of synaptic specializations, but is also seen at extrasynaptic
somatodendritic plasma membranes of target neurons (43). On exposure
to exogenous dopamine, D1-immunoreactivity is internalized from
the cell surface to endosome-like organelles, suggesting that the labeling
on the plasma membrane is indicative of the presence of functional binding
sites (27).
In striatal spiny neurons, dopamine D1 receptors
are known to be co-expressed with NMDA glutamate receptors, which is consistent
with a neuromodulatory role for dopamine D1 receptor agonists in
postsynaptic excitability of glutamatergic targets (20). Whether dopamine D1 and D2
receptors are present in the same striatal neurons is more controversial. Electron microscopic dual labeling studies
have not been able to detect D1
and D2 receptors within the same neuronal profiles in striatum
(37). This finding is consistent with
the results of sensitive in situ
hybridization studies, in which the mRNA for D1 and D2
receptors has been localized to largely separate neuronal populations (54b).
In contrast, there is strong physiological evidence for dual activation of
both receptors in single neurons in this region (99). Additional studies are required to clarify
this issue.
In striatum, dopamine D2 receptors have been shown
physiologically to play a major role in the presynaptic release dopamine,
as well as excitatory amino acids (20). Consistent with this idea, electron microscopy
shows D2-like immunoreactivity in terminals that (i) contain TH
and form thin symmetric synapses, or (ii) lack TH-labeling and form asymmetric,
excitatory-type axospinous synapses (86). In terminals forming asymmetric synapses on spine heads, however,
the D2 receptor labeling was sparse in our original study (86) and non-detectable in a report by Hersch et al.,
(37). It is likely that these results reflect, in
part, low levels of D2 receptors and/or more limited access to
surface receptors in excitatory-type afferents. By varying the labeling conditions, we have now been able to show intense, but discrete labeling of
D2 receptors in axon terminals forming asymmetric synapses in the
striatum (Fig. 3).
In contrast with the presynaptic localization of dopamine D2
receptors, there is general agreement that the receptor proteins are present
in postsynaptic dendrites and dendritic spines (37;86). In dendrites, the labeling is often associated
with tubulovesicular organelles, whereas in dendritic spines the plasma membrane
and postsynaptic densities are most prominently labeled for D2
receptors (Fig. 3).
The immunoreactive postsynaptic densities on spine heads are asymmetric
and typical of glutamatergic terminals (86). This suggests that following release from neighboring
axons, dopamine can modulate glutamatergic excitation by binding to functional
proteins within the postsynaptic densities of dendrites (46). In addition,
activation of D2-like receptors that are expressed in striatal
astrocytes may play a role in dopaminergic modulation of glutamatergic transmission
(9).
GABA is the primary neurotransmitter in spiny striatal neurons
that receive input from dopaminergic terminals (78). These GABAergic neurons also contain D2
receptors (23).
The dopaminergic targets include GABAergic striatal neurons that project
to the substantia nigra (4). These projection neurons contain substance
P and/or dynorphin, while others that project to the ventral pallidum express
Leu5- or Met5-enkephalin, opioid peptides that are also
present in certain striatal targets of dopaminergic terminals (72). Thus, dopaminergic afferents influence the
direct and indirect outputs from the basal ganglia that are largely responsible
for normal motor function (2).
Striatal spiny neurons that are modulated by dopaminergic afferents
are also directly controlled by synaptic inputs from convergent axon terminals,
which include those containing other neurotransmitters and/or neuropeptides.
The detection of convergence within a single plane of section requires that
the two axon terminals are spaced at a similar distance along the dendritic
tree. Furthermore, the relative distance
from the somata is considered to be an indicator of the relative potency of
afferent terminals, with those inputs to spines being less potent than those
to large proximal dendrites (80). Thus, convergence of excitatory-type terminals
and dopaminergic terminals on dendritic spines (89) suggests
that both glutamate and dopamine act mainly in fine-tuning physiological responses
in spiny GABAergic neurons. A similar
function may be served by the endogenous opioid peptides, Leu5-enkephalin
and dynorphin, both of which are present in axon terminals that converge with
dopaminergic terminals on dendrites and dendritic spines in the striatum (72, 106). Support
for this conclusion is provided by the prominent extrasynaptic localization
of μ-opioid
receptors along plasma membranes of dendritic spines that (i) receive input
from striatal dopaminergic terminals (109),
and (ii) contain GABA (100).
