Ultrastructural Localization of the Vesicular Monoamine Transporter-2 in Midbrain Dopaminergic Neurons: Potential Sites for Somatodendritic Storage and Release of Dopamine

Perikarya

Neuronal perikarya in both the SN (Fig. 1A) and the VTA (Fig. 1B) contained extensive immunogold labeling for VMAT2. This immunogold labeling was localized most prominently to tubulovesicular organelles, most of which resembled saccules of SER (Peters et al., 1991; Broadwell and Cataldo, 1983) (Fig.1A,B), but some of which resembled SSVs. In both regions, there was also extensive VMAT2-labeling of the Golgi apparatus, particularly along its lateral saccules (Fig.1A,B). VMAT2-immunoreactive DCVs, some of which were located close to the trans-Golgi network, were also detected in dopaminergic perikarya in the SN and VTA. In some of the labeled SN (Fig. 1A) and VTA perikarya, immunogold particles for VMAT2 were seen along the limiting membranes of multivesicular bodies (MVBs). In contrast, there was no detectable immunolabeling for VMAT2 along the nuclear or plasma membranes of perikarya in either region, and only rare immunogold labeling for VMAT2 along saccules of rough endoplasmic reticulum. The subcellular localization of VMAT2 was most clearly resolved in tissue that was prepared for single immunogold labeling of VMAT2 (Fig. 1A,B), but was similar to that seen in double-labeled tissue, where there was also a diffuse electron-dense peroxidase reaction product for TH.

Dendrites and dendritic spines

In the SN, VMAT2-containing dendritic profiles were distributed throughout the pars compacta and the pars reticulata. Within the VMAT2-labeled dendrites, most of the immunogold labeling was associated with large tubulovesicles that had the morphological appearance of dendritic SER (Peters et al., 1991) (Figs.2A, 3A,B). Less frequently, VMAT2-labeling was localized to the membranes of DCVs (Fig.3C) or to smaller electron-lucent vesicles that resembled SSVs (Fig. 3B). In rare cases, VMAT2-labeled SSVs formed a cluster within the cytoplasm of an SN dendrite (Fig.3B).

In tissue that was dual-labeled for both VMAT2 and TH, most of the VMAT2-labeled dendrites also contained detectable peroxidase reaction product for TH (Fig. 3A). Although the VMAT2-labeled dendrites in the SN were sometimes apposed to and/or received synaptic contacts from unlabeled axon terminals (Fig. 3A) or dendrites (not shown), they were only rarely apposed to other VMAT2- or TH-labeled dendrites.

On several occasions, immunogold labeling for VMAT2 was detected in dendritic spines in the SNR (Fig. 5A,B). Within these spines, VMAT2 was associated prominently with electron-lucent vesicles and tubulovesicles (Fig. 5A,B). The labeled spines were frequently apposed to and/or received asymmetric contacts from unlabeled terminals. In some cases, these unlabeled axon terminals contacted both the spine and the shaft of the same VMAT2-containing dendrite. In sections that were double-labeled with immunogold for VMAT2 and immunoperoxidase for TH, most of the VMAT2-labeled spiny dendrites contained detectable reaction product for both markers (Fig.5A,B).

In the VTA, as in the SN, immunogold labeling for VMAT2 was localized most prominently to tubulovesicles that resembled saccules of SER (Fig. 2B). Less frequently, immunogold labeling was localized to smaller electron-lucent vesicles that resembled SSVs (Fig.4A,B) or to DCVs (Fig. 4A). In several cases, clusters of VMAT2-labeled vesicles were observed near segments of the dendritic plasma membrane that were apposed to and/or formed dendrodendritic synapses onto other dendrites. Some of the apposed dendrites contained (Fig. 4A) and some lacked (Fig.4B) detectable VMAT2 immunoreactivity. In sections that were dual-labeled for both VMAT2 and TH, many of the of the pre- and/or postsynaptic dendrites in which there was immunogold labeling for VMAT2 also contained peroxidase reaction product for TH (Fig. 4A). VMAT2 also was detected occasionally in dendritic spines in the VTA (Fig. 5C). Within these spines, VMAT2 was selectively associated with vesicles and tubulovesicles (Fig.5C).

