Abnormal Development of Glutamatergic Synapses Afferent to Dopaminergic Neurons of the Pink1−/− Mouse Model of Parkinson’s Disease

In a preceding study, we showed that in adult pink1−/− mice, a monogenic animal model of Parkinson’s disease (PD), striatal neurons display aberrant electrical activities that precede the onset of overt clinical manifestations. Here, we tested the hypothesis that the maturation of dopaminergic (DA) neurons of the pink1−/− substantia nigra compacta (SNc) follows, from early stages on, a different developmental trajectory from age-matched wild type (wt) SNc DA neurons. We used immature (postnatal days P2–P10) and young adult (P30–P90) midbrain slices of pink1−/− mice expressing the green fluorescent protein in tyrosine hydroxylase (TH)-positive neurons. We report that the developmental sequence of N-Methyl-D-aspartic acid (NMDA) spontaneous excitatory postsynaptic currents (sEPSCs) is altered in pink1−/− SNc DA neurons, starting from shortly after birth. They lack the transient episode of high NMDA receptor-mediated neuronal activity characteristic of the immature stage of wt SNc DA neurons. The maturation of the membrane resistance of pink1−/− SNc DA neurons is also altered. Collectively, these observations suggest that electrical manifestations occurring shortly after birth in SNc DA neurons might lead to dysfunction in dopamine release and constitute an early pathogenic mechanism of PD.


INTRODUCTION
Dopaminergic (DA) neurons of the substantia nigra compacta (SNc) degenerate progressively during the course of Parkinson's disease (PD; Kordower et al., 2013). Whether and when they dysfunction during the early stages of PD before degenerating remains largely unknown. Addressing this question requires progressive animal models of PD, like genetic models of familial forms of PD. Particularly interesting are pink1-deficient mice, which do not express the PTENinduced kinase 1 (pink1), a ubiquitously expressed mitochondrial kinase consisting of 581 amino acids that encode for a mitochondrial targeting sequence, a transmembrane domain and a Ser/Thr kinase domain (Silvestri et al., 2005;Gandhi et al., 2006;Zhou et al., 2008;Gispert et al., 2009). Pink1 is a mitochondrial quality control factor with functions in repair, fission and autophagic elimination (Deng et al., 2008;Poole et al., 2008;Yang et al., 2008;Gehrke et al., 2015). Pink1 induces mitophagy. Upon mitochondrial damage, pink1 is stabilized on the outer mitochondrial membrane, where it phosphorylates ubiquitin and activates the ubiquitin ligase parkin. This builds ubiquitin chains on mitochondrial outer membrane proteins and leads to removal of damaged mitochondria by autophagy (Narendra et al., 2010;Koyano et al., 2014;Kazlauskaite and Muqit, 2015;Lazarou et al., 2015;Ordureau et al., 2015).
Pink1-deficient mice are a model of the PARK6 variant of PD (mutations in PINK1), the second most common cause of autosomal recessive familial early-onset PD (Bentivoglio et al., 2001;Valente et al., 2004;Bonifati et al., 2005;Gasser, 2009). In this model, the first conspicuous motor abnormalities are manifest at 16 months, offering a large time window to explore earlier subclinical abnormalities (Gispert et al., 2009). During this window, profound dysfunction is observed in the basal ganglia system. Though pink1 −/− DA SNc neurons are preserved during the murine lifespan, they progressively show mitochondrial dysfunction, impaired intracellular calcium signaling leading to functional reduction of the activation of the small K (SK) calcium-activated channels, irregular firing and a greater tendency to fire bursts of action potentials by 1-4 months (Gispert et al., 2009;Bishop et al., 2010). In addition, evoked dopamine release is reduced in the striatum, and corticostriatal synaptic plasticity is disrupted by 3 months. Half the striatal projection neurons (medium spiny neurons, MSNs) show aberrant morphology and generate giant GABAergic currents by 3-6 months. These constitute an electrophysiological signature of dopamine depletion and PD, since they are reversed by chronic levodopa administration or subthalamic nucleus (STN) lesion used to treat PD (Kitada et al., 2007;Gautier et al., 2008;Dehorter et al., 2009Dehorter et al., , 2012Gispert et al., 2009;Wang et al., 2011).
These observations suggest that early sub-clinical manifestations can occur, possibly leading to very early deviations in developmental processes that culminate progressively in motor disturbances. A wide range of experiments suggest that ionic currents, like the brain patterns they generate, follow developmental sequences playing specific roles in the developing brain, which when deviated lead to long-term neurological deleterious sequels (Ben-Ari, 2008). Since α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) receptor activation generates transient high-frequency activity in SNc DA neurons, alterations of their properties during development could produce long-term alterations in SNc DA neurons. Our finding that the developmental sequence of NMDA currents and membrane resistance are altered in pink1 −/− SNc DA neurons could explain dysfunction in dopamine release later in adulthood (Schmitz et al., 2009).