In the striatum, we have also shown that TH-labeled terminals
form symmetric synapses with larger dendrites that are known to receive inhibitory-types
synapses from GABAergic terminals (78). These potentially
reflect the functional sites for dual dopamine and GABA inhibition of striatal
neurons and/or facilitatory interactions between agonists acting at dopamine
D1 and GABAA receptors, as has been shown in the SN
(79). In
addition, since D2 receptors are also present in GABAergic striatal
dendrites, many of which receive synaptic input from GABA containing terminals
(23), D2 receptor activation may play a role
in the postsynaptic inhibition of striatal GABAergic neurons (36).
We have known for more than a decade that striatal dopaminergic
terminals do not form axo-axonic synapses, but are often apposed to other
axons and axon terminals (76). The physiological significance of these axonal
appositions is, however, only now beginning to be well understood by the localization
of presynaptic receptors. As noted
above, axonal appositions are frequently seen between dopaminergic
axons and terminals that form excitatory axospinous synapses and contain dopamine
D2 receptors (89).
Appositions are also often seen between striatal dopaminergic and cholinergic
terminals (70). Furthermore, cholinergic neurons in the striatum
express D2 mRNA (7), but receive
little, if any direct synaptic input from dopaminergic terminals (70). Together,
these, observations suggest that appositional contacts with dopaminergic terminals
are important sites for D2-mediated dopaminergic modulation of
the presynaptic release of glutamate and acetylcholine (47, 55). In addition,
however, the axonal appositions may also contribute to the presynaptic regulation
of dopamine release by these neurotransmitters (22).
Dopaminergic, as well as excitatory-type axon terminals in
the nucleus accumbens are also often apposed to terminals that contain GABA
and form symmetric axodendritic or axospinous synapses (78). Furthermore, dopamine is known to act through
D2 receptors that are present within GABAergic terminals to presynaptically
modulate the striatal release of GABA [see (23) for the ultrastructural localization and reference
to the pertinent literature]. These
observations, together with the sparsity of labeling for dopamine D2
receptors in corticostriatal terminals led Yung et al., (114)
to conclude that the D2 receptor mediated release of glutamate
occurs indirectly through GABAergic neurons.
While we can not exclude this possibility, the now more prominent presynaptic
localization of D2 receptors in excitatory-type terminals favors
a more direct mechanism. In addition,
substance P is known to be present in certain striatal GABAergic neurons and
in terminals that are apposed to TH-immunoreactive terminals in the caudate-putamen
nucleus. Thus, the localization of
D2 receptors within GABAergic terminals might also reflect sites
for modulation of the release of this neuropeptide [see (74) for the dual labeling study of TH and substance P
and a review of the literature on coexistence of substance P and GABA]. Alternatively, the appositional contacts may facilitate activation of presynaptic neurokinin
receptors to modulate dopamine release (32).
Non-Striatal
Mesolimbic Targets
The amygdaloid complex and olfactory tubercle are two of the
primary limbic targets of VTA dopaminergic neurons outside the basal ganglia
(15). Comparison
of TH-immunoreactivity with the labeling for other catecholamine synthesizing
enzymes reveals that most of the axon terminals containing high levels of
TH in the rat amygdaloid complex are dopaminergic (5). These terminals
are small and contain densely packed SSVs. They are either without membrane specializations or form symmetric
synapses with dendrites, many of which receive convergent input from terminals
having the morphology of excitatory or inhibitory amino acids (5,12). These
terminals are most prevalent in the lateral portion of the central nucleus
of the amygdala (CNA). The postsynaptic
targets of dopaminergic terminals in the CNA, like those in the striatum,
are spiny GABAergic projection neurons (12, 50). These results suggests that activation of dopaminergic
receptors in GABAergic projection neurons in the CNA may play a major role
in conditioned fear reactions that are potently modulated by dopamine in this
brain region (53).
Prefrontal Cortex
The mesocortical dopaminergic projections, including those
to the prefrontal cortex are extensively involved in the modulation of cognitive
function [see Mesocorticolimbic
Dopaminergic Neurons: Functional and Regulatory Roles and (33) for references and
review]. Although there are significant
regional variations in synaptic incidence, most cortical terminals form symmetric
synapses on dendritic shafts (85,92). In the monkey, the spines have been verified
to be derived from pyramidal neurons (34). In the prefrontal cortex of rat and monkey,
many of the dopaminergic terminals also synapse on subpopulations of GABAergic
local circuit neurons (87,91,92). On both dendritic spines and shafts, dopaminergic
terminals converge with excitatory, presumably glutamatergic afferents.
