SNc Neurons Research Articles

Микроэлектрофизиологическое изучение соотношения возбудительных и тормозных синаптических процессов в кортиконигральной проекции к SNс на модели болезни Паркинсона

In series experiments on 9 Albino rats (250 g) the comparative analyses of single neuron’s impulse activity of Substantia nigra compacta – SNc at high frequency stimulation (HFS) of the brain primary motor cortex (M1) in norm (107 neurons, n=4) and on the model of Parkison’s disease (PD) (105 neurons, n=5) has been conducted. Analyses of relative degree of depressor and excitatory effects frequency intensity on the base of spikes average frequency diagrams, represented as a disc diagram (in %) resulted in the following conclusions. In SNc neurons on the model of PD the poststimulus manifestations of depressor activity completely absent, but tetanic potentiation in both sequences 1,65- and 2,02-multiple exceeded of those in the norm. The comparative analyses of pre- and poststimulus frequency activity of SNc neurons on the model of PD, led to the conclusion that it is considerably higher in comparison with the norm. It has been revealed vulnerability of depressor poststimulus effects of SNc neurons, even their full absence, that in its turn, testifies about more involvement of SNc in PD.

VGLUT2 Is a Determinant of Dopamine Neuron Resilience in a Rotenone Model of Dopamine Neurodegeneration.

Parkinson's disease (PD) is characterized by progressive dopamine (DA) neuron loss in the SNc. In contrast, DA neurons in the VTA are relatively protected from neurodegeneration, but the underlying mechanisms for this resilience remain poorly understood. Recent work suggests that expression of the vesicular glutamate transporter 2 (VGLUT2) selectively impacts midbrain DA neuron vulnerability. We investigated whether altered DA neuron VGLUT2 expression determines neuronal resilience in rats exposed to rotenone, a mitochondrial complex I inhibitor and toxicant model of PD. We discovered that VTA/SNc DA neurons that expressed VGLUT2 are more resilient to rotenone-induced DA neurodegeneration. Surprisingly, the density of neurons with detectable VGLUT2 expression in the VTA and SNc increases in response to rotenone. Furthermore, dopaminergic terminals within the NAc, where the majority of VGLUT2-expressing DA neurons project, exhibit greater resilience compared with DA terminals in the caudate/putamen. More broadly, VGLUT2-expressing terminals are protected throughout the striatum from rotenone-induced degeneration. Together, our data demonstrate that a distinct subpopulation of VGLUT2-expressing DA neurons are relatively protected from rotenone neurotoxicity. Rotenone-induced upregulation of the glutamatergic machinery in VTA and SNc neurons and their projections may be part of a broader neuroprotective mechanism. These findings offer a putative new target for neuronal resilience that can be manipulated to prevent toxicant-induced DA neurodegeneration in PD.SIGNIFICANCE STATEMENT Environmental exposures to pesticides contribute significantly to pathologic processes that culminate in Parkinson's disease (PD). The pesticide rotenone has been used to generate a PD model that replicates key features of the illness, including dopamine neurodegeneration. To date, longstanding questions remain: are there dopamine neuron subpopulations resilient to rotenone; and if so, what are the molecular determinants of this resilience? Here we show that the subpopulation of midbrain dopaminergic neurons that express the vesicular glutamate transporter 2 (VGLUT2) are more resilient to rotenone-induced neurodegeneration. Rotenone also upregulates VGLUT2 more broadly in the midbrain, suggesting that VGLUT2 expression generally confers increased resilience to rotenone. VGLUT2 may therefore be a new target for boosting neuronal resilience to prevent toxicant-induced DA neurodegeneration in PD.

Mechanism of Pacemaker Activity in Zebrafish DC2/4 Dopaminergic Neurons.