MATERIALS AND METHODS
All experiments were approved by the Institut National de la Santé et de la Recherche Médicale (INSERM) animal care and use agreement (D-13-055-19) and the European community council directive (2010/63/UE). Animals had access to food and water ad libitum and were housed in our institutional animal facilities under a 12 h light/dark cycle at 22-24 • C.

Electrophysiology
All recordings were made in whole-cell voltage-clamp configuration. R m and C m were measured during a 800 ms/−10 mV step from holding potential (−60 mV). R m was calculated using the following formula: R m = ∆V(1/∆I slow − 1/∆I max ), where ∆V is the amplitude of the voltage step, ∆I max is the difference between holding current value and the peak intensity reached by the capacitive current at the start of the hyperpolarizing pulse, and ∆I slow is the difference in current intensity between holding current value and that of the steadystate current measured after the capacitive current. C m was calculated as: C m = τ w /R m where τ w is the weighted membrane time constant (Marcaggi et al., 2003). We measured Ih amplitude by subtracting the amplitude of the current at the end of the 800 ms hyperpolarizing step to −140 mV (V H = −60 mV) from the amplitude of the current 15 ms after the first capacitative current. We measured spontaneous AMPA/Kainate (KA) currents in voltage-clamp mode at V H = −60 mV in the continuous presence of Gabazine (5 µM) to block GABA A receptors. We measured spontaneous NMDA currents in voltage-clamp mode at V H = +40 mV in the continuous presence of Gabazine (5 µM) and NBQX (10 µM) to block GABA A and AMPA/KA receptors, respectively. These currents were stored on a computer using Pclamp8 software (Molecular Devices) and analyzed off-line with a Mini Analysis software (Synaptosoft 6.0), to determine the inter-event intervals (IEIs), amplitude, rise time and decay time of spontaneous currents. The decay of spontaneous synaptic currents was well fitted by a single-exponential function, starting at the peak of the current to the time point when the current had decayed to 99.9% of its peak amplitude. All detected currents were then visually inspected to reject artifactual events. NMDA spontaneous excitatory postsynaptic currents (sEPSCs) occurred either as single events or in bursts. We defined a burst of NMDA sEPSCs as the occurrence of at least three superimposed NMDA sEPSCs and a bursty pattern as at least two bursts/cell/3 min. Miniature AMPA or NMDA currents, recorded in the presence of Tetrodotoxin (TTX; 1 µM), were not studied because they had an extremely low frequency at all ages tested.

