Colocalization in Dopamine Neurons

Ariel Y. Deutch and Andrew J. Bean

It has been a generation since the discovery that multiple chemical messengers can be present in a single neuron. The definition of generation in this case is the conventional temporal one of 20 years. In contrast, the generation in the title of the series Psychopharmacology: Generation of Progress is an intellectual one, which seems to occur about every 5 years. Both definitions apply to colocalization in central neurons, because in the 20-odd years since colocalization has been documented there have been marked advances in our understanding of the varieties of colocalization and the mechanisms that are operative in neurons with multiple messengers.

The presence of multiple transmitters or proteins in single cells also presents other problems in terminology. The term coexistence may be inappropriate because it implies the presence of two or more transmitters in the same vesicle. Moreover, the adjective peaceful is frequently used to modify coexistence, yet there are hints that multiple transmitters may have antagonistic effects, both pre- and postsynaptically. We suggest that colocalization may offer a slight advantage, although it is clearly not ideal.

Colocalization of neuroactive substances occurs in virtually all types of neurons. In this chapter, we review briefly the status of colocalization in central dopamine (DA) neurons. Several recent reviews cover the general field of colocalization from different perspectives and emphasize different aspects (2, 31, 33). By necessity, our review is selective. We have attempted to convey the richness and complexity of colocalization in DA neurons, and to relate in certain cases the significance of colocalization to neuropsychiatric disorders (see also General Overview of Neuropeptides).

Almost concurrent with the discovery (if not acceptance) of chemical neurotransmission, Dale (13) hypothesized that a neuron extends its metabolic activity from the soma to all of its processes. In its most basic form, this principle posits only that metabolic processes that occur in the soma can reach or influence events occurring in distal parts of the neurons. Dale's principle was restated and expanded by Eccles (17) to suggest that a neuron releases the same transmitter at all of its processes. Both of these formulations can accommodate multiple messengers in a single neuron, requiring only that the molecules be distributed in all processes. However, recent data indicate that multiple peptide transmitters are targeted to different processes within a single Aplysia neuron (57); the degree to which such compartmentation is present in mammals is not clear (however, see ref. 30). The presence of the same peptide in different processes indicates that there are exceptions to Dale's principle, and it suggests that single neurons containing multiple transmitters may have the ability to spatially direct different types of output.

The functional significance of colocalization within a single neuron remains unclear. Spatially directed output of different transmitters has been described only in certain simple systems. The difficulties in defining the functional role(s) of colocalization stem from the lack of much basic information concerning both pre- and postsynaptic aspects of neurotransmission, particularly those processes that package and prepare for release chemical messengers. We briefly review these processes below.

Despite the gaps in our knowledge, we are beginning to catch glimpses of the functions of multiple chemical messengers. Using simple systems in which peptide effects are well-defined, peptides have been shown to act at sites distant to the immediate postsynaptic membrane (37). Differences in presynaptic release mechanisms (44, 63) have also provided a clue to the physiological role of colocalized messengers. Evidence gathered from experiments in simple defined systems has suggested that peptide and nonpeptide messengers may provide both temporally and spatially resolved signals (37, 44).

Two pathways are used by neurons to secrete proteins. The constitutive pathway, which is not triggered by extracellular stimulation, is used to secrete membrane components, viral proteins, growth factors, and extracellular matrix molecules; this pathway acts by continuous fusion of Golgi-derived vesicles with the plasma membrane. In contrast, the release of chemical messengers is controlled by extracellular signals and uses the so-called regulated pathway (39). The usual route followed by peptide proteins secreted via the regulated pathway involves synthesis of precursors containing an N-terminal signal sequence, which are targeted to the endoplasmic reticulum (ER) and subsequently translocated into the ER lumen. The complex is then transported from the cis- to the trans-Golgi compartment and to the trans-Golgi network where peptides are packaged into large (~100 nm) dense-core vesicles (DCVs), which are moved to the terminal (39).

