Sex-dependent effects of monomeric α -synuclein on calcium and cell death of lateral hypothalamic mouse neurons are altered by orexin

Parkinson's Disease (PD) patients experience sleeping disorders in addition to the disease-defining symptomology of movement dysfunctions. The prevalence of PD is sex-based and presence of sleeping disorders in PD also shows sex bias with a stronger phenotype in males. In addition to loss of dopamine-containing neurons in the striatum, arousal-related, orexin-containing neurons in the lateral hypothalamus (LH) are lost in PD, which could contribute to state-related disorders. As orexin has been shown to be involved in sleeping disorders and to have neuroprotective effects, we asked whether orexin could protect sleep-related LH neurons from damage putatively from the protein α -synuclein ( α -syn), which is found at high levels in the PD brain and that we have shown is associated with putatively excitotoxic rises in intracellular calcium in brainstem sleep-controlling nuclei, especially in males. Accordingly, we monitored intracellular calcium transients induced by α -syn and whether concurrent exposure to orexin affected those transients in LH cells of the mouse brain slice using calcium imaging. Further, we used an assay of cell death to determine whether LH cell viability was influenced when α -syn and orexin were co-applied when compared to exposure to α -syn alone. We found that excitatory calcium events induced by α -syn were reduced in amplitude and frequency when orexin was co-applied, and when data were evaluated by sex, this effect was found to be greater in females. In addition, α -syn exposure was associated with cell death that was higher in males, and interestingly, reduced cell death was noted when orexin was present, which did not show a sex bias. We interpret our findings to indicate that orexin is protective to α - syn -mediated damage to hypothalamic neurons, and the actions of orexin on α -syn-induced cellular effects differ between sexes, which could underlie sex-based differences in sleeping disorders in PD.


Introduction
Although the etiology of Parkinson's Disease (PD), a disease characterized by presence of neurodegeneration of substantia nigra (SN) dopamine-containing cells is unknown, it is believed to involve the protein α-synuclein (α-syn).The loss of dopaminergic SN cells leads to diagnosis-defining symptomology which are motoric signs, however, during the prodromal phase when motor signs are absent, patients often experience non motor symptoms, which continue after diagnosis.The most common non motor symptoms are disorders of state.PD was found to appear in 38 % of older male patients initially diagnosed with REM sleep behavior disorder (RBD) (Schenck et al., 1996).Nearly 40-65 % of patients with RBD later develop synucleinopathies including PD (Claassen et al., 2010).Following the realization of the association between the sleep disorder RBD and PD, another sleep-wake disorder was associated with PD, excessive daytime somnolence (EDS) (Arnulf et al., 2008;Shen et al., 2018).While the presence of EDS in the PD prodromal period has not been nearly as well characterized as RBD, the risk of developing PD is threefold higher in patients initially diagnosed with EDS (Abbott et al., 2005;Dhawan et al., 2006).The appearance of these disorders of state has led to the hypothesis that degeneration of neurons which control sleep and wake behaviors might actually precede that of the dopamine cells of the SN, which is supported by data showing pathology of the sleep and wake neurons in neurodegenerative diseases (For review, see Borghammer, 2023;Stern and Naidoo, 2015).Interestingly, the prevalence of PD, and the sleeping disorders associated with PD during the prodromal phase and after diagnosis are much higher in males when compared to females in many studies (Martinez-Martin et al., 2012;Ondo et al., 2001;Paus et al., 2003), but not all (Poryazova et al., 2010).Further, after diagnosis of PD, RBD and EDS is present in males at a higher rate than in females diagnosed with PD, which in the case of EDS is not reflected in the sex ratio seen in the general population of non PD patients with EDS (Baldwin et al., 2004;Xiang et al., 2019).When taken together, the data support the hypothesis that sex-based differences in the neural processes underlying the disease exist and may extend to sleep-related nuclei.
Degeneration of the brain stem neurons involved in atonia of REM sleep has been implicated in RBD in PD.Further, their loss could play a role in PD-associated EDS via loss of activity of these neurons in the reticular activating system (RAS).Disease-related changes in activity of the orexin neurons, also called hypocretin, of the lateral hypothalamus could also play a role.Orexin neurons are lost in narcolepsy, a disorder characterized by EDS, and the severity of EDS is correlated with the loss of hypothalamic orexin neurons (Thannickal et al., 2007).Further, decreased levels of orexin peptide have been noted in the ventricular cerebral spinal fluid of PD patients with the lowest levels correlated with severity of the disease, and levels in the prefrontal cortex, one of the target regions of orexin neurons were substantially lower than in controls, suggesting reduced orexinergic transmission (Drouot et al., 2003;Fronczek et al., 2007).These data indicate that orexinergic neurons are significantly diminished at the time of diagnosis and progressed disease, but do not provide evidence of when in the disease process they are vulnerable.Failure to detect protein aggregates in the orexin neurons in a post mortem study in which aggregates were clearly present in the SN lead those investigators to speculate that orexin cells either die by another mechanism, or they die very early in the progression of the disease (Thannickal et al., 2007).As orexin has been shown to be neuroprotective of dopamine SN neurons in mouse models of Parkinson's Disease (Liu et al., 2018), one potentially mechanistically-relevant consequence of an early loss of orexin neurons could be a removal of the protective effect of orexin on neurons at target sites.
While α-syn is believed to play a role in neurodegeneration of PD, the mechanisms underlying this role remain elusive (Ozansoy and Basak,   2013).α-syn is a small protein, which is abundantly expressed in presynaptic neurons and believed to play a role in synaptic events, but its exact function(s) remains unknown (Maroteaux et al., 1988).α-syn is an intrinsically disordered protein, which is therefore very dynamic and easily undergoes conformational changes (Fink, 2006).Depending on the conditions, it can exist in multiple structural variations; e.g.native disordered monomers, membrane interacting monomers with varying α-helical content, misfolded monomers, oligomers and fibrils (Breydo et al., 2012), and of those, oligomers and fibrils are pathological characteristics for PD (For review, see Das and Eliezer, 2019;Lashuel et al., 2013).Presumably, during the prodromal phase of PD, levels of unstructured monomeric α-syn rise.As the disease progresses, the presence of the more aggregated oligomeric and fibril forms increases.We have previously shown that the monomeric form of α-syn induces membrane excitation in males that was associated with rises in calcium in the majority of neurons in two brainstem nuclei that are involved in REM sleep and arousal, the laterodorsal tegmental nucleus (LDT) and the pedunculopontine nucleus (PPT), which differed from actions induced in male SN neurons (Dos Santos et al., 2021).Further, we have shown that monomeric α-syn leads to cell death in the male LDT, which was not seen in male SN (Dos Santos et al., 2021).Females were not included in our first report (Dos Santos et al., 2021).However, in a later study conducted in females, interestingly, inhibitory membrane effects that were associated with fewer rises in calcium were evoked by α-syn in LDT, which indicated a sex-based difference (Dos Santos et al., 2023).
Further, α-syn-associated cell death in the LDT of females was lower than in males (Dos Santos et al., 2023).However, excitatory membrane actions, a shift in dynamics towards higher calcium levels, and increased cell death could be seen in female LDT when GABA transmission was blocked, suggesting presence of a GABAergic protective mechanism (Dos Santos et al., 2023).
As orexin neurons play a significant role in arousal and are lost in PD, and PD patients can exhibit EDS prior to motoric symptoms, in the present study we asked whether the native monomeric form of the α-syn protein exerted actions on lateral hypothalamus (LH) neurons, which we have previously shown were associated with heightened cell death.In addition, in the current work, we directly assayed cell death of LH cells following α-syn exposure.As orexin has been shown to be neuroprotective, we examined whether the cellular actions of α-syn on intracellular calcium were altered when orexin was present, and whether cellular viability differed by comparing and contrasting cell survival following α-syn exposure in presence and absence of orexin.
Finally, we determined whether actions seen on calcium and cell death were present in both sexes.

