Dopamine Receptor Transcript Localization in Human Brain

James H. Meador-Woodruff

The identification of five dopamine receptors in the past decade (12, 15, 19, 61, 63, 64, 67, 70) has led to renewed enthusiasm for the study of the possible role of dopaminergic dysfunction in neuropsychiatric conditions, especially in psychotic disorders and substance abuse. These five dopamine receptors are all members of the superfamily of seven transmembrane domain, G-protein coupled receptors and cluster into D1 and D2-like families of receptors. The assignment of a cloned receptor to one of these families was based on shared pharmacological features, second messenger coupling, and conserved structural features among individual receptors.

The D1-like dopamine receptors consist of the D1 and the D5 receptors. These two receptors share similar pharmacological profiles and are coupled to adenylate cyclase through GS. The D2-like family of dopamine receptors consists of the D2, D3 and D4 receptors (15, 19, 67). These three receptors have high affinities for a number of drugs with antipsychotic properties, although each receptor subtype has unique pharmacological features (59). For example, the D2 and D3 receptors have much higher affinities for raclopride than does the D4 receptor, while the D4 receptor has a particularly high affinity for the atypical neuroleptic clozapine. These three receptors are coupled to second messenger systems through inhibitory G-proteins. Each of these families of receptors has structural features common to its member receptors. Receptors of the D1-like family are intronless in their respective coding regions, have short third intracytosolic loops, and have long C-terminal tails (12, 63, 64, 66, 70). The D2-like receptors are structurally more complex. These receptors contain introns within their coding regions, have fairly lengthy third intracytosolic loops, and short C-terminal tails (15, 19, 53, 61, 67). The presence of introns within the coding regions of the D2-like receptors gives rise to the possibility of isoforms due to alternative splicing of their mRNAs. Alternatively spliced forms of the D2 and D3 receptors have been identified, and repeat sequences have been identified in the D4 receptor, resulting in many different possible forms of this receptor. Additional dopamine receptors are suspected to exist, but they have not been cloned to date. These myriad receptor subtypes hint at the availability to the cell of many possible levels for modulation of the expression of brain dopaminergic tone.

An important determinant in the expression of function of these receptors is neuroanatomical region-specific expression. The anatomical localization of the individual dopamine receptor subtypes has been extensively elucidated in rat brain, initially at the level of mRNA and more recently from work with specific antibodies to visualize receptor proteins in tissue sections (reviewed in refs. 40, 42, 43). Several detailed studies in the rat brain showed that D1 and D2 receptors are expressed in most of the traditional dopaminoceptive regions of the rat brain, including regions associated with motor, limbic, and neuroendocrine functions (14, 29, 32, 33, 39, 40, 44, 45, 51, 68, 69). On the other hand, the novel receptor subtypes D3, D4, and D5 have more restricted distributions. D3 and D4 receptors appear to be localized primarily in limbic regions of rat brain, with little to no expression in the dorsal striatum (5, 29, 46, 53). The D5 receptor has a very limited distribution, primarily in the diencephalon and hippocampus (41). These distinctive distribution patterns suggest that certain receptor subtypes may be associated with specific dopaminergic functions. This has led to new hypotheses about the potential role of these receptors in neuropsychiatric illness and in the mechanisms of action of drugs used to treat these disorders. For example, striatal expression of D1 and D2 receptors suggests that these two subtypes may be the most significant with respect to motor functions modulated by dopamine, while the fact that D3 and D4 receptors are restricted to more limbic structures implies that they may be important in several psychiatric illnesses.

Most recently, there has been significant progress in the elucidation of the neurochemical anatomy of the dopamine receptors in the human brain. While there are many parallels between the distributions of the various dopamine receptors in the rat and human, several very striking differences have been identified. Intriguingly, some of these differences are found in precisely those areas that one might expect to be important in the pathophysiology of neuropsychiatric conditions. In this chapter, the known neurochemical anatomy of the dopamine receptors in the human will be reviewed, and it is summarized in

Data will be presented for the localization of the mRNA encoding each receptor; as the dopamine receptors were cloned, mRNA studies were the only methods available to unambiguously identify each receptor subtype. Rapid progress has been made in the development of highly selective and specific ligands to identify receptor binding sites, however, and several studies are now available in which receptor proteins have been visualized using immunocytochemical techniques. Where available, binding and immunocytochemical anatomical data will be presented as well.

