Susan R. Sesack," Valerie A. Hawrylak," Margaret A. Guido," and Allan 1. Leveyt

*Departments of Neuroscience and Psychiatry University of Pittsburgh

Pittsburgh, Pennsylvania I5260

tDepartment of Neurology Emory University

Atlanta, Georiga 30322

Cellular and Subcellular Localization of the Dopamine Transporter in Rat Cortex

The dopamine transporter (DAT) plays a critical role in regulating the duration of dopamine's synaptic actions and the extent to which dopamine can diffuse in the extracellular space (1). Previous light microscopic imrnunocy- tochemical studies of the rat striatal neuropil have demonstrated a dense local- ization of DAT (2). Furthermore, ultrastructural subcellular studies indicate that most of the DAT protein in striatal dopamine axons is distributed at the periphery of synapses and at nonsynaptic membrane sites ( 3 ) . This extensive distribution of the DAT protein is likely to greatly restrict the extracellular diffusion of dopamine in this brain region.

In rodents, the rostromedial cortex also receives a dopamine innervation, and behavioral studies indicate that this input is important for proper cognitive functioning. Separate dopamine systems derive from A10 or A9 dopamine neurons and terminate, respectively, in deep layers of the prelimbic or superficial layers of the anterior cingulate cortices. Several neurochemical observations suggest that extracellular dopamine in the prelimbic division, or prefrontal cortex (PFC), undergoes less regulation by DAT-mediated uptake, compared with the striatum. (1) Relative to total tissue content, extracellular dopamine levels are 20 times higher in the PFC than in the striatum. (2) In the PFC, levels of the extracellular metabolite, HVA, are higher than the intracellular metabolite, DOPAC, while the reverse is true in the striatum. ( 3 ) Extracellular dopamine diffuses farther in the PFC than in the striatum or other forebrain areas (4). These observations are typically explained on the basis of a reduced availability of DAT, secondary to a lower density of cortical dopamine axons. However, we wished to explore the hypothesis that the neurochemical profile of dopamine overflow and diffusion in the rat PFC is consistent with a restricted distribution of the DAT protein in individual dopamine axons. We tested this hypothesis by using a light and electron microscopic immunocytochemical ap- proach.

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Eleven adult male Sprague-Dawley rats were anesthetized, and 10 animals were perfused with 3.75% acrolein and 2% paraformaldehyde. The remaining animal was perfused with 4% paraformaldehye and 0.2% glutaraldehyde. The brains were removed, postfixed for 30 min, and sectioned at 50 p m on a vibratome. Sections were treated for 30 min with 1% sodium borohydride to improve antigenicity and reduce nonspecific labeling. Sections were incubated for 30 min in a blocking serum consisting of 1% bovine serum albumin and 3% normal goat serum. Steps taken to maximize immunostaining in sections for electron microscopy included the use of 0.04% Triton X-100 or freeze- thaw and, in several cases, the use of two night incubations in primary antibody. Sections for light microscopy included 0.4% Triton.

Two different primary antibodies directed against the N-terminus of the DAT protein were used: a rabbit polyclonal (1 : 100) and a rat monoclonal antibody (1 : 1000). Both were tested for specificity by Western blot analysis against cloned transporter (2) (Levey, personal communication). The immuno- cytochemical staining results with either antibody were comparable, but only the rat antibody was used in quantitative studies. Primary antibody incubation was for 15 hr at room temperature or 40 hr at 4°C. In a preadsorption control experiment, 100 pg/mL of fusion protein antigen was added to the rat primary antibody for 2 hr prior to use. To compare DAT immunoreactivity with another marker for dopamine terminals, adjacent sections from three animals were incubated in rabbit anti-tyrosine hydroxylase (TH) antiserum (1 : 1000). The secondary antibodies employed were biotinylated goat anti-rabbit (1 : 400) or donkey anti-rat (1 : loo), and avidin biotin peroxidase complex (Vectastain Elite) was used (1 : 200). Bound peroxidase was visualized by the addition of 3,3’-diaminobenzidine and H202. Sections for light microscopy were slide mounted, while sections for electron microscopy were osmicated, dehydrated, and plastic embedded.

The electron microscopic results from six rats with the best morphology and most robust staining with the rat antibody were quantified. One to two tissue sections per region per animal were examined, and the surface of the tissue was sampled at random until at least 40 DAT-immunoreactive processes were photographed. Processes were then numbered on the micrographs from upper left to lower right, and a random number generator was used to select 30 processes per region per animal (total of 180 immunoreactive processes per region for DAT and 90 observations for T H in the PFC). The processes were then traced using an image analysis system, which calculated their maximum diameter along the short axis. For processes containing DAT immunoreactivity in only one portion, only the immunoreactive area was traced. For processes with eccentric shape, such as longitudinal sections through both varicose and nonvaricose portions of an axon, the diameter represented the most varicose portion. The data were analyzed statistically by a two-way ANOVA, with main effects being either region and animal (for DAT) or marker and animal (for DAT vs TH in the PFC). The interaction between main effects was also examined in each case.