Animals
All animal procedures were authorized by the Animal Welfare Committee appointed by the Danish Ministry of Justice and in accordance with the European Communities Council Directive of November 1986 (86/609/EEC).Animal handling was conducted by authorized personnel at the Department of Drug Design and Pharmacology at the University of Copenhagen.Studies were carried out on Naval Medical Research Institute (NMRI) wild-type mice aged 14-28 post-natal days (PND) of both sexes that were housed in an open cage system in a temperature and humidity-controlled room (temperature ± 2 • C, humidity 36-58 %) with a 12:12 h light/dark cycle starting from 7 am to 7 pm.The mice were purchased from ScanBur (Charles River, Germany) in litters of 10 pups with a nursing female mother.The mice had ad libitum access to water and food.

Brain slice preparation
Brain slices of a thickness of 250 μm containing the LH (Fig. 1A) were collected from NMRI mice.Mice were anaesthetized with isoflurane (ScanVet Animal Health A/S, Denmark), and the depth of the anesthesia was evaluated by loss of the righting reflex, and failure to respond to a paw pinch.Following decapitation, the section of brain containing the LH area was swiftly removed and submerged in an ice-cold solution of artificial cerebral spinal fluid (aCSF) which contained: NaCl (124 mM), KCl (5 mM), NaHCO 3 (26 mM), Na 2 HPO 4 (1.2 mM), glucose (10 mM), CaCl 2 (2.7 mM), MgSO 4 (1.2 mM) bubbled with carbogen (95 % O 2 /5% CO2) and adjusted to a pH and osmolarity of 7.40 (± 0.05) and Osm/L (± 5), respectively.The LH block was then sectioned into slices of a thickness of 250 μm for calcium imaging or 300 μm for the cell viability assays.Identification of LH was conducted using known hallmarks including locating the visible 3rd ventricle.Relevant anatomical features indicating presence of LH could be collected from 2 to 3 brain slices.Under bright field illumination at 4× magnification, the LH was located lateral to the 3rd ventricle and diagonally ventral to the mammillothalamic tract.Slices containing the LH to be used for calcium imaging were incubated at 32 • C for 15 min.in a water bath and then kept at room temperature for at least 1 h for acclimatization under continuous carbogen bubbling before continuing.Slices containing the LH to be used for cell viability studies were bisected and were not incubated in the water bath but proceeded directly to incubations in relevant drug solutions.responded to α-syn was compared between males and females, there were no significant differences.(B3) There were also no significant differences in the proportion of responding cells which exhibited an increase or a decrease in male LH or female LH.(B4) There were no differences in the amplitude of the increase (B4) or decrease (B5) induced by α-syn between males or females.(C1, 2) Orexin induced changes in fluorescence in the majority of LH cells in males and females as seen in these representative examples of an increase in fluorescence and a decrease in fluorescence from two different cells.(C3) While decreases were elicited in a minority of cells in both males and females, the most common response to orexin was an increase in calcium.There were no differences in amplitude when increases in fluorescence (C4) or decreases in fluorescence (C5) were elicited in males or females.Orx: Orexin; LH: lateral hypothalamus.*: p < 0.05, and ns indicates no significance.

Drug and compound solutions
For calcium imaging, drugs and compounds were applied via the bath and a total volume of 3 mL with a flow rate between 1 and 1.3 mL/ min was used.Final concentrations were prepared from aliquots of frozen stock solutions dissolved in oxygenated aCSF and used immediately.The following drugs or compounds were used for calcium imaging: Monomeric α-synuclein: Expression and characterization of monomeric α-syn was conducted as previously described (Dos Santos et al., 2021, 2022).Stock solutions of 100 μM monomeric α-syn were stored at − 20 • C in aliquots of 10 μL until use at which time the final concentration of 100 nM α-syn was prepared by pipetting 3 μL α-syn (100 μM) to a total volume of 3 mL aCSF.Orexin: Stock solutions of 100 μM orexin were prepared from Orexin A (reported active at human, rat, mouse Orexin receptors -Hello Bio Product #2937), which activates both Orexin A and Orexin B receptors (Scammell and Winrow, 2011) and were stored at − 80 • C in aliquots of 30 μL until use.The final volume of 1 μM orexin was prepared by dilution of 30 μL (100 μM) to a total volume of 3 mL aCSF.Orexin and α-synuclein: A solution of 1 μM orexin and 100 nM α-syn was prepared by pipetting 3 μL α-syn (100 μM) to 3 mL orexin (1 μM).AMPA: Stock solutions of 1 mM AMPA (Tocris, UK) were stored at − 20 • C in aliquots until use.Prior to bath application, the final concentration of 10 μM AMPA was prepared by pipetting 30 μL AMPA (1 mM) to 3 mL aCSF.AMPA and α-synuclein: A solution of 10 μM AMPA and 100 nM α-syn was prepared by pipetting 3 μL α-syn (100 μM) to 3 mL AMPA (10 μM).

Calcium imaging Recordings
In order to monitor changes in fluorescence as an indirect indicator of a change in intracellular calcium, cells in brain slices containing LH were loaded with a Fura 2-AM solution as previously reported (Dos Santos et al., 2021).Slices were incubated in a freshly made solution of 20 μM Fura 2-AM, 0.5 % Pluronic acid (Merck, Denmark) and 500 μL oxygenated aCSF at 32 • C under constant carbogen bubbling.Incubation time for the slices was 1 min for each PND of the animal.For mice younger than 20 PND, an additional 10 mins were added to the time.As the dye is light sensitive and to avoid the risk for photo bleaching of slices, light exposure was minimized by enclosing the incubation chamber in foil.To rinse dye particles, Fura 2-AM loaded slices were submerged in a recording chamber filled with oxygenated aCSF at room temperature for at least 15 mins before drug application.
Calcium imaging recordings were obtained using a cooled CCD camera system (12-bit CCD Sensicam, PCO Imaging, Germany) controlled by a polychromator (Polychrome V, Till Photonics, Germany) with a Xenon light source.The CCD camera was attached to an upright microscope (Olympus BX50WI, Japan) controlled by the imaging software Live Acquisition (Till Photonics, Germany).Slices were perfused at approximately 1-1.3 mL/min using a perfusion pump with aCSF saturated with carbogen.The LH area was located with a 4× zoom objective (Plan N, Olympus, Japan) by bright field illumination and landmarks were identified.After recognition of LH, cells from the area were imaged using a 40× water immersion lens (LUMPlan FI, Olympus, Japan).
Fura 2 binds to free calcium ions and emits at a greater intensity when excited at 340 nm than at 380 nm, making it possible to determine rises and decreases in free calcium as rises or decreases in the F 340nm / F 380nm ratio, respectively.Ratiometric fluorescent imaging was obtained by fast software-controlled switching of the excitation wavelength between 340 nm and 380 nm using a Chroma Fura2 filter set with emission fluorescence detected at 515 nm.Images collected at 340 nm and 380 nm were binned at 2 × 2 and collected with an interval of 250 ms (AMPA) -2000 ms (peptides).Exposure times at the two wavelengths were adjusted to give a maximum fluorescence intensity of around 300-400 grey levels (out of a potential 4096 levels).Regions of interest (ROI) were placed over identified cells and the average fluorescence was monitored within these ROIs, and the ratio of the two wavelengths was obtained online using Live Acquisition.Offline analysis was conducted using the offline tool, Offline Analysis (Till Photonics, Germany).

Data processing and analysis of calcium imaging data
Data were excluded if slices moved during recording, a major change in focus occurred such that cells disappeared within a ROI, or if dye debris were present, which could cross the field leading to disruptions of the recording of fluorescent responses.The ratio of the fluorescence intensities (F) at 340 nm and 380 nm were processed in Microsoft Excel (Microsoft Office) to calculate the percentage of change in fluorescence for each response.The degree to which calcium was changed is indirectly expressed as the difference between fluorescence changes (DF/F) calculated as a percentage (DF/F%).Background fluorescence was determined from a ROI positioned in an area absent of Fura 2 loaded cells and subtracted.Following subtraction of background fluorescence, the difference between the peak and baseline fluorescence which were determined by taking an average of 10 data points was calculated and this was determined as the amplitude of the response.The amplitude was only measured in recordings where clear baselines and peaks could be calculated and fluorescence recovered to near baseline levels.The majority of cells exhibited a kinetic profile defined as a plateau response, which has previously been characterized as a smooth rise to a negative or positive peak and gradual decay back to baseline (Kohlmeier et al., 2004).

