Regulation Of Somatodendritic Dopamine By Uptake Transporters

Plasma membrane uptake of DA by the DA transporter (DAT) is fundamental to the regulation of [DA]0, such that DAT-mediated uptake, coupled with diffusion, defines the sphere of influence of somatodendritically, as well as synaptically released DA (Cragg et al., 2001; Cragg and Rice 2004). Electrophysiological studies suggest a physiological role for uptake in the modulation of somatodendritic [DA]0 (Lacey et al., 1990). Regulation of [DA]0 by uptake differs between the SNc and VTA, however. Ventral tier neurons of the SNc have greater mRNA and protein levels of DA transporter (and D2 DA receptor) than dorsal tier cells in dorsal SNc and VTA (Blanchard et al., 1994; Hurd et al., 1994; Sanghera et al., 1994; Ciliax et al., 1995; Freed et al., 1995). It is relevant to note that ventral tier SNc cells are more susceptible to degeneration in Parkinson's disease than dorsal tier cells in either VTA or SNc (Yamada et al., 1990; Fearnley and Lees, 1991; Gibb and Lees, 1991). This pattern of susceptibility is paralleled in the vulnerability to the DA uptake substrate and toxin, l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine

(MPTP) (German et al., 1988), which can be prevented by DA uptake inhibition. Together, such findings have implicated DA uptake activity or other regionally-specific DA handling mechanisms as possible risk factors in parkinsonian degeneration (Javitch et al., 1985; Sundstrom et al., 1986; Pifl et al., 1993). DA uptake within the midbrain, therefore, is crucial not only for normal DA neuron physiology, but may also contribute to the differential vulnerability of DA cells to pathophysiology.

In vitro experiments indicate that uptake of DA after evoked release or application of exogenous DA in SNc or VTA is primarily via the DAT (Fig. 2) (Cragg et al., 1997b, 2001), consistent with the high density of DAT-expressing cells in these areas (Ciliax et al., 1995; Freed et al., 1995; Nirenberg et al., 1996, 1997). In addition, the DAT is not the only transporter that can transport somatodendritic DA. Uptake of DA by the norepinephrine transporter (NET), albeit modest, is more prominent in VTA than SNc (Cragg et al., 1997b, 2001) and appears to be mediated by a few sparsely packed en passant norepinephrine processes (Cragg et al., 1997b). The role of the DAT on [DA]0 after somatodendritic release may be more marked in SNc than VTA (Cragg et al., 1997b), reflecting differential DAT expression in these regions. Uptake of DA in SNr is much less avid than in either SNc or VTA, enabling DA to diffuse over larger distances without encountering uptake sites (Cragg et al., 2001).

Uptake via the DAT plays an apparently lesser role in the regulation of somatodendritic [DA]0 in the SNc than of axon-terminal in [DA]0 striatum (Cragg et al., 1997b), as shown by a greater increase in [DA]0 during local electrical stimulation in striatum than in SNc when the DAT is blocked. This difference is in keeping with the lower density of the DAT in somatodendritic than axon terminal regions (Donnan et al., 1991; Ciliax et al., 1995; Freed et al., 1995). It can be speculated that a consequence of this apparently less avid regulation of dendritic compared to axon terminal [DA]0, will be a greater sphere of influence of dendritic than for axonal DA (see Cragg et al., 2001; Cragg and Rice, 2004). Nonetheless, uptake via the DAT is an important mechanism of regulating [DA]0 in midbrain (Cragg et al., 1997b, 2001).

Figure 2. Regulation of diffusing DA in VTA by DATs. Left, example DA diffusion profiles and right, simultaneous images of co-diffused Texas Red-labelled dextran after pressure ejection with and without the DAT inhibitor, GBR-12909 (2 nM). DA diffusion profiles ~100 urn from site of ejection show the decrease in uptake constant (k") and the increase in [DA]0 when DATs are inhibited, despite similar ejection volumes (3133 pL). Modified from Cragg et al„ 2001.

(A color version of this figure appears in the signature between pp. 256 and 257.)

Figure 2. Regulation of diffusing DA in VTA by DATs. Left, example DA diffusion profiles and right, simultaneous images of co-diffused Texas Red-labelled dextran after pressure ejection with and without the DAT inhibitor, GBR-12909 (2 nM). DA diffusion profiles ~100 urn from site of ejection show the decrease in uptake constant (k") and the increase in [DA]0 when DATs are inhibited, despite similar ejection volumes (3133 pL). Modified from Cragg et al„ 2001.

(A color version of this figure appears in the signature between pp. 256 and 257.)

