Sex-dependent neuronal effects of α-synuclein reveal that GABAergic transmission is neuroprotective of sleep-controlling neurons

Background Sleep disorders (SDs) are a symptom of the prodromal phase of neurodegenerative disorders that are mechanistically linked to the protein α-synuclein (α-syn) including Parkinson’s disease (PD). SDs during the prodromal phase could result from neurodegeneration induced in state-controlling neurons by accumulation of α-syn predominant early in the disease, and consistent with this, we reported the monomeric form of α-syn (monomeric α-syn; α-synM) caused cell death in the laterodorsal tegmental nucleus (LDT), which controls arousal as well as the sleep and wakefulness state. However, we only examined the male LDT, and since sex is considered a risk factor for the development of α-syn-related diseases including prodromal SDs, the possibility exists of sex-based differences in α-synM effects. Accordingly, we examined the hypothesis that α-synM exerts differential effects on membrane excitability, intracellular calcium, and cell viability in the LDT of females compared to males. Methods Patch clamp electrophysiology, bulk load calcium imaging, and cell death histochemistry were used in LDT brain slices to monitor responses to α-synM and effects of GABA receptor acting agents. Results Consistent with our hypothesis, we found differing effects of α-synM on female LDT neurons when compared to male. In females, α-synM induced a decrease in membrane excitability and heightened reductions in intracellular calcium, which were reliant on functional inhibitory acid transmission, as well as decreased the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs) with a concurrent reduction in action potential firing rate. Cell viability studies showed higher α-synM-mediated neurodegeneration in males compared to females that depended on inhibitory amino acid transmission. Further, presence of GABA receptor agonists was associated with reduced cell death in males. Conclusions When taken together, we conclude that α-synM induces a sex-dependent effect on LDT neurons involving a GABA receptor-mediated mechanism that is neuroprotective. Understanding the potential sex differences in neurodegenerative processes, especially those occurring early in the disease, could enable implementation of sex-based strategies to identify prodromal PD cases, and promote efforts to illuminate new directions for tailored treatment and management of PD.

Sex-dependent neuronal effects of α-synuclein reveal that GABAergic transmission is neuroprotective of sleepcontrolling neurons

Background
Parkinson's disease (PD) is among one of the most widespread neurodegenerative disorders [1,2], and sex is a risk-factor in the development of this disease, as PD is more common in men than in women with an approximated odds ratio of 2:1 [3][4][5][6][7].Although PD is clinically diagnosed by the cardinal motor symptoms, evidence emerging over the past two decades has established sleep disorders (SDs) such as REM sleep behavior disorder (RBD), which is a sleeping disorder characterized by excessive motor behavior during what is normally a period of atonia, and excessive daytime sleepiness (EDS) as markers of the prodromal phase of PD, and these SDs can precede the motor symptoms by years to decades [8][9][10][11][12][13].Sex has been acknowledged as an important determinant of both the susceptibility to neurodegenerative diseases and whether SDs co-occur following diagnosis, and while not well studied, it is likely that sex differences are also present in the prodromal phase of the disease prior to diagnosis, which could include the expression of SDs.
We hypothesized that the appearance of SDs prodromal to the motor symptoms in PD could be due to alteration of cellular function and neurodegeneration in sleep controlling nuclei and that sex differences in cellular effects could be present.Neurodegeneration in several brain nuclei in PD is associated with aggregation of the protein α-synuclein (α-syn), which is the histological hallmark of PD [14].Pathological studies have shown that aggregated α-syn can interfere with several cellular functions in addition to promoting cell toxicity [15].Heightened cell death has been reported in patients with α-synucleinopathies in the laterodorsal tegmentum (LDT) [16], which is a heterogenous nucleus comprised of cholinergic, glutamatergic and GABAergic neurons [17] that are importantly involved in the control of motor atonia during sleep, and arousal during wakefulness [18,19].Further, the co-occurrence of RBD and α-syn pathology was strongly correlated with brainstem cholinergic dysfunction in a predominantly male cohort consistent with α-syn-mediated degeneration of LDT cholinergic systems underlying aberrant behavioral state behavior [16].Taken together, this suggests that α-syn-mediated PD processes include cellular actions in SD-controlling neurons already from abnormal levels of α-syn.
Although several studies have investigated the neuronal effects induced by α-syn, their focus was on actions of forms of the protein which aggregate (oligomeric and fibril).These forms are suspected to be the most damaging to neurons and neural transmission, and so very few studies have reported on cellular effects induced by the disordered, native monomeric form (α-syn M ), which is widely considered to be relatively benign.The aggregation and fibrillation of α-syn may be preceded by dysregulation of expression e.g. as a result of SNCA gene multiplication [20]) and/or clearance [21] leading to abnormal levels of α-syn in the CNS, which then in turn may lead to nucleation and fibril formation [22].We recently found that α-syn M has cellular effects on neurons in the LDT that were associated with heightened cell death, which we speculated could play a role in SDs prodromal to, as well as following, diagnosis of PD.Specifically, we found that α-syn in monomeric form induced excitation, increased intracellular calcium, and heightened neuronal death of neurons of the LDT.In contrast, different effects were induced in the substantia nigra (SN) in that α-syn M elicited membrane inhibition, and greater decreases of intracellular calcium with no evidence of α-syn M -induced cell death [23].However, that investigation was conducted solely in LDT of males.Because the appearance of sex-based differences in α-syn-related disease symptoms, including differences between SDs, suggest different mechanistic actions are involved in disease processes in males and females, we wished to determine whether excitatory cellular effects of α-syn M on LDT, and heightened cell death in males were also present in females.Accordingly, in the present report, we have used electrophysiological and calcium imaging techniques ex vivo to investigate cellular effects of highly purified α-syn M on LDT and SN neurons from females and compared effects to those in male LDT neurons.

Brain slice preparations
A state of anesthesia was induced via inhalation of isoflurane (Baxter A/S, Denmark) and decapitation was conducted when anesthesia had been achieved as assayed by failure to react to a paw pinch.A block of the brain which contained LDT or SN was rapidly removed and submerged in ice-cold artificial cerebrospinal fluid (ACSF).The ACSF solution which contained 124 NaCl, 5 KCl, 1.2 Na 2 HPO 4 •2H 2 O, 2.7 CaCl 2 •2H 2 O, 1.2 MgSO 4 (anhydrous), 10 dextrose, 26 NaHCO 3 in mM was adjusted to a pH of 7.4 and an osmolarity of 298-302 mOsm/kg following saturation with carbogen (95% O 2 /5% CO 2 ).The brain was sectioned in 250 μm thick slices containing the LDT or the SN with a vibratome (Leica VT1200S, Leica Biosystems, Germany).Brain slices were collected and placed in a chamber containing oxygenated ACSF, and incubated at 37 o C for 15 min.To allow the tissue to equilibrate after the incubation period, the slices were kept at room temperature, and carbogen was continuously supplied for at least 1 h prior to further procedures, including exposure of the slice to the monomeric form of α-syn.

