Elsevier

Experimental Neurology

Volume 287, Part 1, January 2017, Pages 21-33
Experimental Neurology

Research Paper
Functional alterations of the dopaminergic and glutamatergic systems in spontaneous α-synuclein overexpressing rats

https://doi.org/10.1016/j.expneurol.2016.10.009Get rights and content

Highlights

  • α-syn accumulates in the mesencephalon, striatum and frontal cortex of m/m rats.

  • SNpc DAergic neurons from m/m rats show normal functional activity.

  • The number of SNpc DAergic neurons from m/m rats is 9% reduced.

  • Dopamine and glutamate release in the striatum from m/m rats is impaired.

  • Loss of striatal DAT- and VGluT1-expressing terminals occurs in m/m rats.

Abstract

The presence of α-synuclein (α-syn) in Lewy bodies and Lewy neurites is an important characteristic of the neurodegenerative processes of substantia nigra pars compacta (SNpc) dopaminergic (DAergic) neurons in Parkinson's disease (PD) and other synucleinopathies.

Here we report that Berlin-Druckrey rats carrying a spontaneous mutation in the 3′ untranslated region of α-syn mRNA (m/m rats) display a marked accumulation of α-syn in the mesencephalic area, striatum and frontal cortex, accompanied to severe dysfunctions in the dorsolateral striatum. Despite a small reduction in the number of SNpc and ventral tegmental area DAergic cells, the surviving dopaminergic neurons of the m/m rats do not show clear-cut alterations of the spontaneous and evoked firing activity, DA responses and somatic amphetamine-induced firing inhibition. Interestingly, mutant DAergic neurons display diminished whole-cell Ih conductance and a reduced frequency of spontaneous excitatory synaptic currents. By contrast, m/m rats show a severe impairment of DA and glutamate release in the dorsolateral striatum, as revealed by amperometric measure of DA currents and by electrophysiological recordings of glutamatergic synaptic events in striatal medium spiny neurons. These functional impairments are paralleled by a decreased expression of the DA transporter and VGluT1 proteins in the same area. Thus, together with α-syn overload in the mesencephalic region, striatum and frontal cortex, the main functional alterations occur in the DAergic and glutamatergic terminals in the dorsal striatum of the m/m rats.

Introduction

Pathological misfolding and aggregation of alpha-synuclein (α-syn) has been genetically linked to familial forms of PD (Polymeropoulos et al., 1997, Krüger et al., 1998, Spillantini et al., 1998, Zarranz et al., 2004), as well as, other related neurodegenerative diseases, which are collectively known as `synucleinopathies'. This term besides PD, also includes dementia with Lewy bodies and multisystem atrophy (Surguchov, 2008, Barker and Williams-Gray, 2016). The latter is mainly characterized by α-syn aggregates in oligodendroglia (Jellinger, 2014). In normal conditions, α-syn is abundant in synaptic terminals where it could modulate vesicle trafficking and neurotransmitter release (Maroteaux et al., 1988, Bellucci et al., 2012, Anwar et al., 2011, Senior et al., 2008, Yavich et al., 2005). On the other hand, misfolded α-syn may exert toxic functions (Norris et al., 2004, Cookson and van der Brug, 2008, Bellucci et al., 2015) or cause loss of defensive properties (da Costa et al., 2000). Accordingly, recent evidence indicates that oligomeric α-syn, forming fibrils found also extracellularly (Martin et al., 2012), is the toxic species that could start the neuropathological processes (Cookson and van der Brug, 2008, da Costa et al., 2000).

Animal models aimed to determine the physiological and pathological role of this protein in the DAergic nigrostriatal neurons have recently been developed but, to date, there is no clear information on the functional alterations caused by α-syn accumulation at somatic, dendritic and terminal level of SNpc DAergic cells. α-syn participates to the synaptic physiology of the DAergic cells (Bellucci et al., 2012, Bellucci et al., 2015), thus, mice lacking α-syn display an alteration in the modalities of DA release, being decreased the refilling of the vesicular content of this catecholamine in readily releasable pools (Chadchankar et al., 2012, Abeliovich et al., 2000).

On the other hand, α-syn over-expression determines less excitatory synaptic vesicle exocytosis in cultured DAergic neurons, which release glutamate beside DA (Nemani et al., 2010). In addition, insertion of a truncated form and increased expression of α-syn in the nigrostriatal system of transgenic mice diminishes vesicle density and reduces DA release (Garcia-Reitböck et al., 2010). This results in a certain degree of motor impairment (Tofaris et al., 2006, Gaugler et al., 2012).