Thus, as described for the striatum, convergence with excitatory-type
afferents is typical of cortical dopaminergic terminals.
In the prefrontal cortex, however, the source of these afferents has
not yet been determined (19).
The dopaminergic terminals in the prefrontal cortex have many
of the same ultrastructural features as those that were described in the striatum,
including their small size and dense packing of SSVs (33,34). When compared to the striatum, however, mesocortical
dopaminergic terminals contain low levels of DAT (87). Furthermore,
the DAT that is present in cortical axons is preferentially distributed to
intervaricose segments, suggesting that following synaptic release in the
cortex, dopamine may be permitted a greater range of extracellular diffusion
and activation of more distant dopaminergic receptors.
Thus, in this region, the cellular localization of dopamine receptors
may be even more critical than in striatum for understanding the functional
cortical circuitry. There are, however, relatively few ultrastructural
studies of dopamine receptor distribution in cortex. In the monkey, D1 and D5
receptors have been differentially localized to dendritic spines and shafts
of cortical pyramidal neurons, respectively (13a). Dopamine D4 receptors also have
been described in the primate as being localized to pryamidal cells and local
circuit neurons that contain parvalbumin (59). Interestingly, the observed distribution of
receptors matches closely the sites of dopaminergic synaptic input to cortical
neurons (87,91,92).
Dopaminergic somata and axon terminals are prevalent throughout
the hypothalamus and play a major role in the control of neuroendocrine function
(see Dopaminergic
Neuronal Systems in the Hypothalamus). These dopaminergic
terminals provide synaptic input to hypothalamic neurons that contain luteinizing
hormone-releasing hormone (LHRH) or GABA (40). Dopamine is also present in efferent projections
from the rostral parvocellular arcuate nucleus and more dorsal periventricular
hypothalamus to the median eminence, where DAT has been localized by light
microscopy to varicose axons (81).
The cytological features of dopaminergic neurons in the mediobasal
hypothalamus have been characterized mainly by using light and electron microscopic
immunocytochemistry for TH (108) and/or dopamine
(66). The somata are small and ovoid with a mean diameter of 10 μm, and contain similar organelles to those seen in the midbrain
cell groups (108). As in the VTA dopaminergic neurons, DCVs in
hypothalamic dopaminergic cells are mainly storage sites for neuropeptides,
particularly neurotensin (8).
Hypothalamic dopaminergic neurons also resemble those in the
midbrain in terms of their associations with astrocytes (108,112). The extensiveness of astrocytic coverage of
dopaminergic perikarya in the hypothalamus is known to be markedly influenced
by gonadal hormones, suggesting a potentially important role in neuroendocrine
regulation (31).
The synaptic input to dopaminergic neurons in the hypothalamus
is largely on dendrites. The afferent
terminals show either asymmetric or symmetric membrane specializations, suggesting
the presence of excitatory or inhibitory amino acids, respectively (108). The presence of GABA in many of the afferents
that form symmetric synapses on TH-immunoreactive hypothalamic dendrites has
been confirmed by electron microscopy (107). In addition, dopaminergic dendrites in the
arcuate nucleus receive monosynaptic input from terminals that contain serotonin
(48), as well as galanin (42). In the mediobasal
hypothalamus, there are also (i) reciprocal synaptic connections between neurotensin
and dopamine containing neurons (57), and
(ii) synaptic input to dopaminergic dendrites from terminals that contain
corticotrophin releasing factor (CRF; 104).
Together, these results indicate that hypothalamic dopaminergic neurons
controlling neuroendocrine functions are influenced by many of the same neurotransmitters
that are present in afferents to midbrain dopaminergic neurons, but may be
more potently modulated by neuropeptides that are enriched in hypothalamic
axon terminals.
Dopamine cells in the retina are distributed fairly evenly
in the inner nuclear and inner plexiform layers [see (110)
for review and references]. Dopaminergic
amacrine cells containing TH immunoreactivity are 10-15 μm in diameter and contain endoplasmic reticulum and other typical
neuronal organelles (49). These cells, as well as interplexiform neurons
often show appositions between dendrites with and without TH labeling (111). In addition,
they receive synaptic input from many axon terminals that originate from bipolar
cells and amacrine terminals, some of which contain GABA (111).