Monomeric α-syn
Human α-syn was recombinantly expressed and purified as described in more detail in our earlier published work using this peptide [23].Briefly, α-syn was cloned into E. Coli BL21DE3 cells using a pET-11a vector construct.Harvested cells were lysed by osmotic shock.Subsequently, boiling and centrifugation were conducted to remove non-heat-stable proteins.Ion-exchange chromatography was used to isolate α-syn, and the monomeric fraction was isolated by size exclusion chromatography SEC; thereafter, the monomers were pooled and kept in PBS buffer stored at -80 o C until application to the slices.

α-syn application
The monomeric form of α-syn (α-syn M ) was stored in solution at -20 o C in aliquots of 10 µl (150 µM) until use at which time it was applied via the bath.To reach a final concentration of 100 nM, an aliquot (150 µM) of 10 µl of α-syn M was diluted in ACSF.After the establishment of baseline holding currents or baseline fluorescence, α-syn M was applied for 3-4 min to monitor effects on membrane holding currents, synaptic activity, action potential firing, and intracellular calcium.For cell viability studies, incubation of the 250 μm brain slice in α-syn M for 7 h was conducted in protocols described below.

Drugs
Action potentials within the slice were blocked by 0.5 mM tetrodotoxin (TTX, Tocris, UK).Glycinergic receptors were blocked with strychnine (2.5 µM; Sigma, Denmark).GABA A and GABA B receptor-mediated responses were blocked by SR-95,531 (gabazine, 10 µM, Sigma, Demark) and CGP 55,845 (10 µM, Tocris, UK), respectively.Muscimol (30 µM, Sigma, Denmark) and baclofen (10 µM, Sigma, Denmark) were used as agonists of the GABA A and GABA B receptors, respectively.Stock solutions were stored in appropriate aliquots at -20 o C prior and diluted in ACSF to final concentrations before use, and all drugs were applied via the bath.

Patch-clamp recordings to monitor changes in membrane currents, synaptic activity, and action potential firing
Whole cell patch clamp recordings were conducted in 250 μm thick brain slices from neurons in the LDT (19 LDT brain slices; 14 mice) or the SN (7 SN brain slices; 6 mice).For recordings done in the LDT, we wished to target cholinergic neurons.Therefore, we selected the recorded neurons based on soma size (medium-to-large cells) and location within the central LDT wherein the concentration of cholinergic neurons is highest [24].To fabricate patch pipette electrodes for recording neuronal electrical activity in LDT and SN brain slices, borosilicate filamented glass capillary tubes (1.5 mm, Sutter Instruments, USA) were pulled after heating to a sharp tip in a horizontal Flaming/Brown micropipette puller (P-97, Sutter Instruments, USA).These glass electrodes were filled with an intracellular solution (144 K-gluconate; 2 KCl; 10 HEPES; 0.2 EGTA; 5 Mg-ATP and 0.3 Na-GTP; in mM), which resulted in a pipette resistance of 6-11 MΩ.A brain slice containing LDT or SN was placed in the recording chamber that was situated in a microscope stage and ACSF saturated with a mixture of 95% oxygen/5% carbon dioxide (carbogen) was continuously perfused over the slice (flow rate 1.2 mL/min).A water immersion objective (60x) 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) were used to visualize the cells.The software, Patchmaster (HEKA; version v2 × 91), was used to control a patch-clamp EPC9 amplifier (HEKA, Germany).Recordings were initiated in voltage-clamp mode to establish high resistance seals (> 1 MΩ) between the patch pipette and the cell membrane, and the holding voltage was kept at -60 mV.After at least a stabilization period of 7 min following membrane breakthrough, data were collected.AxoScope 10.2 (Molecular Devices Corporation, USA) and an Axon miniDigi 1B digitizer (Molecular Devices Corporation) were used to sample membrane effects.To quantify relative changes in holding currents, the holding currents in pA averaged from at least 30 sec of recording before application of α-syn M and the holding currents averaged from at least 30 sec during the maximum effect of α-syn M were subtracted.A change in amplitude of 2 pA from baseline was used as criteria to be considered a response.For firing frequency studies, action potentials were recorded in current-clamp mode before and after α-syn M application.Current was applied to depolarize the neuron sufficiently to induce a sustained firing of action potentials (-45 mV).A period of firing in an epoch of 30 sec immediately prior to and 30 sec after application of α-syn M was selected and interspike intervals were measured and averaged.Interspike intervals were determined by measuring the period of time (in msec) from the initiation of a spike to initiation of the next spike.

Calcium imaging to monitor changes in intracellular calcium
Intracellular loading of cells with a calcium binding dye was performed in 42 LDT F (25 mice) and 15 LDT M (7 mice) 250 μm thick brain slices following standard protocols [25] for single-photon calcium imaging.Recordings were conducted utilizing ratiometric fluorescent calcium indicator dye Fura-2 acetoxymethyl ester (Fura-2 AM).Prior to recordings, slices were rinsed for over 15 min in the recording chamber by continuous perfusion of oxygenated ACSF at a flow rate of 1.2 ml/min to wash out free dye debris and allow temperature equilibration.To localize the LDT, an upright microscope (BX50WI, Olympus, Germany) was used under bright field illumination and visualization guided by characteristic landmarks located close to these nuclei.Individual cells were viewed with a water immersion objective (40x).A cooled CCD fluorescence camera (12-bit Sensicam, PCO Imaging, Germany) attached to the microscope and controlled by the imaging software Live Acquisition (TILL Photonics, Germany) was used to collect paired images that were binned at 2 × 2 pixels on the camera chip in order to optimize spatial and temporal resolution.Live Acquisition controlled rapid switching between the excitation wavelengths of 340 and 380 nm in order to ensure a minimal amount of time passing between collection of the image at each wavelength, which allowed ratiometric calculations.The acquisition interval between each frame pair of 340 and 380 nm was 4 sec.Regions of interest (ROI) were drawn around each Fura-2 loaded cell, as well as around a region of the field of view that contained no dye loaded cells, which was used to measure background fluorescence.Analysis software (Offline Analysis, TILL Photonics, Germany) was used to quantify fluorescence in each ROI by averaging the intensity of the 2 × 2 binned pixels, and background in each channel was subtracted.The 340 and 380 nm channels were ratioed (340 nm:380 nm).Baseline fluorescence was calculated from an average of intensity taken from 10 frames collected before drug application.Following application of α-syn M , changes in fluorescence from that measured at baseline were noted in the majority of cells, and the fluorescence of the peak deflection from baseline was measured by averaging across 10 frames.Data are presented as DF/F% which represents the subtraction of the baseline fluorescence from the maximum change in fluorescence induced by α-syn M application divided by the baseline, followed by conversion to a percentage.Ascendent deflection of fluorescence indicates intracellular calcium elevations with descendent deflections reflecting decreases in intracellular calcium.Actual calcium levels were not calculated due to well-known complications with converting changes in fluorescence in brain slices to calcium concentrations [26,27].

