Neurons In Substantia Nigra Pars Compacta Research Articles

Comparing tonic and phasic dendritic calcium in cholinergic pedunculopontine neurons and dopaminergic substantia nigra neurons.

Several brainstem nuclei degenerate in Parkinson's disease (PD). In addition to the well-characterized dopaminergic neurons of the substantia nigra pars compacta (SNc), the cholinergic neurons of the pedunculopontine nucleus (PPN) also degenerate in PD. One leading hypothesis of selective vulnerability is that pacemaking activity and the activation of low-threshold L-type calcium current are major contributors to tonic calcium load and cellular stress in SNc dopaminergic neurons. However, it is not yet clear whether the vulnerable PPN cholinergic neurons share this property. Therefore, we used two-photon dendritic calcium imaging and whole-cell electrophysiology to evaluate the role of L-type calcium channels in tonic and phasic dendritic calcium signals in PPN and SNc neurons. In addition, we investigated N- and P/Q-type calcium channel regulation of firing properties and dendritic calcium in PPN neurons. We found that blocking L-type channels reduces tonic firing rate and dendritic calcium levels in SNc neurons. By contrast, the tonic calcium load in PPN neurons did not depend on L-, N- or P/Q-type channels. However, we found that blocking either L-type (with nifedipine) or N- and P/Q-type (with omega-conotoxin MVIIC) channels reducesphasic calcium influx in PPN dendrites. Together, these findings show that L-type calcium channels play different roles in the activity of SNc and PPN neurons, and suggest that low-threshold L-type channels are not responsible for tonic calcium levels in PPN cholinergic neurons and are therefore not likely to be a source of selective vulnerability in these cells.

Contributions of the Sodium Leak Channel NALCN to Pacemaking of Medial Ventral Tegmental Area and Substantia Nigra Dopaminergic Neurons.

We tested the role of the sodium leak channel, NALCN, in pacemaking of dopaminergic neuron (DAN) subpopulations from adult male and female mice. In situ hybridization revealed NALCN RNA in all DANs, with lower abundance in medial ventral tegmental area (VTA) relative to substantia nigra pars compacta (SNc). Despite lower relative abundance of NALCN, we found that acute pharmacological blockade of NALCN in medial VTA DANs slowed pacemaking by 49.08%. We also examined the electrophysiological properties of projection-defined VTA DAN subpopulations identified by retrograde labeling. Inhibition of NALCN reduced pacemaking in DANs projecting to medial nucleus accumbens (NAc) and others projecting to lateral NAc by 70.74% and 31.98%, respectively, suggesting that NALCN is a primary driver of pacemaking in VTA DANs. In SNc DANs, potentiating NALCN by lowering extracellular calcium concentration speeded pacemaking in wildtype but not NALCN conditional knockout mice, demonstrating functional presence of NALCN. In contrast to VTA DANs, however, pacemaking in SNc DANs was unaffected by inhibition of NALCN. Instead, we found that inhibition of NALCN increased the gain of frequency-current plots at firing frequencies slower than spontaneous firing. Similarly, inhibition of the hyperpolarization-activated cyclic nucleotide-gated (HCN) conductance increased gain but had little effect on pacemaking. Interestingly, simultaneous inhibition of NALCN and HCN resulted in significant reduction in pacemaker rate. Thus, we found NALCN makes substantial contributions to driving pacemaking in VTA DAN subpopulations. In SNc DANs, NALCN is not critical for pacemaking but inhibition of NALCN makes cells more sensitive to hyperpolarizing stimuli.SIGNIFICANCE STATEMENT Pacemaking in midbrain dopaminergic neurons (DAN) relies on multiple subthreshold conductances, including a sodium leak. Whether the sodium leak channel, NALCN, contributes to pacemaking in DANs located in the VTA and the SNc has not yet been determined. Using electrophysiology and pharmacology, we show that NALCN plays a prominent role in driving pacemaking in projection-defined VTA DAN subpopulations. By contrast, pacemaking in SNc neurons does not rely on NALCN. Instead, the presence of NALCN regulates the excitability of SNc DANs by reducing the gain of the neuron's response to inhibitory stimuli. Together, these findings will inform future efforts to obtain DAN subpopulation-specific treatments for use in neuropsychiatric disorders.

