α -Synuclein Responses in the Laterodorsal Tegmentum,the Pedunculopontine Tegmentum,and the Substantia Nigra: Implications for Early Appearance of Sleep Disorders in Parkinson’s Disease

Recombinant α-syn and size exclusion chromatography (SEC)

Human α-syn was recombinantly expressed and purified as previously described [26] with few modifications. In brief, α-syn was cloned into E. Coli BL21DE3 cells using a pET-11a vector construct. Harvested cells were lysed by osmotic shock and non-heat-stable proteins were removed by boiling and centrifugation. α-syn was isolated by ion-exchange chromatography and the monomer was isolated by SEC. Monomer fractions from SEC were pooled and kept in PBS buffer stored at –80°C.

Fibrillation assay

α-syn in PBS buffer (1.5–2.2 mg/ml) was fibrillated in a microplate reader (FLUOstar Omega. BMG Labtech) at 37°C with 3 mm sterile glass beads and orbital shaking (700 rpm for 280 s in each 360 s cycle). Samples were aliquoted (150μl) in a NUNC 96-well optical polymer-based, clear-bottom black plate (Thermo Fisher scientific 265301) and sealed with clear polyolefin tape (Thermo Fisher scientific 232702). Thioflavin T (ThT; 20μM) was added to three samples, retaining in parallel non-ThT-containing samples for the electrophysiology experiments. ThT emission was recorded at 480±10 nm upon excitation at 450±10 nm. Fibril samples were collected after 7 days, to ensure full fibril maturation, and thereafter stored at room temperature. Although sonication is often used for preparation of pre-formed fibrils, we chose to work with mature fibril formed after 7 days of fibrillation to ensure the stability of the structural states and reproducibility of samples. ThT fluorescence for the 7 day fibrillation assay are shown in Fig. 1 as the mean of a triplicate run, with insert detailing the first 24 h.

Circular dichroism (CD)

The secondary structure of α-syn samples was investigated to confirm monomeric and fibrillar states. CD was measured on monomer and fibrillated α-syn samples, respectively, using a JASCO J-1500 CD spectrometer. Samples were diluted to a final concentration of 0.15 mg/ml (10μM) and placed in a cuvette with a light path of 1 mm. Spectra were collected from 260–190 nm, with a bandwidth of 1 nm, scanning speed of 100 nm/min, and a temperature of 25°C. The final spectra were made as accumulations of three. In Fig. 1, CD spectra of monomeric and fibrillated α-syn are shown indicating random coil structure for monomeric α-syn, consistent with the native, intrinsically disordered form, and β-sheet structure after fibrillation.

Animals

Male NMRI mice (Harlan Mice Laboratories, Denmark) aged 12 to 30 days were used under approved protocols in accordance with European Communities Council Directive (86/609/EEC). At birth, the animals were fostered and housed with a lactating female under controlled temperature (22–23°C) and 45–65% humidity conditions in a 12 : 12 h light-dark cycle, with water and food available ad libitum. At postnatal day 21, the animals were weaned, and group-housed with their same-sex siblings.

Tissue preparation for electrophysiology, calcium imaging and neurodegeneration studies

Following deep anesthesia with inhalation of isoflurane (Baxter A/S, Denmark), a block of the brain containing the LDT, PPT, or SN was excised (Fig. 1A). The brain block was sectioned coronally into 250μm slices using a vibratome (Leica VT1200S, Leica Biosystems, Germany) in ice-cold artificial cerebrospinal fluid (ACSF). The standard ACSF contained (in mM) 124 NaCl, 5 KCl, 1.2 Na₂HPO₄•2H₂O, 2.7 CaCl₂•2H₂O, 1.2 MgSO₄ (anhydrous), 10 dextrose, 26 NaHCO₃ at pH 7.4 and an osmolarity of 298–302 mOsm/kg and was saturated with carbogen (95% O₂/5% CO₂). Within the coronal brain slices, identification of the LDT, PPT and SN for electrophysiology, calcium imaging and neurotoxicity assays was conducted using standard landmarks as shown in Fig. 1. Specifically, the LDT was identified as positioned just lateral to the dorsal tegmentum and ventral to the aqueduct, the PPT was identified by the superior cerebral peduncle, and the SN was identified by the cerebral peduncle. In a subset of slices, immunohistochemistry or electrophysiological protocols confirmed identification of the nuclei.