5. WHAT IS THE MECHANISM OF RELEASE? 5.1. Ca2+ Dependence of Somatodendritic DA Release

The non-axonal source for somatodendritic DA release has led to much speculation about whether release might be mediated by a novel mechanism. However, few studies have contradicted the original proposal by Geffen et al. (1976) that DA release in the SN is vesicular and mediated by exocytosis, as it is in axon terminals in striatum. Indeed, physiological or pharmacological manipulations that alter [DA]0 in striatum generally affect [DA]0 in SN in a parallel manner (Santiago and Westerink, 1991; Heeringa and Abercrombie, 1995; Cragg and Greenfield, 1997; Cragg et al., 1997b; Hoffman and Gerhardt, 1999; Chen and Rice, 2001), although there is evidence for less similarity between DA release in VTA vs. nucleus accumbens (Iravani et al., 1996).

Consistent with the ionic and pharmacologic characteristics of exocytosis, somatodendritic DA release is depolarization- and Ca2+-dependent (Geffen et al., 1976; Cheramy et al., 1981; Rice et al., 1994; Rice et al., 1997; Chen and Rice, 2001) and sensitive to DA depletion by reserpine (Rice et al., 1994; Heeringa and Abercrombie, 1995). Evidence for Ca2+ dependence is often taken as confirmatory of vesicular release, since Ca2+ entry is typically required for exocytosis (Douglas and Rubin, 1963; Catterall 1999; but see Parnas et al., 2000), such that the amount of transmitter released can depend on extracellular Ca2+ ([Ca2+]0) (Dodge and Rahamimoff, 1967). Consistent with characteristics of classical exocytosis, DA release in striatum requires activation of voltage-dependent Ca2+-channels (Dunlap et al., 1995; Phillips and Stamford, 2000); moreover, both basal and evoked [DA]0 increase when [Ca2+]0 is elevated (Moghaddam and Bunney, 1989; Chen and Rice, 2001).

Somatodendritic DA release in SN also requires Ca2+: incubation in Ca2+-free media with EGTA inhibits evoked DA release by >90% (Rice et al., 1994; Rice et al., 1997). However, in contrast to striatal release, DA release in SNc persists in low-Ca2+ media (Rice et al., 1994; Rice et al., 1997; Hoffman and Gerhardt, 1999). Evoked [DA]0 in the SNc in midbrain slices is half-maximal in nominally zero [Ca2+]0 with no EGTA (Fig. 3) (Chen and Rice, 2001). Furthermore, voltage-gated Ca2+-channel blockers have only modest effects on [DA]0 in the SN (Bergquist and Nissbrandt, 2003, 2004). Together, these findings suggest a difference in intracellular Ca2+ availability or sensitivity of somatodendritic versus axonal release. For example, ready release in low [Ca2+]0 might reflect differential sensitivity of Ca2+-dependent release mechanisms, including fusion proteins, between these compartments; the varying forms of synaptotagmin, a key Ca2+-sensing protein responsible for triggering synaptic transmitter release, can differ in Ca2+ affinity by 10-20-fold (SUdhof and Rizo 1996; Sudhof 2002). Differential expression of fusion proteins among cell compartments is not without precedent: Bergquist et al. (2002) recently reported evidence that different synaptobrevin isoforms underlie axonal DA release in striatum versus somatodendritic release in the SN.

In further contrast to striatal DA release, evoked [DA]0 in SNc is maximal in 1.5 mM Ca2+, with no increase in higher [Ca2+]0 (Fig. 3) (Chen and Rice 2001). While this might reflect differences in the readily releasable pool of DA in each region, a possible confounding factor is that the train stimulation used to evoke DA release simultaneously elicits release of other transmitters, including GABA and glutamate, which strongly

Figure 3. Ca2+-dependence of DA release in (A) dorsal striatum, (B) SNc, and (C) both regions; all data are normalized to peak [DA]0 in 1.5 mM [Ca2+]0 as 100%. DA release was elicited in guinea-pig brain slices by local stimulation (10 Hz, 30 pulses) and monitored with FCV (modified from Chen and Rice, 2001).

Figure 3. Ca2+-dependence of DA release in (A) dorsal striatum, (B) SNc, and (C) both regions; all data are normalized to peak [DA]0 in 1.5 mM [Ca2+]0 as 100%. DA release was elicited in guinea-pig brain slices by local stimulation (10 Hz, 30 pulses) and monitored with FCV (modified from Chen and Rice, 2001).

regulate somatodendritic DA release in the SNc (Chen and Rice, 2002; see section 6.2). This raised the particular concern that the true Ca2+-dependence of somatodendritic release might be masked by concurrent increases in what is presumably Ca2+-dependent GABA release. Such Ca2+-dependent GABA release could decrease DA cell excitability and thereby oppose Ca2+-dependent increases in somatodendritic DA release to cause the apparent "clamping" of evoked [DA]0 for [Ca2+]0 > 1.5 mM (Fig. 3C).