Neurotoxicity assay to evaluate cell viability
Propidium iodide (PI; Sigma-Aldrich) was used to identify dead cells and 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was used to mark live cells from LDT F and LDT M slices.A total of 116 slices from 56 mice were used in 3 different protocols in which 250 μm slices were bisected with each half being exposed for 7 h under oxygenation to: (1) ACSF or α-syn M (100 nM), (2) α-syn M (100 nM) or α-syn M (100 nM) plus GABA A , GABA B and glycine receptor antagonists, or (3) α-syn M (100 nM) or α-syn M (100 nM) plus GABA A , GABA B , and glycine receptor agonists.The bisection of the slices ensured that data sets could be compared from tissue taken from the same group of animals, and that protocols were run side by side under the same laboratory conditions.Following incubation, slices were fixed in 4% paraformaldehyde overnight, cryprotected with sucrose saturation (30%), and resectioned to a thickness of 40 μm on a cryostat (Leica CM3050, Triolab, DK).Resectioned slices of 40 μm were incubated for 3 periods of 5 min in a solution which contained 1 µg/ml of both PI and DAPI with a pH of 7.4.To detect fluorescence signals from PI and DAPI, an upright Zeiss microscope coupled to a monochrome CCD camera (Axiocam MRM, Zeiss, Germany) controlled by Axioskop 2 software (AxioVision 4.6, Zeiss) and required filter cubes were used (Zeiss 59 fluorescent filter cube sets, wavelengths PI: 472-578 nm; DAPI: 358-463 nm).
To conduct analysis of collected images, a macro written for ImageJ (National Institutes of Health, Bethesda, MD) was used to automatically count the number of DAPI and PI-labeled cells following background subtraction, application of thresholding, and separation of objects via a watershed algorithm.Cells were selected using the batch processing macro, Analyze Particle; however, selections were manually confirmed.Cell survival was quantified by the proportion of live cells (DAPI positive cells) to the total cell count, which was calculated as the addition of PI positive cells and DAPI positive cells.Cell viability was evaluated in up to 5 areas within the LDT in the 40 μm resectioned slices.For this data set, n a = the number of areas examined/n 40 = the number of resectioned slices.Data was normalized in each of the 3 protocols to the control condition, which was either exposure to ACSF or α-syn M without GABA A , GABA B , and glycine receptor agonists or antagonists.However, to compare cell death between LDT F and LDT M , cell survival was evaluated without normalization.In figure panels depicting PI positive and DAPI positive cells, contrast has been applied equally across entire images.

Data analysis and statistics
Amplitudes of membrane holding currents were measured (the difference between baseline and maximum deflection) using Axoscope 10.5 (Molecular Devices, USA).Spontaneous excitatory synaptic events (sEPSCs) were detected and analyzed using MiniAnalysis (Synaptosoft, USA).Analysis of synaptic events was conducted by selecting 30 sec of the recording just before application of α-syn M and at the peak amplitude of the effect on membrane current.Inter-event intervals and the amplitude of events were averaged across a population of cells and statistically analyzed.Firing frequency was analyzed selecting 30 sec epochs before and after α-syn M application, and intervals between action potentials were measured and averaged.Calcium imaging data including the amplitudes of DF/F% changes were analyzed in Graphpad Prism (version 7.0).The numbers of observations in the data sets analyzed for cell viability are reflected as n a .Results are presented as mean values ± SEM.The figures were prepared using Igor Pro software (Wavemetrics, USA) and GraphPad Prism.Differences in numerical data were tested using a Paired or Unpaired Student's T-test, and differences in categorical data were examined using the Fisher's Exact test.P values are reported in text as 4 decimal points, and a significant difference was determined if alpha was less than 0.05.

α-syn M effects on membrane currents, and synaptic transmission in neurons of sleep and motor controlling nuclei in the female α-syn M induced outward currents in sleep and motor control nuclei in the female LDT
Our previous study showed that α-syn M induced an inward current in LDT neurons in brain slices from male mice (LDT M ) [23].Unexpectedly, in neurons in LDT brain slices from female mice (LDT F ; Fig. 1A), α-syn M (100 nM, 3 min) induced an outward membrane current in all cells examined (amplitude: 54.8 ± 11.3 pA, n = 14).To compare the effect of α-syn M in neurons of a sleep controlling nucleus vs. neurons of a motor control nucleus, we next investigated the neuronal effect of α-syn M in the SN of females (SN F ). Similar to our previous report that showed α-syn M induced inhibitory membrane responses in neurons of the male SN (SN M ), we observed that α-syn M induced inhibitory responses in the membrane of 100% of the neurons examined in SN F (amplitude: 47.2 ± 16.0 pA, n = 11).The average amplitude of the outward current induced in neurons of the LDT F did not differ from that induced in SN F neurons (p = 0.3847; Unpaired Student's T-test; Fig. 1B).