α-syn, lysozyme, AMPA preparation and application

The highly purified monomeric form of α-syn was stored in solution at –80°C in aliquots of 10μl (150μM) until use. Aliquots (150μM) of mature α-syn fibrils were kept at room temperature and used within a maximum of 2 weeks and all efforts were made to treat the solution gently in order to minimize shifts in structural states. In initial experiments, we found that 70 nM of α-syn induced a short-duration, membrane response; whereas, in the same cell, 100 nM induced a longer-lived shift in current amplitude, therefore, we decided to continue with 100 nM, which had been used in other studies [27] (see Fig. 1). The final concentration of α-syn diluted in ACSF was applied for 3–4 min after the establishment of baseline conditions. As lysozyme has a similar molecular weight as α-syn (14 kDa), it aggregates and forms fibrils in solution, and it has been extensively utilized as a control for non-specific peptide effects in α-syn studies [28, 29], effects of lysozyme were evaluated to control for non-specific peptide effects. Lysozyme solution (1 mM) was stored at 5°C in aliquots of 2000μl until use and was diluted in ACSF to a final concentration of 100 nM and applied for 3–4 min via the slice bath, which failed to elicit an excitatory response in LDT neurons (n = 3; Fig. 1C4). Repeatable effects of α-syn in individual neurons indicated that membrane effects were not due to immediate cell death (n = 3; data not shown).

Patch clamp recordings

Borosilicate glass patch pipette electrodes (Sutter Glass, Inner diameter: 0.86 mm, Outer diameter: 1.5 mm) were fabricated using a horizontal puller (P-97, Sutter Instruments, USA) to a tip diameter of 1–3μM, and filled with an intracellular solution containing in mM: 144 K-gluconate; 2 KCl; 10 HEPES; 0.2 EGTA; 5 Mg-ATP and 0.3 Na-GTP, with a resulting pipette impedance of 6–11 MΩ. The brain slice was placed in the recording chamber and 1.2 ml/min of carbogenated ACSF was continuously perfused. Cells were visualized with a 60x water immersion objective coupled to an upright microscope (BX50WI, Olympus; Japan) with an infrared Dodt gradient contrast system (IR-DGC; Luigs & Neumann, Germany) and a CCD camera (CCD-300ETRC; DAGE-MTI, Michigan City, IN). A patch clamp EPC9 amplifier (HEKA, Germany) and the software Pulse (HEKA; version 13.0) were used in voltage clamp mode and the holding voltage was maintained at –60 mV. Following membrane breakthrough, and at least a 7 min stabilization period, data were collected. Recordings of membrane currents were sampled at a rate of 1 kHz using AxoScope 10.2, and an Axon miniDigi 1B digitizer (Molecular Devices Corporation, USA). In some recordings, current-voltage relationships were established by stepping the membrane in 200 ms duration epochs from –110 mV to –30 mV in increments of 10 mV from a holding potential of –60 mV. Differences between control and drug conditions in current amplitudes required to step the membrane to the test voltages reveal a disparity in membrane conductance, and the crossing of the control and drug curves indicate the reversal potential of the conductance activated by monomeric α-syn. Combined patch clamp recordings and calcium imaging were conducted with a different setup in voltage clamp mode with a EPC9 amplifier guided by Pulse (version 9.0); and, membrane current recordings were sampled at 10 kHz using AxoScope 10.2 (Molecular Devices Corporation, USA) and an Axon Digidata 1440A digitizer (Molecular Devices Corporation, USA).

