Alpha-synuclein overexpression in mice alters synaptic communication in the corticostriatal pathway

Abstract

alpha-Synuclein (alpha-Syn) is a presynaptic protein implicated in Parkinson's disease (PD). Mice overexpressing human wildtype (WT) alpha-Syn under the Thy1 promoter show high levels of alpha-Syn in cortical and subcortical regions, exhibit progressive sensorimotor anomalies, as well as non-motor abnormalities and are considered models of pre-manifest PD as there is little evidence of early loss of dopaminergic (DA) neurons. We used whole-cell patch clamp recordings from visually identified striatal medium-sized spiny neurons (MSSNs) in slices from alpha-Syn and WT littermate control mice at 35, 90 and 300 days of age to examine corticostriatal synaptic function. MSSNs displayed significant decreases in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in alpha-Syn mice at all ages. This difference persisted in the presence of tetrodotoxin, indicating it was independent of action potentials. Stimulation thresholds for evoking EPSCs were significantly higher and responses were smaller in alpha-Syn mice. These data suggest a decrease in neurotransmitter release at the corticostriatal synapse. At 90 days the frequency of spontaneous GABA(A) receptor-mediated synaptic currents was decreased in MSSNs but increased in cortical pyramidal neurons. These observations indicate that high levels of expression of alpha-Syn alter corticostriatal synaptic function early and they provide evidence for early synaptic dysfunction in a pre-manifest model of PD. Of importance, these changes are opposite to those found in DA-depletion models, suggesting that before degeneration of DA neurons in the substantia nigra synaptic adaptations occur at the corticostriatal synapse that may initiate subtle preclinical manifestations.

Figure 1

A. Representative traces showing spontaneous synaptic currents from MSSNs from 90 day WT and α-Syn mice in ACSF (V_hold=−70 mV). B. Glutamate receptor-mediated spontaneous EPSCs were isolated by bath applying BIC (20 µM). C. Spontaneous EPSCs were blocked by CNQX (10 µM), a non-NMDA receptor antagonist, demonstrating that they were mediated by non-NMDA ionotropic glutamate receptors. D. Amplitude-frequency distributions of spontaneous EPSCs in ASCF at 35 and 90 days, respectively. Insets show the mean frequency of spontaneous EPSCs. Note the lower frequency of events 6–14 pA in the α-Syn mice compared to the WTs at both ages. Asterisks indicate the mean frequency for WT and α-Syn mice were significantly different. In this and other figures * indicates p<0.05 and ** indicates p<0.01.

Figure 2

A1, B1. Amplitude-frequency distributions of spontaneous EPSCs at 35 and 90 days, respectively. Insets are the mean frequencies of spontaneous EPSCs. A2, B2. Cumulative amplitude distributions of events at each age. A3, B3. Cumulative inter-event distributions at each age. All data obtained in the presence of BIC. In this (A3 and B3) and other figures lines over distributions indicate all differences between bins from α-Syn and WTs are statistically significantly.

Figure 3

A1, B1. Bar graphs show the mean frequencies of mEPSCs and the mean percentage decreases in spontaneous EPSCs in cells at 35 and 90 days in the presence of TTX. A2, B2. Amplitude-frequency distributions of mEPSCs in TTX. Note the lower frequency of small amplitude events in α-Syn mice persisted in the presence of TTX. A3, B3. Cumulative amplitude probability distributions in TTX at each age. A4, B4. Cumulative inter-event interval probability distributions in TTX at each age.

Figure 4

Left graph shows amplitude-frequency distributions of spontaneous EPSCs at 300 days for MSSNs from α-Syn and WT mice. Insets are the mean frequencies of spontaneous EPSCs. Right graph shows cumulative inter-event interval probability distributions in TTX.

Figure 5

Left traces are means of spontaneous EPSCs with amplitudes 6–50 pA from 35 day WT and α-Syn MSSNs in the presence of BIC. Bar graphs show the peak amplitude and area were significantly increased whereas the decay time and duration at half-amplitude were similar in cells from α-Syn mice compared to those of WTs.

