LRRK2‐G2019S mice display alterations in glutamatergic synaptic transmission in midbrain dopamine neurons

Abstract

The progressive degeneration of dopamine (DA) neurons in the substantia nigra compacta (SNc) leads to the emergence of motor symptoms in patients with Parkinson's disease (PD). To propose neuroprotective therapies able to slow or halt the progression of the disease, it is necessary to identify cellular alterations that occur before DA neurons degenerate and before the onset of the motor symptoms that characterize PD. Using electrophysiological, histochemical, and biochemical approaches, we have examined if glutamatergic synaptic transmission in DA neurons in the SNc and in the adjacent ventral tegmental area (VTA) was altered in middle‐aged (10–12 months old) mice with the hG2019S point mutation (G2019S) in the leucine‐rich repeat kinase 2 (LRRK2) gene. G2019S mice showed increased locomotion and exploratory behavior compared with wildtype (WT) littermates, and intact DA neuron integrity. The intrinsic membrane properties and action potential characteristics of DA neurons recorded in brain slices were similar in WT and G2019S mice. Initial glutamate release probability onto SNc‐DA neurons, but not VTA‐DA neurons, was reduced in G2019S mice. We also found reduced protein amounts of the presynaptic marker of glutamatergic terminals, VGLUT1, and of the GluA1 and GluN1 subunits of AMPA and NMDA receptors, respectively, in the ventral midbrain of G2019S mice. These results identify alterations in glutamatergic synaptic transmission in DA neurons of the SNc and VTA before the onset of motor impairments in the LRRK2‐G2019S mouse model of PD.

Neuronal Firing and Glutamatergic Synapses in the Substantia Nigra Pars Reticulata of LRRK2-G2019S Mice

“…Cells that did not meet these criteria, or that met criteria for DA neurons were not further recorded and were not included in the analyses. The recorded neurons are likely to be GABAergic, and not DA, given that their electrophysiological properties were similar to those described in previous studies [ 6 , 13 , 17 , 18 , 19 , 20 , 21 ], and that the GABAergic nature of recorded neurons in the SNr was confirmed by histochemical and molecular analyzes [ 22 , 23 , 24 ]. For voltage-clamp recordings, patch electrodes (3–5 MΩ) were filled with a solution containing (in mM): 140 CsCl, 2 MgCl 2 , 1 CaCl 2 , 10 HEPES, 10 EGTA, 2 MgATP, 0.3 Na 3 GTP, pH adjusted to 7.3 with CsOH.…”

“…Further, in LRRK2-G2019S mice analyzed at postnatal day 21, increased glutamatergic synaptic transmission onto spiny projection neurons was accompanied by hyperexcitability, and these changes were blocked by an LRRK2 kinase inhibitor suggesting their dependency on altered LRRK2 function [ 12 ]. In line with this suggestion, we recently demonstrated that acute treatment with an LRRK2 kinase inhibitor recovered the altered glutamate release on SNc-DA neurons and the increased sEPSC amplitude in DA neurons from the ventral tegmental area to levels similar to those measured in WT mice [ 13 ].…”

“…Next, we examined glutamatergic synapses, and found that the probability of evoked glutamate release was mildly increased in the SNr of G2019S mice. The present study on the SNr is complementary to our recently published work where we examined the expression of glutamatergic markers in the midbrain, as well as the electrophysiological properties and glutamatergic synapses of midbrain DA neurons of middle-aged WT and G2019S mice [ 13 ]. The glutamatergic innervations of the SNc and the SNr are partially segregated: both nuclei receive glutamatergic innervation from the subthalamic nucleus and the pedunculopontine nucleus, whereas the SNr also receives glutamatergic inputs from the mesencephalic locomotor region [ 19 ] and a newly characterized cortical projection [ 29 ].…”

“…These mice were obtained from The Jackson Laboratory (C57BL/6J-Tg(LRRK2-G2019S)2AMjff/J, JAX stock #018785; RRID:IMSR_JAX:018785) and were mated as Noncarrier x Hemizygote. Several studies have previously characterized and validated these mice and demonstrated that the observed neuronal alterations resulted from an increased LRRK2 kinase activity [ 9 , 10 , 11 , 13 , 16 ]. Mice were housed in small groups (2–5 per cage, IVC Mouse – GM500) in a humidity-controlled room with a 12:12 hours light/dark cycle and had free access to food and water.…”

“…In particular, earlier electrophysiological studies demonstrated altered glutamatergic transmission and plasticity in the striatum, the input nucleus of the basal ganglia which expresses high amounts of LRRK2 [ 3 , 12 ]. In a recent study, we demonstrated that glutamatergic synaptic transmission is altered in SNc-DA neurons in G2019S mice (the same type of mice as those used in the present study) when compared with WT mice, before behavioral and neurochemical impairments occur, i.e., in middle aged (10–12 months old) mice [ 13 ]. Because LRRK2 is also expressed in brain regions that provide glutamatergic inputs to the SNr, such as the cortex [ 14 ], altered LRRK2 function in glutamatergic synapses onto SNr GABAergic neurons might contribute to G2019S-linked PD pathology.…”