The explicit yet enigmatic involvement of dopamine in cortical physiology is in part volumetric (beyond the synapse), as is apparently the action of neuroleptics targeting dopamine receptors. The notion that nonsynaptic neuronal membranes would translate extracellular dopamine into receptor-specific spatiotemporal downstream signaling, similar to the chemical synapse, is intriguing. Here, we report that dopamine D5 (but not D1 or D2) receptors in the perisomatic plasma membrane of prefrontal cortical neurons form discrete and exclusively extrasynaptic microdomains with inositol 1,4,5-trisphosphate-gated calcium stores of subsurface cisterns and mitochondria. These findings introduce a novel dopaminoceptive substratum in the brain and a unique D5 receptor-specific signaling paradigm.
- dopamine receptor
- InsP3 receptor
- subsurface cistern
- electron microscopy
- neuronal calcium
- prefrontal cortex
- working memory
In the primate prefrontal cortex (PFC), dopamine (DA) signaling is implicated in complex cognitive operations, the actions of drugs of abuse, and devastating neuropsychiatric disorders, including schizophrenia (Brozoski et al., 1979; Goldman-Rakic, 1987, 2002; Lidow et al., 1998; Carlsson et al., 2001; Weinberger et al., 2001). Yet many of these explicitly specific actions result from DA signals reaching via gradient diffusion receptors on proximal and remote nonsynaptic membranes (Goldman-Rakic et al., 1997; Pickel, 2000) [see reviews edited by Agnati et al. (2000) for a comprehensive account on volume neurotransmission]. Efficacy and cellular compartmentalization of extrasynaptic DA receptors (DARs) is therefore presumed to account for distinctive neuromodulatory and neuropharmacological patterns via specific signal transduction mechanisms assigned to individual receptor subtypes (Goldman-Rakic et al., 1997; Missale et al., 1998).
Three decades after volumetric signaling was first introduced as a potent mechanism of interneuronal communication (for review, see Dismukes, 1977; Beaudet and Descarries, 1978; Agnati et al., 1995b), our understanding of receptors and effector systems mediating volume neurotransmission remains mostly incomplete. This is primarily because these receptors, unlike their synaptic counterparts, do not seem to operate within discrete signaling modules in the brain. These views have recently been challenged by the evolving concept of the signaling microdomain. Elegant physiological and biochemical studies suggest the presence of microdomains that couple exogenous activation with a specific set of transduction molecules on intracellular membranes, thereby facilitating downstream signaling and encoding the stimulus spatiotemporal pattern and the signal transduction mechanisms of individual receptors (for review, see Berridge, 1998; Fagni et al., 2000; Delmas and Brown, 2002; Johenning and Ehrlich, 2002; Augustine et al., 2003). In effect, a signaling microdomain would operate as a signal amplification module to ensure fidelity of transmission and potentially to translate local events into global responses by engaging universal second messengers. Of key significance is the compartmentalization of the local cytosol that sequesters ionic and biochemical pools, exemplified by the dendritic spine, and the phosphoinositide system mobilizing calcium (Ca2+) from intracellular stores via the 1,4,5-trisphosphate receptor (InsP3R) (Berridge, 1993).
Given the importance of volume transmission mechanisms for DA physiology and neuropsychopharmacology (Zoli et al., 1999) and the potential of the signaling microdomain for maintaining input specificity and selectivity of regulation, we evaluated the plausibility of an evolving hypothesis that such modules may in fact exist in neurons to facilitate “diffuse” dopaminoception. We used high-resolution immunoelectron microscopy to study expression patterns of the D1-like DARs (D1-LRs; i.e., D1 and D5 subtypes) and the D2 subtype, the prototypic receptor of the D2-like subfamily (for review, see Missale et al., 1998), in the pyramidal perisomatic region. This compartment, unlike dendritic spines, includes a massive single cytosolic volume and, in the PFC of primates, bears rare association with DAergic afferents (Smiley and Goldman-Rakic, 1993; Sesack et al. 1995). More importantly, the perisomatic plasma membrane is uniquely furnished with subsurface cisterns (SSCs) (see Fig. 1 for an overview), which would ultimately ensure cytosol compartmentalization and serve as scaffold for molecules mediating downstream effects.