Repeated Methamphetamine Administration Results in Axon Loss Prior to Somatic Loss of Substantia Nigra Pars Compacta and Locus Coeruleus Neurons in Male but Not Female Mice.

Methamphetamine (meth) is a neurotoxic psychostimulant that increases monoamine oxidase (MAO)-dependent mitochondrial oxidant stress in axonal but not somatic compartments of substantia nigra pars compacta (SNc) and locus coeruleus (LC) neurons. Chronic meth administration results in the degeneration of SNc and LC neurons in male mice, and MAO inhibition is neuroprotective, suggesting that the deleterious effects of chronic meth begin in axons before advancing to the soma of SNc and LC neurons. To test this hypothesis, mice were administered meth (5 mg/kg) for 14, 21, or 28 days, and SNc and LC axonal lengths and numbers of neurons were quantified. In male mice, the SNc and LC axon lengths decreased with 14, 21, and 28 days of meth, whereas somatic loss was only observed after 28 days of meth; MAO inhibition (phenelzine; 20 mg/kg) prevented axonal and somatic loss of SNc and LC neurons. In contrast, chronic (28-day) meth had no effect on the axon length or numbers of SNc or LC neurons in female mice. The results demonstrate that repeated exposure to meth produces SNc and LC axonal deficits prior to somatic loss in male subjects, consistent with a dying-back pattern of degeneration, whereas female mice are resistant to chronic meth-induced degeneration.

Artemisinin Confers Neuroprotection against 6-OHDA-Induced Neuronal Injury In Vitro and In Vivo through Activation of the ERK1/2 Pathway.

Parkinson's disease (PD) is an age-related, progressive neurodegenerative disease characterized by the gradual and massive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). We have recently reported that artemisinin, an FDA-approved first-line antimalarial drug, possesses a neuroprotective effect. However, the effects and underlying mechanisms of artemisinin on Parkinson's disease remain to be elucidated. In this study, we investigated the neuroprotective effects of artemisinin on 6-OHDA and MPP+ in neuronal cells and animal models, as well as the underlying mechanisms. Our results showed that artemisinin significantly attenuated the loss of cell viability, LDH release, elevated levels of reactive oxygen species (ROS), the collapse of the mitochondria trans-membrane potential and cell apoptosis in PC12 cells. Western blot results showed that artemisinin stimulated the phosphorylation of ERK1/2, its upstream signaling proteins c-Raf and MEK and its downstream target CREB in PC12 cells in a time- and concentration-dependent manner. In addition, the protective effect of artemisinin was significantly reduced when the ERK pathway was blocked using the ERK pathway inhibitor PD98059 or when the expression of ERK was knocked down using sgRNA. These results indicate the essential role of ERK in the protective effect of artemisinin. Similar results were obtained in SH-SY5Y cells and primary cultured neurons treated with 6-OHDA, as well as in cellular models of MPP+ injury. More interestingly, artemisinin attenuated PD-like behavior deficit in mice injected with 6-OHDA evaluated by behavioral tests including swimming test, pole-test, open field exploration and rotarod tests. Moreover, artemisinin also stimulated the phosphorylation of ERK1/2, inhibited apoptosis, and rescued dopaminergic neurons in SNc of these animals. Application of ERK pathway inhibitor PD98059 blocked the protective effect of artemisinin in mice during testing. Taking these results together, it was indicated that artemisinin preserves neuroprotective effects against 6-OHDA and MPP+ induced injury both in vitro and in vivo by the stimulation of the ERK1/2 signaling pathway. Our findings support the potential therapeutic effect of artemisinin in the prevention and treatment of Parkinson's disease.

Rhythmic oscillations in the midbrain dopaminergic nuclei in mice.