Another population of vesicles, the synaptic vesicles (SVs), can be distinguished from DCVs on the basis of size, content, and membrane composition. These small (~50 nm) vesicles are electron-lucent and contain classical transmitters such as DA. SVs are formed from the ER and transported to the terminal, or directly recycled at the terminal, and transmitters are accumulated by specific transporters that are driven by proton pumps of the vesicular membrane (39). Thus, peptide and nonpeptide transmitters are for the most part segregated in their formation and localization in the cell. Recent evidence points to distinct but related molecular mechanisms responsible for the release of DCV and SV contents (7).

The release of multiple messengers, and the molar ratios of release of the colocalized substances, depends upon cellular activity (2, 9, 36, 60). Both firing frequency and the pattern of cell firing govern the relative release of colocalized messengers. Increases in firing frequency promote increases in the ratio of peptide/nonpeptide release (2, 44, 60), while short bursts of high-frequency stimulation may preferentially activate processes responsible for peptide release (44).

Thus, there are two sets of processes that synthesize and package transmitters and that govern the activity-dependent release of these compounds. What brings these two sets of processes together may be differences in the temporal and spatial characteristics of intraterminal calcium levels (56).

The ability to define colocalization requires the unambiguous definition of a uniform population of cells. This has led to immunohistochemical methods being the main approach to defining colocalization, although biochemical techniques have been useful adjunct methods. In contrast, biochemical and electrophysiological methods have in general been more useful for elucidating the functional significance of colocalization.

The problems inherent in immunohistochemical approaches to colocalization have been discussed in several recent reviews (see refs. 31 and 33). Nonetheless, several methodological issues may be worth discussing. Most studies of colocalization focus on the cell body of neurons, because the soma generally contains more of the protein and because of the difficulty in unambiguously defining preterminal axons under light-microscopic conditions. This can be problematic, because different parts of the same neuron do not invariably express a given protein (30, 57). The use of confocal microscopy may lead to a greater emphasis on defining colocalization in axons.

The use of in situ hybridization histochemistry to define specific mRNAs has opened new doors to the study of colocalization, particularly in primates. The underlying assumption is that the mRNA is appropriately translated and post-translationally modified to yield a functional protein. Unfortunately, it is difficult to verify this assumption. Still more problematic is the possibility that certain mRNAs are not expressed under basal conditions, but are expressed only under certain challenge conditions (see refs. 5 and 14).

A factor not often discussed is the nature of the colocalized substances. In cases of colocalization between two presumptive transmitters, we generally assume that the compounds are indeed neurotransmitters. Those criteria that are commonly used to define a transmitter are based on data derived from studies of classical (autonomic) transmitters, perhaps biasing the designation of other categories, such as peptidergic transmitters. Nonetheless, as the numbers of colocalized "transmitters" grow in parallel with the numbers of journal issues, it is appropriate to consider whether a designated molecule functions as a transmitter or has another role.

A large number of studies have addressed the anatomical and physiological aspects of colocalization between DA and transmitters. Much less frequently considered is the colocalization between DA and nontransmitter proteins and peptides, despite the fact that these forms of colocalization may be more prevalent. We have previously suggested an organizational framework in which to place different forms of colocalization (26).

Perhaps the simplest form of colocalization is the presence of DA and a protein or peptide that does not subserve a role as a transmitter, receptor, or transporter. Obviously, among such examples could be enzymes that are ubiquitously present in cells (hexose-6-phosphate) or neurons (neuron-specific enolase) of the mature central nervous system (CNS). The uniform presence of these enzymes suggests that the inclusion of this form of colocalization is not very helpful in attempts to arrive at an organizing scheme. However, a number of proteins and peptides are heterogeneously distributed in the CNS and are found in certain DA neurons. An example is the vitamin D28 calcium-binding protein calbindin (24).