α-syn M alters synaptic events in sleep and motor control nuclei in the female LDT
Our previous study showed that α-syn M altered synaptic transmission in LDT M neurons producing increases in frequency as well as amplitude of spontaneous excitatory postsynaptic currents (sEPSCs).However, in the present study, the opposite effect was seen in LDT F as α-syn M induced a significant decrease of nearly 20% in sEPSC frequency (Control: 7.2 ± 2.7 Hz; α-syn M : 5.8 ± 2.3 Hz; p = 0.0475; n = 5, Paired T-test) and a 21% decrease in the amplitude of sEPSCs was noted which was significant (control: 10.8 ± 1.7 pA; α-syn M : 8.5 ± 1.3 pA; n = 5; p = 0.0253; Paired T-test; Fig. 1C).
In SN F , α-syn M induced a decrease of 14% in amplitude of sEPSCs which was significant when compared to control (control: 6.1 ± 0.8pA; α-syn M : 5.2 ± 0.5pA; n = 4; p = 0.0483; Paired T-test); however, changes induced in the frequency were not significantly different (control: 5.9 ± 1.9 Hz; α-syn M : 7.4 ± 2.8 Hz; n = 4; p = 0.4735; Paired T-test; Fig. 1C).In our previous work, we did not examine the effect of α-syn M on sEPSCs in the SN in the male (SN M ).Therefore, in order to examine sex-based potential differences of α-syn M on sEPSCs in this motor control nucleus, we determined whether α-syn M had effects on synaptic activity in the SN M and found that changes in sEPSCs were qualitatively similar to those seen in SN F .α-syn M induced a decrease of 10% in the amplitude of the current of the sEPSCs (Ctrl: 6.0 ± 1.7 pA; α-syn M : 5.4 ± 1.6 pA; n = 4; p = 0.0449; Paired T-test), and changes induced in the frequency were not significantly different (Ctrl: 11.8 ± 2.2 Hz; α-syn M : 11.7 ± 2.4 Hz; n = 4; p = 0.9162; Paired T-test).In summary, these data indicate that α-syn M induced inhibitory membrane effects on neurons of LDT F , and a reduction in the amplitude and frequency of sEPSCs, which were opposite effects from those we have reported before in LDT M [23].In SN F , α-syn M also induced inhibitory effects on the membrane, which were similar to those effects we have reported before in SN M .Further, α-syn M had similar effects on synaptic events in SN M to those seen in SN F as in both sexes we observed a reduction in the amplitude of sEPSCs with no effect on frequency.Taken together, our findings show that the examined effects of α-syn M on LDT neurons are sex-dependent, whereas, α-syn M effects on SN are independent of sex.

Sex differences in LDT neurons of α-syn M -mediated alteration of intracellular calcium
As α-syn M -induced neuronal effects have been hypothesized to lead to calcium dysregulation [23,28], we previously examined actions of this protein on intracellular calcium levels and found that α-syn M induced changes in calcium in LDT M .We repeated those experiments here and confirmed our earlier findings.Using multiple-cell calcium imaging to monitor changes in Fura 2-AM fluorescence which were induced by α-syn M (100 nM, 3 min), we observed changes in fluorescence in the majority of LDT M cells (97.4%; n = 38/39; Fig. 2B1), and the majority of responses were indicative of an increase in calcium (76.3%; n = 29/38; Fig. 2A1a, B2).We then examined responses in LDT F and found that α-syn M induced changes in intracellular calcium in 100% (n = 89/89) of the examined cells (Fig. 2B1).However, interestingly, the majority of responding LDT F neurons exhibited changes in fluorescence indicative of a decrease in intracellular calcium levels (58.4%, n = 52/89; Fig. 2A2b, B2).When we compared alterations in intracellular calcium induced by α-syn M in LDT M to LDT F , there was no significant difference in the numbers of responding or non-responding neurons (p = 0.3047; Fischer's Exact test; Fig. 2B1).In contrast, there was a difference between males and females in the ratio of increases in calcium to decreases in calcium in response to α-syn M .We observed a significantly higher proportion of cells responding with a decrease in calcium in LDT F when compared to the proportion of cells exhibiting decreases in LDT M (p = 0.0004; Fisher's Exact test; Fig. 2B2).

Evaluation of the mechanism of α-syn M inhibition induced in the membrane of LDT neurons
To gain more information regarding the sex-based difference in the mechanism underlying α-syn M membrane effects, we examined α-syn M actions during blockade of the generation of Na + -dependent action potentials by inclusion in the bath of tetrodotoxin (TTX, 500 nM).Unexpectedly, in presence of TTX, α-syn M induced an inward current (-7.7 ± 2.0 pA, n = 4), and this effect was present in all neurons tested (Fig. 3A, B).
Our findings with TTX indicated that α-syn M -induced outward currents relied on a presynaptic mechanism.To confirm the involvement of a presynaptically-mediated mechanism in the induction of outward currents in the postsynaptic membrane of LDT F neurons, we next applied α-syn M in a reduced calcium solution, which effectively eliminates calcium-dependent synaptic transmission.Because we wished to use a within cell control, we first verified whether a second application of α-syn M to the same cell could result in similar effects on the membrane as those elicited in a first application.Thus, in a subset of LDT F neurons, we reapplied α-syn M following a first application, and we observed that a second inhibitory response was elicited in all neurons tested, which did not significantly vary in amplitude from the outward current obtained in the first application (n = 3).Consistent with the TTX data, in all the cells tested in which the first application elicited an outward current, in the second application in presence of low calcium solution, we did not observe an induction of an outward current, and instead, an inward current was elicited (-16.1 ± 5.5 pA; n = 3; Fig. 3A2, B).Taken together, these data indicate that the inhibitory effect induced by α-syn M on the membrane of LDT F involves presynaptic-dependent mechanisms.
LDT neurons receive a heavy inhibitory input from presynaptic GABAergic terminals both from local LDT neurons but also from projections sourcing from outside the nucleus [29], and, accordingly, GABAergic presynaptic mechanisms could be involved in α-syn M -mediated inhibitory responses in LDT F .Therefore, we investigated α-syn M -induced membrane responses in LDT F in presence of the GABA receptor antagonists, SR-95,531 (10 µM) and CGP-55,845 (10 µM), which block GABA A and GABA B receptors, respectively.Although no evidence has been presented of glycine-mediated inhibition of LDT cells, and we have not noted any glycinergically-mediated spontaneous inhibitory currents (sIPSCs) in our own studies under our recording conditions, we also included strychnine (2.5 µM) in the ACSF to ensure the blockade of any glycine-mediated events.In a population of cells in which the first application of α-syn M induced an outward current, we found that in the presence of GABA and glycine receptor blockade, an inward current was elicited in all tested cells (-16.3 ± 5.2 pA; n = 3; Fig. 3A3, B1, 2).
When taken together, our data indicate that induction of outward current in LDT F neurons is reliant on inhibitory transmission from presynaptic neurons.Blockade of inhibitory transmission revealed an inward membrane current similar to what has been seen in LDT M .Although not tested in the present study, we showed in our previous work that inward currents in LDT M were mediated by a G-protein receptor coupled mechanism in postsynaptic neurons.Although we conducted experiments to examine a role for receptors previously implicated in α-syn M effects, we could not identify the specific receptor involved; however, we speculate that this same excitatory mechanism is being activated in in LDT F but masked by the concurrent induction of outward current induced by α-syn M -mediated stimulation of inhibitory presynaptic transmission.