Dopamine release in the forebrain by midbrain ventral tegmental nucleus (VTA) and substantia nigra pars compacta (SNc) neurons is implicated in reward processing, goal-directed learning, and decision-making. Rhythmic oscillations of neural excitability underlie coordination of network processing, and have been reported in these dopaminergic nuclei at several frequency bands. This paper provides a comparative characterization of several frequencies of oscillations of local field potential and single unit activity, highlighting some behavioral correlates. We recorded from optogenetically identified dopaminergic sites in four mice training in operant olfactory and visual discrimination tasks. Rayleigh and Pairwise Phase Consistency (PPC) analyses revealed some VTA/SNc neurons phase-locked to each frequency range, with fast spiking interneurons (FSIs) prevalent at 1-2.5 Hz (slow) and 4 Hz bands, and dopaminergic neurons predominant in the theta band. More FSIs than dopaminergic neurons were phase-locked in the slow and 4 Hz bands during many task events. The highest incidence of phase-locking in neurons was in the slow and 4 Hz bands, and occurred during the delay between the operant choice and trial outcome (reward or punishment) signals. These data provide a basis for further examination of rhythmic coordination of activity of dopaminergic nuclei with other brain structures, and its impact for adaptive behavior.

The dosage of curcumin to alleviate movement symptoms in a 6-hydroxydopamine-induced Parkinson's disease rat model

BackgroundCurcumin is a natural compound with extensive pharmacological effects. This research is to verify the optimal dose and administration duration efficacy of curcumin in alleviating the movement symptoms of Parkinson's disease (PD). MethodsWistar rats were divided into six groups including control, model, levodopa treatment and low/middle/high (40/80/160 mg/kg/d) curcumin treatment groups. After stereotactic brain injection of 6-hydroxydopamine (6-OHDA), curcumin was given by intragastric administration for 2 weeks. To evaluate the drug effect, the rats received behavioral tests including apomorphine (APO)-induced rotation test, rotarod test and open field test. Then the rats were sacrificed and the brain slices including substantia nigra pars compacta (SNc) were used for immunofluorescence staining. ResultsAfter 6-OHDA injection, the model group showed typical movement symptoms including the severe APO-induced rotation to the healthy side, decreased latency in the rotarod with constant or accelerative mode, and decreased total distance and average speed in the open field test. In the results of immunofluorescence staining, the 6-OHDA induced a severe damage of dopaminergic neurons in SNc. The 160 mg/kg/d treatment of curcumin to intervene for 2 weeks alleviated most of the behavioral disorders but the 40/80 mg/kg/d treatment showed limitations. Then, we compared the effect of 1 week intervention to the 2 weeks with 160 mg/kg/d treatment of curcumin to intervene and results indicated that the treatment of 2 weeks could better alleviate the symptoms. ConclusionsCurcumin alleviated 6-OHDA-induced movement symptoms in a PD rat model. Additionally, the effect of curcumin against PD indicated dose and duration dependent and the intervention of 160 mg/kg/d for 2 weeks showed optimally therapeutic effect.

Electron Tunneling in Ferritin and Associated Biosystems

Ferritin is a 12 nanometer (nm) diameter iron storage protein complex that is found in most plants and animals. A substantial body of evidence has established that electrons can tunnel through and between ferritin protein nanoparticles and that it exhibits Coulomb blockade behavior, which is also seen in quantum dots and nanoparticles. This evidence can be used to understand the behavior of these particles for use in nanoelectronic devices, for biomedical applications and for investigation of quantum biological phenomena. Ferritin also has magnetic properties that make it useful for applications such as memristors and as a contrast agent for magnetic resonance imaging. This article provides a short overview of this evidence, as well as evidence of ferritin structures in vivo and of tunneling in those structures, with an emphasis on ferritin structures in substantia nigra pars compacta (SNc) neurons. Potential biomedical applications that could utilize these ferritin protein nanoparticles are also discussed.

Therapeutic improvement of Parkinson’s disease transplantation therapy through modulating the immune system.