Transporter proteins serve an important role as a means of terminating transmitter action, and therefore are generally (although not invariably) associated with neurons from which their substrate is released. Thus, the dopamine transporter mRNA is present in most (if not all) DA neurons, although the extent to which the actual transporter protein is expressed and functional is not clear (see below). Similarly, neurons can express proteins that serve as autoreceptors or heteroceptors. Among the former are the release-modulating autoreceptor of DA neurons (which biochemical studies suggest is present on all DA neurons) and the synthesis-modulating autoreceptor of DA neurons (expressed in most, but not all, DA neurons). DA neurons also contain a large number of heteroceptors, including ligand-gated ion channel receptors (e.g., nicotinic acetylcholine receptor) and G-protein-coupled receptors (e.g., neurotensin receptor), emphasizing the importance of afferent regulation for DA neurons.

Colocalization of classical transmitters is still another category. For example, several groups of DA neurons also contain g-aminobutyric acid (GABA). Colocalization of classical and nonclassical transmitters represent the form of colocalization most frequently discussed. Peptidergic transmitters are found in a number of different DA cell groups, ranging from the presence of cholecystokinin (CCK) in midbrain DA neurons to growth-hormonereleasing factor (GH-RF) in dopaminergic cells of the arcuate nucleus.

Finally, there are a number of cells that express higher-order combinations of the above-described categories. For example, some midbrain DA neurons contain DA, a peptide (neurotensin or CCK, or both), and a nontransmitter protein (calbindin; see ref. 24).

There are a large number of nontransmitter proteins and peptides that are present in central DA neurons. These range from receptors to proteins involved in calcium sequestration. Although in many cases the functional significance of these forms of colocalization remains to be determined, the functional characteristics of the colocalized molecule in other parts of the nervous system or in non-neural tissues have led to speculations that can be empirically examined.

There are several examples in which transcripts for various transmitters or receptors have been identified in brain, but for which conclusive evidence of colocalization, such as dual staining procedures, is not available. In most brain areas the DA neurons represent but one ingredient in a stew of neurons. However, in the pars compacta of the substantia nigra (SN) the DA neurons are tightly packed, such that {ewc MVIMG, MVIMAGE,!greateq.bmp}90% of pars compacta cells are dopaminergic. Given the characteristic gross structure of the pars compacta, we have included several examples of presumptive colocalization based on presence in this midbrain structure. However, it is important to recognize that these examples are presumptive and that they require confirmatory studies. An excellent illustration of this problem is the fact that although 5-HT2c transcripts are present in cells of the pars compacta, these receptors are expressed uniformly on GABAergic, but not dopaminergic, neurons (19).

There are almost no definitive dual-staining anatomic studies indicating the presence of receptors for classical transmitters on DA neurons. However, anatomical studies of the distribution of receptor mRNAs or proteins, electrophysiological data, and the presence of transmitterspecific terminals all strongly suggest that DA neurons express receptors for certain classical transmitters.

DA receptors serve as autoreceptors to regulate release and synthesis of DA and the activity of DA neurons. Thus, there are release-modulating, synthesis-modulating, and impulse-modulating autoreceptors on DA neurons (see Dopamine Autoreceptor Signal Transduction and Regulation and Biochemical Pharmacology of Midbrain Dopamine Neurons). In situ hybridization studies have detected the presence of the D2 DA receptor mRNA in many DA cell-body regions (see Dopamine Receptor Expression in the Central Nervous System); this is consistent with pharmacological data indicating that autoreceptors in the adult exhibit a D2-like pharmacological profile. The best current data suggest that the D2 receptors, but not the D3 or D4 receptors, are present on DA cells in the rodent and primate (see Dopamine Receptor Expression in the Central Nervous System). If the different functional DA autoreceptors are D2 receptors, this suggests that the type of autoreceptor role manifested is a function of transduction mechanisms rather than a function of the receptor differences.

Although it is clear that there are D2 DA receptor transcripts in midbrain cells, there are no published studies documenting the invariant association of D2 mRNA and DA in neurons using dual-label procedures (see Molecular Biology of the Dopamine Receptor Subtypes and Dopamine Receptor Expression in the Central Nervous System). While the distribution of D2 mRNA-containing cells in the rat suggests an excellent correspondence, preliminary data in the primate suggests that midline DA cells may not express the D2 receptor (J. Meador-Woodruff, personal communication).