We therefore re-examined the Ca2+-dependence of DA release in the SNc (and VTA) using single-pulse stimulation, which elicits DA release that is free from ionotropic GABA or glutamate receptor modulation. With this stimulus, the pattern of Ca2+ sensitivity of evoked DA release in the SNc was similar to that seen with pulse-train stimulation, with ready release in low [Ca2+]0 and a plateau in higher [Ca2+]0 (Moran et al., 2003). This suggests that the Ca2+ dependent somatodendritic release reflects intrinsic rather than extrinsic factors, with the caveat that as yet undefined tonically active inputs might regulate single-pulse release in a Ca2+-dependent manner. In the VTA, single-pulse DA release was seen readily in low [Ca2+]0, as in the SNc, but then evoked [DA]0 progressively increased with increasing [Ca2+]0 throughout the range tested, as in the striatum (Moran et al., 2003). This pattern might reflect a mixture of somatodendritic and axonal DA release predicted by the anatomy of the VTA (Deutch et al., 1988; Bayer and Pickel, 1990).

5.2. Anatomical Issues

Despite existing evidence for exocytotic release of DA in the SNc, this continues to be questioned on the basis of anatomy: synaptic sites for vesicle fusion are rare. Although dendro-dendritic DA synapses are found in the SNc (Wilson et al., 1977), these are absent in the SNr and make up less than 1% of synapses on DA dendrites overall (Groves and Linder, 1983). Moreover, DA release can be elicited from the SNr in isolation (Geffen et al., 1976; Rice et al., 1994), showing that dendro-dendritic synapses are not required for release. Although vesicular release of catecholamines can occur in the absence of synapses, as in adrenal chromaffin cells (Wightman et al., 1991), vesicles are also rare in DA cells of the SNc. Whereas vesicle density is high in DA terminals in striatum (Nirenberg et al., 1996a; 1997), there are few vesicles in DA somata or dendrites (Wilson et al., 1977; Groves and Linder, 1983; Nirenberg et al., 1996a), implying a limited source for vesicular exocytotic release (Nirenberg et al., 1996a).

Storage of somatodendritic DA has been proposed to be in saccules of smooth endoplasmic reticulum (Mercer et al., 1978; Wassef et al., 1981), as well as in vesicles (Wilson et al., 1977; Groves and Linder, 1983). Consistent with dual storage sites, the vesicular monoamine transporter, VMAT2, is expressed in saccules (so-called tubulovesicles) and, less commonly, in vesicles (Nirenberg et al., 1996b). Whether both storage sites contribute to the releasable pool of DA is not known. Both would be susceptible to DA depletion by reserpine, an irreversible inhibitor of VMAT2, such that reserpine sensitivity alone sheds little light on whether release is vesicular or otherwise.

5.3. Reversal of the DAT

The paucity of vesicles and the abundance of DATs in DA dendrites led several groups to speculate that somatodendritic release might be mediated by vesicle-independent reverse transport (Groves and Linder, 1983; Nirenberg et al., 1996a; Leviel, 2001). Indeed, this is the primary mode of release following certain pharmacological manipulations, including veratridine-induced depolarization (Elverfors et al., 1997) or amphetamine-induced DA displacement from intracellular stores (Sulzer et al., 1995; Jones et al., 1998; Torres et al., 2003). Somatodendritic DA release elicited by veratridine requires voltage-dependent Na+ channel opening and subsequent Na+ loading to activate reverse DA transport; interestingly, this release can be blocked by the DAT inhibitor GBR-12909 applied outside the releasing cells (Elverfors et al., 1997). Under normal conditions, cytoplasmic DA concentrations appear to be insufficient to reverse the DAT unless increased by a pharmacological agent, e.g. by amphetamine (Jones et al., 1998).

However, Falkenburger et al. (2001) have reported electrophysiological evidence that stimulation of the subthalamic nucleus can induce dendritic DA release in the SN by a mechanism that can be prevented by low concentrations of GBR-12909. This suggests that under some physiological conditions, somatodendritic release might be mediated by the reversal of the DAT. It is not yet clear how the data of Falkenburger et al. can be reconciled with other in vitro and in vivo studies of somatodendritic DA release that have typically found an increase in [DA]0 in the presence of an uptake blocker (Elverfors and Nissbrandt, 1992; Santiago and Westerink, 1991,1992; Cragg et al., 1997b; Hoffman et al., 1998; Chen and Rice, 2001).

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