Materials and Methods
Histology and immunoreagents. Two adult rhesus monkeys (Macaca mulatta) from the Yale Department of Neurobiology primate colony were anesthetized with sodium pentobarbital (100 mg/kg, i.v.) and perfused transcardially with oxygenated artificial CSF, followed by 4% paraformaldehyde/0.08% glutaraldehyde in phosphate buffer (PB; 100 mm) and aldehyde-free PB. Experimental procedures were conducted in accordance with federal and institutional guidelines. The brains were blocked coronally, vibrosliced at 60 μm, cryoprotected, and stored frozen at -80°C. Prefrontal cortical sections were thawed in PB and preincubated for 45 min in 10% normal goat serum (NGS; Vector Laboratories, Burlingame, CA) and 2% bovine serum albumin (BSA; Vector Laboratories) in Tris-buffered saline (TBS; 50 mm) before being transferred to primary antibodies (Table 1). Nanogold- and biotin-SP-conjugated IgG fragments were purchased from Nanoprobes (Yaphank, NY) and Jackson Immunoresearch (West Grove, PA), respectively. Unlabeled bridging antibodies and peroxidase-antiperoxidase complexes were from Sternberger Monoclonals (Lutherville, MD).
Single gold-based D1R, D2R, D5R immunocytochemistry. Primary antibodies diluted in TBS plus 2% NGS (i.e., N-TBS) were applied for 36 hr at 4°C. Sections were then incubated for 2 hr in 1:500 species-specific biotinylated F(ab′)2 and for another 2 hr in 1:200 goat anti-biotin Fab′ conjugated to 1.4 nm gold cluster. Immunocomplexes were fixed for 5 min in 1% buffered glutaraldehyde and, after a thorough wash in ultrapure water, nanogold was enhanced for 8 min with a silver autometallographic developer (HQ Silver; Nanoprobes).
Dual gold-based and peroxidase-gold-based D5R/InsP3R immunocytochemistry. Primary antibodies in N-TBS were simultaneously applied for 48 hr at 4°C. To visualize the D5R, sections were preincubated for 30 min in N-TBS supplemented with 0.1% acetylated BSA (Aurion, Wageningen, The Netherlands), 0.1% fish skin gelatin and 0.07% Tween 20 (i.e., gold buffer), and transferred for 3 hr in nanogold-conjugated goat anti-rabbit Fab′ (1:200 in gold buffer). After wash in ultrapure water and 20 mm sodium citrate, nanogold was enhanced for 12 min on ice with a gold developer (GoldEnhance; Nanoprobes). Subsequently, nanogoldconjugated goat anti-mouse Fab′ (1:200) was applied for 4 hr to probe the InsP3R primary antibody. Sections were finally postfixed in glutaraldehyde and transferred for 4 min to the gold developer to enhance the InsP3R-bound nanogold but also to enhance further (i.e., 12 + 4 min in total) the D5R gold signal of the first series (two-step autometallography). This sequential enhancement produced distinct, nonoverlapping particle-size groups (see Methodological Considerations). Alternatively, D5Rs were labeled in the second series using species-specific bridging antibodies and peroxidase-antiperoxidase tertiary complexes (1:200 in N-TBS for 2 hr each). Peroxidase activity was visualized in 0.025% diaminobenzidine (DAB) in TBS with the addition of 0.007% hydrogen peroxide. In this case, InsP3Rs were labeled as detailed above, with gold enhancement ranging from 3 to 8 min at 22°C.