In the human striatum, D1, D2, and D3 receptor mRNAs are expressed at moderately high levels. D1 and D2 receptor mRNAs are homogeneously distributed throughout the dorsal striatum and nucleus accumbens (34, 43, 45). On the other hand, D3 receptor mRNA is expressed in a striking gradient, with prominent labeling apparent in the accumbens and the ventral aspect of the putamen, and minimal labeling in dorsal striatum (34, 43, 46). This may suggest a specific role for the D3 receptor in limbic functions. D1, D2, and D3 receptor mRNA distributions in the human are very similar to those in rat brain (40, 42, 43).

The correspondence of the distributions of mRNAs to binding sites has been somewhat difficult to determine. Most early studies in which the localization of dopamine receptor binding sites was examined depended upon ligands which we now know have high affinities not only to a molecularly defined receptor but to the entire family of receptors. For example, [3H]spiroperidol has often been used to label D2-like receptors, but it is now clear that this compound labels D2 , D3, and D4 sites as well. A number of early studies found extensive labeling of all regions of the human striatum with D1-like ligands (especially SCH 23390 [9, 11]) and with D2-like compounds (such as [3H]spiroperidol [11], [125I]epidepride [26], and [3H]CV 205-502 [7]). Although D1 and D2 -like binding are expressed in both dorsal and ventral aspects of the human striatum, they are also relatively segregated into the striosomal and matrix compartments, respectively. This enrichment of receptors into compartments is particularly well defined in the dorsal striatum. When it became apparent that these receptors represented families of receptors, selective ligands are beginning to be identified and developed. At this point, there are several compounds that preferentially label the D3 receptor, especially [3H]PD 128907 (21), [3H]7-OH-DPAT (23, 28), and [125I]epidepride under suitable incubation conditions (50). The distribution of D3 receptor binding sites parallels the distribution of the mRNA for this receptor in human striatum, also with a significant dorsoventral gradient. The use of specific antisera and immunocytochemical techniques has revealed that D1 and D2 receptor proteins are expressed in patterns identical to the distributions of their respective transcripts (31).

Unlike D1, D2 and D3 receptors, D4 and D5 receptors have minimal levels of expression in the human striatum. The possible expression of D4 receptors in the striatum has been an area of recent controversy. D4 mRNA expression is negligible in the human striatum; if present, it is in minute quantities relative to the transcripts encoding the D1, D2, and D3 receptors (34, 37). There is a single report demonstrating D4 receptor mRNA in the rat striatum using in situ hybridization, and this report indicated that D4 mRNA was restricted to the ventral aspect of the striatum (53). Other laboratories (including ours) have been unable to replicate this finding (10, 40, 42, 43). In order to measure striatal D4 mRNA in regulatory studies in the rat, this transcript requires PCR amplification for multiple cycles (30 in one report), suggesting that it is present in the rat striatum but at extremely low levels of expression (57).

The lack of expression of D4 receptor mRNA in the human striatum is interesting given several reports of high levels of expression of D4 binding sites in the human striatum, and the apparent upregulation of striatal D4 binding sites in schizophrenia. These binding studies depended on an indirect method involving subtracting Bmax values for two different radioligands with different affinities for the D2, D3, and D4 receptors. These reports suggested that nearly half of all "D2-like" binding in the striatum is due to D4 binding sites (49, 58, 62). Not all groups have been able to demonstrate D4-like binding in human striatum using these subtraction techniques (28, 55). However, as specific D4 ligands and anti-D4 antibodies (48) have become available, it appears that there is minimal "true" D4 receptor binding in the primate or human striatum. There does appear to be a dramatic increase in the binding of a dopamine-like receptor site in the schizophrenic striatum; it now appears that this site is not a D4 receptor. The identification of this site remains to be elucidated, but it may be a sigma site (22).

D5 receptor mRNA is not found in the striatum of the human brain (34, 43). Interestingly, however, low levels of D5 receptor transcript have been found in the brain of the old-world monkey (2, 8, 24), and it has been detected using specific antisera and immunological techniques in the monkey striatum as well (4). As in the case of D4 transcripts, the localization of this mRNA in rat striatum is controversial. Most investigators have not found significant levels of this mRNA in the rat striatum (10, 40, 42, 43), but a few reports suggested that it may be present in low levels in rodents (30, 63).

In summary, in the human striatum, significant levels of dopamine receptor expression are restricted to the D1, D2, and D3 receptors, a finding that generally parallels that of the rodent brain. These observations suggest that while D1 and D2 receptors are likely involved in both motor and limbic functions mediated in the striatum, the D3 receptor may have a uniquely limbic function in this brain region, given its expression in a dorsoventral gradient with enrichment in the accumbens. These findings are summarized in and representative anatomical images are presented in .