Identification of cell phenotype

For post hoc identification of the recorded cells as being present within the perimeter of the cholinergic LDT and PPT, or to phenotypically identify recorded neurons, in some recordings, Alexa-594 was included in the intracellular solution, and immunohistochemistry for brain-derived nitric oxide synthase (bNOS), which is a validated marker of cholinergic neurons in the mouse LDT and PPT was performed [30]. While ChAT immunohistochemistry is often used to identify cholinergic neurons, in our studies we have found that it is not as accurate as bNOS immunohistochemistry in brain slices that have not been optimally prepared for antibody detection. Optimal conditions include collection from a brain which has been rinsed, and perfused with paraformaldehyde via transcardial perfusion just prior to cessation of cardiac activity. Our slices are not prepared in this manner, and they incubate for hours in ACSF, with or without α-syn or lysozyme exposure prior to paraformaldehyde fixation. We have previously confirmed that in the mouse brain stem, bNOS is just as reliable a marker for cholinergic neurons as ChAT [30]. Accordingly, after the recordings, slices were submerged in 4% paraformaldehyde and stored overnight. Slices were then cryoprotected by saturation in a 30% sucrose solution and resectioned at 40μm. Immunohistochemical labeling for bNOS was conducted following previously reported protocols on the resectioned tissue [30]. Supplementary Figure 1A shows representative examples of cells recorded in this study (Alexa-594-positive), which were identified as cholinergic or non-cholinergic based on presence or absence of bNOS. In order to determine the phenotype of neurons in the SN, we evaluated firing frequency, degree of presence of an I_h current, and spike cessation during electrophysiological recordings in current clamp recording mode [31], as shown in Supplementary Figure 1B. To evaluate firing frequency, current was applied which depolarized putative dopamine-containing neurons sufficiently (–50 mV to –45 mV) to induce a regular, slow firing of action potentials (< 4–5 Hz). To determine presence of I_h a large hyperpolarizing pulse (–150 pA; 1-2 sec duration) was used to evaluate appearance of a prominent ‘sag’ which is a shift in the voltage of the membrane back towards a more depolarized potential. To evaluate blockade of action potential firing, a large amplitude depolarizing current step (+150 pA; 1-2 sec duration) was applied and cessation of action potentials was monitored.

Multiple cell and single cell calcium imaging

Single-photon, multiple-cell calcium imaging based on the ratiometric fluorescent calcium indicator dye (excitation wavelengths 340 and 380 nM), fura-2 acetoxymethyl ester (fura-2 AM) was conducted. Dye loading of brain slices was performed as in previous studies [32, 33]. For single-cell calcium imaging, bis-Fura 2 (25μM, Molecular Probes) replaced EGTA in the intracellular pipette solution. For both single and multiple cell calcium imaging, changes in fluorescence emitted within selected regions of interest encompassing cells were detected using a cooled CCD fluorescence camera system (12-bit Sensicam, PCO Imaging, Germany) controlled by the imaging software Live Acquisition (TILL Photonics, Germany).

Neurotoxicity assays (DAPI/PI and bNOS)

Coronal slices containing LDT or SN were incubated for a period of 7½ hours in monomeric α-syn (100 nM), or in ACSF under the same time protocols while carbogen (95% O₂/5% CO₂) was continuously supplied. For studies examining the effect of fibril α-syn (500 nM), bisected slices were used. Another investigator who was blinded to slice treatment conducted the identification of cell viability (C.R.R.L). The slices were incubated for 3 periods of 5 minutes in 1μg/ml solution of both propidium iodide (PI; Sigma-Aldrich) to identify dead cells and DAPI, which stains live cells (Sigma-Aldrich) with a pH of 7.4. Fluorescent signals were detected by an upright Zeiss microscope (Zeiss 59 fluorescent filter cube sets, wavelengths PI: 472–578 nm; DAPI: 358–463 nm) coupled to a monochrome CCD camera (Axiocam MRM, Zeiss, Germany) controlled by Axioskop 2 software (AxioVision 4.6, Zeiss). ImageJ software (National Institutes of Health, Bethesda, MD) was used to analyze the collected images and count the number of DAPI and PI-labeled cells. The total cell count was calculated as PI-positive plus DAPI-positive cells. In a separate population of bisected slices, in order to test non-specific peptide effects on cell death, we incubated LDT and PPT bisected slices in lysozyme (500 nM) or monomeric α-syn (500 nM) for 4–5 hours; thereafter, PI was added, and dead cells were counted in both slice hemi-sections. The proportion of dead cells in the monomeric α-syn exposed half was normalized to cell death seen in the hemi-slice exposed to lysozyme.