Electron microscopy. Sections were postfixed in 1% buffered osmium tetroxide (15 and 30 min for silver- and gold-enhanced material, respectively), treated with ethanolic uranyl acetate en block, and finally embedded in Durcupan epoxy resin (Fluka, Steinheim, Switzerland) and polymerized at 58°C for 48 hr under vacuum. Layers II-IV and V-VI of the dorsolateral PFC (Walker's area 46) were sampled for thin sectioning and ultrastructural analysis under a JEM-1010 (Jeol, Tokyo, Japan) transmission electron microscope operated at 80 KV, with or without lead counterstaining.
Distinction between pyramidal and nonpyramidal neurons at the level of the perisomatic region was made on the basis of the established ultrastructural criteria for neuron-class identification in the neocortex, including the unique synaptology of each class (i.e., symmetric axo-somatic and proximal axo-dendritic synapses for pyramids versus both symmetric and asymmetric synapses for interneurons) (Colonnier, 1981; Peters and Kara, 1985a,b).
Antibodies. The full characterization of the affinity-purified DAR antibodies has been detailed previously (Boundy et al., 1993; Levey et al., 1993; Khan et al., 2000; Centonze et al., 2003). The immunogen peptide for each subtype-specific antibody (Table 1) shows no sequence homology with other identified DARs. The D5R antibody (aa 377-477) has been characterized also in D1R null (-/-) mice and shown not to cross-react with the homologous D1R subtype (Centonze et al., 2003). Various groups have provided additional characterization and applied these antibodies for the study of DARs in rodents, cats, humans, and nonhuman primates with various fixation protocols (Smiley et al., 1994; Bergson et al., 1995b; Yung et al., 1995; Rivera et al., 2002; Wang and Pickel, 2002). Both D5R antibodies labeled selectively plasmalemma/SSC complexes and revealed in the neuropil (data not shown) an expression pattern complementary to that of the D1R, as described with antibodies raised against the 375-477 residue of the human D5R protein (Bergson et al., 1995b). The affinity-purified InsP3R monoclonal antibodies (Bourguignon et al., 1993, 1994) yielded indistinguishable labeling patterns in both the neuropil and the perisomatic region, targeting selectively the cytoplasmic face of reticular endomembranes as predicted from the transmembrane topology of the immunogen peptide (i.e., cytoplasmic C terminus sequence of the InsP3R) (see Fig. 5A-D). To confirm the specificity of immunolabeling for the InsP3R, we used an affinity-purified polyclonal InsP3R antibody raised against a different amino acid sequence (aa 2596-2608 vs 2492-2502) (Table 1) of the human InsP3R C-terminal domain (Bourguignon et al., 1994; Wang et al., 2004). Nanogold immunocytochemistry with thisantibody revealed strong but sporadic InsP3R expression in dendritic spine apparata (supplemental Fig. 1 A, available at www.jneurosci.org; compare with Fig. 5A) and prominentimmunolabeling of reticular endomembranes (supplemental Fig. 1 B-D, available at www.jneurosci.org), which is consistent with the labeling patterns of the monoclonal InsP3R antibodies (Fig. 5B-D) (see Results) and previous descriptions (Sharp et al., 1993). For all immunoprocedures, we monitored specificity and selectivity with a series of omission-substitution controls, including reversal of the dual labeling sequence (see Fig. 6C) and prolonged gold enhancement (10 min at 22°C or 16 min on ice) after omitting the nanogold conjugates.
Methodological considerations. The single anddual gold-based immunoprocedures yielded distinctive particulate signals. The DAR single immunolabeling produced particle aggregates (Figs. 2 D-G, 3A-E) by introducing to the immunocomplex the long form SP-biotinylated antibody and multiple nanogold-tertiary antibodies. In dual immunolabeling, in contrast, a two-layer procedure with a stoichiometry close to 1 (i.e., 1 nanogold/immunocomplex) resulted in single immunoparticles (Fig. 4 A-D, compare with Fig. 2 E-G). In effect, the multiparticle approach combined with the more sensitive silver autometallography generally increased detection sensitivity, whereas the single-particle procedure provided higher spatial resolution because of the smaller immunocomplex and particle size. Finally, note the use of Tween 20 permeabilization in single-particle immunoprocedures for increasing the penetration of immunoprobes and facilitating labeling between tightly apposed membranes (Fig. 4 A-C).