Statistics
Results are given as mean ± standard error of the mean. The non-parametric Mann-Whitney test (Graphpad Prism 6 software, San Diego, CA, USA) was used to compare results from Pink1 −/− to wt SNc DA neurons (at P2-3, P4-10 and P30+). Since amplitude, IEI, rise time and decay time of sEPSCs were not normally distributed, we also calculated the median value of these parameters (± standard error of the mean of medians) for each cell (data not shown). Statistical significance was not different when comparing means or medians. We pooled the results obtained between P4 and P10 because there was no significant difference between the full set of results obtained at P4-5 and at P8-10 for AMPA-and NMDA-mediated sEPSCs (data not shown). For example, there was no significant difference in frequency (P = 0.3; Mann-Whitney test), and amplitude (P = 0.5; Mann-Whitney test) of AMPA sEPSCs. Similarly, there was no significant difference in IEI, (P = 0.35; Mann-Whitney test) and amplitude (P = 0.52; Mann-Whitney test) of single NMDA sEPSCs. We used the χ 2 or Fisher's exact test to compare proportions. For each test performed, the P value was provided and the statistical significance was set at P ≤ 0.05. In all figures: * P < 0.05, * * P < 0.01 and * * * P < 0.001.

The wt Developmental Sequence of NMDAR-Mediated Spontaneous Currents is Abolished in Pink1 −/− SNc DA Neurons
In this set of experiments, we first observed at P4-10 a drastic change in the developmental sequence of NMDA sEPSCs in pink1 −/− compared to wt SNc DA neurons. To determine whether this developmental sequence had shifted towards younger ages, we recorded NMDA sEPSCs at P2-3 in both wt and mutated mice.