By light microscopy, DAT-immunoreactive fibers were abundantly ex- pressed in the neuropil of the dorsolateral striatum beneath the corpus callosum. This immunostaining was absent from sections incubated in primary antibody

Cellular, Subcellular Localization of DAT in Rat Cortex I73

preadsorbed with the fusion protein antigen. The anterior cingulate cortex also showed robust immunostaining for DAT, particularly in clusters of axons in layer Ill. However, the immediately adjacent prelimbic PFC showed sparse labeling for DAT that was dramatically lower than that seen in the anterior cingulate cortex. Furthermore, many of the axons that were present were diffi- cult to visualize without differential interference contrast optics. This weak immunolabeling was observed despite the use of two night incubations in primary antibody that contained a high concentration of detergent to enhance penetration. Finally, this degree of immunostaining for DAT was considerably weaker than that seen with other markers for dopamine axons, such as TH antibodies. These light microscopic observations suggest that individual dopa- mine axons in the rat PFC are relatively lacking in the DAT protein.

By electron microscopic examination of the dorsolateral striatum, peroxi- dase immunoreactivity for DAT was abundantly expressed in axon varicosities, some of which made punctate symmetric synapses on spines or distal dendrites. In the superficial layers of the anterior cingulate cortex, immunoreactivity for DAT was also frequently localized to axon varicosities. In addition, DAT immunolabeling was observed in numerous small-diameter axons. In the deep layers of the prelimbic PFC, immunoreactivity for DAT was localized almost exclusively to small-diameter axons. In a few cases, varicose axons cut in a longitudinal plane showed DAT immunoreactivity only in the preterminal re- gions and not in varicose segments that formed synapses. These results were observed consistently in all animals, regardless of the fixative used, the primary antibody employed, the use of one- or two-night incubations in antibody, or the use of Triton detergent or freeze-thaw to enhance antibody penetration. As a positive control for the ability to detect proteins in dopamine axon varicosities, immunoreactivity for TH was assessed in adjacent sections of the PFC from three animals. TH labeling was seen in intervaricose axons, as well as in numerous varicosities, some of which formed synapses on spines and small dendrites.

The quantitative analysis of process diameter by region and marker pro- duced the following means and standard deviations: DAT-PFC, 0.137 i 0.049;

0.088. By ANOVA, there was a significant overall effect of region ( p < .0001) and no significant effect of animal. However, a significant interaction between region and animal ( p < .012) suggested that some of the region effect might be explained by animal differences. Post-hoc analyses using Tukey’s studentized range test on all pair-wise comparisons with a simultaneous significance of p < .05 revealed a significant difference in diameter between the PFC and striatum in all six animals. For the anterior cingulate cortex, significant differ- ences with the PFC were observed in three of the six animals, while significant differences with the striatum were seen in only two of the six animals. For the ANOVA comparing markers, there was an overall significant effect of marker ( p < .0001) with no effect of animal and no interaction effect.

The combined results of light and electron microscopic immunocytochemi- cal studies suggest that dopamine axon varicosities in the PFC express a relative paucity of immunoreactivity for the DAT protein. This finding is consistent with in situ hybridization studies showing lower mRNA levels for DAT in the A10 relative to the A9 dopamine cell groups (5). The fact that the dopamine

DAT-CING, 0.180 5 0.074; DAT-STR, 0.218 ? 0.084; TH-PFC, 0.215 2

I74 Susan R. Sesack et a/.

inputs to the dorsolateral striatum and anterior cingulate cortex both originate from A9 dopamine neurons is consistent with their observed greater content of DAT protein. The data also suggest that DAT protein contained in dopamine axons in the PFC is localized at a distance from synaptic release sites. Although the exact subcellular distribution was not determined by immunogold labeling, the more sensitive avidin-biotin peroxidase method failed to reveal DAT protein within most dopamine axon varicosities in this region. However, it should be noted that intervaricose segments of axons can sometimes form synapses, in which case the DAT protein would be localized closer to the release site. Finally, an alternative interpretation of our data is that dopamine axon varicosities in the PFC express a biochemically modified DAT protein that has an altered antigenicity. Such a protein also might be expected to function differently from the striatal DAT. However, the fact that our findings were consistent with two different antibodies argues against this latter explanation of the data. Nevertheless, additional antibodies should be tested once they become available.

In conclusion, dopamine’s high extracellular levels (relative to tissue con- tent) and considerable diffusion in the PFC may be secondary to restricted localization and/or altered function of the DAT protein within individual axon varicosities. While these data appear to suggest a greater paracrine role for dopamine in the PFC, compared with the striatum and other forebrain regions, dopamine’s actual sphere of influence may be limited by the low density of its receptors in the cortex.

Acknowledgment

Supported by U. S. Public Health Service grant MH50314.

References

1. Giros, B., Jaber, M., Jones, S. R., Wightman, R. M., and Caron, M. G. (1996). Hyperlocomo- tion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 379, 606-612.

2. Ciliax, B. J., Heilman, C., Demchyshyn, L. L., Pristupa, Z. B., Ince, E., Hersch, S. M., Niznik, H. B., Land evey, A. I . (1995). The dopamine transporter: Iinrnunocytochemical characterization and localization in brain. J . Neurosci. 15, 1714-1723.

3 . Nirenberg, M. J., Vaughan, R. A., Uhl, G. R., Kuhar, M. J., and Pickel, V. M. (1996). The dopamine transporter is localized to dendritic and axonal plasma membranes of nigrostriatal dopaminergic neurons. J . Neurosci. 16, 436-447.

4. Garris, P., and Wightman, R. M. (1994). Different kinetics govern dopaminergic transrnis- sion in the amygdala, prefrontal cortex, and striatum: An in vivo voltammetric study. J . Neuroscz. 14, 442-450.

5. Shimada, S., Kitayama, S., Walther, D., and Uhl, G. (1992). Dopamine transporter mRNA: Dense expression in ventral midbrain neurons. MoE. Bruin. Res. 13, 359-362.