By quantitative analysis, there were significantly more gold particles for VMAT2 per unit area within TH-immunoreactive dendrites in the VTA (1.78 ± 0.10/μm²) than in TH-immunoreactive dendritic profiles in either the SNC (1.30 ± 0.07/μm²; p < 0.0001) or the SNR (1.22 ± 0.07/μm²; p < 0.0001). In contrast, when TH-immunoreactive dendrites in the SNC and SNR were compared, there was no statistical difference in the density of immunogold labeling for VMAT2 (p = 0.47).

Axons and axon terminals

Immunogold labeling for VMAT2 was frequently detected in axon terminals and in small, unmyelinated axons in the SN (Fig.6A,B) and VTA (Fig. 6C,D). Within the labeled axonal processes, gold particles for VMAT2 were localized predominantly to the membranes of SSVs (Fig. 6A,C,D) or DCVs (Fig. 6A,B,D) and only infrequently to larger tubulovesicles (Fig. 6C). In rare cases, one or more immunogold particles seemed to contact the cytoplasmic surface of the plasma membrane of an axon terminal that contained VMAT2-labeled vesicles (Fig.6A).

In sections that were dually labeled with immunogold for VMAT2 and immunoperoxidase for TH, a small number of unmyelinated axons in the SN and the VTA contained immunoreaction product for both markers. The majority of the VMAT2-containing unmyelinated axons in the SN (Fig.6A,B) and the VTA (Fig. 6C,D), however, did not contain detectable immunoreaction product for TH. This was the case even when other TH-immunoreactive structures were clearly observed within adjacent profiles within the same tissue section (Fig.6A–C). The VMAT2-labeled axon terminals in both regions were frequently apposed to, and in some cases formed synaptic junctions with, TH-labeled dendrites, as illustrated in the VTA (Fig.6C) and SN (Fig. 6A).

Methodological considerations

The subcellular localization of VMAT2 to saccules of Golgi tubulovesicles of SER and vesicles in the SN and VTA is consistent with electron microscopic studies of uptake of false neurotransmitters and radiolabeled monoamines, which have identified these organelles as potential sites of monoamine storage (Cuello and Iversen, 1978; Mercer et al., 1979; Groves and Linder, 1983). The immunocytochemical methods used in the present study, however, offer several potential advantages over the methods used in previous experiments. First, the immunogold–silver method offers greater spatial resolution than autoradiography, thereby permitting identification of the specific organelles that are potential sites of monoamine storage in the SN and VTA. Second, because dopamine is the only catecholamine that is present in perikarya and dendrites in these regions (Pickel and Sesack, 1995), dual labeling for TH, a specific marker for catecholaminergic neurons, has permitted us to identify the VMAT2-containing cells as dopaminergic neurons. Third, because VMAT2 is an intrinsic membrane protein of vesicles and other subcellular organelles, immunocytochemical localization of this transporter provides a novel approach for identifying potential monoamine storage sites that is not limited by the potential for nonspecific uptake of exogenous radiolabeled or false neurotransmitters. Although the immunocytochemical methods used in the present study cannot discriminate between functional and nonfunctional sites of expression of VMAT2, the interpretation of our findings in light of those of previous uptake and autoradiographic studies provides compelling new information regarding the probable sites of reserpine-sensitive somatodendritic storage of dopamine in midbrain dopaminergic neurons.