Cell viability assay
In this study, DNA fragmentation and membrane integrity were assayed by using nuclear staining with the DNA specific fluorescent stains 4,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) (Cummings and Schnellmann, 2004) as previously described (Dos Santos et al., 2021Santos et al., , 2022)).This allowed us to determine the number of viable cells in our treated slices.Live/fixed cells accumulate DAPI but not PI; whereas, PI accumulates in necrotic, dead cells indicating a loss of membrane integrity (Cummings and Schnellmann, 2004).The two nuclear stains exhibit different maximum excitation and emission wavelengths, as PI-stained nuclei are visualized using 493 nm excitation and 636 nm emission wavelengths, and DAPI is visualized using 350 nm excitation and 470 nm emission wavelengths, which allows for dual wavelength imaging (Cummings and Schnellmann, 2004).
Following bisection, each LH hemi slice of 300 μm thickness was incubated at room temperature (20-22 • C) in 1 mL of aCSF, 100 nM α-syn, or a solution of 1 μM orexin and 100 nM α-syn under constant carbogen bubbling for 7.5 h, which was defined as an acute exposure to drug.Two studies were conducted.In the first, one half of the slice was incubated for 7.5 h in a solution of 100 nM α-syn in aCSF, and the other half was incubated in aCSF for the same duration, which served as the matched control.In the second study, one half of the slice was incubated for 7.5 h in a solution of 1 μM orexin and 100 nM α-syn, and the other half was incubated in 100 nM α-syn for the same duration, which served as the matched control.
Following incubation, slices were fixated in 4 % paraformaldehyde (PFA) for 4 h before changing the medium to 30 % sucrose to stop the fixation induced by PFA and induce cryoprotection.Slices were kept in sucrose for at least 24 h at 5 • C until further sectioning of slices.

S. Bohid et al.
Sectioning of slices at 40 μm was performed on a Leica cryostat (CM3050S, Germany) following mounting in O.C.T (Tissue-Tek®, Netherlands).Slices of 40 μm thickness were mounted on glass slides and dried.Slides were submerged for 5 min in an aluminum foil covered staining dish containing a PBS solution with the fluorescent markers PI and DAPI.Washing under protection from light exposure was conducted, and slides were placed to dry before being covered with coverslips using a quick-hardening mounting medium (Eukitt®, Sigma-Aldrich).

Data processing and analysis of cell viability data
Imaging of PI and DAPI positive cells in the LH was obtained using a fluorescence microscope (Axioskop 2, Zeiss, Germany) equipped with a monochrome CCD digital camera (AxioCam MRM, Zeiss, Germany).Following localization of LH, the fluorescence of DAPI and PI was viewed using Axiovision Software (Zeiss, Germany).Exposure time was adjusted to maximize contrast between the fluorescence of DAPI and PI cells and background without suppressing cells exhibiting lower fluorescence intensity.Data analyses of acquired images were performed using the software ImageJ/FIJI (NIH, USA).Positively labelled cells were semi-automatically counted by first converting all images to binary images to enhance the visualization of fluorescent stained cells against the background by enabling thresholding, which subtracted background and adjusted brightness in order to enhance the contrast between fluorescent stained cells and background.A watershed algorithm was then used to separate cells, which allowed counting of individual cells.The final step was automatic cell counting, with manual checking, which was performed under illumination of DAPI positive and PI positive fluorescence separately from the same LH area within a slice.Cell count was then transferred to Microsoft Excel to calculate the cell viability.Cell viability percentages were calculated as the ratio of living cells (DAPI positive) to the total number of cells (DAPI positive + PI positive).

Statistical analysis
GraphPad Prism 8 was used to conduct all statistical analysis and create all graphs.For both calcium imaging and cell viability studies, data were first analyzed irrespective of sex, and then data were divided into male and female groups with figures mostly reflecting data analyzed when groups were divided because sex-differences were noted.
Prior to comparisons of differences between mean amplitudes of DF/ F%, identification of outliers using the ROUT method was performed on all responding cells before statistical analysis.Outliers were removed from data sets according to the aggressivity constant (Q-value), which was set to 1 %.As data were also checked for outliers after being divided by sex, sometimes outliers were identified following this division, which means the n for the males and females added together does not always equal the n for the population when sex is combined.
For calcium imaging data, the number of cells used in the statistical analysis is denoted with "n" unless otherwise stated.Recordings were conducted in an additional 185 cells not included in the 'n'; however, analysis was not conducted of those recordings primarily due to artefacts from Fura 2-AM particles.For comparison of mean amplitudes of DF/F% rises or decreases, the Student's unpaired t-test (two tailed) was used for comparison of two populations, while a one-way ANOVA was used for comparisons of three or more populations.In case of a significant difference using the one-way ANOVA, the test was followed by post hoc analysis using Tukey's honestly significant difference (HSD) test.For comparison of categorical outcomes (differences between the proportion of cells responding or not responding, and the proportion of cells exhibiting increases or decreases in amplitude), a Fisher's exact test was used for small-sized samples, while a Chi-square test was used for larger samples, followed by a Fisher's exact test when significance was detected when conducting multiple categorical comparisons.
When compounds were co-applied, a population study was conducted to compare expected outcomes with observed outcomes.
Expected values for proportions responding or for proportions showing increases or decreases in fluorescence when compounds were co-applied were mathematically calculated by adding together the response ratios seen when compounds (i.e.AMPA, orexin and α-syn) were individually applied.This expected value (based on summation of numerical outcomes of the individual effects of each compound) was statistically compared to the observed numerical outcomes when compounds were co-applied using a Fisher's exact-test or a Chi-Square text.For graphs, data were plotted as percentages, but numerical outcomes are presented in Results text.Expected values for response amplitudes in DF/F% were mathematically calculated by using the individual amplitudes in DF/F% from each cell when AMPA, orexin or α-syn were applied, rather than adding the average mean obtained for each drug.This required that the number of observations for the category with the majority of observations be reduced by use of a systematic sampling method to equal that of the category with a minority of observations.In some cases, this resulted in an expected mean that was different from those obtained by instead adding the mean amplitude obtained for each drug.The expected value was statistically compared to the value obtained when the two compounds were co-applied using a Student's t-test, or an ANOVA.
For analysis of the cell viability data, the number of slices used in the statistical analysis is denoted by "n".Results were normalized in one half of the slice to their matched controls (the other half of the slice).The value of viability in the matched control was characterized as 100 %.A Student's paired t-test was used for comparison of pooled data from male and female cell viability percentages because each hemi slice could be compared to its matched control half, while a Student's unpaired t-test (two-tailed) was used for statistical difference testing between sexes.Data sets were considered significantly different if p < 0.05 with differing p values indicated by asterisks in figures: *: p < 0.05, **: p < 0.01, ***: p < 0.001 and ****: p < 0.0001.NS denotes comparisons where a lack of significance was detected.

Results
The data included in the calcium imaging results comprise recordings on 842 cells from 63 naïve.Of the 63 mice, 33 were male, and the remaining 30 were female.Data from the cell viability study source from examination of 41 slices of a 300 μm thickness from 10 mice.Of those 10 mice, 6 were males and the remaining 4 were female.