To compare relative presence of bNOS-positive cells in the LDT and PPT, coronal LDT slices were bisected and one half was incubated in control solution, and the other half in one of two monomeric α-syn containing solutions (200 nM or 500 nM) for 7 h. Following immunohistochemical procedures, cells were determined to be cholinergic based on presence of bNOS. All bNOS-positive cells within the border of the LDT or PPT were counted, and for presentation in paired plots, the numbers of positive cells in the α-syn-exposed hemi-slice were normalized to control, which was the numbers of positive cells in the other half of the same slice.

Real-time cell viability

Slices containing the LDT were bisected, individual cells were visualized, and patch clamp and calcium imaging recordings were initiated in the same cell. Monomeric α-syn (500 nM) was bath applied, and after filling the recording chamber, perfusion was halted to ensure continuous exposure to α-syn. As a control, the same protocols were conducted on the other half of the slice with the exception of application of peptide. After completing the calcium imaging recording, PI was added to the recording chamber (2μl; 0.1μg/ml), and real-time increases in fluorescent signal of PI were measured by time-lapse imaging using the excitation wavelength of 545 nm and the appropriate filter set (Chroma, USA).

Data analysis and statistics

Calcium imaging data were analyzed in Igor Pro 6 (Wavemetrics, USA). Response amplitude and polarity were quantified with changes in fluorescence below 4% considered too small to reliably discern from background noise. The ratiometric measures of changes in fluorescence are shown in graphs as % DF/F. DF/F is the difference (D) between the average of a series of data points at the maximum change in fluorescence following drug application and the average baseline fluorescence (F) relative to F, with ascendant deflection indicating intracellular calcium elevation. Amplitudes of membrane currents were measured (the difference between baseline and maximum deflection) by using AxoScope 10.5 (Molecular Devices, USA). In Figs. 1 and 2, the axis label, Response Frequency equals the percentage of tested cells which responded with membrane current or changes in calcium, and the axis label, Evoked Polarity indicates the polarity (inward or outward) of the current or increase or decrease in calcium elicited in the population of responding cells, respectively. Statistical analyses of data were performed in Prism (version 7.0, GraphPad, USA). Results are presented as mean values±SEM with the degrees of freedom (df). The figures were prepared using Igor Pro software, Graphpad Prism and Matlab R2018b. The n reported reflects numbers of single neurons recorded and represents neurons collected from at least 3 different animals, which sourced from different litters. Sample sizes were determined from past experiences with electrophysiological recordings in order to limit unnecessary loss of animal life in adherence to 3R guidelines. Differences in numerical data were tested using a one-way ANOVA with Tukey’s post hoc test, or a two-way, Paired or Unpaired Student’s t-test, and reporting of the f or t statistic is included as per APA style guidelines. Differences in categorical data were examined using the Fisher’s exact test, two-way Chi Square test, or a three-way Chi Square test that in cases of overall significance, was followed by a post hoc Fisher’s exact test to identify which contingency table cells differed significantly from expected outcomes. The alpha level for statistical significance was set at 0.05, and the exact p value is presented.

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Fig. 2

A) Representative image under bright field illumination and under fluorescence of fura-2 loaded neurons within LDT (A1), PPT (A2) and SN are presented (A3). Sample effects of monomeric and fibril α-syn on fluorescence indicative of alterations in intracellular calcium levels in LDT, PPT and SN are shown (B1-6). Summary data from the population of recorded cells showed that the proportion of cells responding to monomeric α-syn (C1), and the valence of calcium effects (C2) differed significantly between the sleep-related nuclei and SN. However, when nuclei were exposed to fibril α-syn, there were no differences between the three nuclei in the proportion of neurons responding (C3), nor of the valence of the response (C4).