Dopaminergic perikarya: potential sites of membrane synthesis and recycling

The pattern of VMAT2-immunolabeling along saccules of Golgi, tubulovesicular organelles, DCVs, and occasional MVBs in TH-immunoreactive (dopaminergic) perikarya in the SN and VTA was similar to the distribution of VMAT2-immunolabeling that we showed previously in the nuclei of the solitary tract (NTS) (Nirenberg et al., 1995). These labeled organelles, which were usually distant from the perikaryal plasma membrane, probably include sites of synthesis, transport, and recycling of VMAT2-containing membranes. The Golgi apparatus, for example, is known to be involved in both the post-translational modification and recycling of vesicular membrane proteins such as VMAT2 (Griffiths and Simons, 1986; Kelly, 1993) and in the synthesis of immature DCVs (Tooze, 1991). Similarly, SSV precursor membranes, which are synthesized in neuronal perikarya, are believed to be tubulovesicular in morphology (Schwarzenbrunner et al., 1990; Jahn and Südhof, 1993; Mundigl and De Camilli, 1994). Furthermore, MVBs have been implicated in the recycling and degradation of vesicular membrane proteins (Jahn and Südhof, 1993; Wittich et al., 1994;Bannon et al., 1995).

Although the VMAT2-labeled perikaryal organelles may include sites of nonfunctional expression of VMAT2 or its C-terminal domain, experiments using the uptake of false neurotransmitters have shown evidence that the trans-Golgi apparatus, DCVs, and tubulovesicles that resemble SER in SN perikarya can sequester monoamines (Wilson et al., 1977; Mercer et al., 1979). Thus, it is likely that at least some of the VMAT2-labeled saccules of Golgi and SER seen in dopaminergic perikarya are also sites of reserpine-sensitive storage of dopamine.

Dopaminergic dendrites: potential sites of dopamine storage and release

We have shown that small numbers of VMAT2-containing SSVs are present in the cytoplasm of dopaminergic dendrites and dendritic spines in both the SN and VTA. These findings are consistent with those of false neurotransmitter uptake studies, which have demonstrated that the dendrites of dopaminergic neurons in the SN contain relatively few synaptic vesicles with a functional monoamine uptake carrier (Sotelo, 1971; Groves and Linder, 1983). We have also seen isolated DCVs in dopaminergic dendrites in the SN and VTA. Previous studies have shown that dendritic DCVs in the SN can specifically take up 5-hydroxydopamine (Sotelo, 1971; Groves and Linder, 1983). Together, these observations suggest that both SSVs and DCVs are involved in the reserpine-sensitive storage and calcium-dependent release of dopamine from midbrain dopaminergic dendrites.

Dopaminergic perikarya and dendrites in the SN and VTA were also filled with extensive networks of VMAT2-labeled tubulovesicles. In fact, some of the organelles that have met our morphological criteria for SSVs may have been tubulovesicular structures, which can be indistinguishable from SSVs when examined in cross-section (Ayala, 1994). The prominent localization of VMAT2 to dendritic tubulovesicles is similar to that which we have demonstrated in the NTS (Nirenberg et al., 1995). Some of these tubulovesicles may represent nonfunctional sites of storage, transport, or recycling of VMAT2-containing membranes; however, there is also convincing evidence that tubulovesicular organelles can store dopamine (Wilson et al., 1977) and in fact may be the major site of somatodendritic dopamine storage (Mercer et al., 1979). The characteristic morphological appearance of these tubulovesicles strongly suggests that they are composed of saccules of SER, as has been presumed in earlier studies (Hattori et al., 1979; Mercer et al., 1979; Wassef et al., 1981). It is also possible, however, that these tubulovesicles represent a novel type of membranous organelle, such as the one that was identified recently in PC12 cells by its expression of the facilitated glucose transporter GLUT4 (Herman et al., 1994).

