Impaired Functional Connectivity of Cortico-Amygdala Pathway Can Drive Social Behavior Deficits in Synucleinopathies

The small molecule protein α-synuclein forms insoluble aggregates in a group of neurological disorders, including Parkinson’s disease and dementia with Lewy bodies (DLB), which are collectively called synucleinopathies. In PD and DLB, the amygdala has been identified as a particularly susceptible region in the brain for the deposition of Lewy-like α-synuclein aggregates. Though α-synuclein aggregation is closely associated with neurodegeneration, there is a poor correlation between neurodegeneration in the amygdala and the clinical features of PD/DLB. We hypothesize that, prior to neurodegeneration, α-synuclein aggregation disrupts functional cortical modulation of the amygdala circuits, leading to emotion dysregulation in synucleinopathies. In the present study, we combined electrophysiology, optogenetics, mouse model of synucleinopathies, and behavioral analysis to test this hypothesis. Using an α-synuclein preformed fibrils (PFFs)-based mouse model of synucleinopathies, we reported dynamic changes in the levels of α-synuclein pathology in the basolateral amygdala (BLA). Such dynamic changes of pathology associated with a decreased cortico-BLA connection strength prior to a significant loss of cortical axon terminals. In parallel to the reduced cortico-BLA connection, PFFs-injected mice manifested impaired social preference behavior. The impaired sociability of PFFs-injected mice could be rescued by chemogenetic stimulation of cortico-BLA inputs. Altogether, we presented a series of evidence to delineate key circuit events associated with α-synuclein pathology development in the amygdala circuits. The present work highlights the necessity of a thorough investigation of functional consequences of α-synuclein aggregation to advance our understand of pathophysiology of synucleinopathies and development of effective therapies.

Experimental work suggests that gradual accumulaKon of α-Syn aggregates can underlie the pathogenesis and progression of synucleinopathies [3][4][5].ParKcularly, accumulaKon of pathologic α-Syn aggregates has been reported to impair nigrostriatal dopaminergic neurotransmission and closely relate to the neurodegeneraKon, leading to the manifestaKon of motor symptoms in PD [4,6,7].In addiKon, α-Syn pathology is proposed to spread between cells and brain regions [3,5,8,9].Consequently, a large body of studies have focused on understanding the geneKc and microenvironmental factors that may promote or prevent the formaKon of pathologic α-Syn aggregaKon, as they perhaps can provide us with strategies to regulate the temporal development and spaKal distribuKon of α-Syn pathology across brain regions and in that way modify disease progression [10][11][12][13][14][15].FormaKon of α-Syn aggregates in the brain trigger series of circuitry events, including but not limited to, the degeneraKon of vulnerable neuronal populaKons.For example, compelling evidence also suggests that α-Syn aggregates disrupt synapKc structure, funcKon, and plasKcity in suscepKble brain regions, like the nigrostriatal pathway [4,[16][17][18].Motor dysfuncKon in PD is closely linked with dopamine depleKon in the basal ganglia and the subsequent funcKonal maladaptaKon within the basal ganglia-thalamocorKcal network [19,20].
Similar pathophysiological processes are likely to occur in other brain regions that are vulnerable to the formaKon of α-synuclein aggregates, in which funcKonal circuit changes, not necessarily neurodegeneraKon, are causally linked to symptom manifestaKon.
Considering the criKcal role of the amygdala in emoKon regulaKon, heavy load of Lewy-like pathology in the amygdala and the subsequent neuronal loss are expected to causally associate with depression and/or anxiety in PD/DLB [27][28][29][30].However, such a linear relaKonship between neurodegeneraKon and clinical features has not been supported by studies using human Kssues of PD/DLB [24] or its animal models [25,31].Building on the research about the nigrostriatal dopaminergic system, it is plausible to hypothesize that impaired amygdala circuit funcKon, but not significant neurodegeneraKon, is sufficient to cause emoKon dysregulaKon in synucleinopathies.In the present work, we combined electrophysiological, immunohistochemical, and behavioral approaches to test this hypothesis.Using an intrastriatal α-synuclein preformed fibrils (PFFs) model, we demonstrated that: 1) α-synuclein aggregates selecKvely eliminate corKcal afferents to the basolateral amygdala (BLA); 2) α-synuclein aggregaKon induces funcKonal impairment of corKco-BLA inputs, prior to robust synapse loss; 3) neuronal loss in the BLA occurs at late stages as α-synuclein aggregates accumulate; and 4) social behavioral deficits associate with corKco-BLA synapKc impairments, occurring prior to synapKc or neuronal loss in the BLA, and can be alleviated by funcKonal restoraKon of corKco-BLA connecKon strength.Our work here provides novel insights into the amygdala circuit dysfuncKon associated with α-Syn pathology formaKon and possible biological mechanisms underlying emoKon dysregulaKon in PD and DLB.