All five dopamine receptor transcripts are seen throughout the human neocortex. At this time, detailed surveys have been published on prefrontal, motor, temporal, and primary visual cortices. In prefrontal and temporal cortices, the mRNAs encoding the D1 and D4 receptors are the most abundant of the five receptor transcripts (34). In these areas, D1 receptor mRNA is particularly enriched in infragranular layers, although fainter labeling is observed in more superficial layers. A similar pattern is seen for D4 receptor mRNA: this transcript is observed in both infra- and supragranular layers of these cortical regions. D2, D3, and D5 mRNAs are present at lower levels than those encoding D1 and D4 in these cortical areas. D2 receptor mRNA is the most abundant of these rarer cortical transcripts and can be seen in both superficial and deep layers. D3 and D5 receptor mRNAs are even less abundant and are predominantly expressed in deeper layers. An alternatively spliced form of D3 receptor mRNA has been detected in some human cortical regions, and this modified form may be altered in schizophrenic brain (56). Detailed examination of the distribution of dopamine receptor transcripts in primate and human motor cortex has been limited to D1, D2, and D5 receptor mRNAs, and these results are similar to what has been reported in prefrontal and temporal regions (24).

Specific antisera have recently been developed for several of the dopamine receptors. D1 and D5 receptor proteins are identifiable in many regions of primate and human neocortex, with immunocytochemical labeling apparent in both infra- and supragranular layers, in parallel with both the pattern of D1-like mRNA and binding sites in cortex (4, 6, 60). D4 receptor protein has been identified in multiple regions of the primate neocortex; interestingly, it appears to be concentrated within cortical GABAergic interneurons (48).

Striking laminar patterns of expression of these receptor mRNAs have been reported in primary visual cortex, associated with the unique cytoarchitecture of this cortical region (34). D1 receptor mRNA is distributed in both infra- and supragranular layers, with particular enrichment in deeper layers, as seen in other regions of the cortex. There is a gradient of infragranular labeling, with higher expression of D1 mRNA in layer VI, compared with layer V. The other four transcripts are seen in three discrete bands: a deep zone consisting of lamina VI; a superficial zone containing laminae II, III, and IVa; and a well-defined middle zone that corresponds to lamina IVc. As in the case of the other regions of cortex that have been studied, D1 and D4 mRNA appear to be the most abundant of the dopamine receptor transcripts. The distributions of cortical dopamine receptor transcripts in the human are summarized in , and representative anatomical images are presented in .

The distribution of dopamine receptor mRNAs in human cortex are significantly different from what has been previously reported in the rat brain, likely reflecting reported differences between rodents and primates in the dopaminergic innervation of the cortex (3). D1 and D2 receptor mRNAs have been reported in the rat neocortex (40, 42, 43). In the human, all five of these mRNAs are expressed in the cortex at much higher levels than in the rat. While the distributions of D1 and D2 receptor mRNA in the rat and human appear similar, the expression of D3, D4, and D5 are strikingly different. Neither D3 nor D4 receptor mRNA have been consistently demonstrated in the rat neocortex. In the human, however, both D3 and D4 mRNAs are found in multiple cortical regions. D3 receptor mRNA is clearly the rarest of the dopamine receptor transcripts in human neocortex. On the other hand, D4 receptor mRNA is enriched in human cortex, and this may be the predominant form of "D2-like" mRNA expression in most regions surveyed to date. D5 receptor mRNA has a pattern similar to that of the D2 receptor, which is both unusual and unexpected, given its lack of cortical distribution in the rat (41).

One of the striking findings of the neuroanatomical distributions of the five dopamine receptor mRNAs in the rat was that the hippocampus was the single anatomical location in the brain that expressed all five of these mRNAs (40, 42, 43). All five of the dopamine receptor mRNAs are expressed in the human medial temporal lobe, but they are present in unique patterns of distribution. All five are also detectable in entorhinal and adjacent temporal neocortex, again in unique distribution patterns (36).

D1 receptor mRNA is concentrated primarily in deeper layers of the temporal neocortex (as noted earlier) with moderate levels of expression in the perirhinal cortex, the subiculum, and the pyramidal cell layer of CA1. Expression of this receptor message in other medial temporal lobe regions is negligible.

In contrast, D2 receptor mRNA is seen throughout the medial temporal lobe. The granular cell layer of the dentate gyrus has a high level of expression. Other regions express low to moderate amounts of this transcript. The pyramidal cell layers of CA1–CA4 all have modest levels of expression, as does the subiculum, presubiculum and parasubiculum. Entorhinal, perirhinal and temporal neocortex have modest levels of expression as well.