Given the limited detection of VMAT2-containing vesicles, the VMAT2-containing tubulovesicular organelles are likely to represent the major site of reserpine-sensitive dopamine storage in the SN and VTA. Thus, these tubulovesicular organelles probably contribute more significantly than SSVs or DCVs to somatodendritic dopamine release (Mercer et al., 1979; Wassef et al., 1981). The somatodendritic release of dopamine from these tubulovesicular organelles might potentially involve an exocytosis-like mechanism (Llinás, 1979). Alternatively, dopamine might be released from these organelles into the dendritic cytoplasm, causing an increase in the local dopamine concentration and thereby permitting dendritic release of dopamine through reversal of the plasmalemmal dopamine transporter (Bannon et al., 1995). This possibility is supported by our recent demonstration that the dopamine transporter is localized to the plasma membranes of dopaminergic dendrites and dendritic spines in the SN (Nirenberg et al., 1996). A similar mechanism for dopamine release through reverse dopamine transport has been demonstrated in vitro under pharmacological conditions in which the plasmalemmal dopamine gradient has been reversed (Bernardini et al., 1991; Sulzer et al., 1993).

Nondopaminergic terminals: potential sites of storage and release of other monoamines

In addition to the TH-immunoreactive perikarya and dendrites, we have also observed numerous VMAT2-immunoreactive axon terminals in both the SN and the VTA. These VMAT2-containing axonal processes almost never contained detectable TH-immunolabeling, but were frequently apposed to and/or formed synaptic contacts with dopaminergic (TH-immunoreactive) dendrites in both the SN and the VTA. The presence of VMAT2 immunolabeling and absence of detectable TH-immunolabeling in these axon terminals suggests that they contain monoamines other than catecholamines. In particular, serotonergic neurons in the raphe nuclei are known to make dense projections to both dopaminergic and nondopaminergic dendrites in the SN (Fibiger and Miller, 1977;Nedergaard et al., 1988) and VTA (Herve et al., 1987; Van Bockstaele et al., 1994). Interestingly, there is also evidence that serotonergic inputs to the SN, like those to the striatum and accumbens nucleus, may facilitate the dendritic release of dopamine from midbrain dopaminergic neurons by causing local changes in the dendritic calcium potential (Williams and Davies, 1983; Nedergaard et al., 1988; Jacocks and Cox, 1992). The rare unmyelinated axon terminals that contained reaction product for both VMAT2 and TH may be derived from dopaminergic neurons in the A8 dopamine cell group that have minor projections to the SN and the VTA (Deutch et al., 1988).

Differential localization of VMAT2 in the SN and VTA: functional implications

Dopaminergic neurons in the SN and VTA are known to differ markedly in their roles in motor and motivational functions, their biochemical and pharmacological properties, and their susceptibility to exogenous neurotoxins and neurodegenerative disease (for review, seeRoth and Elsworth, 1995). The present study is the first, however, to show that there are higher levels of VMAT2 per unit area in dopaminergic dendrites in the VTA than in either the SNC or the SNR. These findings suggest that dopaminergic neurons in the VTA contain a larger pool of reserpine-sensitive dopamine storage and release sites than those in the SN. Interestingly, in vitro studies have shown that there are higher levels of somatodendritic dopamine release in the VTA as compared with the SN (Rice et al., 1994). Together, these findings suggest that the greater release of dopamine in the VTA as compared with the SN is at least partially attributable to the presence of higher levels of storage and release of dopamine from individual dendrites in this region.

Recent evidence has also shown that vesicular monoamine transporters can confer neuroprotection by sequestering dopamine neurotoxins such as 1-methyl-4-phenylpyridinium (MPP⁺) that have been used in experimental models for Parkinson’s disease (Liu et al., 1992a,b; Stern-Bach et al., 1992; Edwards, 1993; Liu et al., 1994). Thus, our observation that there are higher levels of VMAT2 in dopaminergic dendrites in the VTA as compared with those in the SN may also explain the greater susceptibility of nigrostriatal dopaminergic neurons to neurotoxic insult. Additional studies are necessary to determine whether there are comparable differences in the expression of VMAT2 in the human SN and VTA that might potentially contribute to the greater vulnerability of the nigrostriatal dopaminergic neurons to drug-induced parkinsonism and idiopathic Parkinson’s disease (Edwards, 1993).