α-syn fibrillation and characterization

α-syn monomer fractions were frozen in solution directly from size exclusion chromatography to avoid aggregate formation. A Thioflavin T (ThT) assay was used to monitor the fibrillation of α-syn, and displayed a typical sigmoidal curve indicating fibril formation (Fig. 1B1). The secondary structure of α-syn was investigated with CD showing the expected random coil for monomeric α-syn and β-sheet structure for the fibril form (Fig. 1B2).

Membrane responses

The monomeric and fibril forms of α-syn induced membrane currents in the majority of LDT, PPT and SN neurons. Surprisingly, the polarity of the elicited currents was opposite in the sleep-regulating LDT and PPT neurons to those elicited in the motor-controlling SN neurons. Inward currents were induced in the sleep-regulating nuclei (Fig. 1C1, 2), whereas, outward currents were elicited in SN neurons (Fig. 1C3). The proportion of LDT neurons and PPT cells responding to monomeric α-syn (100 nM) was greater than the proportion of SN neurons responding to this form of the protein (LDT: n = 35/35 and PPT: n = 8/8 vs. SN: n = 6/11; Fisher’s test; df:1, p = 0.003; df:1, p = 0.04, respectively, Fig 1C5). The fibril form of α-syn produced a significantly higher response rate in the SN than the monomeric form (Percentage Responding: Monomer: 54.5%, n = 6/11; Fibril: 100%, n = 7/7, Chi Square, df: 1, p = 0.0358, Fig. 1D4), and the amplitudes of the currents were significantly greater (Monomer: 15.1±3.9 pA, n = 6; Fibril: 33.7±4.1 pA, n = 7; Unpaired Student’s t-test, t(11) = 3.18, p = 0.008; Fig. 1D6). Within the LDT and the PPT, the proportions of cells responding to, and the amplitudes of inward current induced by α-syn were not significantly different for the two forms of the protein (Fibril LDT: n = 7; PPT: n = 8; Unpaired Student’s t-test, proportion responding: t(40) = 0.81, p = 0.7; Amplitude: t(14): 0.58, p = 0.5, respectively, Fig. 1D4, 6).

Calcium responses

α-syn altered the concentration of intracellular calcium in the majority of cells examined. Monomeric α-syn induced responses in most calcium indicator-labeled cells in the LDT (n = 56/68) and PPT (n = 34/41) (Fig. 2A, B). While the fibril form elicited calcium changes in even larger proportions of cells, this difference was not significant (LDT: n = 60/62, PPT: n = 56/60, Fisher’s test, df: 1, p = 0.0958; df: 1, p = 0.1155 Fig. 2C). Among responding LDT and PPT cells, the majority of changes in fluorescence induced by either form of α-syn indicated calcium increases (Monomeric: LDT: n = 38/56; PPT: n = 20/34; Fibril: LDT: n = 43/60; PPT: n = 34/56; Fig. 2C). Monomeric α-syn elicited calcium responses in a significantly lower proportion of cells in the SN (n = 34/60) than in LDT or PPT (Fisher’s test, df: 1, p = 0.001; df: 1, p = 0.0278; Fig. 2C). Further, a relatively higher proportion of cells in SN exhibited calcium decreases (n = 24/34) than in LDT or PPT (Fisher’s test, df: 1, p = 0.0005; df: 1; p = 0.0272; Fig. 2C). Interestingly, when the fibril form was applied, there were no significant differences between the proportions of cells responding in the SN (n = 51/56) from those in the LDT or PPT (Chi Square test, df: 2, p = 0.429; Fig. 2C). Further, of those SN cells responding to the fibril form, more showed increases in fluorescence (n = 39/51) than decreases (n = 13/51) (Fisher’s test, df: 1, p < 0.00001, n = 39/51), and the distribution of the direction of responses did not differ between nuclei (Chi Square Test, df: 2, p = 0.237, Fig. 2C). Recovery to baseline and rises in calcium induced by a subsequent application of 1μM AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; 3 ml) confirmed in LDT cells that changes in calcium by α-syn were not due to acute cell death. The average amplitude of AMPA-mediated rises was 37.3±5.9% DF/F (n = 5; data not shown), which is similar to that seen in other studies (% DF/F: 34.7±6.5; [33]).

