Geneneration of triple synuclein null (TKO) mice
At all stages of the breeding programme (see Materials and Methods) an expected Mendelian frequency of TKO mice on pure C57Bl6J genetic background was observed in weaned litters. TKO mice, studied up to 14 months of age, were phenotypically indistinguishable from wild type mice generated within the same breeding programme and no differences in size, weight or gross anatomy of the brain of TKO and wild type mice were observed.
Because of the well-known links between α-synuclein and dopamine dysfunction in Parkinson’s disease, and the prominent expression and presynaptic localisation of all three synuclein family members in dopaminergic neurons of the SNpc (data will be available online if the manuscript is accepted, also see Abeliovich et al., 2000
) we carried out in-depth studies on the midbrain dopaminergic systems of TKO mice.
Normal numbers of dopaminergic neurons in the SNpc and normal expression of synaptic markers in the striatum of TKO mice
Stereological counts of midbrain dopaminergic neurons, identified by immunostaining for tyrosine hydroxylase (TH), revealed no difference in the number of neurons in the SNpc and ventral tegmental area (VTA) of TKO mice compared to wild type mice (). Immunostaining of the striatum with markers of dopaminergic terminals, TH or DAT (), or general synaptic marker synapsin IIa (), and quantitative Western blot analysis of striatal tissue proteins (, quantification of data will be available online if the manuscript is accepted) also revealed no differences in morphology or levels of all studied synaptic markers.
Triple synuclein null mutant mice possess a normal complement of dopaminergic neurons in the SNpc and VTA, normal rate of in vivo TH activity but reduced level of dopamine and its metabolites in the striatum
Normal morphology and expression of synaptic markers in the striatum of triple synuclein null mutant mice
Decreased levels of dopamine and its metabolites in the striatum of TKO mice
HPLC analysis of tissue monoamines revealed that the dopamine content in the dorsal striatum of 4-month old male TKO mice was substantially decreased compared to wild type animals (). The levels of the major dopamine metabolites, DOPAC and HVA, were less affected, resulting in increases of DOPAC/dopamine and HVA/dopamine ratios ( and additional data will be available online if the manuscript is accepted). To determine whether decreased striatal dopamine level in mutant mice is a consequence of reduced activity of TH, the rate-limiting enzyme in dopamine synthesis, we inhibited aromatic L-amino acid decarboxylase (AADC), the enzyme immediately downstream of TH in the synthetic pathway, by treating mice with 3-hydroxybenzylhydrazine (NSD-1015). Striatal extracts were prepared 45 minutes after i.p. injection of 100 mg/kg of the inhibitor and the level of L-DOPA was assessed by HPLC. No difference in L-DOPA accumulation, which is an indicator of in vivo TH activity, was found between wild type and TKO mice (). Together, these data suggest that in the absence of neuronal loss or obvious biochemical or morphological changes of dopaminergic synapses (also confirmed by electron microscopy analysis, see below), TKO mice display signs of synaptic dysfunction in the nigrostriatal system at the level of regulation of dopamine availability. Therefore we studied performance of TKO mice in behaviour tests, which results might be affected by alterations in dopamine neurotransmission.
Hyperdopaminergic-like behaviour of TKO mice
The inverted grid and static rods tests were used to assess balance and coordination of 4-month old TKO mice. The performance of both male and female adult mutant mice was very similar to the performance of age/gender-matched wild type mice ( and additional data will be available online if the manuscript is accepted). However, ageing mutant mice gradually lose the ability to stay on the inverted grid (). In contrast, the accelerated rotarod test, in which the animal’s endurance capacity affects the results, revealed significantly compromised performance of TKO mice already at the age of 4 months ().
Performance of wild type and triple synuclein null mutant mice in balance/coordination and exploratory behaviour tests and their activity in novel non-anxiogenic environment
Wild type and TKO animals behaved in a similar manner in the bright open field and elevated plus maze tests (data will be available online if the manuscript is accepted), suggesting that the lack of synucleins does not affect mouse anxiety.
Several tests revealed significant differences in the activity in novel non-anxiogenic environment and exploratory behaviour of wild type and TKO mice. In the non-anxiogenic open-field mutant mice were more active () and reared more often (). In the holeboard test, the number of nose pokes into holes was significantly greater for mutant than wild type mice (), suggesting increased overall exploratory behaviour. TKO mice also showed a trend towards a decreased spontaneous alternation in a T-maze (). Finally, monitoring of animal activity in the home-like cage demonstrated that TKO mice respond to changes in the environment with a substantially greater increase in locomotion than wild type mice (). However, after adaptation to the new environment, the locomotor activity of mice was similar for both genotypes ().