Zebrafish models are used increasingly to study the molecular pathogenesis of Parkinson's disease (PD), owing to the extensive array of techniques available for their experimental manipulation and analysis. The ascending dopaminergic projection from the posterior tuberculum (TPp; diencephalic populations DC2 and DC4) to the subpallium is considered the zebrafish correlate of the mammalian nigrostriatal projection, but little is known about the neurophysiology of zebrafish DC2/4 neurons. This is an important knowledge gap, because autonomous activity in mammalian substantia nigra (SNc) dopaminergic neurons contributes to their vulnerability in PD models. Using a new transgenic zebrafish line to label living dopaminergic neurons, and a novel brain slice preparation, we conducted whole-cell patch clamp recordings of DC2/4 neurons from adult zebrafish of both sexes. Zebrafish DC2/4 neurons share many physiological properties with mammalian dopaminergic neurons, including the cell-autonomous generation of action potentials. However, in contrast to mammalian dopaminergic neurons, the pacemaker driving intrinsic rhythmic activity in zebrafish DC2/4 neurons does not involve calcium conductances, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, or sodium leak currents. Instead, voltage clamp recordings and computational models show that interactions between three components - a small, predominantly potassium, leak conductance, voltage-gated sodium channels, and voltage-gated potassium channels - are sufficient for pacemaker activity in zebrafish DC2/4 neurons. These results contribute to understanding the comparative physiology of the dopaminergic system and provide a conceptual basis for interpreting data derived from zebrafish PD models. The findings further suggest new experimental opportunities to address the role of dopaminergic pacemaker activity in the pathogenesis of PD.SIGNIFICANCE STATEMENT Posterior tuberculum (TPp) DC2/4 dopaminergic neurons are considered the zebrafish correlate of mammalian substantia nigra (SNc) neurons, whose degeneration causes the motor signs of Parkinson's disease (PD). Our study shows that DC2/4 and SNc neurons share a number of electrophysiological properties, including depolarized membrane potential, high input resistance, and continual, cell-autonomous pacemaker activity, that strengthen the basis for the increasing use of zebrafish models to study the molecular pathogenesis of PD. The mechanisms driving pacemaker activity differ between DC2/4 and SNc neurons, providing: (1) experimental opportunities to dissociate the contributions of intrinsic activity and underlying pacemaker currents to pathogenesis; and (2) essential information for the design and interpretation of studies using zebrafish PD models.

Therapeutic effects of Wharton’s jelly-derived Mesenchymal Stromal Cells on behaviors, EEG changes and IGF-1 in rat model of the Parkinson’s disease

Human Wharton's jelly-derived Mesenchymal Stromal Cells (hWJ-MSCs) have shown beneficial effects in improving the dopaminergic cells in the Parkinson’s disease (PD). In the present study, the effects of hWJ-MSCs on hyperalgesia, anxiety deficiency and Pallidal local electroencephalogram (EEG) impairment, alone and combined with L-dopa, were examined in a rat model of PD.Adult male Wistar rats were divided into five groups: 1) sham, 2) PD, 3) PD + C (Cell therapy), 4) PD + C+D (Drug), and 5) PD + D. PD was induced by injection of 6-OHDA (16 μg/2 μl into medial forebrain bundle (MFB)). PD + C group received hWJ-MSCs (1 × 106 cells, intravenous (i.v.)) twice post PD induction. PD + C+D groups received hWJ-MSCs combined with L-Dopa/Carbidopa, (10/30 mg/kg, intraperitoneally (i.p.)). PD + D group received L-Dopa/Carbidopa alone. Four months later, analgesia, anxiety-like behaviors, were evaluated and Pallidal local EEG was recorded. Level of insulin-like growth factor 1 (IGF-1) was measured in the striatum and dopaminergic neurons were counted in substantia nigra (SNc).According to data, MFB-lesioned rats showed hyperalgesia in tail flick, anxiety-like symptoms in cognitive tests, impairment of electrical power of pallidal local EEG as field potential, count of dopaminergic neurons in SNc and level of IGF-1 in striatum. These complications restored significantly by MSCs treatment (p < 0.001).Our findings confirm that chronic treatment with hWJ-MSC, alone and in combination with L-Dopa, improved nociception and cognitive deficit in PD rats which may be the result of increasing IGF-1 and protect the viability of dopaminergic neurons.