The peroxidase-gold methodology produced readily distinguishable immunocytochemical signals (amorphous precipitates vs particulate gold) (Fig. 6 A, inset). However, immunoperoxidase, which can be a valuable auxiliary marker for initially exploring and confirming patterns of receptor expression, performs poorly when spatial resolution is required at the subcellular level (Novikoff, 1980). Therefore, only immunolabeling with gold probes was used for quantification throughout the study.
The sequential enhancement of nanogold with autometallography is based on the descriptions of Bienz and Egger (1998) for colloidal gold and was developed in our laboratory for receptor colocalization and independently reported by another group for cytoskeletal and vesicular proteins (Yi et al., 2001). To determine the optimal conditions for this experimental system, gold conjugates were sequentially introduced and gold enhanced, either being linked to antigens (supplemental Fig. 2C-G, available at www.jneurosci.org) or fixed directly on tissues with glutaraldehyde (supplemental Fig. 2 A, B, available at www.jneurosci.org).
This study pertains to the neuronal perisomatic region (i.e., perikaryon, axon initial segment, and proximal primary dendrites) known to include reticular specializations in the form of SSCs (see Fig. 1 for an overview). The smallest dendrite containing an SSC in our material had a diameter (minor axis) of 1.2 μm and displayed rough endoplasmic reticular profiles (data not shown), which would accordingly correspond to a primary shaft, and generally SSCs were present in more proximal primary branches (>1.6 μm) continuous with the perikaryon. The SSCs were almost exclusively localized in the perisomatic region of pyramidal cells, whereas they occurred rarely in perikarya and were virtually absent from the proximal processes of interneurons (see Materials and Methods for identification criteria). These rarely occurring SSCs in nonpyramidal neurons were never seen in association with DARs [see Khan et al. (2001) for D2R, Muly et al. (1998) for D1R]. In addition, both the D5R protein and D5R mRNA are localized predominantly in pyramidal neurons (Bergson et al., 1995a; Ariano et al., 1997; Ciliax et al., 2000). Therefore, in the following sections, we describe the subcellular expression patterns of DAR subtypes in the pyramidal perisomatic region.
The D1Rs comprised a highly immunoreactive cytoplasmic pool in specific association with the Golgi apparatus in perikarya and dendrites (Fig. 2A). Plasmalemmal expression, predominant in dendritic spines of the neuropil (Smiley et al., 1994), was not identified in the perisomatic region. In contrast, D2Rs were not generally localized in the perisomatic region, with the exception of certain pyramids expressing weak immunoreactivity in rough endoplasmic reticular (RER) membranes, the Golgi, and in association with endosomes (Fig. 2B), consistent with the rare presence of D2Rs in PFC efferents (Wang and Pickel, 2002). In these cells, nonsynaptic D2Rs were additionally distributed on “uncomplexed” portions of the plasmalemma (i.e., lacking juxtaposed endomembranes; see below) (Fig. 2C). Overall, the perisomatic region expressed high levels of D5R immunoreactivity, with the exception of the axon initial segment. Indeed, labeling was rarely seen beyond the axon hillock (data not shown). The D5R immunoparticles labeled RER profiles deeper in the cytoplasm and formed distinct clusters below the cell surface (Fig. 2D-G) often in association with clathrin-coated vesicles (Fig. 2D). More importantly, the specific clustering of D5Rs subjacent to the plasma membrane invariably predicted the presence of a juxtaposed SSC (Figs. 2D-G, 3, 4). In addition to the cytoplasmic pool, we observed and examined in detail a plasmalemmal D5R component.