Increased levels of electrically evoked dopamine in the dorsal striatum of TKO mice
Together these data suggest that despite a decrease in striatal dopamine levels, TKO mice exhibit behaviour typical of hyperdopaminergic animals (Zhuang et al., 2001
), suggesting that synaptic mechanisms regulating dopamine release or uptake might be modified in the absence of synucleins. We used fast-scan cyclic voltammetry (FCV) at carbon-fibre microelectrodes to assess subsecond release and uptake of dopamine in the dorsal (caudate putamen; CPu) and ventral (nucleus accumbens; NAc) striatum. In the CPu, mean peak extracellular concentrations of dopamine ([DA]o
) evoked by single pulses or burst stimuli (4 pulses, 100 Hz) across a large number of recording sites were ~1.5-fold greater in TKO than wild type mice ( and additional data that will be available online if the manuscript is accepted). By contrast, in the NAc no difference in evoked [DA]o
was detected between the two genotypes ( and additional data that will be available online if the manuscript is accepted). The frequency-dependence of dopamine release at given recording sites assessed across a full range of frequencies observed for dopaminergic neurons in vivo
(1 to 100 Hz) was similar in both genotypes ( and additional data that will be available online if the manuscript is accepted) and consistent with previous observations in CPu and NAc (Exley et al., 2008
). Analysis by HPLC of dopamine content in CPu and NAc subdissected from slices used for FCV confirmed that dopamine content in CPu was ~30% lower in triple synuclein null mice (; as seen in fresh tissue, ) and identified that in the NAc, dopamine content was not different between genotypes ().
Electrically evoked dopamine transients and regulation of dopamine signals by firing frequency in wild type and triple synuclein null mutant mice measured by FCV
Increased evoked dopamine in the dorsal striatum is not due to differences in nicotinic receptor function or the Ca2+-dependence of release
Since in CPu of TKO mice, dopamine content is two thirds of wild-type, but release is ~1.5-fold greater, deletion of all three synucleins results in a more than 2-fold increase in the releasability of the available dopamine. Because striatal acetylcholine (ACh) is a critical regulator of dopamine release probability, we assessed how dopaminergic synapses of TKO mice perform independently of ACh input. The nicotinic acetylcholine receptors (nAChRs) antagonist dihydro-β-erythroidine hydrobromide (DHβE) modified evoked [DA]o in a frequency-dependent manner (), but similarly in both genotypes. Furthermore, in the absence of nAChR activity, the underlying range of [DA]o released by 4p at 100 Hz versus 1 Hz was a ~3-4-fold range in both genotypes (), consistent with a similar release probability in both genotypes. Therefore, the observed increase in dopamine releasability in CPu of triple synuclein null mice is not readily attributable to an upregulation of either nAChR function or underlying dopamine release probability. We assessed whether the higher evoked [DA]o despite reduced dopamine content in CPu of triple synuclein null mice may be due to a modified sensitivity of the exocytotic machinery to calcium. Varying extracellular calcium (in the presence of DHβE to eliminate confounding effects on Ca2+-dependent ACh release), modified [DA]o evoked by a single pulse in both genotypes as predicted, but in a manner that was not significantly different (). Furthermore, the ratio of [DA]o evoked by a burst (4p/100Hz) versus one pulse (4p:1p) decreased as expected with increasing [Ca2+]o but with no difference between genotypes () indicating no difference in the relative probability of dopamine release between genotypes.
Regulation of dopamine signaling by cholinergic input, calcium and uptake probability are similar in the CPu of wild type and triple synuclein null mutant mice
Increased levels of evoked dopamine in the dorsal striatum is not due to differences in uptake of dopamine
Functional interactions between α-synuclein and DAT have been suggested previously (Lee et al., 2001
; Wersinger and Sidhu, 2003
; Fountaine et al., 2008
). Therefore, we investigated whether higher evoked [DA]o
in TKO mice could be attributed to lower dopamine uptake. In extracellular dopamine transients that were matched for peak [DA]o
(1.0 ± 0.1 μM), the decay rate was similar in each genotype (). Because the decay phase of the dopamine transients is a function of dopamine uptake by DAT, this suggests the rate of reuptake was not different in TKO mice compared to wild type control mice.
Attenuated responses of TKO mice to psychostimulants
Our studies of TKO mice revealed a paradoxical combination of reduced striatal dopamine content but a hyperdopaminergic phenotype associated with a greater realisability but unchanged re-uptake of this neurotransmitter.