Inhibitory amino acids are involved in the decrease of intracellular calcium
As we had seen that membrane current effects of α-syn M involved inhibitory transmission, we examined whether similar mechanisms were also involved in the decrease of intracellular calcium seen in response to α-syn M in the majority of neurons of the LDT F .During blockade of GABA and glycine receptors, while decreases in fluorescence indicative of reductions in calcium were still elicited, the amplitude of the decrease in fluorescence was significantly smaller (36%) compared to that elicited in control conditions (control: 57.1 ± 3.0% DF/F, n = 52; blockers: 36.2 ± 2.3% DF/F, n = 49; p = 0.0001; Paired T-test; Fig. 3C).Taken together, while they suggest that other mechanisms might be involved in the reductions in intracellular calcium induced by α-syn M , these data provide evidence that inhibitory amino acids, most likely GABA contribute to α-syn M -mediated calcium decreases in LDT F and provide further support that inhibitory transmission targeting postsynaptic LDT cells is activated by α-syn M .

α-syn M Reduces the excitability of LDT neurons in the female
We previously reported that α-syn M enhanced the firing frequency of neurons within LDT M .However, the inhibitory effect induced by α-syn M on the membrane of LDT F in conjunction with the reduction in amplitude and frequency of EPSCs could reduce neuronal excitability in the female.To directly investigate the functional effect of α-syn M -mediated actions on LDT F neurons which could affect the output of these cells, we examined the firing frequency in current clamp mode following depolarization of the membrane of LDT F neurons sufficiently to induce action potentials (V M : -45.0 ± 5.0 mV) before and after application of α-syn M .Under these conditions, α-syn M reduced the firing frequency by 36.5%  4A, B).These data suggest that in direct contrast to findings in LDT M , functional actions of α-syn M include reductions in neuronal excitability of neurons in LDT F , which would be expected to alter output of these cells to target regions.

α-syn M induces a lower cell death of LDT neurons in females compared to males
In our previous report, α-syn M -mediated excitation of the membrane of neurons with a concurrent rise of intracellular calcium was suspected to induce excitotoxicity, which was supported by a heightened cell death over control in LDT M [34].As α-syn M -induced inhibitory membrane current, and increases in calcium were less prominent in LDT F neurons, we hypothesized that neurodegeneration induced by α-syn M would also exhibit a sex-based difference.First, we needed to determine whether α-syn M induced cell death in LDT F above control.Accordingly, we evaluated cell survival in LDT F hemi slices in which one half had been exposed for 7 h to ACSF and the other half to 7 h in α-syn M .We found a relatively lower cell survival in the half of the slice exposed to α-syn M when normalized to survival seen in control (Cell Survival: Control: ACSF: 100 ± 0.9%, n a = 217/n 40 = 57; α-syn M : 92.8 ± 1.5%, n a = 183/n 40 = 48; Fig. 5A).
Next, we compared cell survival in the halves of the LDT F exposed to α-syn M for 7 h to halves of brain slices of the LDT M that had been similarly exposed to 7 h of α-syn M .Supporting our hypothesis of a sex difference in α-syn M cellular effects, we noted a significantly greater cell survival in the LDT F when compared to cell survival seen in the LDT M (Cell Survival: Female: 87.4 ± 0.6%, n a = 183/n 40 = 48; Males: 79.9 ± 0.6%, n a = 168/n 40 = 66, p < 0.0001; Unpaired Student's T-test; Fig. 5A).These results indicate that α-syn M induces toxic effects in neurons of the LDT F to a lesser degree than in LDT M .

Endogenous neuroprotection against α-syn M induced degeneration in LDT neurons from female involved inhibitory transmission
As we showed a sex-based differential effect of α-syn M on neuronal mortality in the LDT which was reminiscent of the sex-based difference in membrane excitability and changes in intracellular calcium levels induced by this protein which were affected by GABA and glycine receptor antagonists, we reasoned that the differential effect on neurodegeneration could involve inhibitory amino To compare the population data, bisected slices were used, and the proportion of surviving cells observed in the half of the bisected slice exposed to α-syn M was considered the baseline, and the number of surviving cells in the other half of the bisected slice exposed to α-syn M + G-ANT was normalized to this baseline.(C) Fluorescent images of LDT M slices exposed to ⍺-syn M or to α-syn M in presence of 7 h of GABA A and GABA B receptor agonists (G-AGO).As can be seen from the population data shown in bar graphs to the right, the presence of the GABA and glycine receptor agonists in the LDT M was associated with significantly greater cell survival following exposure to α-syn M (Cell Survival α-syn M : n a = 168/n 40 = 66, Cell Survival α-syn M + G-AGO: n a = 127 /n 40 = 48; p = 0.0334; Mann-Whitney Test).In this protocol, the proportion of surviving cells observed in the half of the bisected slice exposed to α-syn M was considered the baseline, and the number of surviving cells in the other half of the bisected slice exposed to α-syn M + G-AGO was normalized to this baseline.LDT F : the laterodorsal tegmental nucleus of female; LDT M : the laterodorsal tegmental nucleus of male.G-ANT: contains SR-95,531 (gabazine, 10 µM), CGP 55,845 (10 µM) and strychnine (2.5 µM) to block GABA A , GABA B , and glycine receptor-mediated responses, respectively.G-AGO: contains muscimol (30 µM) and baclofen (10 µM), which are agonists of GABA A and GABA B receptors, respectively.The scale bar in all images corresponds to 50 μm under 40x magnification.Contrast has been added equally across all the images.*p < 0.05, **** p < 0.0001 acid activity which was protective in the LDT F .To examine this hypothesis, we exposed LDT F cells to α-syn M for 7 h in the presence of antagonists of GABA A , GABA B and glycine receptors (G-ANT) and normalized cell viability to that in the other halves of the bisected slices that were exposed only to α-syn M .The relative degree of LDT F of cell survival associated with α-syn M in the presence of G-ANT was significantly lower than in absence of blockers of receptors of inhibitory amino acids (Cell survival: α-syn M : 100.0 ± 2.4%, n a = 199/n 40 = 44, α-syn M + G-ANT: 88.8 ± 1.5%, n a = 169/n 40 = 35; p = 0.0001; Mann-Whitney Test; Fig. 5B).These results indicate that the neuroprotective effects seen in the female brain against α-syn M -mediated toxicity involve functional inhibitory amino acid transmission.