For the three-layer immunoprocedures, membrane-bound immunoparticles were defined as within 25 nm from the inner aspect of the plasmalemma. Proximity criteria were based on the size of the immunocomplex used for probing the intracellularly protruding C terminus of the receptor protein; equivalent to one IgG and two Fab′ fragments, including the long-form biotin conjugate (see Materials and Methods). In two-layer immunoprocedures, D5R particles that appeared on the cytoplasmic face of the plasma membrane were considered plasmalemmal. We identified 618 membrane-bound immunoparticles in 26 perisomatic profiles of individual pyramids midsectioned through the apical dendrite (final magnification, 60,000×). Ninety-three percent (577 particles) were distributed on membranes that overlay or flanked a juxtaposed SSC, at a distance no more than 80 nm of its margins (Figs. 2F,G, 3A,B,D, 4A-D). Spatial specificity of D5R expression was also manifest among the plasmalemma-SSC complexes of a single neuron, and occasionally highly immunopositive and immunonegative complexes were merely 0.5 μm apart (Fig. 2D). The latter appeared characteristically devoid of the subjacent D5R cytoplasmic cluster, which also suggests that this cluster could represent an exchanging D5R pool locally associated with an overlying plasmalemma-SSC complex.
To determine whether this spatially restricted expression of D5Rs along the perisomatic membrane is in response to synaptic input or other specific cellular apposition, we examined D5R plasmalemma-SSC complexes (called D5R microdomains hereafter) in single and successive sections (Table 2). Although the more frequently encountered appositions involved axons often forming asymmetric synapses with profiles other than the perisomatic membrane (Fig. 3C,D) (Alonso-Nanclares et al., 2004), the D5R microdomain was never directly associated with the symmetric synapses that typically converge on the pyramidal perisomatic region (Beaulieu and Colonnier, 1985) (Figs. 2D, 3A,B). This is of particular interest because cisternal structures have indeed been shown to accompany symmetric synapses (GABAergic) on the axon initial segment of hippocampal pyramids (Benedeczky et al., 1994) (Fig. 5D). In two single cases, we identified microdomains in somato-somatic plasma membrane juxtapositions (Fig. 6H).
Given the central role of reticular endomembranes (and the SSC) in Ca2+ homeostasis (Park et al., 2000; Meldolesi, 2001) and the capacity of D1-LRs to mobilize Ca2+ stores (for review, see Missale et al., 1998), we next examined whether the D5R microdomain could be a potential physical substrate for the phosphoinositide system by also expressing InsP3Rs. In the neuropil (Fig. 5A,B), InsP3Rs were sporadically expressed in dendritic spine apparata and in slender vesicular structures within dendrites and rarely axonal varicosities. In the perisomatic region, we detected InsP3Rs on reticular endomembranes and rarely on Golgi complexes (Fig. 5C,D) (for Golgi as a Ca2+-signaling apparatus, see Pinton et al., 1998). Coexpression of D5Rs and InsP3Rs in individual microdomains was confirmed with different dual immunoprocedures (i.e., gold-gold- and peroxidase-gold-based; see Materials and Methods). Despite the inherent limitations of immunoperoxidase (discussed in Materials and Methods), DAB immunocytochemistry for the D5R labeled intensely plasmalemma-SSC complexes and often the subjacent cytoplasm, whereas precipitation over uncomplexed membranes and the juxtaposed cellular profiles was minimal or absent (Fig. 6A, compare with the gold labeling pattern in B-H). The InsP3R immunoparticles labeled the lateral vacuolar expansions as well as central portions of the SSC limiting membranes (Fig. 6A-H).