Abstract We report clinically the implantation of patient-derived midbrain dopaminergic progenitor cells (mDAPs), differentiated in vitrofrom autologous iPSCs, in a patient with idiopathic Parkinson's disease (PD). The patient-specific mDAPs were produced under Good Manufacturing Practice conditions and characterized as having the phenotypic properties of substantia nigra pars compacta neurons; testing in a humanized mouse model indicated an absence of immunogenicity to these cells. The cells were implanted into the putamen (left hemisphere followed by right hemisphere, 6 months apart) of a patient with PD, without the need for immunosuppression. Clinical measures of symptoms of PD after surgery stabilized or improved at 18 to 24 months after implantation. Although our previous data (Schweitzer*, Park* et al., NEJM, 382(20) p1926–1932, 2020) showed that using autologous mDAPs can avoid graft rejection, cell survival and functional recovery in the first patient as assessed by 18F-DOPA PET scan analysis and clinical motor assessments was modest, suggesting that survival of mDA neurons may have been suboptimal even in autologous implantation. We have developed a safer and more effective treatment by not only increasing mDA neuron survival by simultaneously controlling the causative immune response, but also by modulating the problematic proliferative property, which is a characteristic of progenitor cells after implantation. In conclusion, our new study suggests that initial, specific mDA neuron survival in these grafts is strongly affected by the host innate immune response, while long-term graft survival is subject to the adaptive immune response, elucidating a two-phase involvement of the immune system for graft survival. This work was supported by NIH grants (NS070577 and OD024622), the Parkinson’s Cell Therapy Research Fund at McLean Hospital and Massachusetts General Hospital, and the Masson Family Endowed Scholar in Neurosurgery, and the George A. Lopez, MD Endowed Chair in Neurosurgery.

IRAK-M deficiency exacerbates dopaminergic neuronal damage in a mouse model of sub-acute Parkinson's disease.

Emerging evidence has proved that inflammatory responses aggravate the pathological progression of Parkinson's disease. This study aimed to identify the role of Interleukin-1 receptor-associated kinase-M (IRAK-M) as an important negative regulator of innate immunity, in the pathological progression of Parkinson's disease. In the present study, a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection was administered to prepare the acute and sub-acute Parkinson's disease mouse models. Western blot analysis was utilized to examine the protein expressions of tyrosine hydroxylase and IRAK-M. The mRNA expression levels of IRAK-M, interleukin (IL)-6, IL-β, and cyclooxygenase-2 were evaluated via using reverse transcription quantitative PCR (RT-qPCR). The expression of tyrosine hydroxylase-positive neurons in corpus striatum and substantia nigra pars compacta (SNc) tissues was detected using immunohistochemistry. The results showed that the protein and mRNA levels of IRAK-M were considerably upregulated in corpus striatum and SNc tissues in the sub-acute Parkinson's disease model. Furthermore, IRAK-M knockout significantly enhanced the MPTP-induced loss of tyrosine hydroxylase-positive fibers in corpus striatum and tyrosine hydroxylase-positive neurons in SNc, and intensified the effect of MPTP on the activation of microglial cells and the expression of inflammatory cytokines. In addition, sub-acute Parkinson's disease mice with IRAK-M deletion exhibited worse motor abilities than those of wild-type littermates. Overall, the present study suggested that IRAK-M reduces dopaminergic neuron damage in sub-acute Parkinson's disease by suppressing inflammation, which may provide a new therapeutic target for Parkinson's disease treatment.