α-synuclein induces changes in calcium in the lateral hypothalamus in male and female
Our first step was to determine whether α-syn induced changes in intracellular calcium in LH cells as, to the best of our knowledge, no one has applied α-syn to LH cells and monitored alterations in activity, including activity which regulates intracellular calcium.Accordingly, we applied 100 nM α-syn to the bath to mouse brain slices containing the LH and monitored changes in fluorescence in Fura 2 loaded LH cells (Fig. 1A).Changes in fluorescence that exhibited a plateau response were seen in the majority of LH cells examined (88 %; n = 107/122).Of the responding cells, 77 % of them exhibited fluorescent changes indicative of rises in intracellular calcium, whereas decreases were seen in the remainder, which was a significant difference (n = 82/107; Fisher's exact test; p < 0.0001; data not shown).Increases and decreases elicited by α-syn were characterized by a smooth rise to peak, and a gradual return to baseline, which we have previously called a plateau response (Fig. 1B1) (Kohlmeier et al., 2004).The amplitude of the increase across the population of cells was 32.6 ± 2.5 %DF/F (n = 76), which was similar to the average amplitude of the change in fluorescence when decreases were elicited (23.2 ± 2.4 %DF/F; n = 24).
As we had previously seen that calcium responses to α-syn exhibited sex differences in the LDT (Dos Santos et al., 2023), we examined potential sex-based differences in response to the protein in the LH.
Although α-syn induced changes in calcium in the majority of LH cells in S. Bohid et al. the male and female, there was a significant difference in the numbers of cells responding, as the proportions of responding cells in male and female mice were 81 % and 96 %, respectively (Male: n = 56/69, Female: n = 51/53; Fisher's exact test; p = 0.0127; Fig. 1B2).However, while the proportion responding was higher in females, there were no significant differences in the distribution of the polarity of the responses between the sexes (Increases: Male: n = 40/56; Female: n = 42/51; Fisher's exact test; p-value = 0.2530; Fig. 1B3), which is in contrast to what has been seen in the LDT, where calcium decreases are the most predominant response in females, whereas increases in calcium represent the majority of responses in males (Dos Santos et al., 2023).There were no significant differences in amplitudes of calcium rises as males exhibited an average amplitude of 33.0 ± 4.5 %DF/F in response to α-syn, and LH cells from female mice showed an amplitude rise of 34.3 ± 2.7 %DF/F (Male: n = 40; Female: n = 37; Student's unpaired t-test, p = 0.8051; Fig. 1B4).The amplitude of decreases in calcium was also not significantly different between the sexes (Male: 20.4 ± 3.1 %DF/F, n = 16; Female: 28.8 ± 3.1 %DF/F, n = 8; Student's unpaired t-test, p = 0.1019; Fig. 1B5).

Orexin induces changes in calcium in the lateral hypothalamus in male and female
Based on literature, we expected orexin to lead to rises in intracellular calcium, and consistent with an earlier report using Fura 2 imaging (van den Pol et al., 1998), we found that 1 μM orexin induced changes in fluorescence in 87 % of the cells examined in the LH area (n = 90/103), and the predominant response was an increase in calcium as 89 % of the responding cells exhibited rises in fluorescence (n = 80/90; Fisher's exact test; p < 0.0001; data not shown).Both increases and decreases in calcium exhibited the plateau kinetic, which presented with an average amplitude of 28.6 ± 2.0 %DF/F (n = 71) and 38.1 ± 6.3 %DF/F, (n = 10), respectively (Fig. 1C1).
Next, we analyzed our data according to sex and found that there were no significant differences in the proportions of cells responding to orexin between males and females, (Male: 90 %, n = 45/50; Female: 85 %, n = 45/53; Fisher's exact test; p = 0.5569; Fig. 1C2).Further, there were no significant differences in the proportion of increases vs. decreases between the sexes and rises in calcium were the most common response in both males and females as this response occurred in 88 % of the responding cells in both sexes (Male: n = 40/45; Female: n = 40/45, Fisher's test; p > 0.9999; Fig. 1C3).There were no significant differences between sex in the amplitude of the rise in calcium (Male: 29.2 ± 2.4 % DF/F, n = 36; Female: 26.2 ± 2.7 %DF/F, n = 34; Student's unpaired ttest, p = 0.4221; Fig. 1C4).Similarly, although decreases were rare, when elicited, the amplitude of the decrease was not significantly different between the sexes (Males: 36.2 ± 7.5 %DF/F, n = 5; Females: 40.1 ± 11.1 %DF/F, n = 5; Student's unpaired t-test, p = 0.7763; Fig. 1C5).

Co-application of orexin and α-synuclein resulted in rises in calcium
which were smaller than would be expected based on individual responses to either peptide 3.3.1.AMPA and α-syn effects on calcium are additive in females and in males they are synergistic (Control Study) Our next step was to conduct an occlusion experiment to evaluate whether an interaction between α-syn and orexin was occurring by determining whether the change in fluorescence expected by adding the individual responses to each peptide was not different from what was observed when the two peptides were co-applied.However, we first conducted a control experiment to see whether rises should be additive if two processes were not interacting.We previously showed in the LDT that α-syn does not result in membrane actions via effects at AMPA receptors (Dos Santos et al., 2022), therefore, effects of these two compounds on inducing rises in calcium when co-applied should be additive.
As expected, bath application of 10 μM AMPA resulted in rises in calcium exhibiting a plateau kinetic (Fig. 2A) in 100 % of the LH cells examined (n = 79/79) with an average amplitude of 40.5 ± 3.3 %DF/F (n = 67).This effect did not significantly differ between males or females as 100 % of the cells tested in the male and female responded with rises in calcium exhibiting the plateau kinetic (Fisher's exact test for proportion responding and polarity; p > 0.9999; Fig. 2B1, B2).However, interestingly, the amplitude of the rise did show a sex-based difference with an average of 28.9 ± 3.0 %DF/F in males, and 46.1 ± 4.7 %DF/F in females (Male: n = 25; Female: n = 41; Student's unpaired t-test, p = 0.0102; total n = 66 as one data point was removed in outlier tests; Fig. 2B3).This suggests some sex-based difference in processes mediating AMPA-induced calcium, but while interesting, this point was tangential to our study, and we did not pursue it further.
Co-application of 10 μM AMPA and 100 nM α-syn via the bath induced a change in calcium in 100 % of cells examined (n = 70/70).Coapplication induced increases in calcium levels in 99 % of responding cells which exhibited a plateau-like profile (n = 69/70).When comparing the observed results with those expected, we found that there were no significant differences between the proportion of cells responding with AMPA alone, and the proportion responding to coapplication of AMPA and α-syn (Fisher's exact test; p > 0.9999).
Further, of the responding cells, there were no significant differences in the proportion of cells exhibiting increases or decreases as the responding cells virtually all exhibited increases when both compounds were applied (Fisher's exact test; p > 0.9999).When comparing the expected amplitudes in the population of cells irrespective of sex with those which were observed, there were no significant differences (Average Amplitude: Expected: 84.7 ± 5.5, n = 67; Observed: 99.0 ± 6.1, n = 70, Student's unpaired t-test, p = 0.0821; Fig. 3A1).
When examined according to sex, co-application of 10 μM AMPA and 100 nM α-syn induced responses in nearly 100 % of LH cells in both male and female (Males: 98 %; Females: 100 %), and there were no significant differences between the sexes in regard to the proportion of cells responding and the polarity of the response (Fisher's exact test; p > 0.9999; data not shown).However, there was a sex difference in the amplitude of the rise elicited as co-application of AMPA and α-syn induced an amplitude of 114.5 ± 6.8 %DF/F in males (n = 41), and 65.8.1 ± 6.8 %DF/F in females (n = 21), which was significantly different between the sexes (Student's unpaired t-test, p < 0.0001; Fig. 3A2).Further, when comparing the expected vs. observed amplitude results in each sex, it was noted in males that the observed amplitude of co-application was significantly greater than the expected amplitude (Average Amplitude: Expected: 71.6 ± 6.1% DF/F, n = 25; Observed: 114.5 ± 6.8% DF/F, n = 41, Tukey's HSD test: p < 0.0001; Fig. 3A2); whereas, in females, there was no significant difference between the amplitude expected and the amplitude observed (Average Amplitude: Expected: 80.5 ± 6.5 %DF/F, n = 40; Observed: 65.8 ± 6.8 %DF/F, n = 27, Tukey's HSD test: p = 0.3859; Fig. 3A2).The differences between sexes which were observed were surprising as comparing expected values based on adding observed amplitudes resulting from application of α-syn and AMPA alone did not show a statistical difference (Average Amplitude: Male: 71.6 ± 6.1% DF/F, n = 25; Female: 80.5 ± 5.6% DF/F, n = 40, Tukey's HSD test: p = 0.7793; Fig. 3A2).While this sex-based difference was interesting and could suggest sexbased differences in synergy between the processes activated by AMPA receptor stimulation and currently-unidentified mechanisms activated by α-syn, we did not pursue this sex-based finding further as we wished to use this assay to eliminate occlusion, and as we had not seen that the two compounds occluded each other in either sex, we went further in our co-application studies with orexin and α-syn.