Microelectrophysiological Studies of the Ratio of Excitatory to Inhibitory Synaptic Processes in the Corticonigral Projection in a Model of Parkinson’s

Experiments on 23 white male rats (250 g) analyzed the spike activity of individual neurons in the substantia nigra pars compacta (SNc, 242 neurons, n = 11) and substantia nigra pars reticulata (SNr, 289 neurons, n = 12) during high-frequency stimulation of the primary motor cortex (M1)in normal animals and in animals with a rotenone model of Parkinson’s disease (BP). SNc neurons in the model of PD showed a complete absence of depressor effects induced by stimulation, though tetanic potentiation was accompanied by posttetanic potentiation and depression at levels 1.65 and 2.02 times greater than in normal animals. In SNr neurons in normal animals, tetanic potentiation, accompanied by post-tetanic potentiation and depression, was 2.37 times greater than tetanic depression, while in the model of PD the levels of both depressor and excitatory activity induced by stimulation were below normal. Spike activity frequency in SNc and SNr neurons preceding and accompanying stimulation was significantly greater than normal in the model of PD. This is evidence for excitotoxicity accompanying neurodegenerative damage, which is completed by neuron apoptosis and death. In SNr neurons, both depressor and excitatory reactions accompanying stimulation were markedly dominant over those in SNc neurons, which is evidence for more extensive cortical projections to the SNr. Furthermore, SNc neurons demonstrated greater susceptibility to pathological changes due to poststimulus depressor effects than SNr neurons, with formation of more marked excitatory effects, which is evidence of a greater involvement of the SNc in PD. In the model of PD, lacking stimulation-induced depressor effects and more marked excitatory effects in SNc neurons, SNr neurons retained their depressor reactions and relatively decreased excitatory reactions, which is evidence of a lower level of susceptibility of SNr neurons to excitotoxicity, extreme increases in the excitability of surviving neurons compensating for the lack of excitation of dead cells.

Functional Dissection of Basal Ganglia Inhibitory Inputs onto Substantia Nigra Dopaminergic Neurons

Substantia nigra (SNc) dopaminergic neurons respond to aversive stimuli with inhibitory pauses in firing followed by transient rebound activation. We tested integration of inhibitory synaptic inputs onto SNc neurons from genetically defined populations in dorsal striatum (striosome and matrix) and external globus pallidus (GPe; parvalbumin- and Lhx6-positive), and examined their contribution to pause-rebound firing. Activation of striosome projections, which target "dendron bouquets" in the pars reticulata (SNr), consistently quiets firing and relief from striosome inhibition triggers rebound activity. Striosomal inhibitory postsynaptic currents (IPSCs) display a prominent GABA-B receptor-mediated component that strengthens the impact of SNr dendrite synapses on somatic excitability and enables rebounding. By contrast, GPe projections activate GABA-A receptors on the soma and proximal dendrites but do not result in rebounding. Lastly, optical mapping shows that dorsal striatum selectively inhibits the ventral population of SNc neurons, which are intrinsically capable of rebounding. Therefore, we define a distinct striatonigral circuit for generating dopamine rebound.

Микроэлектрофизиологическое изучение соотношения возбудительных и тормозных синаптических процессов в кортико-нигральной проекции на модели болезни Паркинсона

The impulse activity of single neurons in substantia nigra pars compacta - SNc (242 neurons, n=11) and substantia nigra pars reticulate - SNr (289 neurons, n=12) under high frequency stimulation of primary motor cortex (M1) was analyzed on 23 white male rats in control and rotenone model of Parkinson’s disease (PD). In SNc neurons in PD model there was no stimulation-induced depression, however, the posttetatic potentiation and depression following the tetanic potentiation, were 1.65 and 2.02 times higher than in control. In control SNr neurons the tetanic potentiation followed by the posttetanic potentiation and depression was 2.37 times higher than the tetanic depression, whereas in the PD model both depression and potentiation levels were subnormal. In PD model, both SNc and SNr neurons exhibited significantly higher spike frequency before and during the stimulation in comparison with control. This indicates the presence of the excitotoxicity accompanied by the neurodegenerative damage followed by the apoptosis and the neuronal death. In SNr neurons both depression and potentiation after the stimulation were much more prominent than in SNc neurons, which indicates that the cortical projections on the SNr are more pronounced. Moreover, the SNc neurons demonstrated higher succeptibility to the pathological changes in the poststimulus depression, leading to the more pronounced potentiation in those neurons in comparison with the SNr neurons, which indicates the more prominent involvement of the former in the PD pathogenesis. In the PD model, with no stimulation-induced depression and more prominent potentiation in the SNc neurons, the SNr neurons maintain the depression and show relatively lowered potentiation, which indicates their lower succeptibility to the excitotoxicity, and the excessive excitability of the remaining neurons as a compensation for the dead ones.