Immunohistochemical studies of the nicotinic cholinergic receptor (see Neuronal Nicotinic Acetylcholine Receptors: Novel Targets for CNS Therapeutics) have revealed dense immunoreactivity of cells in the pars compacta of the SN and ventral tegmental area (VTA) (15). Studies of the distribution of mRNAs encoding different subunits of the nicotinic acetylcholine receptor also suggest the presence of a1, a3, a4, a5, and b2 subunits on A9 and A10 DA neurons (62). While most of the subunits that form heteromeric channels are present in the midbrain DA neurons, it appears that some are not (e.g., a2 and a7). The precise assembly of the subunits may confer different receptor properties on different DA neurons.

The GABAA receptor complex is a multimeric ion channel assembly with several associated allosteric modulatory sites. There are over 10 subunits, which are differentially expressed; different subunit compositions of the receptor confer different functional properties (59). The a3, a4, b3, and b4 are present in the pars compacta, suggesting that these subunits may be expressed in A9 DA cells (64). The use of subunit specific antibodies has led to the conclusion that DA cells of the pars compacta express a GABAA receptor that consists of subunits a3 and g2 (22). A large body of data indicates the presence of GABAA receptors on both DA and non-DA neurons in the SN and VTA.

Receptors for excitatory amino acids, including glutamate and aspartate, are present on most neurons in the CNS, including DA cells (see Excitatory Amino Acid Neurotransmission). Both N-methyl-D-aspartate (NMDA) and non-NMDA excitatory amino acid receptors are thought to be present in certain DA neurons. For example, the GluR1 AMPA family receptor is expressed in VTA and pars compacta neurons, and both flip and flop forms of the GluR1 mRNA are seen in the SN and VTA (46). Other AMPA family receptors (GluR2/3 and GluR4) are also present in the SN and VTA. Pharmacological studies also indicate that these excitatory amino acid receptors are present on DA neurons, but more conclusive dual-staining procedures are required to confirm this (see Excitatory Amino Acid Neurotransmission).

There is a paucity of information on the direct electrophysiological effects of serotonin on central DA neurons, and there is a similar lack of anatomical data suggesting the presence of serotonin receptors on DA neurons. There are over a dozen serotonin receptors (the rate at which new ones are cloned suggests that it is prudent not to specify an exact number), some of which are present only in certain species (see Serotonin Receptor Subtypes and Ligands). 5-HT2c (previously designated 5-HT1c) receptor transcripts are present in cells of the pars compacta, suggestive of colocalization in the A9 DA neurons. However, Chesselet and colleagues (19) have found that those pars compacta cells that express 5-HT2c mRNA are exclusively nondopaminergic.

Electrophysiological and biochemical pharmacology as well as autoradiographic studies suggest the presence of a number of peptidergic receptors on DA neurons. In many cases, in situ hybridization and immunohistochemistry, or studies using lesion methods, have confirmed the presence of peptidergic receptors on DA neurons (see General Overview of Neuropeptides).

Three tachykinin receptors (NK1, NK2, and NK3) respond to central tachykinins, including substance P, neurokinin A, and neurokinin B (41). A high density of NK1 receptor sites has been demonstrated by autoradiographic studies, and in situ hybridization suggests that these receptors are present on DA neurons of the pars compacta. There are substantial species differences in expression of tachykinin receptors, both across different rodents as well as between rodents and primates. The presence of a few scattered tachykinin-containing neurons in the VTA of the rat suggests the possibility that these cells exhibit tachykinin autoreceptors; further studies will be required to determine if these tachykinin-containing cells are also dopaminergic.

Neurotensin (NT) is colocalized with certain midbrain DA neurons and DA neurons in the arcuate nucleus (20), as are NT-containing axon terminals. Autoradiographic studies have indicated a high density of NT receptors in the SN that are associated with DA neurons, as have recent in situ hybridization data (18, 65). It is not clear if the coexistent DA–NT cells express NT receptors. Surprisingly, although several lines of evidence indicate that NT receptors are expressed on nigral DA neurons, the NT innervation of the SN is directed to the non-DA neurons (65), suggesting a ligand–receptor mismatch (see also The Neurobiology of Neurotensin).