Animals
C57BL/6J mice at 2-3-month-old (Jax#:000664, RRID: IMSR_JAX:000664) of both sexes were obtained through the Van Andel InsKtute vivarium and used in this study.All animal studies were reviewed and approved by the InsKtuKonal Animal Care and Use Commicee at Van Andel InsKtute (animal use protocol #: 22-02-007) and in accordance with the standards of NIH for care and use of animals.Animals were housed under a 12:12 h light-dark cycle, up to four animals per cage with access to water and food ad libitum.

Prepara.on and valida.on of α-Syn preformed fibrils
The mouse α-Syn protein was purified using Escherichia coli BL21 codon plus RIPL cells (RRID:CVCL_M639), which was then dialyzed using a buffer containing 10 mM Tris and 50 mM NaCl (pH 7.5).A high-capacity endotoxin removal kit (PI88276) was used to remove the endotoxins, the levels of which were assessed using an endotoxin quanKficaKon kit (A39552).
The protein concentraKon was esKmated using absorbance at 280 nm with an exKncKon coefficient of 7450 M −1 cm −1 .Purified mouse α-Syn monomer protein was used to generate mouse α-Syn preformed fibrils (PFFs).Specifically, monomeric α-Syn protein was diluted to 5 mg/mL in the buffer, 150 mM KCl, 50 mM Tris-HCl and incubated at 37°C with shaking for 7 days, as described previously [32].Aner the incubaKon, sample was centrifuged for 10 min at 13,200 rpm.The protein pellet was re-suspended in half of the iniKal volume of the soluKon.To esKmate the fibril concentraKon, 5 μl of fibril soluKon was incubated with 5 μL of 8 M guanidinium chloride at room temperature for one hour.Aner incubaKon, the concentraKon of PFF was measured using absorbance at 280 nm and diluted the PFF at 5 mg/mL and 22-25 μl aliquots were stored at −80°C unKl use.Prior to the injecKon, PFF aliquot (22-25 μl at 5 mg/mL) was thawed at room temperature and sonicated using Qsonica 700W cup horn sonicator at 30% amplitude using 3 seconds on/2 seconds off cycle for 15 min at 15°C.The size of sonicated PFF (30-70 nm segments) was esKmated and confirmed using the dynamic light scacering (DynaPro NanoStar from Wyac technology).Detailed of generaKng and validaKng α-Syn PFFs can be found here: dx.doi.org/10.17504/protocols.io.bhhrj356.
Animals were returned to a cage placed on a warm pad aner surgery and then housed in their home cage unKl experiments.Details of this protocol can be found at: dx.doi.org/10.17504/protocols.io.rm7vzye28lx1/v1

Animal behaviors
To assess motor funcKon, animals were subject to locomoKon and rotarods tests.For open field locomoKon test, the animal was placed in the empty open field (40 x 40 x 30 cm, W x L x H) and allowed to explore for 10 min.Animal's locomotor acKvity was conKnuously recorded and analyzed using ANYmaze sonware (RRID:SCR_014289, StoelKng Co., Wood Dale, IL; hcp://www.sandiegoinstruments.com/any-maze-video-tracking/).For rotarod test, the animal was placed on an acceleraKng rod (speed of 4-40 rpm in 5 min, San Diego Instruments, CA) and the latency of fall was monitored and recorded.Detailed protocol of motor acKvity assays can be found here: hcps://www.protocols.io/view/open-field-locomoKon-test-e6nvwjxmdlmk/v1.
To assess animals' social interacKon behavior, mice were placed to three-chamber apparatus (MazeEngineers, Skokie, IL) with len, middle, and right chambers (20 x 13.5 cm 2 ).A round wire cage (10 cm in diameter, 20 cm in height) was placed in the len and right chambers for novel sKmulus mouse.The sKmulus mice were habituated to the wire cages for 20 min before the tesKng day.The tesKng mice were habituated to the behavior room for at least 30 min before the experiment.Three sessions were conducted during the tesKng day, including the habituaKon, the session 1, and the session 2. The tesKng mice were habituated to the middle chamber for 5 min before opening the doors to allow the mice freely explore the three chambers for 10 min.During the first session, the tesKng mice were allowed to explore the three chambers for a total of 10 min with one empty cage and the presence of a sKmulus mouse in the other wire cage.In the second session, the same tesKng mouse was allowed to explore the three chambers for another 10 min with the presence of the familiar mouse in one cage and a second never-met sKmulus mouse in another wire cage.Mice movements were tracking and the Kme spent in each chamber was quanKfied by AnyMaze sonware (RRID:SCR_014289, StoelKng Co.).Between each tesKng, the chamber was thoroughly cleaned with 70% ethanol to eliminate the odor of mice.