Orexin and α-syn effects on calcium occlude one another
After establishing that effects of two compounds which putatively work on different receptor and effector systems do not occlude one another, we examined the effects of co-application of 1 μM orexin and 100 nM α-syn.Co-application of orexin and α-syn altered the intracellular calcium level in 88 % of the cells examined in the LH (n = 124/141) and fluorescent changes were indicative of both rises and decreases in calcium (Fig. 3B1).When the two peptides were co-applied there was not a significant difference in distribution between the two response polarities (Increases: 56 % of responding cells, n = 70; Decreases: 44 % of responding cells, n = 54; Fisher's exact test; p = 0.0717).
When divided by sex, there were no significant differences between male and female mice in the proportion of responding cells when orexin and α-sync were co-applied (Male: 87 %, n = 65/75; Female: 89 %, n = 59/66; Fisher's exact test; p = 0.7964; Fig. 3B2).Further, there were no significant differences in the distribution of the proportion of response polarities between the sexes (Male: n = 41/65 increases; Female: n = 29/ 59; Fisher's exact test; p = 0.1476; Fig. 3B3).However, from examination of the data, it appeared that there had been a shift in the proportion of increases to decreases in both sexes when compared to the relative ratio obtained from application of α-syn or application of orexin individually (Fig. 3B3 vs Fig. 1B3, Fig. 1C3).Therefore, we compared the expected polarity ratio with that observed.
When data was not divided by sex, when we compared the observed results of co-application of orexin and α-syn with the results we would expect if effects were simply additive, we found several differences.The number of cells exhibiting increases in calcium was smaller after coapplication of orexin and α-syn than was expected based on the numbers responding to either drug alone (Expected: n = 162/197, Observed: n = 70/124, Fisher's test: p < 0.0001; Data not shown).There was a signficant difference in the proportion of cells which showed decreases when orexin and α-syn were co-applied, with a greater proportion of decreases when both were present (Expected: 18 %, Observed 44 % Fisher's test: p < 0.0001; Data not shown).However, there was no significant difference between expected and observed in the proportion of cells responding (Expected: n = 197/225, Observed: n = 124/141, Fisher's test: p > 0.9999).There was a significant difference between the expected value and the observed value in the amplitude when rises were elicited (Average Amplitude: Expected: 69.5 ± 3.8 %DF/F, n = 71; Observed: 22.4 ± 2.0 %DF/F, n = 64, Student's unpaired t-test, p < 0.0001; Data not shown).Additionally, there was a significant difference between expected and observed in the amplitude of cells that exhibited decreases in calcium levels (Average Amplitude: Expected: 53.9 ± 4.8 % DF/F, n = 9; Observed: 27.8 ± 2.5 %DF/F, n = 52, Student's unpaired ttest, p = 0.0001; Data not shown).Because these data could have been driven by one sex, we determined whether differences were sex-based.When data were analyzed between males and females, interesting sex-based differences emerged.
When increases were elicited following co-application of α-syn and orexin, concurrent application resulted in a significantly smaller amplitude of calcium rises in females when compared to the amplitude elicited in males with co-application (Male: 35.9 ± 4.1 %DF/F, n = 40; Female: 12.1 ± 1.3 %DF/F, n = 26; Student's unpaired t-test, p < 0.0001; Fig. 3C1).This was surprising as there were no sex-based differences in the amplitude when α-syn or orexin were applied individually (Fig. 1B4,   C4).Further, there was no difference when adding together the amplitudes resulting from individual application of each peptide in either sex (Average amplitude based on addition of individual peptide effects: Male: 64.7 ± 5.7, n = 36; Female: 61.5 ± 3.9, n = 34, Tukey's HSD test: p = 0.9538).There were no significant sex-based differences in the amplitude when decreases were elicited by co-application (Average Amplitude: Male: 30.9 ± 5.5%DF/F, n = 23; Female: 27.5 ± 2.6 %DF/F, n = 30, Student's unpaired t-test, p = 0.5464; Fig. 3C2).
Sex differences were neither expected nor observed in the proportion of responding cells when both orexin and α-syn  3C4).In males, there were 21 % fewer rises and 16 % more decreases than expected, and in females there were 58 % fewer rises, and the number of decreases was 3.5-fold greater than expected, which represented a significant sex-based difference with a relatively greater shift towards calcium decreases in females (Fisher's test: p < 0.0001).
As further evidence of an interaction between the two peptides in both males and females, when the combination of orexin and α-syn elicited rises in amplitude of calcium, the amplitude was significantly smaller than expected (Male Average Amplitude: Expected: 64.7 ± 5.7 %DF/F, n = 36; Observed: 35.9 ± 4.1 %DF/F, n = 40, Tukey's HSD test: p < 0.0001; Female Average Amplitude: Expected: 61.5 ± 3.9 %DF/F, n = 34; Observed: 12.1 ± 1.3 %DF/F, n = 26, Tukey's HSD test: p < 0.0001; Fig. 3C1).When decreases were elicited in males, while the average amplitude of the observed decrease was smaller than that of the expected, this difference was not significant (Average Amplitude:  between responding cells and non-responding cells when orexin and α-syn were co-applied was not significantly different than the expected ratio (checkered columns) based on the addition of the response rates seen when orexin or α-syn were individually applied.(C4) However, the ratio of the polarity of the response observed by the co-application when compared to the expected polarity ratio of co-application was different in that fewer cells responded with increases in calcium and more cells responded with decreases than was expected based on adding individual effects, which was a phenomenon seen in both males and females.Orx Expected: 47.5 ± 6.8 %DF/F, n = 5; Observed: 30.9 ± 5.4 %DF/F, n = 23, Tukey's HSD test: p = 0.3488; Fig. 3C2).When decreases were elicited in females, interestingly, there was a significant difference in the observed amplitude from what was expected, as the expected effect was greater than the observed effect of co-application (Average Amplitude: Expected: 66.1 ± 10.3 %DF/F, n = 5; Observed: 27.5 ± 2.6 %DF/F, n = 30, Tukey's HSD test: p = 0.0011; Fig. 3C2).This sex-based difference was not anticipated as no significant differences were present in expected and observed amplitudes when decreases were elicited between males and females (Expected Average Amplitude: Male: 47.5 ± 6.8 %D/ F, n = 5; Female: 66.1 ± 10.3 %D/F, n = 5, Tukey's HSD test: p = 0.4661; Observed Average Amplitude: Male: 30.9 ± 5.4 %DF/F, n = 23; Female: 27.5 ± 2.6 %DF/F, n = 30, Tukey's HSD test: p = 0.9285).In summary, our calcium imaging data show that when orexin and α-syn are co-applied, there is a shift in the distribution of the polarity of responses in both sexes with the co-application inducing more decreases in calcium than would be expected from simple additive effects with a relatively larger shift in females, and when increases were induced, the amplitude of these increases was smaller in both sexes than expected.Finally, there was only a significant impact on the amplitudes of calcium decreases of the co-application of the two peptides seen in females, further suggesting a sex-based difference in the degree of interaction between mechanisms stimulated by co-application of orexin and α-syn when compared to mechanisms activated by either alone.When taken together, these results suggest in both sexes that the addition of orexin leads to reductions in the numbers of cells exhibiting calcium rises and in those cells which do exhibit a rise, the amplitude of the α-syn-associated rise in intracellular calcium is reduced.was significantly lower in the hemi slices exposed to α-syn.There were 11.4 ± 2.1 % fewer living cells compared to control in the slices exposed to α-syn.(B2) Cell viability fractions showed a significant difference between the sexes in that greater cell survival was seen in female hemi slices exposed to α-syn.(C1) Comparison of cell viability in slices exposed to orexin (1 μM) and α-syn (100 nM) revealed a significantly lower cell death as shown in normalized data in control conditions under which the slice was exposed to α-syn and in conditions with co incubation with orexin and α-syn (Orx + α-syn: 8.1 ± 1.6 % increase from control).There was no sexbased difference in the cell survival induced by orexin as there were no significantly differences between cell viability percentages between males and females.***: p < 0.001, ****: p < 0.0001, and ns indicates no significance.The final set of experiments was aimed to investigate effects of α-syn, orexin, and the combination of these two proteins on cell death of LH cells.Using bisected slices which paired each half slice with its matched hemi slice as control, cell viability was studied in LH after incubation in either α-syn (100 nM) or control solution of ACSF for 7 ½ h (Fig. 3A1).
Cell viability fractions were calculated as the ratio of living cells (DAPI positive) to the total number of cells seen in fluorescent images (DAPI positive + PI positive) (Fig. 4A2).α-syn exposed LH slices showed a greater proportion of cell death (Fig. 4A2a) compared to control slices (Fig. 4A2b).There was an 11.4 ± 2.1 % difference in the cell viability of the treated slices with a greater fraction of dead cells present in the α-syn exposed LH slice (Cell viability normalized: ACSF: 100.0 ± 0.6 %, n = 22; α-syn: 88.6 ± 2.1 %, n = 22, Paired t-test: p < 0.0001; Fig. 4B1).
Exposure to α-syn showed a significant difference in the cell viability fractions between male and female mice with the cell viability fraction being greater in females compared to males (Student's unpaired t-test, p = 0.0002).Exposure to α-syn induced less death in females compared to males as the cell viability percentage was significantly different between the sexes (Cell viability percentage: Male: 79.6 ± 2.4 %, n = 8; Female: 93.8 ± 1.9 %, n = 14, Student's unpaired t-test, p = 0.0002; Fig. 4B2).