There are two forms of CCK receptors, CCK-A and CCK-B, both showing considerable species variability in distribution. The regulation of the mesotelencephalic DA neurons through CCK receptors has been extensively examined using pharmacological approaches. Many of these data suggest that CCK modulates DA function primarily through CCK-A receptors. However, a recent in situ hybridization study indicates that CCK-B, but not CCK-A, transcripts are present in the pars compacta and VTA (35), suggesting colocalization; dual staining procedures have not yet been performed.

Because CCK is found in most nigral DA neurons in the rat (see below), the presence of the CCK-B receptor in many neurons of the pars compacta raises the possibility that the CCK-B site functions as an autoreceptor on these neurons (see also Cholecystokinin).

A large body of data indicates that opioid peptides regulate DA neurons in the hypothalamus and midbrain. Autoradiographic data indicate a moderate density of k receptors throughout the hypothalamus, including the arcuate nucleus. In the pars compacta and VTA, there is a high density of m-opioid receptors but a low density of k sites (45). However, the m receptors revealed by autoradiography in the pars compacta appear to be primarily associated with afferents, because m transcripts are present only in scattered neurons in the SN and VTA (58). In contrast, an mRNA encoding for the k receptor is abundant in the pars compacta (48). Thus, the present data suggest that k receptors are expressed in many DA neurons of the SN; this interpretation requires confirmation using dual-labeling procedures (see also Neuropharmacology of Endogenous Opioid Peptides).

Autoradiographic studies and the availability of antisera to the glucocorticoid receptor led to the finding that although glucocorticoid receptors are distributed almost homogeneously across the CNS if one considered very low levels, certain neurons stand out sharply by expressing large amounts of the protein. The monoaminergic cells of the brain are among the sites in which the glucocorticoid receptor is present in high amounts. All DA neurons in the hypothalamic arcuate nucleus (A12 cell group), the majority of cells in the A13 cell group (zona incerta), and all of the A14 cells (periventricular hypothalamus) expressed the glucocorticoid receptor (29). In the midbrain, 40–70% of the DA cells expressed the glucocorticoid receptor, with A10 DA cells in the VTA having strong immunoreactivity. Although there has been a wide appreciation of the role of glucocorticoids in the hypothalamo-hypophyseal axis, less is known about how glucocorticoids impact on the dopaminergic function in the mesotelencephalic system. Recent data suggest that glucocorticoids may play an important permissive role in the development of sensitization of DA neurons to drugs of abuse (see also Neuroendocrine Interactions and Stress).

There are several receptors for which pharmacological data indicate a role in regulating central DA neurons, particularly in the hypothalamus; indeed, there are data to support virtually every peptide as a regulator in this region. In virtually all of these studies, it is impossible to parcel out direct and indirect effects. Because conclusive data concerning colocalization with these receptors are lacking, we will not review these data.

A large number of transport molecules have recently been cloned. These do not appear to share a singlemembrane topography, but they all function in the reuptake of released transmitter.

Dopamine neurons express a membrane reuptake process that is the major means of terminating dopaminergic transmission. The DA transporter (DAT) has recently been cloned, and the DAT mRNA is expressed at detectable levels in most, but not all, DA neurons (1, 47). Pharmacological data suggest that a functional DAT is not present on tuberoinfundibular DA neurons; DAT transcripts are either absent or below detection threshold in DA neurons of the ventrolateral arcuate nucleus (47). There have been suggestions that distinct DATs may arise from regionally specific differences in post-translational processes, particularly glycosylation; there are no current data to indicate the presence of more than one DAT mRNA. Interestingly, the DA transporter has reasonable affinities for norepinephrine, serotonin, and epinephrine as well as for DA (see The Dopamine Transporter: Potential Involvement in Neuropsychiatric Disorders).

In addition to the membrane DA transporter, a vesicular monoamine transporter has recently been cloned (43). This transporter is present in DA neurons, and it is thought to be of importance in packaging DA into the vesicle for subsequent release. In addition, the sequestering of DA by the vesicular monoamine transporter may be of importance in preventing DA from attack by free radicals.