Time-course of α-Syn pathology development in the BLA
We injected α-Syn PFFs into the dorsal striatum of wildtype C57BL6 mice to induce the accumulaKon of α-Syn pathology in the brain, including the cerebral corKcal regions and the amygdala [3,25,31].In the PFFs-based models, fibrillar α-Syn species are reported to be internalized at the axon terminals, which then recruit endogenous α-Syn to form insoluble inclusions and eventually result in neurodegeneraKon [3,11,16].As expected, we detected robust phospho-Ser129 (pS129 + ) α-Syn immunoreacKvity in the BLA of PFFs-injected mice (Figure 1A-D), but not in the BLA of PBS-or monomer-injected controls ("controls" hereinaner).α-Syn pathology in the BLA included both pS129 + Lewy neurite (LN)-like and large Lewy body (LB)-like aggregates (Figure 1A-D), which are likely the α-Syn pathology within corKcal afferents of the BLA and the cytoplasmic aggregates within glutamatergic principal neurons of the BLA, respecKvely [31,34].Next, we characterized the dynamic changes of α-Syn pathology abundance in the BLA at 1, 3, 6, and 12 months-post-injecKon (mpi).We measured the proporKon of the BLA region covered by LN-and LB-like pS129 + aggregaKon, as well as their combinaKon as the total α-Syn pathology (i.e., pS129 + LN/LB-like aggregates, Figure 1E) [25,26].Specifically, moderate level of pS129 + α-Syn pathology was detected in the BLA as early as at 1 mpi, which was dominated by the LN-like pS129 + aggregates (Figure 1A, E).The level of total pS129 + α-Syn pathology conKnually grew beyond 1 mpi, peaking at 3 mpi (Figure 1B, E).Aner 3 mpi, the amount of total α-Syn pathology in the BLA remained at a relaKvely high at the 6 mpi Kmepoint but decreased significantly at 12 mpi (Figure 1C, D, E).ParKcularly, at 12 mpi only few LB-like pS129 + aggregates could be seen in the BLA (Figure 1D, E).
Post-mortem studies of paKents with advanced PD reported that the amygdala exhibits significant Lewy body pathology and reduced cell density [24].Consistently, using unbiased stereological counKng of pan-neuronal marker NeuN, we detected a significant reducKon of the NeuN density in the BLA at 12 mpi relaKve to controls, but not at 6 mpi (Figure 1F, G, see also [31]).These data are consistent with the loss of corKcal glutamatergic neurons and midbrain dopaminergic neurons driven by the formaKon and maturaKon of LB-like pS129 + aggregates [3,11,35].Together the above results suggest that robust reducKon in the LB-like pS129 + aggregates at 12 mpi was, at least parKally, due to neuronal loss in the BLA.