Activation of GABAergic transmission in male brain induces neuroprotection against α-syn M -induced neurodegeneration
Next, we hypothesized that activation of GABA receptors could be neuroprotective against α-syn M -mediated toxicity in LDT M .To examine this hypothesis, we exposed LDT M neurons to α-syn M for 7 h in the presence of the GABA A , and GABA B agonists, muscimol and baclofen (G-AGO), and cell death was compared to that in the other halves of the bisected slices exposed only to α-syn M .Remarkably, when exposed to α-syn M the degree of cell survival seen in the halves of the slice treated with GABA receptor agonists was significantly higher than that in the other halves of the bisected slices exposed only to α-syn M (Cell Survival: ⍺-syn M : 100.0 ± 2.3%, n a = 168/n 40 = 66, α-syn M + G-AGO; 106.0 ± 2.7%, n a = 127 /n 40 = 48, p = 0.0334; Mann-Whitney Test; Fig. 5C).These results provide further support for the conclusion that in LDT F , a GABAergic-mediated mechanism protects against α-syn M -induced toxicity, and, excitingly, indicate that activation of GABA mechanisms could protect neurons of the LDT M from α-syn M -mediated neurodegeneration.

Discussion
We found that in contrast to what we previously saw in LDT M , effects of α-syn M on the membrane of LDT F neurons were inhibitory, and we recorded decreases in excitatory synaptic events, reductions in firing rate, and relatively more decreases in intracellular calcium.Further, cell death associated with α-syn M was lower in females than in males.Changes in membrane currents and synaptic excitability noted in the SN did not exhibit a sex-based difference, suggesting nucleus specificity of α-syn M -mediated effects.Inhibitory membrane currents and reductions in calcium induced in LDT F were found to involve inhibitory transmission, which when blocked revealed membrane excitation similar to that seen in LDT M .Finally, consistent with a protective role of inhibitory signaling, blockade of GABA A , GABA B , and glycine neurotransmission in the LDT of the female resulted in greater cell death and activation of GAB-Aergic receptors reduced α-syn M -mediated neurodegeneration in the LDT of the male.
While the polarity of α-syn M -induced membrane effects on LDT F neurons was opposite from that seen in our earlier study conducted in LDT M , when presynaptic input was blocked, an excitatory membrane response similar to that seen in LDT M neurons was revealed.This leads us to the interpretation that α-syn M induces a dual effect on the membrane of neurons of the LDT F with the summation resulting in inhibition of membrane currents of the postsynaptic neuron.The mechanism underlying the occlusion of excitatory membrane actions putatively involved GABAreleasing, presynaptic neurons, although glycinergic mechanisms were not ruled out.Sex-based differences were also found in the polarity of calcium responses between male and female in that a decrease of intracellular calcium was observed in the majority of neurons of the LDT F , whereas in LDT M , the majority of responses were indicative of rises in intracellular calcium.Similar to the membrane responses, the decrease in intracellular calcium induced by α-syn M in female involved inhibitory amino acids, suggesting sexbased differences in inhibitory transmission in the LDT.Cell death associated with α-syn M was lower in females but increased when GABA was blocked suggesting that GABA is involved in inhibiting neurodegenerative processes.Consistent with this, the presence of GABA receptor acting agents was able to prevent neurodegeneration in the male LDT.
Besides suggesting potential targets to inhibit neurodegeneration, our data suggest presence of a GABAergic system in the female LDT, which leads to reductions in excitatory cellular effects and cell death and that this system does not function similarly in the male LDT.While not examined specifically within the LDT, the GABAergic system has shown sexual dimorphism in the brain.In both young and adult mice, expression of proteins involved in GABA synthesis and metabolism, as well as presence of GABA A receptors have shown sex-based differences [30][31][32][33][34][35].Further, the numbers of GABAergic neurons, as well as the responsiveness to GABA-acting drugs, have been shown to be associated with sex [36,37].The sex-specific phenotypic and functional differences in the GABAergic system may play key roles in the differential sensitivity of the LDT M and LDT F to α-syn M [31,34,38,39].
Our study has several limitations.Patch clamp recordings and multiple-cell calcium imaging with Fura-AM is difficult in slices from old animals, and thus it remains unknown if the sex-dependent effect of α-syn M continues across ontogeny, which is relevant to neurodegeneration which is expected to increase across age.Further, we did not identify the phenotype of cells that were protected in presence of GABA A , and GABA B receptor agonists.Since loss of cholinergic cells in the LDT and the neighboring pedunculopontine tegmentum has been one neuropathological feature noted in α-syn-related diseases [40,41], we tried to target large neurons with the cholinergic profile [24], however, while we do not believe we recorded from many, if any, GABA cells as they are much smaller [17,24], non-cholinergic neurons could have been included.Nevertheless, while we did not identify the LDT F cell phenotypes exhibiting inhibitory membrane responses to α-syn M , we did show in our earlier work that excitatory cellular responses in the LDT M were transmitter phenotype-independent [42].Finally, while we did examine a nucleus which was central in SDs, given the global nature of sleep, it is almost certain that networks of nuclei, and not just activity in one nucleus mediate aberrant sleep behaviors seen in neurodegenerative diseases.Accordingly, future studies should examine effects across a larger age span, identify cellular phenotype, and conduct recordings across multiple sleep-controlling nuclei.
Despite the limitations, our work is based on multiple strengths, which differ from other investigations.Most studies of cellular effects of α-syn have been conducted using the oligomeric form of α-syn, and thus our data provides important information about effects of the monomeric form.Further, while the focus of much work has been on targeting α-syn intracellular exposure, we have utilized extracellular exposure.Additionally, in many studies, concentrations applied have been higher than those seen during pathological conditions (from 0.5 µM to 5 µM) [43]; however, we have used nanomolar concentrations of highly purified α-syn M , which more accurately reflects the clinical condition.Moreover, few studies have used ex vivo brain tissue but rather cultured cells; thus, our findings more directly add to the body of knowledge of effects in native mammalian tissue.Finally, to the best of our knowledge, no one has previously reported a sex-based difference in cellular effects of α-syn M on any neuronal type in ex vivo studies.α-syn M was shown to induce an inhibitory effect in synaptic transmission in Calyx of Held; however, while both male and female rats were used, data were not analyzed for potential sex-based differences [43].Taken together, this constitutes the first report to show that α-syn M at a concentration reflective of clinical exposures induces a sex-dependent, cellular effect in mammalian neurons.Furthermore, as no difference in membrane effects was observed between LDT F and SN F , but we did see differences between the LDT M and SN M in earlier work [23], this suggests that α-syn M induces sexdependent effects in specific brain nuclei.Future studies of α-syn M effects should consider sex as a factor as well as regional differences.