Next, we sought to quantify the extent of colocalization of D5Rs and InsP3Rs in the pyramidal perisomatic region. However, note that this is a semiquantitative approach, for several factors discussed in the following section may influence differentially the immunolocalization of individual antigens. Because the number of SSCs in individual perisomatic profiles varies depending on the length of visible plasma membrane, we examined perisomatic regions midsectioned through the nucleus and the apical primary dendrite. In D5R-InsP3R-immunolabeled material, we identified a total of 210 plasmalemma-SSC complexes (referred to as complexes) in 24 perisomatic profiles (8.8 ± 2.7 complexes/profile; mean ± SD; range, 4-14). The majority (158 complexes; 75%) expressed InsP3R immunoreactivity on SSCs as described in the previous section. D5R immunoreactivity was identified in 96 of the complexes (4 ± 1.7 complexes/profile; mean ± SD; range, 1-9), equal to 46% of total complexes. With the exception of two perisomatic profiles containing exclusively D5R complexes, immunopositive and immunonegative complexes for the D5R generally coexisted in individual profiles (Fig. 2D). Eighty-one (84%) of D5R complexes expressed InsP3Rs immunoreactivity on SSCs. The remaining were immunonegative (Fig. 6H) or more frequently displayed InsP3Rs in association with other endomembranes in the vicinity of the SSC.
Having established the association of D5Rs with the InsP3R-expressing cisternal endomembranes, we finally investigated whether the InsP3-sensitive stores of the microdomain could be additionally linked to the mitochondria, an important cytoplasmic component of the intracellular Ca2+ network (Rizzuto et al., 1994, 1998; Babcock et al., 1997). In 21% (n = 100) of single-sectioned and 32% (n = 46) of serially sectioned D5R microdomains (Fig. 3, compare C and D), a single mitochondrion was directly apposed to the SSC, separated by a sparsely bridged 12-20 nm electron-lucent zone (Fig. 7A-C). In favorably sectioned appositions (Fig. 7C), we observed InsP3R immunoparticles within the minute cleft separating the cisternal from the mitochondrial outer membrane, apparently labeling the cisternal face because mitochondria are not known nor were they observed here to express InsP3Rs.
Although the perisomatic occurrence of the SSC has been documented in central and peripheral neurons (see Fig. 1 for review) and corroborated here for the macaque PFC, there is no compelling evidence until now, with respect to SSCs and neuronal classification. The original descriptions either do not distinguish between pyramidal and nonpyramidal neurons (Rosenbluth, 1962) or report SSCs primarily in pyramids (Buschmann, 1979). Here, we confirmed the predominant localization of SSCs in pyramidal cells as opposed to interneurons. Moreover, the rarely occurring SSCs in nonpyramidal perikarya were never associated with DARs.
Within pyramidal neurons (Fig. 2), the DAR subtypes formed cytoplasmic pools (D1R, D2R, and D5R) and a membrane-bound component (D2R and D5R). The latter would represent the “functional” component of the perisomatic region, because active receptors signal from the plasma membrane and only these receptors would be available to extracellular DA. Another possibility to consider is that DARs could be additionally localized within postsynaptic densities of DAergic synapses preventing immunodetection with the pre-embedding method, as repeatedly demonstrated by Somogyi's group for ionotropic glutamate receptors [see Baude et al. (1995) for discussion on pre-embedding vs post-embedding approaches]. Even for these receptors, however, one would expect the pre-embedding method to occasionally visualize relatively “unmasked” antigens, especially at the edges of the synapse (Baude et al., 1995), which was never observed here. Although it seems unlikely, considering the rare occurrence of perisomatic DAergic input (Goldman-Rakic et al., 1997), we cannot exclude this possibility. Likewise, we cannot preclude a biased immunodetection because of physical barriers (i.e., the tightly apposed plasmalemma and cisternal membranes) or other factors that may interfere with the dual immunolabeling, such as loss of gold signal of the first labeling series, reduced antigenicity in the second series, nonuniform preservation of antigenicity for D5Rs and InsP3Rs after fixation or simply different potencies of the individual antibodies. Therefore, one should bear in mind these inherent limitations when interpreting immunocytochemical findings.