Loss of cor.cal afferents contributes to the decreased pS129 + neurites in the BLA
Glutamatergic inputs from the cerebral cortex and the thalamus are the major excitatory drive of the BLA neuronal acKvity and they form synapses with the spines and dendrites of BLA principal neurons.InteracKon of corKcal and thalamic inputs is criKcal to proper manifestaKon of emoKon and behavior.Our recent study suggest that corKcal and thalamic axons show disKnct funcKonal vulnerability to α-Syn aggregaKon at 1 month post PFFs injecKons when there was no overt loss of glutamatergic axonal terminals.It remains to be determined whether and when α-Syn aggregates induce loss of corKcal and/or thalamic inputs in the BLA.Thus, we rigorously examined corKcal and thalamic inputs to the BLA at addiKonal Kme points in both controls and PFFs-injected mice.
CorKcal and thalamic axon terminals can be disKnguished by their preferenKal expression of vesicular glutamate transporter (vGluT) 1 and 2, respecKvely.Using opKcal dissector methods, we stereologically quanKfied the density of vGluT1 + puncta in the BLA, starKng from 3 mpi when the level of LN-like pS129 + α-Syn aggregaKon reached a peak (Figure 1E).Although pS129 + α-Syn was tremendous in the BLA at 3 mpi, there was no change in the density of vGluT1 + puncta (Figure 2A).However, a significant reducKon in the density of vGluT1 + puncta in the BLA was detected at 6 mpi (Figure 2B), indicaKng a gradual loss of corKco-BLA axonal projecKons at later Kme points, though a downregulaKon of vGluT1 protein itself cannot be excluded.When it came to thalamic inputs to the BLA, we found no change in the density of vGluT2 + puncta in the BLA of PFFs injected mice relaKve to respecKve controls at 3 and 6 mpi (Figure 2C, D).These data indicate that thalamic-BLA axonal projecKons were not altered by the α-Syn pathology in the brain.Taken together, we concluded that the reducKon of LN-like pS129 + aggregates in the BLA between 3 mpi and 6 mpi was mainly due to a loss of corKcal axonal inputs, but not the thalamic afferents.
Given the corKcal inputs mainly target dendriKc spines of BLA principal neurons, loss of corKcal axonal terminals is expected to be accompanied by a reducKon of postsynapKc spines.Thus, we further quanKfied the density of dendriKc spines of the biocyKn labeled BLA principal neurons that were retrogradely labeled by intrastriatal injecKon of Retrobeads and PFFs mixture (Figure 3A, B).We found that BLA principal neurons exhibited a reduced density of dendriKc spines at 3 and 6 mpi (Figure 3C-F).
Taken together, α-Syn aggregaKon triggers loss of presynapKc corKcal axon terminals and postsynapKc dendriKc spines of BLA principal neurons, which is likely to result in a loss of corKco-BLA synapses as α-Syn pathology accumulates in the brain, e.g. at 6 mpi.

α-Syn pathology disrupts cor.co-BLA func.onal connec.vity prior to loss of synapses
Aner establishing a Kme course of neuronal and synapKc losses in the BLA, we next assessed the funcKonal connecKvity between the cerebral cortex and the BLA.The medial prefrontal cortex (mFPC) and the BLA form interconnected microcircuits that play a criKcal role in regulaKng social behavior [36][37][38].Both the mPFC and the BLA accumulate robust α-Syn pathology following intrastriatal PFFs injecKon [3,31], the funcKonal connecKvity of mPFC-BLA pathway is expected to be impaired.Thus, we assessed how α-Syn pathology affects mFPC-BLA connecKon at 3 and 6 mpi when the abundance of α-Syn pathology remained high but has caused no or subtle degeneraKon of BLA neurons and presynapKc corKcal axonal terminals (Figures 1 and 2).We virally expressed AAV9-hSyn-ChR2(H134R)-eYFP in the layer 5 projecKon neurons of the mPFC, allowing a selecKve acKvaKon of mPFC-BLA inputs (Figure 4A).We optogeneKcally sKmulated ChR2(H134R)-expressing axon terminals in the BLA (Figure 4A) and recorded the evoked excitatory postsynapKc currents (EPSCs) in the retrogradely labeled BLA principal neurons under the voltage-clamp mode from controls and PFFs-injected mice (Figure 4B, C).The amplitude of optogeneKcally-evoked, monosynapKc EPSCs were quanKfied to assess the connecKon strength of mPFC-BLA synapses.A significant reducKon of the amplitude of mPFC-BLA EPSCs was detected in PFFs-injected mice relaKve to control at both 3 and 6 mpi (Figure 4B, C), indicaKng a reduced connecKon strength of the mPFC-BLA synapses.
The reduced connecKon strength of mPFC-BLA synapses was accompanied by a decreased raKo of amplitude of EPSCs mediated by AMPA receptors and NMDA receptors (i.e., AMPA/NMDAR raKo) in PFFs-injected mice relaKve to controls at both 3 and 6 mpi (Figure 4D-F).Moreover, the raKo of EPSC2/EPSC1 at the mPFC-BLA synapses evoked by paired optogeneKc sKmulaKon was also decreased in PFFs-injected mice relaKve to controls at both 3 and 6 mpi (Figure 4G-I), indicaKng an enhanced iniKal release probability of neurotransmicer at mPFC-BLA input.Together with the Kme course of synapKc marker changes (Figures 2 and 3), these data demonstrated that α-Syn pathology decreases the connecKon strength of corKco-BLA pathway at 3 mpi prior to a robust loss of corKco-BLA synapses at 6 mpi.