Functional implications
The observed sex-based, different cellular responses likely have pervasive functional implications for behaviors and symptoms when neurons of the LDT are exposed to α-syn M .We have a working hypothesis that prodromal SDs in PD could be due to early dysfunction of sleep controlling nuclei, including the LDT, which has been implicated in RBD and EDS [23,42,44,45].
Central to this hypothesis, we suspect that as levels of the monomeric form of α-syn rise, cellular effects are exerted on LDT neurons, which include enhancement of cellular excitability and increases in levels of intracellular calcium.Such effects are not elicited in the SN by the monomeric form, and it is believed to be the later appearing forms of aggregated oligomeric and fibril α-syn which produces neurodegeneration in the SN [23,[46][47][48][49][50].Sustained elevation of excitability and calcium levels in the LDT induced by α-syn M could trigger neurodegenerative processes when cells are excited for extended periods, and when calcium levels remain high [51,52].Consistent with this, we have shown that the effects induced by α-syn M were associated with neuronal death in LDT M neurons.However, in SN M neurons in which α-syn M induced an inhibitory membrane effect, and the predominant response was a decrease in intracellular calcium, no differences in neuronal survival were noted, which shows that α-syn M cellular actions do not necessarily lead to degeneration as we saw in the LDT [23].
Interestingly, in the LDT F , α-syn M induced very similar effects on the membrane and synaptic events to those seen in the SN M leading us to suggest that these effects are neuroprotective in the female LDT.This tenant is supported by greater cell viability in the LDT of the female compared to that in the male following α-syn M exposure.The neuroprotective mechanisms involved inhibitory amino acid transmission as the blockade of GABA and glycine receptors revealed an excitatory effect in the LDT F similar to that seen in LDT M , which we hypothesize could underlie cellular degeneration.The outward current was sufficient to mask the concurrent excitatory effect and presumably limit putative damage from α-syn M -mediated excitation.Further, treatment with GABA receptor agonists resulted in reductions in cell death in the LDT M lending further support to the interpretation that GABA signaling is neuroprotective.We did not identify the source of the putative, protective GABAergic effect in LDT F ; however, elucidation of the source of this GABA tone in LDT F is of great interest and will be a focus of future studies.Also likely contributing to α-syn M -induced neurodegeneration in the male LDT were the sex-dependent differences in the firing frequency, as a reduction in firing was induced in the LDT F by α-syn M , whereas an enhancement in firing was seen in our earlier study in the LDT M [42].Over the long-term, increases of neuronal discharge can produce overexcitability-induced cell death since high-levels of excitability and firing elevates glutamate exposure, which results in alterations of intracellular calcium levels.This can trigger apoptotic events and collapse of mitochondrial functions which are all processes contributing to neuronal death [53][54][55][56][57][58].Accordingly, the α-syn M -induced reduction in neuronal firing seen in LDT F could exert a protective effect.
One implication of our findings is that processes controlled by the LDT that could be affected in PD are less likely to be affected in females.While speculative, our data do support clinical findings related to occurrence of LDTinvolved sleeping disorders seen prodromal to PD. RBD and EDS appear to be more common in PD males as well as in males during the prodromal phase.The majority of patients diagnosed with RBD are male, with the reported percentage of females in these studies ranging from 10 to 17.5% of all diagnosed cases [9,10,45,[59][60][61][62].Although very few studies have focused on differences in SD symptoms between men and women, sex differences in RBD symptomatology have been reported with aggressive and violent motor active RBD behaviors appearing more commonly in men than in women [63,64].EDS is characterized by the incapacity of the individual to stay awake during the circadian day due to excessive sleepiness.Several studies have examined the risk of development of PD in EDS patients; however, the majority of investigations which have shown an association between EDS in the prodromal phase of PD have been conducted in males [13,65].In one of the few studies to include both sexes, a higher risk of development of PD was documented in those expressing sleepiness during the day; however, the data were not analyzed to compare the risk in males vs. females, and the majority of the cohort who exhibited EDS were males, which reflects the sex-based odds ratio of PD in the general population [66].In several studies of PD diagnosed patients, EDS has been shown to be more common among PD-affected men than women [65,67,68].

Conclusion
Taken together, our data lead us to conclude that the cellular effects exerted by α-syn M are neuroprotective in the LDT F and could be sufficient to delay α-syn M -mediated cell death in this nucleus, perhaps ceasing when the relevant GAB-Aergic neurons perish as neuronal loss proceeds throughout the PD affected brain.Output from the LDT to rostral and caudal targets is implicated in control of arousal during wakefulness, governance of the sleep and wakefulness cycle, and maintenance of motor atonia, which is a hallmark of REM sleep [18,69].Thus, if GABAergic neurotransmission plays a neuroprotective role by leading to outward currents and reductions in intracellular calcium, thereby counterbalancing negative effects induced by PD processes, this could block loss of cells in the LDT that produce motor atonia during REM sleep and an aroused EEG during wakefulness and sleep, and thereby lead to the lower frequency of RBD and EDS symptoms see in female when compared to those seen in male patients with PD [10,70,71].
As we observed no sex-based difference in cellular responses in the SN, our findings cannot account for the higher incidence of PD in males compared to females; however, we do suggest a mechanistic basis for the higher prevalence of SDs among male vs. female PD patients.Thus, our findings represent an important step toward the identification of sex differences in the mechanisms underlying the pathology of α-syn-associated neurogenerative diseases.Such identification offers the potential of targeting inhibitory mechanisms as neuroprotective strategies in neurodegenerative diseases, and to speed efforts for development of new directions for PD treatment and management in the prodromal phase of these diseases.