Presence of orexin is associated with a reduced a-syn mediated cell death in the LH
The final cell viability assay was designed to examine whether orexin exhibits a neuroprotective effect on cell viability in LH slices after incubation in α-syn (100 nM).Accordingly, cell viability of hemi slices treated with a solution of orexin (1 μM) and α-syn (100 nM) was compared to viability seen in the other matched half of the slice treated with a solution of α-syn (100 nM) alone for 7 ½ h.Orexin and α-syn exposed LH slices showed a reduced proportion of cell death compared to control slices exposed to α-syn (8.1 ± 1.6 % cell death vs 11.4 ± 2.1 %; Student's paired t-test; n = 26, p < 0.0001; Cell viability Normalized: Control: 100.0 ± 0.8 %, n = 26; Orx + α-syn: 108.1 ± 1.6 %, n = 26, Student's paired t-test: p < 0.0001; Fig. 4C1).This effect was not sexbased as exposure to orexin and α-syn showed no significant sex difference in the cell viability percentage between males and females (Cell viability percentage normalized to α-syn: Male: 106.5 ± 2.4 %, n = 15; Female: 110.4 ± 1.8 %, n = 11, Student's unpaired t-test, p = 0.2354; Fig. 4C2).

Discussion
The results from this study are the first to show that the native, monomeric form of α-syn induces an increase in intracellular calcium in the majority of LH cells.Further, we confirm what has been seen in other reports that orexin induces rises in calcium in the majority of LH cells, and extend these data by showing no sex-based differences when using Fura 2 imaging.When α-syn and orexin were co-applied, rises in calcium were still induced in LH cells, but interestingly, the rises were smaller than would be expected based on a simple additive effect if the rises induced when the compounds were applied individually were summated.By delving further into the data, we found subtle but interesting differences according to sex, which could have physiological implications.When taken together with our previous hypothesis that α-syninduced rises in calcium could be involved in α-syn-mediated excitotoxic-related cell death, we speculated that the orexin-mediated effects on α-syn-induced rises in calcium could confer a protection to both male and female LH cells, with perhaps a greater effect on viability in females.Using cell viability protocols to examine the ratios of dead cell to live cells, we found that α-syn induced a degree of cell death above control in the LH that was higher in males, and the combination of orexin and α-syn resulted in heightening viability, which did not show a sex-based difference.Our data suggest an interaction between processes induced by α-syn and orexin leading to calcium rises; however, in our study we were not able to elucidate the cellular mechanisms underlying this interaction.Our data are discussed in light of the appearance of sleeping and arousal disorders co morbid in PD in which the LH plays a role and emphasize the need for sex being considered a factor in studies evaluating cellular effects of peptides.

Monomeric α-syn induces rises in calcium in LH cells
While we are the first to show the phenomenon in native, mammalian LH cells ex vivo, rises in calcium have been induced in other cell types by the monomeric form of α-syn.In primary neurons, as well as hippocampal and cortical brain slices, application of monomeric α-syn led to rises in calcium (Angelova et al., 2016).We saw rises in α-syninduced calcium in the majority of LDT neurons and PPT neurons in mouse brain slices in males, although decreases were also noted as was seen in the LH (Dos Santos et al., 2021).Interestingly, while rises were also induced in the SN, there was a difference in the distribution of the directionality of the change in calcium between SN cells and those in the LDT and PPT as a greater proportion of SN cells responded with decreases when compared to the proportion which responded similarly in the LDT and PPT.
The source of the α-syn-induced calcium seen in these studies was not determined.From studies examining the interaction of α-syn with cells, most of which have been conducted in reduced preparations, it is known that α-syn binds to phospholipid membranes, which results in a multimeric form assuming an α helical conformation (Killinger et al., 2019;Mori et al., 2020).This conformational change may be followed by the opening of an ionic conductance across the membrane, enhancement of activity via voltage gated calcium channels (VGCCs), intracellular release of calcium and/or pore formation (Adamczyk and Strosznajder, 2006;Tosatto et al., 2012;Zakharov et al., 2007).Within the LDT and PPT the mechanism behind the rise in calcium was explored in brain slices.Pore formation, activity at glutamate receptors, an involvement of SERCA-pump maintained intracellular calcium stores, and VGCCs were ruled out.Using fluorescently tagged α-syn, effects of the protein on excitability were shown to involve membrane interaction with no indication of internalization being required, and using pharmacology, evidence for role of a G-protein coupled receptor was provided for the rises in calcium (Dos Santos et al., 2022).However, identification of the mechanism leading to rises was not possible in that study.Future experiments will be conducted to elucidate the mechanism by which α-syn induces calcium rises in LH cells, as well as in cells of the LDT, PPT and SN, and whether the mechanisms differ across nuclei.

Orexin induces rises in calcium in LH cells
Our findings that orexin induces rises in calcium in LH neurons were not unexpected.Previous work had shown that application of orexin to identified, orexinergic LH neurons in brain slices induces cellular depolarization and increases in excitatory synaptic events (Li et al., 2002), and orexin induced rises in intracellular calcium in LH cell culture (van den Pol et al., 1998).In the present work, the mechanisms by which rises in calcium were induced in LH neurons by orexin were not elucidated.Speculation about the source of calcium is complicated by the fact that with bulk load calcium imaging, we could not be sure of the phenotype of recorded cells.The LH contains orexinergic neurons, but also melanocortin-containing cells (MCH).Ablation studies in mice suggest that while the numbers of MCH neurons is higher than the number of orexin cells in the LH, the difference is nearer to a few hundred individual cells rather than a 2-fold or more difference suggesting that the liklihood of imaging from orexin vs MCH cells is not vastly different (Hung et al., 2020).Patch clamp recordings revealed that orexin does not have direct actions on the membrane of orexinergic LH cells and that S. Bohid et al. excitatory actions were due to effects on presynaptic glutamate cells, which heightened the release of glutamate acting at putative AMPA and NMDA receptors (Li et al., 2002).In LH cell culture, orexin-induced rises in calcium were independent of intracellular calcium release, and were due to flux of calcium across the membrane, which was blocked by Cd 2+ indicating that orexin was inducing calcium rises via effects on voltage gated calcium channels (VGCCs) (van den Pol et al., 1998).Accordingly, in our slices, orexin-mediated calcium rises induced in the orexinergic cells likely result from a combination of passage of calcium through activated glutamate receptors, and via either direct activation of VGCCs or indirectly via membrane depolarization.Further, our slices likely contained MCH-containing cells and orexin has been shown to have effects on AAV-identified, MCH-containing LH neurons resulting in both direct and indirectly mediated excitation, which would result in rises in calcium (van den Pol et al., 2004).Although the responses to orexin of the other cell types in the LH have not been explicitly studied, rises in calcium would be expected as in addition to inducing flux across the membrane and enhancement of glutamate release, orexin receptors have been shown in other cell types to be linked to PLC activation and formation of IP 3 , which could lead to intracellular release of calcium independent of effects on the membrane (Kukkonen and Leonard, 2014;Larsson et al., 2005;Lund et al., 2000;Nasman et al., 2006) (For review, see Wang et al., 2018).Interestingly, we also saw reductions in calcium induced by orexin in a minority of cells, which was unexpected based on previous studies.However, as it is well known that activation of MCH neurons can result in MCH-mediated inhibition of excitability of orexin cells (van den Pol et al., 2004), it is possible that retention of the LH circuitry in the slice resulted in presynaptic MCH activation leading to inhibition of postsynaptic cells, thereby resulting in reductions in calcium.When taken together, our data showing rises in calcium induced in the majority of cells within the LH is in accordance with the cell type specific studies which have been conducted in this nucleus.