DISCUSSION
The main result shown here is that pink1 −/− SNc DA neurons lack the characteristic wt developmental sequence of NMDA sEPSCs identified between P2 and P10, which consists of large amplitude and high frequency events associated to a characteristic bursting pattern. The absence of this high NMDA activity at immature stage drastically changes the developmental profile of NMDA sEPSCs in pink1 −/− SNc DA neurons. This may have consequences on calcium influxes into developing dendrites and somas. In contrast, there is a similar developmental profile of AMPA sEPSCs, i.e., immature AMPA sEPSCs with larger amplitudes and longer IEIs than young adult ones, in pink1 −/− and wt (Pearlstein et al., 2015) SNc DA neurons. This abnormal skip in the NMDA developmental sequence recorded in vitro in coronal slices most probably concerns glutamatergic synapses between midbrain glutamatergic neurons located in the Ventral tegmental area (VTA) and/or in the SNc and SNc DA neurons. Maturation of the morphological properties of wt SNc DA neurons is achieved during the very early prenatal period in mice (Ferrari et al., 2012), and rats (Tepper et al., 1994;Park et al., 2000). We found comparable morphological maturation for pink1 −/− SNc DA neurons, with their dendritic tree already mature at P4-10. In contrast, intrinsic membrane properties developed differently. The membrane resistance of pink1 −/− SNc DA neurons did not diminish between P4-10 and P30+ in the manner observed for wt neurons (Pearlstein et al., 2015), suggesting the maintenance of immature features in pink1 −/− SNc DA neurons. This paves the way for alterations in both intrinsic and evoked activities in pink1 −/− SNc DA neurons, as high input resistance facilitates the generation of oscillations. To date, the nature of the intrinsic current underlying these changes has not been determined. Cationic H channels do not appear to be involved: the amplitude of I h at P30+ is similar. Further experiments are required to shed light on these differences, which are clearly not the consequence of morphological features, since dendritic arborizations and soma surfaces were similar in wt and pink1 −/− SNc DA neurons.
The absence of large and frequent NMDA events with a bursty pattern at immature stage (P2 to P10) was the striking difference between pink1 −/− and wt SNc DA neurons. Even later, at young adult stage (P30-P90), synaptic NMDA activity remained limited, with smaller and less frequent NMDA sEPSCs. The absence of bursts of NMDA sEPSCs and the lack of effect of DQP 1105, a preferential antagonist of NR2D-containing NMDA receptors, confirm that the glutamatergic synapses afferent to pink1 −/− SNc DA neurons studied here in coronal slices are different from the wt ones. In particular the presence of bursts in control SNc DA neurons was correlated to the presence of slowly-decaying NMDA events. This strongly suggests that the dynamic remodeling of NMDA receptor subunit composition described in midbrain regions (Monyer et al., 1994;Dunah et al., 1996;Wenzel et al., 1996;Laurie et al., 1997;Liu and Wong-Riley, 2010) during postnatal development, with GluN2D subunits no longer present in young adult wt mice, is altered in pink1 −/− SNc DA neurons, at least for the glutamatergic synapses studied here (see paragraph below). We propose that from P2 to P90, the NMDA sEPSCs of pink1 −/− SNc DA neurons that we recorded in the present study result from the activation of diheteromeric GluN1/GluN2B receptors (Dunah et al., 1998;Jones and Gibb, 2005;Brothwell et al., 2008;Suarez et al., 2010;Huang and Gibb, 2014), since all NMDA sEPSCs were antagonized by Ro 25-6981 but not by DQP 1105.
The most abundant afferent glutamatergic inputs to SNc DA neurons come from the subthalamic and pedunculopontine nuclei (Hammond et al., 1978(Hammond et al., , 1983Kita and Kitai, 1987;Smith and Grace, 1992;Iribe et al., 1999;Ammari et al., 2009). However, given that in the presence of TTX, ongoing NMDA receptor-mediated sEPSCs were absent, AMPA and NMDA spontaneous events were generated by afferent action potential firing, implying that somas of afferent glutamatergic inputs were present in the slice. In coronal slices, STN or pedunculopontine somas are absent, which makes the VTA (A10) and SNc (A9) the most likely candidate glutamatergic neuronal sources (Kawano et al., 2006;Yamaguchi et al., 2007Yamaguchi et al., , 2011Yamaguchi et al., , 2013Stuber et al., 2010;Tecuapetla et al., 2010;Zhang et al., 2015). Interestingly, several studies linked polymorphisms of the GRIN2B gene (GluN2B in rodents) to the increased risk of impulse control behavior in PD patients under DA treatment (Lee et al., 2009;Zainal Abidin et al., 2015). Summing up, our previous studies in pink1 −/− mice showed an excess of cortical synchronization at immature stage (P15-20), followed by giant GABAergic currents in striatal MSNs (3-6 month-old) that are reversed either by STN high frequency stimulation (Carron et al., 2014), or chronic STN lesion or levodopa treatment . These features are therefore relevant to PD and might provide biological markers of the early stages of the disease, which in this model is known to begin at 16 months (Gispert et al., 2009). This would correspond to very early insults in human brain development since postnatal days 4-10 in mice correspond in humans to the last trimester of gestation and P20 to delivery.
The underlying ''neuroarcheology'' concept (Ben-Ari, 2008) has now been confirmed in several developmental disorders in which genetic mutations like early environmental insults have been shown to impact the development of brain networks. We propose that the pink1 mutation deviates the developmental sequence of NMDA currents at a very early stage, leading to altered formation of functional neuronal ensembles that could later culminate in clinical manifestations. Preclinical nigrostriatal dysfunction has been previously identified in cotwins with idiopathic PD and in asymptomatic carriers of a single mutant PARK6 allele. Some of these subjects developed parkinsonian signs a few years later (Piccini et al., 1997(Piccini et al., , 1999Khan et al., 2002). Presynaptic DA dysfunction may be a central pathogenic precursor of PD, before leading to frank loss of nigral DA neurons (Shen, 2010).

AUTHOR CONTRIBUTIONS
EP, FJM, LS and DF performed the experiments. EP and CH analyzed the data. CH designed the study and wrote the article.

ACKNOWLEDGMENTS
Authors acknowledge the Gispert/Auburger team at the university Frankfurt am Main for providing the pink1-KO mice, discussing the data and their critical reading of the manuscript. We are grateful for the help of G. Chazal for immunocytochemistry and the support of the animal house facility headed by S. Pellegrino.