There are suggestive data concerning the presence of several other transporters in nigral DA neurons. These include the GAT-3 (previously termed GAT-B) GABA transporter (12), one form of the alternatively spliced glycine transporter (10), and SV2, a synaptic vesicle transporter of unknown function (8).

There are several examples of proteins which are heterogeneously distributed in the CNS but which are not transmitters, receptors, or transporters and which are present in DA neurons. We briefly discuss a few well-documented cases.

Probably the best known of these is acetylcholinesterase (AChE), which conventional histochemical methods showed to be present in high concentrations in catecholaminergic neurons, prominently including the A9 DA cells of the SN (see ref. 27). AChE in nigral DA neurons does not appear to function (at least predominantly) as a metabolic enzyme for acetylcholine, although there is a sparse cholinergic input to this region. AChE has been documented to be released from dendrites of these DA neurons in response to certain stimuli (27). The specific functions subserved by AChE in DA neurons remain unclear (27).

Calbindin is present in distinct subsets of midbrain DA neurons (23, 24). Neurons in the dorsal tier of the pars compacta, which project to the islandic (patch) compartment of the striatum, contain calbindin, as do certain DA neurons in the VTA (23). The functional role of calbindin (other than regulation of intracellular calcium stores) in these neurons remains obscure, although there have been speculations that the enzyme may play a neuroprotective role (see below).

Microsomal mixed function oxidases that are of importance in xenobiotic metabolism are found in high concentration in the liver. Antibodies generated against the purified hepatic enzyme NADPH cytochrome P450 reductase stain monoaminergic cells of the brainstem, including the midbrain DA neurons and the A11, A12, and A13 cells of the hypothalamus (28). Although most DA neurons in these regions are immunoreactive for the enzyme, there are also non-DA cells that express NADPH cytochrome P450 reductase. The functional significance of this colocalized protein is not known, although its hepatic function suggests that it may play a role in metabolism of, and hence protection from, certain toxins.

NAD(P)H:quinone oxidoreductase (DT diaphorase) is a dicoumarol-sensitive enzyme that catalyzes the reduction of NADH and NADPH in the presence of quinones. DT diaphorase is present in a subset of midbrain DA neurons, as well as in glial cells (52). The function of this enzyme in DA neurons is not known, but because it reduces catecholamine quinones to hydroquinones it may be of importance in protecting DA neurons from toxic free radicals.

One of the most interesting groups of proteins that are colocalized with DA neurons are neurotrophic factors. Several neurotrophic factors, including acidic and basic fibroblast growth factors (6), brain-derived neurotrophic factor (53), and neurotropin-3 (53), are expressed in midbrain DA neurons. In contrast, other trophic factors, including nerve growth factor and NT-4, are not present in detectable amounts, and still others (ciliary neurotrophic factor, transforming growth factor a) have not been examined (53). Glial-derived growth factor is presumably not present in DA neurons, but this remains to be determined.

Although the proposed function of growth factors is obvious, the presence of growth factors in DA neurons leads to several questions. Growth factors are thought to provide trophic support for afferent neurons. Because several of the growth factors appear to be of benefit in preventing DA cell death or enhancing cell survival in various paradigms, it is not clear if the function of the growth factors present in DA cells is to support afferents (e.g., striatonigral neurons) or to support the same DA neurons that elaborate the growth factors. Alternatively, the growth factors could provide trophic support for adjacent DA cells or intramesencephalic DA afferents.

There are perhaps fewer examples of coexistence of DA and classical transmitters than any other category. This may simply be due to there being fewer so-called "classical" transmitters than "modern" (and even "post-modern," e.g., nitric oxide) transmitters.

The only well-documented classical transmitter that is present in central DA neurons is GABA. Colocalized DA–GABA neurons are found in the arcuate nucleus of the hypothalamus and in the periglomerular cells of the olfactory bulb (20, 21). Despite the fact that there are few cases of colocalization of classical transmitters and DA, DA–GABA colocalization in the olfactory bulb is seen across several species, being present in insects and reptiles as well as in mammals (16, 40).