We have established that D5Rs exhibit strict spatial specificity along the pyramidal perisomatic plasmalemma and form discrete and exclusively extrasynaptic microdomains. Moreover, this expression pattern appears to be unique to the D5R, as manifested not only between subtypes of distinct DAR subfamilies (D5Rs but not D2Rs) but also between the structurally and pharmacologically homologous subtypes of the D1-LR subfamily (D5Rs but not D1Rs). To the best of our knowledge, the D5R microdomain is the first evidence for the existence of intricately structured neurotransmission modules in the brain exhibiting spatial-, receptor-, and cell-class/compartment specificity beyond the chemical synapse.
Evidently, such modules would never meet the exquisite “one-to-one” spatiotemporal requirement of the synapse. Therefore, they should not be viewed as an alternative to “wired” communication to compensate for the rare occurrence of perisomatic DA synapses but rather as a synergistic mechanism for the modulation of cellular assemblies (for review, see Agnati et al., 2000). Consistent with these views, the dopaminoceptive microdomain is strategically positioned to influence the neuronal excitability centrally by influencing directly the signal relay compartment of the cortical principal cell, as opposed to DAergic modulation of individual synapses on distal dendrites mediated primarily via the D1R (for review, see Goldman-Rakic et al., 2000). The selective expression of the high-affinity D5R (its affinity for DA is an order of magnitude higher than that of the D1R) (Sunahara et al., 1991) will then favor perisomatic dopaminoception at relatively lower transmitter levels and potentially long-range volumetric signaling. Inevitably, the dopaminoceptive microdomain should be regarded as a potent substratum for D1-LR pharmacological manipulation. It is noteworthy that chronic treatment with typical and atypical antipsychotics downregulates D5R mRNA in the PFC (Lidow et al., 1997), although it is not currently known whether this involves neuropilar or perisomatic D5Rs.
The question naturally arises as to whether the D5Rs could be organized in discrete microdomains independently of extrinsic signals (i.e., in response to the intracellular milieu) or, conversely, whether extrinsic signals may trigger clustering of the D5R and juxtaposition of cisternal membranes. Apparently, the clustered D5R expression is not in response to synapses (either DAergic or heterosynapses), and local extrasynaptic release of DA across the microdomain might seem unlikely considering the frequent juxtaposition of non-DAergic profiles (i.e., glutamatergic-like axons, dendrites, glia) (Table 2) (however, see the DAergic-like en passant axon in Fig. 6B). In fact, analyses of the DAergic innervation of the frontal cortex have confirmed the rare association of DA axons with the perisomatic region (Smiley and Goldman-Rakic, 1993; Sesack et al. 1995; for review, see Goldman-Rakic et al., 1997). Nonetheless, this evidence does not necessarily imply that the perisomatic membrane is uniformly exposed to “circulating” DA. Glial barriers or heterogeneous distribution of DA transporters within individual axons (Lewis et al., 2001) could favor diffusion anisotropies (Barbour and Häusser, 1997; Gonon et al., 2000; Syková, 2001), which may ultimately result in spatially restricted elevations of DA levels (“hot spots”) along the perisomatic membrane. These DAergic axons could be located in the relative vicinity of a microdomain or even more remotely within the parenchyma. Whether the nonuniformity of the plasmalemma with regard to extracellular DA may apply in vivo is only speculative. What is very interesting, however, is that both the SSC and mitochondria are most certainly dynamic structures, unlike their static images captured in electron microscopy. Early ultrastructural studies have demonstrated that SSCs are capable of plastic changes after axotomy and during development (Sumner, 1975; Buschmann, 1979), and recent evidence indicates that local Ca2+ dynamics may affect physical coupling between the smooth endoplasmic reticulum and mitochondria (Wang et al., 2000). Finally, we should consider the possibility of a dynamic trafficking between the plasmalemmal component and the cytoplasmic pool of the D5R, as might indicate its selective clustering bellow the microdomain, often in association with clathrin-coated vesicles (Fig. 2D-G). Compared with the D1R, which undergoes dynamin-dependent endocytosis involving clathrin-coated pits (Bloch et al., 1999; Vickery and Zastrow, 1999), little is known about the D5R regarding its handling after agonist stimulation. Although it appears to undergo agonist-induced internalization, similar to the D1R, recent evidence suggests that after endocytosis the D5R is shorted for degradation in the lysosomes (Heydorn et al., 2004).