Concerning cellular integrity, it has been reported that an increased production of α-syn by viral infection causes DAergic cell loss (Lundblad et al., 2012, Gaugler et al., 2012). Of note, bacterial artificial chromosome (BAC) transgenic rats expressing the human full-length wild-type α-syn, develop intracellular α-syn-containing insoluble precipitates which cause alteration of the DAergic system. Thus, these animals show early changes in novelty-seeking, avoidance and smell, followed, at later stages, by progressive motor deficits closely resembling human PD (Nuber et al., 2013).

Recently, a spontaneous autosomal recessive rat model has been discovered, characterized by multisystemic neurodegeneration that rapidly recapitulates some pathological features of PD and possibly, dementia with Lewy bodies (Stoica et al., 2012). During the first weeks of age, these rats display an increase in α-syn in the whole brain, modifications of dendritic spines in striatal medium spiny neurons and a severe loss of tyrosine-hydroxylase positive terminals in the striatum. These pathological aspects could be linked to the presence of a point mutation in the 3′ untranslated region (UTR) of the α-syn mRNA that may increase or deregulate transcription of the protein (Stoica et al., 2012).

In the attempt to assess if the morphological and biochemical modifications occurring in this rat model of neurodegenerative disease are linked to functional alterations in the basal ganglia circuitry and, particularly, of the nigrostriatal DAergic and cortico-striatal glutamatergic systems, here we performed in vitro electrophysiological and amperometric recordings plus stereological cell count of TH-stained neurons in the midbrain. Additionally, we quantified the expression levels of dopamine and vesicular glutamate transporters 1 and 2 in striatal synaptic terminals. We found that at advanced stages of the disease besides abnormal α-syn accumulation in the mesencephalic area, striatum and frontal cortex, there is a severe functional alteration of the DAergic and glutamatergic neurotransmission within the dorsal striatum, whereas only minor abnormalities are found in the mesencephalic area.

Section snippets

Animals

All experiments were carried out in accordance with the international guidelines on the ethical use of animals from the EU Directive 2010/63/EU for animal experiments and the Ethics Committee of the University of Tor Vergata, Rome, Italy. The spontaneously inherited autosomal recessive rat model for neurodegeneration was provided by Prof. Stoica, Texas AM University (Stoica et al., 2012). Affected offspring (m/m) were identified by the gray color of their head coat and by the appearance of

Intrinsic membrane properties of SNpc DAergic neurons

Whole cell patch clamp recordings of SNpc DAergic neurons were performed to assess possible differences in active and passive membrane properties between cells obtained from +/+ and m/m rats during disease progression, from P15 to P27.

All neurons from both animal groups displayed similar spontaneous firing activity (1.96 ± 0.14 Hz, N = 31, +/+; 1.88 ± 0.14 Hz, N = 34, m/m, P = 0.66; Fig. 1 A) and cell capacitance (99.02 ± 4.35 pF, N = 42 +/+, 99.32 ± 4.12 pF, N = 47 m/m, P = 0.99). Since no age-related differences of

Discussion

The present results indicate that the motor abnormalities previously described in the spontaneous α-synuclein overexpressing rats (Stoica et al., 2012) marginally depend from a dysfunction of DAergic neurons within the SNpc but principally from severely impaired DA and glutamate release in the dorsal striatum. In fact, our electrophysiological investigations in the ventral mesencephalon demonstrated that, even at advanced stages of the disease, SNpc DAergic neurons appear rather “intact”,

Conclusions

As already reported for other animal models of synucleopatic diseases, the damage to striatal DAergic terminals in the m/m rats is far more extensive than the alterations of DAergic neurons in the SNpc (Cheng et al., 2010).

In this spontaneous mutated rat strain, the consistent diminution of striatal DA and glutamate release implies that accumulation of α-syn causes a constant and heavy impairment of the nigro- and cortico-striatal pathways that very likely take part in the poor life outcome.

Acknowledgements

We are grateful to Prof. Stoica for providing the animals and for his supervision to α-syn measurements in the rat brain areas. This work was supported by grants from the Italian Ministry of Health to N.B.M. (Ricerca Corrente 2013-15), to M.D.A. (Project Code GR-2011-02351457) and to M.T.V. (Project Code GR-2010-2310524). The authors have no conflict of interest to disclose.

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