Figure 6

A. Spontaneous IPSCs mediated by activation of MSSN GABA_A receptors. V_hold=+20 mV in ACSF. B. BIC application eliminated the events, demonstrating they were mediated by GABA_A receptors. C1. Amplitude-frequency distributions of spontaneous IPSCs were similar in the cells from 35 day WT and α-Syn mice. Insets are the mean frequencies. D1. Amplitude-frequency distributions of spontaneous EPSCs in ASCF at 90 days. Insets show the mean frequencies of spontaneous EPSCs. Note the lower frequencies of events 10–70 pA in the α-Syn mice compared to the WTs. C2, D2. Cumulative amplitude probability distributions were similar between the two genotypes at both ages. C3, D3. Cumulative inter-event interval probability distributions were similar at 35 days but were significantly different at 90 days.

Figure 7

A. Spontaneous IPSCs mediated by activation of GABA_A receptors in cortical pyramidal neurons were recorded by holding the membrane at +20 mV in standard ACSF. Inset shows bursting from the same cell at a faster time scale. B. BIC application eliminated the events, demonstrating they were mediated by GABA_A receptors. C. Left bar graph shows that mean frequency of spontaneous IPSCs was higher in cells from α-Syn mice compared to WTs, but the difference was not statistically significant. Right graph shows amplitude-frequency distributions of spontaneous IPSCs. Significant differences between α-Syn and WT mice occurred in events of 20–30 pA. D. Cumulative amplitudes probability distributions were similar. E. Cumulative inter-event interval probability distributions were significantly different for short duration events.

Figure 8

A. Representative non-NMDA receptor-mediated EPSCs evoked by an increasing series of stimulation intensities and recorded in MSSNs from α-Syn and WT mice at 90 days in ACSF in the presence of PIC (100 µM). B. Mean stimulation threshold was significantly higher in α-Syn mice than in WTs. C. Mean peak EPSC amplitudes were significantly reduced in α-Syn mice compared to those of WTs. D. Mean EPSC areas were significantly reduced in α-Syn mice compared to those of WTs.

Figure 9

A. Representative NMDA receptor-mediated EPSCs evoked by an increasing series of stimulation intensities and recorded from MSSNs from α-Syn and WT mice at 90 days in ACSF in the presence of CNQX (10 µM) and PIC (100 µM). B. Mean stimulation threshold was significantly higher in α-Syn mice than in WTs. C. Mean peak EPSC amplitudes were significantly reduced in α-Syn mice compared to those of WTs. D. Mean EPSC areas were significantly reduced in α-Syn mice compared to those of WTs. E. Mean EPSC half-amplitude durations were significantly shorter in α-Syn mice than in WTs. F. Graph shows mean peak non-NMDA receptor-mediated EPSC amplitudes evoked by an increasing series of stimulation intensities and recorded in MSSNs from α-Syn and WT mice at 300 days in ACSF in the presence of BIC (20 µM). Mean peak EPSC amplitudes were significantly reduced in α-Syn mice compared to those of WTs at stimulation intensities of 0.25 to 0.6 mA. G. Graph shows mean peak NMDA receptor-mediated EPSCs evoked by an increasing series of stimulation intensities and recorded from MSSNs from α-Syn and WT mice at 300 days in ACSF in the presence of CNQX (10 µM) and BIC (20 µM). Mean peak EPSC amplitudes were significantly reduced in α-Syn mice compared to those of WTs at stimulation intensities of 0.2 to 0.6 mA.

Figure 10

A. Graphs show mean NMDA concentration-response function obtained from acutely dissociated MSSNs from 90 day α-Syn and WT mice in Mg²⁺-free ACSF in the presence of TTX (0.3 µM). B. Graphs show mean AMPA-CTZ concentration-response function obtained from acutely dissociated MSSNs from 90 day α-Syn and WT mice in Mg²⁺-free ACSF in the presence of TTX (0.3 µM).