Role of alpha-synuclein in presynaptic dopamine recruitment

Abstract

Real-time monitoring of stimulated dopamine release in mice with different alpha-synuclein expression was used to study the role of alpha-synuclein in presynaptic dopamine recruitment. Repeated electrical stimulations of ascending dopaminergic pathways decreased the capacity of the readily releasable pool (RRP) and temporarily increased its refilling rate, significantly slowing the rate of dopamine decline in mice with normally expressed alpha-synuclein. Mice with alpha-synuclein null mutation demonstrated a permanent increase of the refilling rate. This increase maintained stable dopamine release during stimulation (which induced dopamine decline in other animals) and served as an adaptation to altered dopamine compartmentalization. Mice without alpha-synuclein and with overexpression of human A30P mutated alpha-synuclein had a lower capacity of the dopamine storage pool than other animals. Reducing capacity of the storage pool in transgenic A30P mice led to paradoxical effects of l-dopa, which elevated dopamine release in response to single stimulation but decreased the refilling rate of the RRP.

Figure 1.

Different aspects of presynaptic dopamine storage and mobilization in control C57BL/6J mice (b6m⁺). a, Original recording of evoked dopamine (DA) release in the caudate nucleus after repeated electrical stimulations (2 sec in length; 50 Hz; 6 stimulations at 5 sec intervals) of the MFB, organized in three bursts. Arrowheads depict responses to the first stimulation in each burst. Dopamine decline at marked stimulations indicates exhaustion of the RRP by the previous burst. b, Summary of the data on repeated stimulations in b6m⁺ mice, given as percentage of dopamine release (mean ± SEM; n = 5). The reference point (hatched bar) is the first stimulation in the first burst. This presentation of data clearly indicates that dopamine generally declines after massive stimulation. However, inside each burst, exhaustion of the RRP facilitates dopamine release. c, When the reference point is the first stimulation in each burst, progressive facilitation of dopamine release is also seen in the second and third bursts. A facilitation of release is attributed to increase in the refilling rate of the RRP. Each data point is mean ± SEM (n = 6).

Figure 2.

Probing for the presence of the mouse endogenous α-syn gene or the human α-syn transgene in the four mouse lines. The mouse genomic α-syn (aSY) DNA fragment (130 bp) and the human α-syn transgene fragment (137 bp) amplified by PCR in separate reactions are shown together with positive control gene NR1 fragment (508 bp). Products were separated on a 1.8% agarose gel, so that wells with even numbers had the PCR for the mouse endogenous α-syn gene (m) and wells with odd numbers had the PCR for the human α-syn transgene (h).

Figure 3.

The capacity of the RRP determined by dopamine (DA) decline after repeated stimulation of the medial forebrain bundle. Three bursts of stimulations were applied to the MFB according to the stimulation protocol described in Figure 1 and Materials and Methods. These massive stimulations exhausted the RRP and to some extent the dopamine storage and induced a decline in the evoked dopamine levels in all of the mouse lines except b6m^-. Data are given as percentage of dopamine release after the first stimulation in the first burst. Each data point represents the relative level of dopamine after the first stimulation in each consecutive burst (numbers 1-3 on the horizontal axis). The inset shows dopamine release after single 2 sec, 50 Hz stimulation, expressed in molar concentrations (means ± SEM; n = 5-8 per group).

Figure 4.

Comparison of presynaptic dopamine compartmentalization in C57BL/6J α-syn knock-out (b6m^-) and control (b6m⁺) mice. Right, Original recordings of evoked dopamine (DA) release after repeated stimulations in a representative mouse from each line. Examples were chosen on the basis of similar initial molar levels of extracellular dopamine after the first stimulation (marked by asterisk). The numbers show molar dopamine concentrations at the first peak of each burst. Left, Summaries of results in these two mouse lines. Data are presented as percentage in reference to the first peak of the first burst (marked by an asterisk). Each data point is mean ± SEM (n = 5-6 per group).

Figure 5.

Activation of the refilling rate of the RRP in the four mouse lines in response to repeated stimulations. Evoked dopamine (DA) release after repeated stimulation in the first and the third bursts shown as group data (each data point is mean ± SEM; n = 5-9 per group). Data are presented as percentage in reference to the first peak of each burst and thus describe the dynamics of dopamine release within the burst.

Figure 6.

The effects of αMpT (250 mg/kg) on the refilling rate of the RRP in transgenic mice with overexpression of human mutated A30P α-syn (h⁺m⁺) and in control C57BL/6 (b6m⁺) mice. Evoked dopamine (DA) release after repeated stimulations shown for the first burst before and the first burst 60 min after the administration of drug as group data (each data point is mean ± SEM; n = 6-9 per group). The dynamics of dopamine release within the burst are presented as a percentage, as in Figure 5. αMpT treatment exhausted the RRP and decreased evoked dopamine release. Insets show the effects of drug on dopamine release evoked by 2 sec, 50 Hz single stimulation in molar concentrations as means ± SEM (open bar, before treatment in b6m⁺ and h⁺m⁺ mice; filled bar, after treatment in b6m⁺ and h⁺m⁺ mice). The treatment increased the RRP refilling rate in b6m⁺ mice and did not change the dynamics of dopamine release in transgenic mice, which corresponds with the effects of repeated stimulation in transgenic and knock-out mice in the previous experiment shown in Figure 5.

Figure 7.

The effects of l-dopa (30 mg/kg) on the refilling rate of the RRP in transgenic (h⁺m⁺) and control (b6m⁺) mice. Data are presented in the same manner as in Figure 6 (n = 5 per group). The effects of l-dopa on facilitation of dopamine (DA) release during the first burst were analyzed 40 min after treatment (l-dopa was injected 20 min after carbidopa). Treatment increased evoked dopamine release after single stimulation (insets), did not change the refilling rate of the RRP in b6m⁺ mice, and decreased it in h⁺m⁺ mice.