Extracellular matrix protein anosmin-1 overexpression alters dopaminergic phenotype in the CNS and the PNS with no pathogenic consequences in a MPTP model of Parkinson’s disease

The development and survival of dopaminergic neurons are influenced by the fibroblast growth factor (FGF) pathway. Anosmin-1 (A1) is an extracellular matrix protein that acts as a major regulator of this signaling pathway, controlling FGF diffusion, and receptor interaction and shuttling. In particular, previous work showed that A1 overexpression results in more dopaminergic neurons in the olfactory bulb. Prompted by those intriguing results, in this study, we investigated the effects of A1 overexpression on different populations of catecholaminergic neurons in the central (CNS) and the peripheral nervous systems (PNS). We found that A1 overexpression increases the number of dopaminergic substantia nigra pars compacta (SNpc) neurons and alters the striosome/matrix organization of the striatum. Interestingly, these numerical and morphological changes in the nigrostriatal pathway of A1-mice did not confer an altered susceptibility to experimental MPTP-parkinsonism with respect to wild-type controls. Moreover, the study of the effects of A1 overexpression was extended to different dopaminergic tissues associated with the PNS, detecting a significant reduction in the number of dopaminergic chemosensitive carotid body glomus cells in A1-mice. Overall, our work shows that A1 regulates the development and survival of dopaminergic neurons in different nuclei of the mammalian nervous system.

Differential vulnerability of locus coeruleus and dorsal raphe neurons to chronic methamphetamine-induced degeneration.

Methamphetamine (meth) increases monoamine oxidase (MAO)-dependent mitochondrial stress in axons of substantia nigra pars compacta (SNc), and ventral tegmental area (VTA) dopamine neurons. Chronic administration of meth results in SNc degeneration and MAO inhibition is neuroprotective, whereas, the VTA is resistant to degeneration. This differential vulnerability is attributed, at least in part, to the presence of L-type Ca2+ channel-dependent mitochondrial stress in SNc but not VTA dopamine neurons. MAO is also expressed in other monoaminergic neurons such as noradrenergic locus coeruleus (LC) and serotonergic dorsal raphe (DR) neurons. The impact of meth on mitochondrial stress in LC and DR neurons is unknown. In the current study we used a genetically encoded redox biosensor to investigate meth-induced MAO-dependent mitochondrial stress in LC and DR neurons. Similar to SNc and VTA neurons, meth increased MAO-dependent mitochondrial stress in axonal but not somatic compartments of LC norepinephrine and DR serotonin neurons. Chronic meth administration (5 mg/kg; 28-day) resulted in degeneration of LC neurons and MAO inhibition was neuroprotective whereas DR neurons were resistant to degeneration. Activating L-type Ca2+ channels increased mitochondrial stress in LC but not DR axons and inhibiting L-type Ca2+ channels in vivo with isradipine prevented meth-induced LC degeneration. These data suggest that similar to recent findings in SNc and VTA dopamine neurons, the differential vulnerability between LC and DR neurons can be attributed to the presence of L-type Ca2+ channel-dependent mitochondrial stress. Taken together, the present study demonstrates that both meth-induced MAO- and L-type Ca2+ channel-dependent mitochondrial stress are necessary for chronic meth-induced neurodegeneration.

A Multi-Scale Computational Model of Levodopa-Induced Toxicity in Parkinson's Disease.

Parkinson's disease (PD) is caused by the progressive loss of dopaminergic cells in substantia nigra pars compacta (SNc). The root cause of this cell loss in PD is still not decisively elucidated. A recent line of thinking has traced the cause of PD neurodegeneration to metabolic deficiency. Levodopa (L-DOPA), a precursor of dopamine, used as a symptom-relieving treatment for PD, leads to positive and negative outcomes. Several researchers inferred that L-DOPA might be harmful to SNc cells due to oxidative stress. The role of L-DOPA in the course of the PD pathogenesis is still debatable. We hypothesize that energy deficiency can lead to L-DOPA-induced toxicity in two ways: by promoting dopamine-induced oxidative stress and by exacerbating excitotoxicity in SNc. We present a systems-level computational model of SNc-striatum, which will help us understand the mechanism behind neurodegeneration postulated above and provide insights into developing disease-modifying therapeutics. It was observed that SNc terminals are more vulnerable to energy deficiency than SNc somas. During L-DOPA therapy, it was observed that higher L-DOPA dosage results in increased loss of terminals in SNc. It was also observed that co-administration of L-DOPA and glutathione (antioxidant) evades L-DOPA-induced toxicity in SNc neurons. Our proposed model of the SNc-striatum system is the first of its kind, where SNc neurons were modeled at a biophysical level, and striatal neurons were modeled at a spiking level. We show that our proposed model was able to capture L-DOPA-induced toxicity in SNc, caused by energy deficiency.