Social interac.on behavior is impaired by α-Syn pathology and rescued by chemogene.c ac.va.on of mPFC-BLA pathway
Given the funcKonal disconnecKon between the mPFC and the BLA at 3 mpi (Figure 5), we next acempted to determine whether the impaired mPFC-BLA projecKon affects amygdaladependent behavior.We performed behavioral tests using both controls and PFFs-injected mice at 3 mpi when the α-Syn pathology remained at peak levels but has not yet resulted in significant neuronal or synapKc losses in the BLA (Figures 2 and 3), making funcKonal rescue sKll possible.We chose the 3-chamber sociability test, as the performance in this test is dependent on an intact amygdala microcircuit as well the interacKon between the BLA and the mPFC [36,39,40].Each test began with 10 min habituaKon to the behavior apparatus with two empty cages.During the habituaKon, both controls and PFF-injected mice spent comparable amount of Kme in the len and right chambers of the empty cage.This result indicates that neither controls nor PFFs-injected mice exhibited baseline preference of either chamber.Next, a sKmulus mouse was placed into one empty cage (S1, Figure 5A), and the test mouse was then allowed to explore both chambers (session I in Figure 5A).Both controls and PFF-injected mice spent significantly more Kme with the never-before-met sKmulus mouse S1 than that with the nonsocial empty cage (Figure 5A, B, D).This result suggests that PFFs-injected mice exhibit normal sociability relaKve to controls.Next, we studied social novelty preference of PFFs-injected mice.
To do this, we added a second novel sKmulus mouse 2 (S2) into the empty cage in the presence of the familiar sKmulus S1 to evaluate the test mouse's interest in the familiar social sKmulus versus the novel one when both were present (see the session 2 in Figure 5A, C).While controls spent much more Kme with the novel sKmulus (S2) relaKve to the familiar one (S1), PFFsinjected mice spent similar amount of Kme with either the familiar sKmulus (S1) or the novel sKmulus (S2) (Figure 5C, E).
It is important to note that relaKve to controls, PFFs-injected mice at 3 mpi showed comparable general motor acKvity in locomoKon test and rotarod test and similar levels of anxiety elevated plus maze as measured by the Kme spent in open arms (Supplementary Figure 1).These data were consistent with previous report [3,25,31] and suggested that PFFs-injected mice do not develop parkinsonian motor deficits or enhanced anxiety, which is important as such behaviors could confound the social interacKon behavioral test.Taken together, these results suggested that PFFs-injected mice exhibited impaired social novelty preference and were not able to disKnguish between familiar-and novel-sKmuli.novelty preference test aner DCZ injecKon, they showed strong preference to S2 versus S1 (Figure 5J), suggesKng that their impaired social novelty preference has been rescued by chemogeneKc sKmulaKon of mPFC-BLA connecKon.Altogether, the above data suggest that the social novelty preference was disrupted by α-Syn pathology in PFFs-injected mice at 3 mpi.Importantly, such disrupKon correlated with impaired mPFC-BLA synapKc connecKon, which was then rescued by chemogeneKc sKmulaKon of the mPFC-BLA connecKon in the PFFs-injected mice.

Discussion
In the present study, we performed longitudinal analyses of anatomical and physiological changes associated with the formaKon of α-Syn aggregaKon, and provided novel insight into how α-Syn aggregaKon impairs the connecKvity and funcKon of amygdala microcircuits.While most research in the field has focused on the processes that iniKate, facilitate, and/or exacerbate α-Syn pathology formaKon and propagaKon, the present work highlights the less explored, yet important, circuitry dysfuncKon associated with α-Syn aggregaKon that can underlie some of the symptoms manifested by individuals afflicted by synucleinopathies.These studies are of clinical relevance as strategies to prevent worsening of α-Syn pathology and to repair the damaged circuits could make the current treatments for diseases like PD and DLB more effecKve.

Synap.c dysfunc.on occurs earlier than neurodegenera.on in the BLA
We demonstrated that α-Syn aggregaKon compromises synapKc funcKon before structural changes occur, including the loss of presynapKc axon terminal markers.In the context of our recent work, even though funcKonal deficits of corKco-BLA synapse may occur as early as 1 mpi [34], loss of corKco-BLA presynapKc marker vGluT1 was only detected at the 6 mpi Kmepoint (Figure 2).Moreover, BLA neurons are also prone to accumulaKng α-Syn aggregates, which perhaps results in the loss of spines at 3 mpi (Figure 4) and their degeneraKon at much later stages, i.e., 12 mpi.These observaKons are consistent with reports from the nigrostriatal system in parkinsonian animals, showing deficits in dopamine release and degeneraKon of axons prior to severe degeneraKon of dopaminergic neurons [4,6,17,41,42].
It is important to note that while BLA neurons exhibited loss of dendriKc spines, the iniKal release probability at corKco-BLA synapses increased at 3 and 6 mpi (Figure 4).This observaKon is consistent with earlier findings from cultured hippocampal neurons, in which the loss of postsynapKc spines and a paradoxical increase of presynapKc glutamate release were reported [43].Froula et al suggested that the enhanced presynapKc release probability might be due to an increased size of readily releasable pool of neurotransmicers.

