Abstract

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Section 5

Discussion
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There have been many new findings in neurotransporter research over the past several years. One of the most interesting findings has come from recent electron microscopic immunohistochemical studies which suggest that transporters are indeed found on the surface membrane of neurons as expected, but they are not found within the synapse itself. They are localized to the plasma membrane of axons and preterminal axons, but they are not found in the part of the nerve terminal membrane which forms the synapse as can be seen in Figures 1 and 2. A variety of evidence for this has been slowly accumulating. In this communication, we will summarize this evidence which is limited but from several laboratories. Our focus is on the plasma membrane transporter and not the vesicular transporter, and most evidence deals with the dopamine transporter (DAT).

The importance of transporters in removing released neurotransmitter is well established.¹ Blockade of uptake has clearly demonstrable cellular, physiologic and behavioral effects. Important therapeutic drugs such as antidepressants and antihyperactivity medications target transporters, as do the psychostimulants, cocaine and amphetamine. Because of this, it has been assumed that transporters would be found in the synapse, a location that might be most efficient for removing released neurotransmitter. The findings that some transporters are also in glia and other cells (in retrospect, a harbinger of perisynaptic transporters) seemed important, but of lesser importance than putative synaptic transporters. Recently, electron microscopic localization studies suggest that transporters are located perisynaptically rather than intrasynaptically.

The most studied transporter in this regard is the dopamine transporter (DAT). Three laboratories using different polyclonal and monoclonal antibodies have found that DAT is on dendrites, axons and nerve terminals, but not in the part of the nerve terminal membrane that is in the synapse. Nirenberg et al.,² Hersch et al.,³ and Sesack et al.⁴ used antibodies directed at amino terminal peptides from DAT, and Hersch et al. (personal communication) found similar results using an antibody directed against a portion of the glycosylated, large extracellular loop of DAT. Conti et al.⁵, studying neuronal glutamate transporters (EAAC1), did not find the transporter anywhere on the nerve terminal, but rather on pre- and postsynaptic cell bodies and glia.

Could technical problems account for the lack of detection of intrasynaptic transporter by immunohistochemical methods? This certainly seems possible. If the antigenic sites are not available to the antibody, i. e., if there are penetration problems, then synaptic transporter might not be detected. Also, it seems possible that synaptic transporter may exist in functionally important levels, but this level is too low to be detected by the procedures used. However, the fact that different labs using different antibodies to different portions of the protein obtain the same results would argue against, but not completely rule out these possibilities. But, there are some other findings that are compatible with a nonsynaptic localization of transporters.

Garris et al.⁶ studied removal of dopamine by electrochemical detection from the nucleus accumbens after stimulation of ascending dopaminergic fibers. Their data and calculations suggest that dopamine is not captured in the synaptic cleft but diffuses out the synapse before reuptake by perisynaptic transporters. They suggested a model where functionally relevant transporter would be extrasynaptic. The notion of perisynaptic transporters as opposed to synaptic transporters seems reasonably supported.

Transporters are clearly of major functional importance. But is there any functional significance to such a model where transporters are perisynaptic rather than intrasynaptic? It seems that a major implication would be that diffusion of neurotransmitter out of the synapse would be more important than previously thought. Neurotransmitter would first diffuse out of the synapse and then be recaptured by transporters. Another implication of this would be that neurotransmitter would be more available to extrasynaptic receptors. Paracrine or volume transmission is thought to be important in some cases.^7,8 Also, the nerve terminal membrane in the synapse is the site of release and therefore the site of many release-associated proteins and protein recycling; the partitioning of transporter to perisynaptic areas would simplify the process in that membrane transporter proteins would not "interfere" with the release process.

In summary, emerging EM immunohistochemical data suggests that at least dopamine transporters are not found IN the synapse but rather AROUND or in perisynaptic parts of the axonal and nerve terminal membrane. Other anatomical and physiologic findings support this. If these findings continue to be supported for DAT and other transporters, then diffusion of neurotransmitter out the synapse prior to reuptake will be more important than previously thought. This would not diminish the overall importance of reuptake but would alter our view of synaptic processes. It also may not be true for all neurotransmitter transporters. The possible lack of synaptic transporters could have functional consequences such as making neurotransmitter more available to extrasynaptic receptors. Indeed, a relevant question at this time would be "What is the evidence that transporters are in fact IN the synapse rather than surrounding it?"

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