Fig. 1 α
Fig. 1 α-syn M induced an inhibitory outward current and modulated synaptic transmission in LDT neurons in the female.A) (Left) Cartoon schematic of the sagittal mouse brain to show the block of the brain containing the LDT.A LDT coronal brain slice taken from this block is shown below in inset.(A) (Right) Coronal brain slice cartoon modified from [72] to show in greater detail in insets to the sides the location of the LDT (indicated by white arrow in panel to the left).(B) Sample of membrane response to α-syn M , which induced inhibitory, outward currents in the female in the LDT (B1).An outward inhibitory current was also elicited in SN neurons (B2).(B) Graphs of holding currents before and after application of α-syn M to LDT F and SN F neurons showed a significant increase in positive holding current indicating that α-syn M induced outward currents.The amplitude of the outward current evoked by α-syn M in LDT F and SN F neurons was not different (LDT F : n = 14, SN F : n = 11; p = 0.3847; Unpaired Student's T-test) as shown by the plots of the individual amplitude of current induced in both nuclei.Bar chart showing that the proportion of recorded cells responding to α-syn M with induction of outward current did not differ significantly between the LDT F and SN F (LDT F : n = 14 sampled/14 responded, SN F : n = 11 sampled/11 responded; p = 1.000;Fisher's Exact Test).(C) α-syn M modulated synaptic events in neurons recorded within LDT F and SN F .(C1-C2) samples of recordings showing frequency of synaptic events in control and in presence of α-syn M in both LDT F and SN F .(Rightmost panels) Single sEPSCs (spontaneous excitatory postsynaptic currents) in a LDT F and in a SN F neuron are shown with a high-gain time and amplitude scale under control conditions and in presence of α-syn M illustrating the reduction in amplitude in both nuclei when α-syn M was present.Data presented in paired plots summarize findings from the population of recorded cells, which revealed that α-syn M induced a significant decrease in amplitude of EPSCs in LDT F (n = 5; p = 0.0253; Paired T-test) and SN F neurons (n = 4; p = 0.0483; Paired T-test) and elicited a significant decrease in the frequency of sEPSCs in LDT F neurons (n = 5; p = 0.0475; Paired T-test), which was a change not seen in sEPSCs in the SN (n = 4; p = 0.4735; Paired T-test).LDT: Laterodorsal tegmental nucleus; 4 V: 4th ventricle; IC: Inferior colliculus; DTgP: Dorsal tegmental nucleus, pericentral: DRN: dorsal raphe nucleus; LC: Locus coeruleus; LDT F : Laterodorsal tegmental nucleus of female; SN F : Substantia nigra of female.* Indicates p < 0.05, *** Indicates p < 0.001

Fig. 2
Fig. 2 Sample of changes in fluorescence (DF/F%) induced by α-syn M , which are indicative of alterations in intracellular calcium levels in LDT M and LDT F .(A) In both sexes, changes in response of the fluorescence to α-syn M exhibited two different polarities, which suggested increases (A1a, A2a) or decreases (A1b, A2b) in intracellular calcium levels, respectively.Inset in A2 is a fluorescent image under 380 nm wavelength light of one of the LDT F brain slices used in this study in which two Fura 2-AM filled cells indicated with red arrows can be seen.Regions of interest were drawn around each cell and average fluorescent intensity (F) within each region of interest was plotted against time.White scale bar indicates 20 μm.(B) Histograms summarizing the data from the population of recorded cells indicating that (B1) the frequency of responses to α-syn M did not significantly differ between the two sexes (LDT M : n = 38/39, LDT F : n = 89/89; p = 0.3047; Fischer's Exact test), (B2) whereas the distribution of response polarity differed significantly between the sexes with a greater proportion of responses suggesting decreases in calcium being elicited in females than males (LDT M : n of decreases = 9/38, LDT F : n of decreases = 52/89; p = 0.0004; Fisher's Exact test).LDT M : Laterodorsal tegmental nucleus of male; LDT F : Laterodorsal tegmental nucleus of female.*** Indicates p < 0.001

Fig. 3
Fig.3 An excitatory inward current was revealed when α-syn M was applied during blockade of presynaptic transmission, or antagonism of GABA A , GABA B and glycine receptors, suggesting that α-syn M induces an outward membrane current in LDT F neurons due to actions at presynaptic inhibitory neurons.A) Sample of membrane responses to α-syn M in which an inward current is revealed when α-syn M is applied in presence of (A1) TTX, (A2) low calcium solution, or (A3) a cocktail of SR-95,531 + CGP-55,845 + strychnine, which block GABA A , GABA B and glycine receptors, respectively).(B) Histograms from a population of cells recorded in which there were significant changes in the (B1) polarity of the evoked membrane current responses to α-syn M in presence of TTX, low calcium solution or GABA and glycine receptor antagonists when compared to control responses (Fisher's Exact test).(B2) The amplitude of the current evoked by α-syn M in control conditions and under conditions of synaptic blockade and inhibitory receptor antagonists is shown revealing the change in polarity of the α-syn M induced current.(B2, Inset) Graphs of holding currents before and after application of α-syn M to LDT F showed that in presence of TTX, low calcium solution, and GABA A , GABA B , and glycine receptor antagonists, holding currents became significantly more negative after application of α-syn M , which reflected the α-syn M -mediated induction of inward currents.(C) The decrease in intracellular calcium in LDT F is mediated, at least in part, by inhibitory receptors as illustrated in this histogram showing data from the population of LDT F cells recorded that showed a significantly smaller decrease in the amplitude of intracellular calcium induced by α-syn M in presence of SR-95,531, CGP-55,845 and strychnine when compared to control conditions (Control: n = 52, GABA/Gly antagonists: n = 49; p = 0.0001; Paired T-test).LDT F : Laterodorsal tegmental nucleus of female.* Indicates p < 0.05, ** Indicates p < 0.01, *** Indicates p < 0.001

Fig. 4 αFig. 5 (
Fig. 4 α-syn M induces significant changes in the firing frequency in neurons recorded within LDT F .(A) Representative examples of current-clamp recordings of LDT neurons in which action potentials were induced by holding the cell at -45 mV under control conditions (top) and in presence of α-syn M .(bottom).(B) The reduction in firing frequency induced by α-syn M was significant as shown in the bar graphs depicting the average firing rate from the population of recorded LDT F neurons (n = 3; p = 0.0498; Paired T-test).LDT F : Laterodorsal tegmental nucleus of female * Indicates p < 0.05