Central dopaminergic neurons show many ultrastructural similarities,
but also important differences, that provide vital clues to their function.
Most, if not all, dopaminergic somata and dendrites receive major input
from GABAergic axon terminals that are distinguished on the basis of their
content of peptide containing DCVs, which suggests that GABAergic inhibition
of dopaminergic neurons is selectively modulated
by specific types of co-released peptides. Dopaminergic neurons also typically show appositions between dendrites
having the vesicular storage organelles, reuptake transporters and receptors
for dopamine. These dendritic sites
are likely to be critically involved in autoregulation and synchronization
of dopaminergic neuronal activity. In most terminal fields, dopaminergic axons form symmetric synapses
with dendritic spine necks and dendritic shafts, but the reuptake of dopamine
can occur at a distance from these synapses, as indicated by the extrasynaptic
localization of DAT. In addition,
many varicose axons do not form synapses. Together, these observations indicate that dopaminergic terminals
communicate with other neurons through both conventional synaptic and volume
modes of transmission.
The differential ultrastructure and subcellular distribution
of functional proteins in subpopulations of dopaminergic neurons provide information
that is directly relevant to the pathogenesis and treatment of Parkinson’s
disease (18,35) and schizophrenia (25,28); Dopamine
Receptor Transcript Localization in Human Brain. The degenerative
changes in human SN neurons that are seen in Parkinson’s reflect increased
lipid peroxidation, altered iron metabolism, and impaired mitochondrial function
(45). The presence of Lewy bodies in nigral
somata and dendrites is one of the ultrastructural characteristics of this
disorder, but in addition, these organelles are also seen in periventricular
hypothalamic dopaminergic neurons from the autopsied brains of patients with
Parkinson’s. These observations implicate
the dopaminergic neurons in both the motor and endocrine abnormalities that
are seen in this neurodegenerative disease.
In the rodent model of Parkinson’s, 6-hydroxydopamine (6-OHDA)
is one of the drugs most commonly used (44). This drug enters dopaminergic neurons though
the DAT and leads to the generation of superoxide anions that are largely
responsible for its toxicity (6). Thus, neurons expressing higher levels of DAT
are likely to be more vulnerable to 6-OHDA toxicity (63). Accordingly, we have shown that individual
TH-immunoreactive terminals in the motor associated core of the nucleus accumbens
have significantly higher levels of DAT than those in the shell region, which
are minimally affected in Parkinson’s disease (63). The extensiveness
of glial coverage of subpopulations of dopaminergic neurons may also directly
contribute to their differential neurotoxin sensitivity (98a).
Functional recovery in young animals following partial 6-OHDA
lesions has been attributed to collateral sprouting (51). The most
marked ultrastructural changes in the striatum of adult animals that receive
neonatal 6-OHDA lesions is an increase in the volume of the residual dopamine
axon terminals. These terminals often
show no recognizable membrane specializations and contain densely packed SSVs
(75). Thus, each
of the remaining terminals has the potential for heightened vesicular storage
and release of dopamine affecting the activity of all neighboring neurons
that express functionally relevant dopamine receptors. Unilateral 6-OHDA lesions of nigrostriatal
afferents also evokes neuronal plasticity in the caudate-putamen nucleus of
adult animals, but the changes are seen mainly as a loss of dendritic spines
and asymmetric axospinous synapses (44).
Loss of dendritic spines in the caudate-putamen nucleus is also produced by chronic administration of haloperidol, a D2 receptor antagonist that produces dyskinesia in addition to its beneficial antipsychotic actions [see (103) for review]. The beneficial and motor effects of haloperidol now appear to be a direct reflections of interactions involving glutamatergic systems, and more specifically the NMDA subtype of glutamate receptor, which we have shown to be expressed in higher levels in the residual dendritic spines within the caudate-putamen nucleus of animals that receive chronic haloperidol treatment (82). These observations indicate the physiological and clinical significance of those ultrastructural features of dopaminergic neurons that signify dopaminergic transmission and specific targeting to dendritic spines that receive input from excitatory afferents.
We wish to thank Drs. A.L
Svingos and J.J. Rodríguez for comments on the manuscript and June
Chan for her input on the immunogold labeling and preparation of illustrations.
Grants from NIMH (MH40342, MH48776 and 00078) and NIDA (DA04600) provided
support for collection of data that was reviewed.
published 2000