In addition to GABA, there is some evidence that glutamate may be colocalized with DA neurons in the midbrain. Midbrain DA neurons are strongly immunoreactive for phosphate-activated glutaminase (38), which is thought to be the biosynthetic enzyme for the transmitter pool of glutamate. The colocalization of DA and glutamate awaits verification using other approaches.

Probably the best-known form of colocalization is that of the DA and a neuropeptide transmitter. There are a large number of peptidergic transmitters that are found in DA neurons; in many cases two peptides are present with DA in single cells. Despite the fact that peptidergic neurons in the arcuate nucleus of the hypothalamus are so frequent as to resemble a pointilistic construction, perhaps the best-characterized colocalized populations of DA–peptide cells are those in the ventral mesencephalon.

NT–DA-containing neurons are found in the mesencephalon and hypothalamus of the rat. There is a colocalized population of neurons in the arcuate nucleus (20), and NT cells are present in the A10 DA neurons of the VTA (32). NT is also found in CCK-containing DA neurons in the VTA (55). NT-containing neurons of the VTA are clearly seen in the rat using immunohistochemical methods, but in contrast are not apparent in primate species (15). A recent in situ hybridization paper has revealed that the NT/neuromedin N (NMN) transcript is present in a small population of cells in the ventral mesencephalon of primates, including that of humans (4). It is not known if the NT/NMN mRNA is translated to the mature peptide species in primates.

NT–DA colocalized cells in the VTA project to a number of forebrain targets, including the prefrontal cortex (PFC). In fact, all NT axons in the PFC have been suggested to contain DA (see ref. 14). The presence of a colocalized population of NT-containing axons in the PFC, a region that does not receive NT-containing innervations from regions other than the ventral midbrain, has allowed the in vivo investigation of features governing the release of colocalized neurons (2).

One other aspect of NT–DA colocalization in midbrain DA cells suggests a novel function of colocalization. Under certain conditions, NT injected directly into the striatum is taken up by nigrostriatal DA terminals and retrogradely transported to the SN (11) via a rapid transport process. The mechanism through which NT is taken up by the DA terminals is not clear, but may represent internalization of a ligand–receptor complex. The number of pars compacta cells expressing TH mRNA after intrastriatal injection of either NT or its active fragment NT8–13 has been reported to be increased by ~40% (11). This is clearly an unusual role for a colocalized peptide, particularly in light of the suggestion that the retrogradely transported peptide increases the number of neurons expressing TH mRNA rather than increasing the abundance of the transcript per cell (see ref. 14). In any event, this represents a novel form of colocalization and if verified may suggest a trophic role for NT (see The Neurobiology of Neurotensin).

CCK-containing DA neurons are present in the A8, A9, and A10 cell groups of the midbrain (34). As noted above, many of these cells also contain NT and thus innervate the same forebrain targets. Also similar to NT, there are species differences in the localization of CCK (see below). Initially, CCK was thought to represent a relatively small subpopulation of A9 cells. However, the use of fixatives that preserve the antigen better and the availability of several different antibodies to CCK have revealed that most nigral DA neurons of the rat express CCK. There are considerable species differences in the degree to which CCK is expressed in DA neurons (50) (see Cholecystokinin).

Several other peptides have been demonstrated to coexist in central DA neurons, particularly in the arcuate nucleus. Among these are galanin, GH-RF, and the opioid peptides dynorphin, met-enkephalin, and leu-enkephalin (20). In addition, somatostatin-containing DA cells are present in the A13 cell group of the zona incerta (20), as are some DA cells that contain calcitonin-gene related peptide (49). There are several cases of A12 DA neurons containing more than one peptide (20).

DA cells are scattered through the supramammillary nucleus at the mes-diencephalic border. Several peptides have been found in these DA neurons, including peptide histidine isoleucine/vasoactive intestinal peptide (PHI/VIP), substance P, and CCK (54); a few of these cells contain DA, CCK, and PHI/VIP.

