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Neuropsychopharmacology: The Fifth Generation of Progress

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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.

METHODOLOGICAL ADVANCES

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.

Tyrosine Hydroxylase and Dopamine

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.

Monoamine Transporter†† ††††††††

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).

Dopamine Transporter

A single gene encoding a sodium- and chloride-dependentDAT 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 andproteins 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.

Subcellular Organelles and Plasma Membranes

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 ofVMAT2-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 fordopamineD2 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.

Afferent Input

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 terminalsform 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].

Astrocytic Associations

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).

STRIATAL DOPAMINERGIC TERMINALS

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).

Receptor Localization

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 detectD1 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).

GABAergic Postsynaptic Targets

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).

Presynaptic Appositions††

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 notedabove, 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, theappositional contacts may facilitate activation of presynaptic neurokinin receptors to modulate dopamine release (32).

COMPARATIVE MORPHOLOGY IN OTHER DOPAMINERGIC SYSTEMS

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 ofSSVs (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).

Hypothalamic Neurons

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.

Retina

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 are10-15 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).

Dopaminergic amacrine cells have highly collateralized and varicose axons extending widely beyond the dendritic tree (49).The axon terminals are morphologically similar to those in the striatum, and form thin symmetric synaptic junctions on dendrites (39).In the distal retina of amphibians, axonal profiles derived from interplexiform cells appose the surfaces of rod inner segments without forming recognizable junctions, supporting physiological evidence for involvement of dopamine in modulatory feedback to rods (30).Interestingly, the terminal-like processes of these cells in the inner plexiform layer do form conventional synapses. Furthermore, dopamine interplexiform cells in teleost and mammalian species form synaptic junctions in the distal retina (30).In retinal horizontal cells, dopamine reduces electrical coupling and increases their sensitivity to glutamate (67). The dopaminergic terminals responsible for dopamine release at these sites are large, filled with SSVs, and are devoid of synaptic specializations (67).Together, these observations suggest that, as was described for dopaminergic terminals in the striatum, those in the retina communicate through classical synapses, as well as volume transmission (14).

Functional Implications

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 ofdopaminergic 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 Parkinsonís 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 Parkinsonís 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 Parkinsonís.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 Parkinsonís, 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 Parkinsonís 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.

ACKNOWLEDGMENTS

We wish to thank Drs. A.LSvingos and J.J. RodrŪguez 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