Dopamine Dysregulation and Altered Responses to Drugs Affecting Dopaminergic Transmission in a New Dopamine Transporter Knockout (DAT-KO) Rat Model

Under normal conditions, dopamine (DA) clearance after release largely depends on uptake by the DA transporter (DAT). DAT expression/activity is reduced in some neuropsychiatric and neurological disorders. Our aim was to characterize the behavioral, neurochemical and electrophysiological effects of eliminating DAT in a novel knockout rat model we generated using CRISPR/Cas9. Consistent with existing DAT-KO models, our DAT-KO rats displayed increased locomotion, paradoxical calming by amphetamine, and reduced kinetics of DA clearance after stimulated release. Reduced DA kinetics were demonstrated using fast-scan cyclic voltammetry in brain slices containing the striatum or substantia nigra pars compacta (SNc) and in the dorsal striatum in vivo. Cocaine enhanced DA release in wild-type (WT) but not DAT-KO rats. Basal extracellular DA concentration measured with fast-scan controlled-adsorption voltammetry was higher in DAT-KO rats both in the striatum and SNc and was enhanced by L-DOPA (particularly after pharmacological block of monoamine oxidase), confirming that DA release after L-DOPA is not due to DAT reversal. The baseline firing frequency of SNc neurons was similar in both genotypes. However, D2 receptor-mediated inhibition of firing (by quinpirole or L-DOPA) was blunted in DAT-KO rats, while GABAB-mediated inhibition was preserved. We have also provided new data for the DAT-KO rat regarding the effects of slowing DA diffusion with dextran and blocking organic cation transporter 3 with corticosterone. Together, our results validate our DAT-KO rat and provide new insights into the mechanisms of chronic dysregulation of the DA system by addressing several unresolved issues in previous studies with other DAT-KO models.

"Reframing" dopamine signaling at the intersection of glial networks in the aged Parkinsonian brain as innate Nrf2/Wnt driver: Therapeutical implications.

Dopamine (DA) signaling via G protein‐coupled receptors is a multifunctional neurotransmitter and neuroendocrine–immune modulator. The DA nigrostriatal pathway, which controls the motor coordination, progressively degenerates in Parkinson's disease (PD), a most common neurodegenerative disorder (ND) characterized by a selective, age‐dependent loss of substantia nigra pars compacta (SNpc) neurons, where DA itself is a primary source of oxidative stress and mitochondrial impairment, intersecting astrocyte and microglial inflammatory networks. Importantly, glia acts as a preferential neuroendocrine–immune DA target, in turn, counter‐modulating inflammatory processes. With a major focus on DA intersection within the astrocyte–microglial inflammatory network in PD vulnerability, we herein first summarize the characteristics of DA signaling systems, the propensity of DA neurons to oxidative stress, and glial inflammatory triggers dictating the vulnerability to PD. Reciprocally, DA modulation of astrocytes and microglial reactivity, coupled to the synergic impact of gene–environment interactions, then constitute a further level of control regulating midbrain DA neuron (mDAn) survival/death. Not surprisingly, within this circuitry, DA converges to modulate nuclear factor erythroid 2‐like 2 (Nrf2), the master regulator of cellular defense against oxidative stress and inflammation, and Wingless (Wnt)/β‐catenin signaling, a key pathway for mDAn neurogenesis, neuroprotection, and immunomodulation, adding to the already complex “signaling puzzle,” a novel actor in mDAn–glial regulatory machinery. Here, we propose an autoregulatory feedback system allowing DA to act as an endogenous Nrf2/Wnt innate modulator and trace the importance of DA receptor agonists applied to the clinic as immune modifiers.