Monomeric α-syn enhanced cell death in the LH
In our study, we saw heightened cell death in the LH associated with exposure to monomeric α-syn.Although the oligomeric and fibril forms of α-syn are thought to mediate the neurotoxicity seen in PD that leads to neurodegeneration due to various downstream mechanisms (Fusco et al., 2017;Lashuel et al., 2013), we have shown that the monomeric form of α-syn can also be damaging in some cell types.In the LDT, monomeric α-syn was shown to induce cell death (Dos Santos et al., 2021).This was in contrast to findings in primary co-cultures in which the oligomeric form of α-syn caused a calcium influx-dependent cell death, however, exposure of these cells to the monomeric form of α-syn did not heighten death at 15 min or at 6 h as evaluated by capase-3 activation (Angelova et al., 2016).The discrepancy in the findings between the studies could be due to technical differences including cell preparation (culture vs ex vivo), differences in detecting cell death as we used disruption of membrane integrity as a marker of death rather than capase-3 activation, or to duration of exposure as we incubated in monomeric α-syn for 7.5 h, which was longer than the 15 min or 6 h in other studies.These differences should be explored to determine if they explain the discrepancy.If they do not, then a more interesting possibility could be evaluated, which is that the difference could be due to cell phenotype.This interpretation is supported by findings in our laboratory that exposure of SN cells to α-syn did not increase cell death when using an identical protocol to that used when examining effects on LDT cells (Dos Santos et al., 2021Santos et al., , 2022)), and that used to expose LH cells in the present report.Interestingly, the α-syn associated cell death in LH was relatively greater in males than in females, which is similar to our findings in the LDT (Dos Santos et al., 2023).While the mechanisms underlying this difference were not explored, we have hypothesized that the higher degree of cell death seen in males in the LDT was due, in part, to rises in calcium induced by α-syn that were greater than those seen in females as sustained rises of intracellular calcium are known to initiate processes which lead to excitotoxicity (Prentice et al., 2015).In support of this interpretation, extracellular calcium was found to be critical for oligomeric-induced cell toxicity in culture (Angelova et al., 2016).Further, in the LDT, we provided evidence of a GABAergic protective mechanism present in females which reduced calcium transients and cell death, suggesting a mechanistic link between the two processes (Dos Santos et al., 2023).However, a GABAergic mechanism limiting the extent of α-syn-mediated calcium rises does not appear to play a role in the LH as the amplitudes of the rises induced by α-syn were not significantly different between males and females.One possibility for greater viability in females is that the higher levels of estrogens in females are protective of α-syn-linked damage, as estrogens have been shown to have neuroprotective effects and estrogen has been previously hypothesized to contribute to sex-based differences in PD (Bourque et al., 2012;Rajsombath et al., 2019).While the rises in calcium could play a role in α-syn-mediated LH cell death, further work is necessary to determine the mechanism(s) underlying the greater α-syn-mediated cell death in the LH of males when compared to females.

Orexin protected against cell death
Regardless of the mechanism underlying the cell death in both male and female LH, orexin was able to enhance LH cell viability in both sexes.While a novel finding in the LH, this was not surprising since orexin has been previously suggested to play a neuroprotective role.Application of orexin was found to protect dopamine neurons from MPTP +-induced, mitochondrial involved cell damage (Liu et al., 2018).The mechanism underlying this protection is unknown, but was thought to involve activation of the orexin type I receptor, rises in calcium, and increases in levels of the protein BDNF.Orexin was also shown to protect human neuroblastoma SH-SY5Y cells from 6-OHDA and MPP+ toxicity through induction of anti-apoptotic and antioxidant pathways (Esmaeili-Mahani et al., 2013;Feng et al., 2014;Pasban-Aliabadi et al., 2017).While we did not determine the mechanism by which orexin protected against α-syn-mediated LH cell death, our working hypothesis remains that orexin-mediated reductions in α-syn calcium rises could play a role.While rises in calcium were still induced by the combination of orexin and α-syn in LH cells, the rises were reduced from that expected if the effects of the peptides individually were added, suggesting some occlusion was occurring from the combination.The smaller rises in calcium could be expected to result in reduction in the ability to express calcium-dependent death genes (Steller, 1995).However, a confound of our hypothesis is that LH cell viability in presence of orexin did not show a sex-based difference, whereas α-syn-mediated calcium changes were more affected by orexin in females when compared to males.The α-synmediated rise induced in females when the two peptides were combined was significantly smaller in amplitude than that seen in males, which was not expected based on the results of rises induced by application of α-syn and orexin alone as they did not show a sex-based difference.
Further, there was a relatively larger shift towards decreases in calcium in females when both peptides were applied.If the rises in calcium play a role in cell death, it would be predicted that the greater effect of orexin in reducing rises would be apparent in sex-based differences in cell survival, which we did not see.Future studies need to be conducted to clarify the role of the calcium rises in α-syn-associated cell death.However, while the role played by α-syn-mediated calcium rises in heightened cell death and the mechanisms underlying the protection of LH cells by orexin remain to be determined, it is interesting to speculate why larger effects on α-syn changes in calcium were mediated by orexin in females.The mechanism(s) underlying the occlusion of the rise in calcium in males and females is unknown.However, rises induced in LDT neurons by α-syn did not rely on the players shown to be involved in orexin-mediated activation of LH cells, specifically, glutamate receptors or VCGGs (Dos Santos et al., 2022), which raises the possibility of a lack of a shared mechanism at the level of the membrane.However perhaps intracellular players activated subsequent to calcium rises induced by orexin and α-syn interact and result in attenuating individual effects of the peptides that lead to calcium rises.If the interaction of the two intracellular pathways activated by orexin and α-syn negatively interact and result in reduction of individual actions, smaller rises could be explained by higher-levels of orexin receptors in the LH of the female, which could lead to heightened stimulation of the mechanisms counteracting α-syn-mediated calcium rises.