Although more studies, both anatomical and physiological, would be needed to elucidate these key issues, it is obvious that ligands (endogenous DA and pharmacological analogs) would reach the D5R microdomain, as well as other dopaminoceptive membranes, via volumetric diffusion (Zoli et al., 1999; Pickel, 2000). Even unoccupied D5Rs may retain substantial capacity for activating adenylyl cyclase by behaving as constitutively active receptors (Tiberi and Caron, 1994). Yet, unlike any other brain dopaminoceptive membranes, the D5R microdomain presents a unique cellular feature: the distinctive alignment of the D5R-expressing membranes with the InsP3R-furnished SSC and the mitochondrion, implying their involvement in similarly restricted intracellular signaling mechanisms to maintain input specificity and selectivity of regulation. It has been suggested, for example, that InsP3Rs expressed in the reticulum-mitochondrion interface, as shown here, would generate the essential Ca2+ hot spots for the low-affinity, high-capacity mitochondrial Ca2+ uniporter to operate, leading to sharp rise of matrix Ca2+ levels in response to InsP3 (Rizzuto et al., 1994, 1998). In essence, the D5R microdomain constitutes the equivalent of a nonprojecting “somatic spine” by sequestering the cytosol and by supplying the reticular scaffold, reminiscent of the spine apparatus, for specific macromolecular arrays, including the InsP3R, to assemble and function (Berridge, 1998; Blackstone and Sheng, 1999; Delmas et al., 2002; Augustine et al., 2003).
Previous studies in behaving primates have established that the D1-LRs are critically involved in working memory operations (Sawaguchi and Goldman-Rakic, 1994; Williams and Goldman-Rakic, 1995; Castner et al., 2000), even though the lack of specific ligands for D1R and D5R subtypes has hitherto precluded consideration of their potentially distinct roles within the cortical microcircuitry (Gao and Goldman-Rakic, 2003). Moreover, their signal transduction mechanisms (for review, see Missale et al., 1998) have most commonly been linked to activation of adenylyl cyclase. In addition to the cAMP pathway, however, D1-LR activation increases the formation of inositol phosphates in the brain (Mahan et al., 1990; Undie and Friedman, 1990) and stimulates Ca2+ release from InsP3-sensitive stores in perikarya of dissociated cortical neurons (Lezcano and Bergson, 2002). It appears that the involvement of D1-LRs in the phosphatidylinositol turnover is independent of the cAMP pathway and is likely attributed to a subtype distinct from the prototypic D1R (Friedman et al., 1997). The discovery here of a specialized microdomain that facilitates selective physical coupling of the D5R and the phosphoinositide system introduces a unique signaling paradigm for the volumetric regulation of cortical functions by DA and identifies new targets (Agnati et al., 1995a) that could be involved in the numerous neuropsychiatric disorders affecting the brain DAergic system. This link can be particularly tempting in light of the recently discovered Ca2+-binding, DAR-interacting proteins and the proposed involvement of Ca2+ in the pathophysiology of schizophrenia (Bergson et al., 2003; Lidow, 2003).
This work was supported by National Institutes of Health Grant MH44866 (P.S.G.-R.). This study is dedicated to the memory of Patricia Shoer Goldman-Rakic. C.D.P. is indebted to R. Moratalla and Z. Khan for generously providing the D5R antibodies, K. Szigeti for technical assistance, and C. Bergson, B. Ehrlich, T. Koos, M. Yeckel, and P. Rakic for critical comments and discussion.
Correspondence should be addressed to Dr. Constantinos D. Paspalas, Yale University School of Medicine, Department of Neurobiology, Sterling Hall of Medicine B408, 333 Cedar Street, New Haven, CT 06510. E-mail:.
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↵† Deceased July 31, 2003.