D3, D4, and D5 receptor messages have distributions similar to that of D2 receptor mRNA. Fairly high levels of expression of all are observed in the granular cell layer of the dentate gyrus, with modest levels seen in the pyramidal cell layer of CA1–CA4, and lower levels in the subiculum and related structures. Each of these transcripts are modestly expressed in entorhinal, perirhinal, and neocortical areas. The distributions of dopamine receptor transcripts in medial temporal lobe structures are summarized in , and representative anatomical images in the hippocampus and surrounding structures are presented in .

The distribution of dopamine receptor binding sites has also been examined in the human medial temporal lobe, with the primary focus on D2-like receptors. In general, D2-like binding exists in the same distribution as the corresponding transcripts but is generally in a different lamina of each region (18, 27). Recently, D2 receptor binding was found to be enriched in columns in the human temporal neocortex, suggesting a modular organization of dopaminergic innervation in this cortical area (17). As dopamine receptor antibodies have become available, receptor proteins are being identified in hippocampal areas in patterns that parallel those of their corresponding mRNAs (4, 48).

The midbrain dopamine cell groups consist of discrete zones of cells that synthesize dopamine. The primary cell groups are the retrorubral fields (A8), the pars compacta of the substantia nigra (A9), and the ventral tegmental area (A10). A8 and A10 have extensive projections to cortical and limbic structures, while the substantia nigra projects primarily to the striatum. Dopamine receptors are also expressed in these structures. Given that these receptors are located in cells that are also synthesizing dopamine, they represent dopamine autoreceptors. The identification of a transcript in a cell that also expresses a marker of dopamine synthesis (usually tyrosine hydroxylase) thus marks an mRNA species that likely encodes a receptor that functions as an autoreceptor in that particular cell.

In the human midbrain, tyrosine hydroxylase-positive cells are readily identifiable throughout the entire extent of A8, A9, and A10. On the other hand, dopamine receptor mRNAs can be found only in A9: dopamine synthesizing cells in A8 and A10 have negligible levels of dopamine receptor mRNA expression in the human (20, 25, 35). Originally, the D2 receptor was identified as the dopamine receptor subtype that subserved autoreceptor functions. Consistent with this, the primary dopamine receptor transcript that is identifiable in the human substantia nigra is D2 mRNA. D3 mRNA is also present, but at considerably reduced levels relative to D2. No D4 mRNA is seen in the human midbrain. Unexpectedly, in both the old-world monkey and in the human, D5 receptor mRNA can also be identified within A9 (2, 8); the significance of this finding is not yet clear. The distributions of dopamine receptor transcripts in human midbrain are summarized in , and representative anatomical images in the substantia nigra are presented in .

In the rat, D2 mRNA is also the predominant transcript expressed in dopamine synthesizing regions; D3 mRNA has been identified in these cell groups, albeit at much lower levels (35, 40, 42, 43). Unlike the human, however, transcripts are found in all of these dopamine cell groups and are not restricted to A9. This is a striking species difference in the expression of dopamine receptors, in this case at the level of autoreceptor expression. Based on rat anatomy, A9 has been viewed as giving rise to the nigrostriatal system, which subserves motor functions, with A8 and A10 projecting primarily to limbic structures. Accordingly, a parsimonious interpretation is that, in the human, the dopaminergic circuitry associated with motor function has autoreceptors, while the limbic circuitry does not. This is an overly simplistic interpretation, as work has revealed significant overlap in these projections, particularly in the primate (1, 13, 47, 52, 65). Most A10 fibers probably terminate in limbic regions in the human, but some likely have motor targets. Similarly, some of the A9 projections have limbic targets, including frontal cortex (16, 53). Accordingly, this finding should probably not be construed to indicate that all limbic regions in the human brain lack dopamine autoreceptors; rather, it is those projections arising from A8 and A10 lack autoreceptors. Nonetheless, this remains an exciting finding that requires continued study and may be an area that can be targeted for therapeutic intervention in some psychiatric illnesses.

The identification of five genes encoding dopamine receptors has been an exciting development in the neuropsychiatric community, of importance to our understanding of the pathophysiology and treatment strategies for various neuropsychiatric conditions. The combination of pharmacology coupled with the neuroanatomy of the products of these genes is important as we continue to develop and refine our understanding of this receptor family. This is a rapidly moving field, with the development of novel compounds to specifically label each receptor site, of specific antisera to label each receptor protein, and the recent development of compounds with specific and selective receptor affinities that are being targeted as possible therapeutic agents. The continued study of both the neurochemistry and anatomy of these receptor subtypes will continue to provide a growing base of knowledge for our understanding of brain neurotransmission and its dysregulation in neuropsychiatric conditions.

ACKNOWLEDGMENTS

The enthusiastic assistance of Scott P. Damask with preparation of the figures contained in this chapter is appreciated. This work was supported by grants from NIMH (MH53327) and the Stanley Foundation.

published 2000