Functional implications and significance
Our data suggest that the monomeric form of α-syn leads to rises in intracellular calcium, which are associated with heightened ex vivo cell death in the LH.As sleeping disorders such as EDS in which the LH could play a role appear early in the progression of PD, we propose that death induced by the early form of α-syn would lead to cell loss in the LH, resulting in contributing to the early appearance of these disorders.A non-mutually exclusive possibility is that loss of serotonergic, adrenergic and cholinergic neurons that control behavioral state are also lost with those of the LH, and their degeneration contribute to the early appearance of disorders of state, which is a possibility supported by our recent data in the brainstem cholinergic cell groups and findings from other studies (Bohnen and Albin, 2011;Dos Santos et al., 2021, 2022;Halliday et al., 1990;Paulus and Jellinger, 1991).As these cells groups receive input from LH orexinergic cells, the loss of orexin-containing cells early in PD could contribute to hastening the loss of these other cell types.As cells die within the LH in PD, putatively due to rising levels of α-syn and increased presence of the more putatively damaging forms of the protein due to conformational changes or aggregation of the monomeric form, the loss of orexin-containing cells would exacerbate loss of neurons at sites to which orexinergic LH neurons project.Thus, loss of synaptic effects of LH neurons on projection nuclei and removal of the neuroprotective effects of orexin on monoaminergic and cholinergic cell groups would be expected to facilitate disorders of state control.
The differential cell death seen in females could result in a longer preservation of orexinergic LH neurons upon α-syn exposure than that seen in males, which could partly explain the sex-based difference that females exhibit fewer sleep related issues during PD's prodromal phase.
Longer preservation could involve a sex-based difference in orexinergic tone.Supporting this interpretation, higher levels of mRNA for orexin, and orexin-A have been noted in females, which were revealed to be most apparent in the lateral and posterior hypothalamus, and later confirmed in cFOS activation studies and monitoring of levels in CSF (Grafe et al., 2017;Johren et al., 2002;Taheri et al., 1999).Protection conferred by estrogen and orexin was shown to be amplified through the positive interaction known to exist between estrogen levels and numbers of activated orexin neurons (Grafe and Bhatnagar, 2020).Levels of orexin have also been found to be higher in female patients experiencing neurodegenerative disease involving aberrant proteins, including Lewy Body disease (Schmidt et al., 2013;Wennstrom et al., 2012).The potential protective effect of orexin raises the possibility that orexin-based therapies could be useful in management of sleeping disorders seen in PD.Further, PD occurs at a higher rate in males, which could suggest that orexin-based therapies could be relevant for the prodromal phase of PD when symptoms even beyond those of sleeping disorders are apparent, including monoaminergic-controlled behaviors.If orexin levels are maintained, this could potentially slow neurodegeneration in other PD-affected regions including the SN.This is an interesting consideration as dopamine neurons in the striatum have been shown to exhibit sex-based differences in vulnerability (for review, see (Gillies et al., 2014)), suggestive of greater susceptibility to PD-related degenerative processes, which in some cases appeared to not involve mechanisms underlying protection by estrogen, but which might in theory be affected by orexin-acting agents (Rodriguez-Navarro et al., 2008).

Conclusions
When taken together, our findings could provide, in part, a mechanistic explanation for the sex-based differences in the appearance of state-related behaviors in the progression of PD and suggest a potential treatment approach for both males and female PD patients based on heightening orexin transmission.Future work should examine the mechanisms underlying the interaction between the mechanisms underlying changes in calcium induced by orexin and α-syn.Since the discovery of the orexins, drug development has focused on the potential therapeutic effects of targeting this system for insomnia with an orexin receptor antagonist (Scammell and Winrow, 2011).Therapies such as cell transplantation, gene therapy and use of specific orexin agonists, which could compensate for the loss of orexin neurons in narcolepsy have been proposed (Equihua-Benitez et al., 2020;Kantor et al., 2013;Mieda et al., 2004;Weinhold et al., 2014).As orexins are neuropeptides and do not cross the blood brain barrier, promising candidates for management of the prodromal phase of PD may be non-peptide orexin receptor agonists, which have been shown to be effective in mouse models (Barateau and Dauvilliers, 2019;Irukayama-Tomobe et al., 2017).Very interestingly, while hepatotoxic effects resulted in termination of a Phase 2 trial of an orexin receptor type 2 agonist, reductions in sleepiness and cataplexy were seen in narcolepsy patients deficient of orexin (Dauvilliers et al., 2023), which raises hopes that formulation of a non-toxic orexin receptor agonist will be pursued that, in future, could be examined for its neuroprotective, as well as arousing effects in PD.

Fig. 1 .
Fig. 1.Application of α-syn or orexin induces changes in calcium in the majority of LH neurons in both males and females.(A) Image of a coronal brain slice from a mouse brain containing the lateral hypothalamus under 4× magnification and bright field illumination.The LH is marked with a dashed circle.The third ventricle is visible at the arrow.(Bottom Left) Under 40× magnification, cells in the LH can be visualized under bright filed illumination.(Bottom Right) Under 380 nm fluorescent light, the Fura 2 loaded cells can be seen.Scale across bright field and fluorescent images is 50 μm.Regions of interest (ROIs) are drawn around these cells and the average fluorescence within each ROI is displayed as DF/F%.(B1) The average fluorescence of two different cells is shown before and after application of α-synuclein (α-syn).As can be seen, α-syn induced increases in fluorescence in some cells reflected as upward going deflections of DF/F%, whereas decreases in fluorescence were elicited in others as shown by decreases in DF/F%.(B2) When the proportion of cells which

Fig. 2 .
Fig. 2. AMPA induces increases in fluorescence in all LH cells tested in males and females, but fluorescent changes are larger in females.(A) Application of 10 μMAMPA induces changes in intracellular calcium in males and females with the plateau kinetic, which did not appear to differ between males or females.(B1) There was no significant difference between the proportion of responding or non-responding cells in male or female mice (Male: n = 29/29, Female: n = 50/50, Fisher's test: p > 0.9999).(B2) There was also no significant difference between the distribution of response polarity between the sexes (Male: n = 29/29, Female: n = 50/50, Fisher's test: p > 0.9999).(B3) There was a significant difference in the average amplitude of the calcium increase in (Male: n = 25; Female: n = 41; Student's unpaired t-test, p = 0.0102; total n = 66 as one data point was removed in outlier tests).

Fig. 3 .
Fig.3.Orexin and α-syn co-application resulted in rises in calcium but they were smaller than expected in females and males.(A1) AMPA induced rises in intracellular calcium that were additive with rises induced by α-syn, and the increases in calcium showed no significant attenuation suggesting that occlusion of effects was not occurring as shown in the population data, which showed no difference between observed responses and expected responses when data from males and females were combined.(A2) However, rises induced by AMPA and α-syn did exhibit sex differences as rises in males were greater than would be expected by simple addition of individual effects, and these rises were significantly greater than those observed in females.(B1) Co-administration of orexin and α-syn still induced rises and decreases in calcium as shown by two representative fluorescent recordings from two different LH cells.(B2) The distribution of cells which responded to the combination of orexin and α-syn was not different between males or females, and (B3) the proportion of cells that showed increases or decreases was also not different between males and females.(C1) The amplitude of the observed rises in calcium induced by co-administration of orexin and α-syn were significantly different in males and females from the amplitude that would be expected based on individual application of the two peptides.In addition, the amplitude of the rise induced by orexin and α-syn in females was significantly smaller than that seen in males, although, based on results from individual application of each peptide, the amplitude was not expected to be different between the sexes.(C2) The amplitude of the decrease in calcium observed was only significantly different in females further suggesting further that sex-based differences in the interaction of effects of the two peptides exist.(C3) The observed ratio (solid columns) : orexin; Male_EXP: expected response in males from summation of individual responses are indicated by blue checkered boxe; Male_OBS indicates the actual response observed when compounds were co-applied in males and are indicated by the blue solid box; Female_EXP: response expected in females when individual responses are added are indicated by red checkered boxes; Female_OBS: Observed responses recorded when compounds were co-applied are indicated by solid red boxes.*: p < 0.05, **: p < 0.01, ****: p < 0.0001, and ns indicates no significance.

Fig. 4 .
Fig. 4. Orexin protects from α-syn-mediated cell death.(A) Shown is a cartoon illustrating the incubation process of bisected LH slices from mice for cell viability studies.(A1a) LH slices of a 300 μm thickness were (A1b) bisected with a scalpel and immediately placed in tubes (A1c) with 1 mL of either artificial cerebral spinal fluid (aCSF), 100 nM monomeric α-synuclein (α-syn) or a solution of 1 μM orexin and 100 nM monomeric α-synuclein (Orx + α-syn) under constant carbogen bubbling.(A2a) Merged fluorescent image showing PI positive cells (red) and DAPI positive cells (blue) in an α-syn exposed hemi slice under a 4× objective.(A2b) Merged fluorescent image under 4× optics showing PI positive cells (red) and DAPI positive cells (blue) in the control hemi slice.As can be seen, more PI positive cells are present in the α-syn exposed slice, suggesting greater cell death in the α-syn-exposed condition.Scale in both images is 50 μm.(B) Normalization of the data to the matched controls showed across the population of slices that the ratio of living cells (DAPI positive) to the total number of cells (DAPI positive + PI positive) α-synuclein mediates cell death in the LH, and orexin reduces this death 3.4.1.3.4.1Monomericα-synuclein mediates lateral hypothalamic cell death