2021 Workshop: Neurodegenerative Diseases in the Gut-Brain Axis—Parkinson's Disease

2021 Workshop: Neurodegenerative Diseases in the Gut-Brain Axis—Parkinson's Disease

Interleukin-6 triggers toxic neuronal iron sequestration in response to pathological α-synuclein.

SUMMARYα-synuclein (α-syn) aggregation and accumulation drive neurodegeneration in Parkinson’s disease (PD). The substantia nigra of patients with PD contains excess iron, yet the underlying mechanism accounting for this iron accumulation is unclear. Here, we show that misfolded α-syn activates microglia, which release interleukin 6 (IL-6). IL-6, via its trans-signaling pathway, induces changes in the neuronal iron transcriptome that promote ferrous iron uptake and decrease cellular iron export via a pathway we term the cellular iron sequestration response, or CISR. The brains of patients with PD exhibit molecular signatures of the IL-6-mediated CISR. Genetic deletion of IL-6, or treatment with the iron chelator deferiprone, reduces pathological α-syn toxicity in a mouse model of sporadic PD. These data suggest that IL-6-induced CISR leads to toxic neuronal iron accumulation, contributing to synuclein-induced neurodegeneration.

A Whole-Brain Connectivity Map of VTA and SNc Glutamatergic and GABAergic Neurons in Mice.

The glutamatergic and GABAergic neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) mediated diverse brain functions. However, their whole-brain neural connectivity has not been comprehensively mapped. Here we used the virus tracers to characterize the whole-brain inputs and outputs of glutamatergic and GABAergic neurons in VTA and SNc. We found that these neurons received similar inputs from upstream brain regions, but some quantitative differences were also observed. Neocortex and dorsal striatum provided a greater share of input to VTA glutamatergic neurons. Periaqueductal gray and lateral hypothalamic area preferentially innervated VTA GABAergic neurons. Specifically, superior colliculus provided the largest input to SNc glutamatergic neurons. Compared to input patterns, the output patterns of glutamatergic and GABAergic neurons in the VTA and SNc showed significant preference to different brain regions. Our results laid the anatomical foundation for understanding the functions of cell-type-specific neurons in VTA and SNc.

Daidzein exerts neuroprotective activity against MPTP-induced Parkinson's disease in experimental mice and lipopolysaccharide-induced BV2 microglial cells.

Parkinson's disease (PD) ranks as the second most neurodegenerative disease characterized by loss of neurons, bradykinesia, anosmia, sleep disorder, and motor deficiency with increased global prevalence. Here, we have analyzed daidzein's neuroprotective functions in in vitro and in vivo models of PD. BV2 microglial cells induced with lipopolysaccharide (LPS) and C57BL6 mice induced with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) were used in this study to investigate neuroprotective functions of daidzein. BV2 cells induced with LPS do not exert and significant (p < 0.05) reduction in cell viability up to concentration range (5-100 µM/ml). Furthermore, LPS exposed BV2 microglia exhibited significantly (p < 0.05) increased NO production, pro-inflammatory mediators PGE2, interleukin-6 (IL6), and interleukin-1β (IL-1β) levels. Treatment with daidzein (10, 25, and 50 µM/ml) to LPS-induced BV2 microglia exhibited significantly (p < 0.05) decreased NO, pro-inflammatory mediators PGE2, IL6, and IlL-1β. Similar to the in vitro results, C57BL6 mice induced with MPTP showed defects in motor functions as observed from altered forelimb and hindlimb footprint analyses, grip strength, and perturbed motor coordination observed via rotarod tests. Additionally, levels of dopamine were significantly reduced, andpro-inflammatory mediators tumor necrosis factor alpha (TNF-α), IL-1β, IL6 were found to be increased in MPTP-induced C57BL6 PD mice. Administering daidzein significantly restored the functional levels of dopamine and pro-inflammatory mediators TNF-α, IL-1β, IL6 to near normal physiology as seen in healthy C57BL6 mice controls. Similarly, daidzein treatment to PD mice also restored the histological architecture to near normal levels as in control mice. Together, our results collectively endorse the neuroprotective functions of daidzein as observed from our initial studies, and further studies aimed at investigating daidzein's ability in regulating the catecholamine synthesis pathway to protect substantia nigra pars compacta (SNpc) neurons arein focus.