It is easy to understand the significance of colocalization for neuropsychiatric disorders if one simply thinks of receptor changes in various disorders. For example, there are changes in D2 DA receptors that are seen in Parkinson's disease, including the loss of D2 receptors related to the late-stage atrophy of dendritic spines on dopaminoceptive neurons; this results in patients who are refractory to DA replacement therapy. Less well characterized but even better known is the proposed increase in the density of striatal D2 receptors in schizophrenic subjects. In addition to changes in receptor systems, several recent postmortem studies have led to findings that graphically illustrate the potential significance of colocalization in DA neurons. We have chosen examples from schizophrenia and Parkinson's disease (PD).

The presence of both CCK and NT in certain midbrain DA neurons has long fueled speculation that there may be alterations in both peptidergic and dopaminergic function in schizophrenia. An initial report concerning the distribution of CCK mRNA in the human brain noted that nonhuman primates do not express discernible amounts of the transcript in the SN (50); this finding fits well with reports NT-like immunoreactive neurons could not be demonstrated in the several monkey species (14). Subsequent careful studies in the human midbrain by Hökfelt and colleagues (51) fortuitously used tissue from schizophrenic subjects and found moderate expression of the CCK in the nigra, but in contrast noted that CCK mRNA is present in low abundance or below the threshold for detection in control subjects. Animal studies suggest that this difference is not due to the effects of chronic neuroleptic treatment; moreover, age, sex, and postmortem interval do not appear to contribute (51).

As mentioned above, there are species differences in NT–DA colocalization in the midbrain: NT-like immunoreactive neurons are not seen in the ventral mesencephalon of nonhuman primates (14). Again, the use of probes to the human neurotensin/neuromedin N (NT/M) gene led to the finding that there is a small population of human midbrain DA neurons that express NT/M (4). However, in contrast to CCK, there is no significant difference in the number of cells expressing NT/N and DA (or melanin) in the SN of schizophrenics (4).

While these studies illustrate the potential significance of colocalization for clinical conditions, they paradoxically alert us to the difficulties in attempting to define the extent and role of neuronal colocalization. There are clear species differences present that unfortunately limit the ability to extrapolate from nonhuman subjects, particularly rodents. Another problem is that although the gene for a particular colocalized transmitter may be expressed, it remains unclear if the corresponding protein product is translated. Even more difficult to assess are the factors that promote transcription. For example, the degree to which CCK mRNA is expressed in the midbrain of humans appears to be quite low under normal conditions, yet given the appropriate state or trait, the CCK gene appears to be induced. It is not clear to what degree this reflects a state problem (i.e., the contribution of antipsychotic drugs) or a trait problem (schizophrenia). Thus, the study of one issue that on the surface appears relatively simple—namely, the degree to which CCK is expressed in the midbrain DA neurons of humans—opens up issues of (a) species dependency, state, and trait characteristics of the subject from whom the tissue is obtained, (b) mRNA stability and translation, and (c) factors that regulate gene expression.

In contrast to the coexistence of CCK or NT and DA, calbindin in midbrain DA neurons does not appear to play a direct transmitter role. Instead, calbindin appears to be of significance in sequestration of intracellular calcium stores and thus appears to be of potential importance as a neuroprotective agent. Several studies have documented that calbindin-containing DA neurons in the SN of nonhuman subjects are preferentially spared following systemic administration of the neurotoxin MPTP (42). Studies of the midbrain from PD patients have led to the same finding: There is a preferential sparing of those neurons that contain calbindin (25, 61). Although there is scant evidence for direct calcium-mediated excitotoxicity in PD, these observations that calbindin-containing DA cells are resistant to the neuropathological process in PD may lead to new ideas concerning the pathophysiology of the disorder.

We are indebted to Tomas Hökfelt for frequent and illuminating discussions and encouragement. We also thank Richard Scheller for his support and encouragement. This work was supported by grant MH-45124 and by the Howard Hughes Medical Institute, the National Parkinson Foundation Center of Excellence at Yale University, and the Veterans Administration National Centers for Schizophrenia Research and for Post-Traumatic Stress Disorder, West Haven VA Medical Center.

published 2000