Additional data confirm that presynaptic dopamine stores are reduced in the striatum of mutant mice and also argue against a hypothesis of upregulation of postsynaptic transduction mechanisms rather than enhanced presynaptic dopamine releasability. We found that injection of 4 mg/kg dAMPH, which displaces dopamine stored in synaptic vesicles into extracellular space and whose effects depends on the size of presynaptic dopamine stores, but not regulated exocytosis, stimulated locomotor activity of TKO mice to a lesser and slower extent than of wild type () or single synuclein KO mice (our unpublished data), whereas this activity was significantly greater in mutants when animals were first placed in a novel non-anxiogenic environment. In contrast, the locomotor response of TKO mice to injection of 10 mg/kg cocaine, which blocks dopamine uptake but does not induce reverse transport of dopamine from presynaptic terminal stores, was similar to wild type mice (). The difference in the effect of dAMPH and cocaine on TKO mice supports the hypothesis that dopaminergic presynaptic terminals of these mice have reduced levels of the neurotransmitter stored in synaptic vesicles. Notably, boosting of presynaptic dopamine storage by injection of 50 mg/kg of methyl L-DOPA 20 minutes before placing animal in a novel non-anxiogenic environment did not change the behaviour of TKO mice prior to amphetamine injection but restored the level of their dAMPH-induced locomotor activity to the level of wild type mice ().
Behaviour of wild type and triple synuclein null mutant mice after pharmacological challenging of dopamine neurotransmission
Direct activation of postsynaptic D1/D2 dopamine receptors by apomorphine (APO), shows that a transiently induced characteristic “climbing” behaviour (Protais et al., 1976
) was slightly less marked in TKO mice than in wild type mice (), suggesting that postsynaptic dopamine signalling is in fact modestly attenuated in the absence of synucleins, which is similar to other mouse lines with a hyperdopaminergic phenotypes and probably represent mechanisms in which downregulation of postsynaptic D1/D2 heteroreceptors compensates for increased levels of dopamine in the synaptic cleft (Zhuang et al., 2001
). This result further supports the hypothesis that presynaptic mechanisms are responsible for hyperdopaminergic phenotype of TKO mice.
No gross ultrastructural changes of striatal dopaminergic axons in TKO mice
Quantitative electron microscopic analyses of dopaminergic profiles in the dorsal striatum of TKO and wild type mice performed on sections immunostained for TH using two techniques () indicated the overall density of TH-positive profiles, the number of mitochondria per structure and synaptic incidence were not different between two genotypes. Analysis of all TH-immunogold profiles, as well as the subgroup forming synaptic specialisations showed trends towards reduction in some parameters in TKO mice such as the cross-sectional area of TH-positive profiles (18.6% smaller in all profiles; 35.6% smaller in synapse-forming profiles), the number of synaptic vesicles per profile (6.2% and 38.5% less) and also in the average distance of a synaptic vesicle from the plasma membrane (5.2% and 14.4% smaller; ) but trends towards opposite outcomes in all versus synapse-forming structures for other parameters, for example for the density of vesicles (9.5% increase across all versus 14.3% decrease in synapse-forming) and the average inter-vesicle distance, an indication of clustering (24.8% decrease, , versus 3.0% increase). In TH-immunogold profiles that formed synapses the average distance of vesicles to the active zone was smaller in the mutant animals (22.4% decreased, ). However, differences in these or other studied parameters (data will be available online if the manuscript is accepted) were not statistically significant suggesting that the ultrastructure of dopaminergic synapses in the striatum is not substantially affected by the absence of synucleins.
Electron microscopic analyses of dopaminergic structures within the dorsal striatum in wild type and triple synuclein null mutant mice
Normal abundance of SNARE complexes in the dorsal striatum of TKO mice
Recent in vivo
studies demonstrated that synucleins might regulate neurotransmitter release by regulating synaptic SNARE complex assembly and/or distribution of SNARE proteins within synapses (Chandra et al., 2005
; Burre et al., 2010
; Garcia-Reitbock et al., 2010
). Analysis of total brain lysates revealed attenuated assembly of SNARE complexes in TKO mice, which was mirrored by a positive effect of α-synuclein overexpression on this process in cultured hippocampal neurons (Burre et al., 2010
). In contrast, accumulation of α-synuclein in striatal dopaminergic terminals of transgenic mice resulted in reduced dopamine release (Garcia-Reitbock et al., 2010
). Overall, no uniform pattern emerges from these studies, suggesting that different types of synapses may respond differently to changes in synucleins availability. To reveal whether the absence of synucleins affects SNARE complex assembly in the dorsal striatum we quantified amount of vSNARE VAMP2/synaptobrevin co-immunoprecipitated with tSNARE SNAP-25 from the lysates of freshly dissected dorsal striatum tissue and found no difference between wild type and TKO animals (). We also observed no changes in distribution of SNAP-25 or CSP, a co-chaperone of SNARE complex assembly, in the striatum of TKO mice (data will be available online if the manuscript is accepted).
Quantification of SNARE complexes in the dorsal striatum of wild type and triple synuclein null mutant mice