Neurotoxicity assays

As we hypothesized that continuous exposure to the monomeric form of α-syn could have adverse effects on LDT and PPT neurons, but not on SN cells, we examined neuronal death induced by relatively brief, but continuous exposure to α-syn via DAPI/PI labeling or bNOS immunohistochemistry. For DAPI/PI labeling, LDT and SN slices were incubated in monomeric α-syn (100 nM) or control solution for 7½ hours and cell viability fractions were calculated as the ratio of living cells (DAPI-positive) to the total number of cells (DAPI-positive+PI-positive). Relative to control slices, α-syn-exposed LDT slices exhibited a greater proportion of dead cells (Control: n = 4; α-syn: n = 7; Unpaired Student’s t-test, t(9) = 2.52, p = 0.03; Fig. 3A). Further, in bNOS immunohistochemistry studies, fewer surviving cholinergic cells (bNOS-positive cells) in α-syn-exposed slices were noted when compared to the other half incubated in control solution (Fig. 3B). Similarly indicative of heightened cell death in the PPT, we found a lower proportion of bNOS-positive cells in monomeric α-syn-exposed hemi-slices when compared to the other half of the slice, which was incubated for an equivalent period in absence of α-syn (n = 6; Paired Student’s t-test, t(5) = 3.331, p = 0.02; Fig 3B). However, SN slices showed no indication of heightened cell death over control (Control: n = 8; α-syn: n = 7; Unpaired Student’s t-test, t(13) = 0.04, p = 0.96, Fig. 3A. Cell death seen in LDT, PPT and SN slices after exposure to the fibril form of α-syn was significantly higher in the LDT (n = 6; Paired Student’s t-test, t(5) = 2.415, p = 0.03), PPT (n = 6; Paired Student’s t-test, t(5) = 2.350, p = 0.03), and the SN (n = 6; Paired Student’s t-test, t(5) = 2.185, p = 0.04) compared to control solution exposure (Fig. 3A).

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Fig. 3

A) Monomeric α-syn facilitated cell death in the LDT and the PPT, but not the SN, whereas the fibril form induced greater cell death in all three nuclei. Fluorescent images of LDT (A1) and SN brain slices (A2) exposed to monomeric α-syn showing dead cells in red (PI-positive, left) and live cells in blue (DAPI-positive, middle) with the two fields overlaid (right). As shown in the bar graphs to the right (top), there was a significantly greater percentage of cell death over control in the population of LDT cells exposed to monomeric α-syn; whereas in the SN, cell death was not significantly different between control and α-syn-exposed slices. (A, right bottom) Bar graphs show DAPI/PI-assayed cell death induced by the fibril form of α-syn, which was significantly greater than in control studies in the LDT, PPT and SN. B) Quantification of bNOS-positive cells in bisected LDT and PPT slices revealed a smaller number of cholinergic neurons within monomeric α-syn-exposed half slices (B2, 4) when compared to the number in the equivalent region in the other half of the slice incubated in control solution (B1, 3) for the same time period. A count of bNOS-positive cells in the LDT revealed 14.7% fewer cholinergic cells in the monomeric α-syn-incubated, hemi-slices (200 nM; n = 2) relative to control, and 47.3% fewer when incubated in 500 nM (n = 2), which constituted a significant difference (Paired Student’s t-test; df: 6; p = 0.04 ^*p < 0.05). Within the PPT, 35% fewer cholinergic cells were noted in the half of the bisected slice which was exposed to 200 nM monomeric α-syn compared to the control half (n = 6; Paired Student’s t-test, t(5) = 3.331, p = 0.02). C) Fluorescent images of PI cells in the LDT (top panels) and PPT (bottom panels) in hemi-slices exposed to lysozyme (C1, 3) or monomeric α-syn (C2, 4) showing heightened cell death in the half of the slice exposed to monomeric α-syn. Paired plots are shown to the right, which demonstrated elevated cell death in cells exposed to monomeric α-syn compared to those exposed to lysozyme. These data support the conclusion that cell death seen in the LDT and PPT was not due to general protein effects, but rather was due to specific actions of monomeric α-syn.