Synap.c dysfunc.on is sufficient to drive the manifesta.on of behavioral phenotypes
At the early stage of α-Syn based models, funcKonal alteraKons of nigrostriatal pathway have been reported to underlie the manifestaKon of parkinsonian motor deficits [4,17,44,45].In the present study, we found that, prior to the loss of BLA neurons, mPFC-BLA synapses exhibited funcKonal impairment in PFFs-injected mice at 3 mpi, which temporally correlated with the manifestaKon of defected social interacKon behavior (Figures 4 and 5).InteresKngly, the impaired social novelty preference behavior was rescued by chemogeneKc sKmulaKon of the defected mPFC-BLA pathway.Together, these findings support the hypothesis that synapKc dysfuncKon is sufficient to drive the manifestaKon of behavioral phenotypes in synucleinopathies.The intrastriatal PFFs-injected mice exhibited very specific deficits in emoKon related behavior.For example, while they exhibited deficits in social novelty preference (Figure 5) and social dominance behavior [31], no change in their anxiety levels in the elevated plus maze test were detected [25,31].Though compelling evidence suggests that both social behavior and anxiety are Kghtly regulated by the amygdala, it is plausible that performance in the elevated plus maze test may involves disKnct amygdala subcircuits from those acKvated during the 3-chamber social interacKon test [46,47].
Cell type-specific α-Syn expression and its implica.ons in circuit dysfunc.onIn a complex circuit, not all afferents within the same neurochemical category are equally affected by α-Syn aggregaKon.For example, vGluT2 + glutamatergic axons arising from the thalamus are more resilient relaKve to vGluT1 + axons in the BLA (Figure 2).This observaKon is largely consistent with lower Snca mRNA levels in thalamic neurons as well as α-Syn expression in their axon terminals [34,48].Moreover, evidence in the literature consistently reports that Lewy body-like pS129 + somaKc aggregates are more abundant in cerebral corKcal regions relaKve to thalamic regions in intrastriatal PFFs models [3,25,26,34,49], suggesKng that the cerebral cortex and thalamus are affected differently by α-Syn pathology in diseased state.Of relevance to the present work, it is known that cooperaKve acKvity and plasKcity of corKcal and thalamic inputs are essenKal to normal amygdala funcKon in the formaKon and long-term storage of emoKon memory [50,51].It remains to be determined how the disrupted balance or cooperaKon of corKcal and thalamic inputs to the BLA might impair emoKon encoding and regulaKon by the amygdala circuits.Remarkably, human imaging studies reported that PD individuals diagnosed with depression showed a reduced acKvaKon of the amygdala in the presence of aversive sKmulaKons as well as a disconnected corKcal (but not the thalamic) inputs to the amygdala [27,30].Our findings of weakened corKcal, but not thalamic inputs, to the BLA might be of relevance to this clinical observaKon in PD individuals exhibiKng neuropsychiatric symptoms.
The complicated interacKon and impact of α-Syn pathology on circuit funcKon can also parKally explain why emoKon dysregulaKon, including anxiety and depression, can occur at any stages of the disease in PD or DLB individuals (e.g., even before the onset of motor symptoms), which might vary depending on where the α-Syn pathology starts and how it can affect specific circuits.While the present studies focus on the impact of α-Syn pathology on the amygdala funcKon and related behavior, we must acknowledge that degeneraKon of key neuromodulators and their inputs to the amygdala (e.g., dopaminergic and noradrenergic systems) also play key roles in the development of neuropsychiatric symptoms in PD and DLB.

Conclusion
We presented a combinaKon of anatomical and physiological evidence to demonstrate a series of circuitry events occurred in the amygdala circuits associated with α-Syn pathology in mouse brain.Our findings not only support several well-accepted concepts in the field (e.g., synapKc deficits prior to neurodegeneraKon), but also highlight the fact that we need to consider circuit complexity before we can gain a thorough understanding on the contribuKon of α-Syn to brain funcKon in health and in diseased states.