The essential role of transcription factor Pitx3 in preventing mesodiencephalic dopaminergic neurodegeneration and maintaining neuronal subtype identities during aging

Pituitary homeobox 3 (Pitx3) is required for the terminal differentiation of nigrostriatal dopaminergic neurons during neuronal development. However, whether Pitx3 contributes to the normal physiological function and cell-type identity of adult neurons remains unknown. To explore the role of Pitx3 in maintaining mature neurons, we selectively deleted Pitx3 in the mesodiencephalic dopaminergic (mdDA) neurons of Pitx3fl/fl/DATCreERT2 bigenic mice using a tamoxifen inducible CreERT2/loxp gene-targeting system. Pitx3fl/fl/DATCreERT2 mice developed age-dependent progressive motor deficits, concomitant with a rapid reduction of striatal dopamine (DA) content and a profound loss of mdDA neurons in the substantia nigra pars compacta (SNc) but not in the adjacent ventral tegmental area (VTA), recapitulating the canonical neuropathological features of Parkinson’s disease (PD). Mechanistic studies showed that Pitx3-deficiency significantly increased the number of cleaved caspase-3+ cells in SNc, which likely underwent neurodegeneration. Meanwhile, the vulnerability of SNc mdDA neurons was increased in Pitx3fl/fl/DATCreERT2 mice, as indicated by an early decline in glial cell line-derived neurotrophic factor (GDNF) and aldehyde dehydrogenase 1a1 (Aldh1a1) levels. Noticeably, somatic accumulation of α-synuclein (α-syn) was also significantly increased in the Pitx3-deficient neurons. Together, our data demonstrate that the loss of Pitx3 in fully differentiated mdDA neurons results in progressive neurodegeneration, indicating the importance of the Pitx3 gene in adult neuronal survival. Our findings also suggest that distinct Pitx3-dependent pathways exist in SNc and VTA mdDA neurons, correlating with the differential vulnerability of SNc and VTA mdDA neurons in the absence of Pitx3.

Chronic methamphetamine-induced neurodegeneration: Differential vulnerability of ventral tegmental area and substantia nigra pars compacta dopamine neurons

Methamphetamine (meth) increases monoamine oxidase (MAO)-dependent mitochondrial stress in substantia nigra pars compacta (SNc) axons; chronic administration produces SNc degeneration that is prevented by MAO inhibition suggesting that MAO-dependent axonal mitochondrial stress is a causal factor. To test whether meth similarly increases mitochondrial stress in ventral tegmental area (VTA) axons, we used a genetically encoded redox biosensor to assess mitochondrial stress ex vivo. Meth increased MAO-dependent mitochondrial stress in both SNc and VTA axons. However, despite having the same meth-induced stress as SNc neurons, VTA neurons were resistant to chronic meth-induced degeneration indicating that meth-induced MAO-dependent mitochondrial stress in axons was necessary but not sufficient for degeneration. To determine whether L-type Ca2+ channel-dependent stress differentiates SNc and VTA axons, as reported in the soma, the L-type Ca2+ channel activator Bay K8644 was used. Opening L-type Ca2+ channels increased axonal mitochondrial stress in SNc but not VTA axons. To first determine whether mitochondrial stress was necessary for SNc degeneration, mice were treated with the mitochondrial antioxidant mitoTEMPO. Chronic meth-induced SNc degeneration was prevented by mitoTEMPO thereby confirming the necessity of mitochondrial stress. Similar to results with the antioxidant, both MAO inhibition and L-type Ca2+ channel inhibition also prevented SNc degeneration. Taken together the presented data demonstrate that both MAO- and L-type Ca2+ channel-dependent mitochondrial stress is necessary for chronic meth-induced degeneration.