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Fig. 4

Real-time recordings of cell death were conducted in LDT neurons in which membrane responses and calcium rises were induced by α-syn. A) Representative example of the responses of a LDT neuron, which is shown in bright field in (A1), and in fluorescence in (A2), to application of monomeric α-syn. As can be seen, patch clamp recordings revealed induction of an inward membrane current (A1), and fluorescence changes indicative of an increase in intracellular calcium concentration (A2) following application of α-syn. Real-time visualization of the PI fluorescence signal in the same cell revealed cell death at 1 : 55 h following α-syn exposure (A3). B) Similar recordings were conducted in LDT neurons in control conditions, as illustrated by this example of a patch clamp recorded cell shown under bright field (B1), and under fluorescent optics in (B2). Recordings from control conditions at equivalent time points as in treatment sessions were conducted. We did not see any changes in membrane current or intracellular calcium levels similar to those observed following α-syn exposure. However, it should be noted that in control conditions, when the perfusion was halted, we observed a change in the membrane current in one recorded cell suggesting mechanical disturbance. Live cell imaging from the cell recorded under control conditions indicated that cell death occurred at 3 : 30 h (B3). C) Analysis showed that the same population of LDT cells which exhibited significantly different α-syn-mediated inward currents (Unpaired Student’s t-test, t(5) = 4.94, p = 0.004) (C1) and rises in fluorescence (Unpaired Student’s t-test, t(5) = 2.78, p = 0.04) (C2) from control also demonstrated significantly shorter cell lifetimes compared to control LDT neurons to which α-syn was not applied (C3).

In order to examine whether heightened cell death was specific to monomeric α-syn or merely due to high protein levels, we applied lysozyme to LDT and PPT slices. We found a significantly greater number of PI-positive cells in LDT and PPT hemi-slices exposed to monomeric α-syn, than in the other half of the same slice which had been exposed to lysozyme, suggesting that effects on cell death were specific to α-syn, and were not due to non-specific protein effects (LDT: 165% cell mortality rate increase, n = 8, Paired Student’s t-test, t(7) = 1.915, p = 0.04; PPT: 91% cell mortality rate increase: n = 8; Paired Student’s t-test, t(7) = 2.015, p = 0.04; Fig. 3C).

Single cell recordings of membrane effect, calcium influx and real-time, cell viability assay

As we observed effects on membrane currents, calcium levels, as well as heightened cell death by monomeric α-syn in separate populations of cells in sleep control nuclei, we next examined whether those effects co-occurred in the same cell. As these studies were difficult to execute because they required long recording times, we only conducted them in the LDT to confirm that the membrane events, calcium changes, and cell death co-occurred in the same neuron. Thus, in LDT slices exposed to a high concentration of monomeric α-syn (500 nM), we tracked neuronal viability in real-time using PI fluorescence and, within the same neuron, we monitored the membrane current and intracellular calcium response. As control, the same protocols were conducted in the other half of the slice, which was not exposed to α-syn. In all neurons tested, α-syn induced inward currents (–140.5±11.3 pA, n = 4), and an increase in intracellular calcium levels (39.7 ± 10.7 % DF/F, n = 4), which were significantly different from control conditions (Fig. 4A-C). Subsequent real-time recordings of PI fluorescence showed a significantly shorter lifetime of the cells which were α-syn-exposed compared to control cells (α-syn: 93.7±14.3 min; n = 4, control: 163.3±29.6 min, n = 3, Unpaired Student’s t-test; t(5) = 2.3; p = 0.04; Fig. 4C3).