Quantum dot conjugated saporin activates microglia and induces selective substantia nigra degeneration

Parkinson’s disease (PD) is characterized by profound microglial driven inflammatory processes and the loss of dopamine neurons of the substantia nigra (SNc). Both microglia and dopamine neurons that are affected in the SNc are particularly vulnerable to environmental toxicants and finding more selective ways of targeting these cell types is of importance. Quantum dots (QDs) might be a useful vehicle for selectively delivering toxicants to microglia and owing to their fluorescent capability, they can be microscopically tracked within the cell. Accordingly, we assessed the impact of QDs alone and QDs conjugated to the ribosomal toxin, saporin, upon SNc microglia and dopamine neurons. We found that intra-SNc infused QDs selectively entered microglia and induced morphological changes consistent with an activated state. QDs conjugated to saporin also caused a significant loss of dopamine neurons and motor coordination (on a rotarod test) deficits, along with an increase in the inflammatory microglial actin regulatory factors, WAVE2. These data suggest that QDs might be a viable route for toxicant delivery and also has an added advantage of being fluorescently visible. Ultimately, we found SNc neurons to be exceptionally vulnerable to QD-saporin and suggest that this could be a novel targeted approach to model PD-like inflammatory pathology.

Biophysical Properties of Somatic and Axonal Voltage-Gated Sodium Channels in Midbrain Dopaminergic Neurons.

Spiking activities of midbrain dopaminergic neurons are critical for key brain functions including motor control and affective behaviors. Voltage-gated Na+ channels determine neuronal excitability and action potential (AP) generation. Previous studies on dopaminergic neuron excitability mainly focused on Na+ channels at the somatodendritic compartments. Properties of axonal Na+ channels, however, remain largely unknown. Using patch-clamp recording from somatic nucleated patches and isolated axonal blebs from the axon initial segment (AIS) of dopaminergic neurons in mouse midbrain slices, we found that AIS channel density is approximately 4–9 fold higher than that at the soma. Similar voltage dependence of channel activation and inactivation was observed between somatic and axonal channels in both SNc and VTA cells, except that SNc somatic channels inactivate at more hyperpolarized membrane potentials (Vm). In both SNc and VTA, axonal channels take longer time to inactivate at a subthreshold depolarization Vm level, but are faster to recover from inactivation than somatic channels. Moreover, we found that immunosignals of Nav1.2 accumulate at the AIS of dopaminergic neurons. In contrast, Nav1.1 and Nav1.6 immunosignals are not detectible. Together, our results reveal a high density of Na+ channels at the AIS and their molecular identity. In general, somatic and axonal channels of both SNc and VTA dopaminergic neurons share similar biophysical properties. The relatively delayed inactivation onset and faster recovery from inactivation of axonal Na+ channels may ensure AP initiation at high frequencies and faithful signal conduction along the axon.

Individual Dopaminergic Neurons of Lamprey SNc/VTA Project to Both the Striatum and Optic Tectum but Restrict Co-release of Glutamate to Striatum Only

Dopaminergic neurons in the substantia nigra (SNc) innervate both striatum and the superior colliculus in mammals, as well as its homolog the optic tectum in lampreys, belonging to the oldest group of living vertebrates [1-3]. In the lamprey, we have previously shown that the same neuron sends axonal branches to both striatum and the optic tectum [3]. Here, we show that most neurons in the lamprey SNc and ventral tegmental area (VTA) (also referred to as the nucleus of the posterior tuberculum) express not only tyrosine hydroxylase (TH), in lamprey a marker of dopaminergic neurons [4], but also the vesicular glutamate transporter (vGluT), suggesting that glutamate is a co-transmitter. Remarkably, the axonal branches that project to striatum elicit both dopaminergic and glutamatergic synaptic effects on striatal neurons, whereas the axonal projections to the optic tectum only evoke dopaminergic effects. Thus, axonal branches from the same neuron can use two transmitters in one branch and only one in the other. Previous studies suggest that, along an individual dopaminergic axon, there can be microdomains of either TH or vGluT [5-8]. In addition, the present results demonstrate that entire axonal branches to one target structure can differ from that of branches to another target, both originating from the same dopamine neuron. This implies that a given dopamine neuron can exert different effects on two different target structures. The combined release of dopamine and glutamate may be appropriate in striatum, whereas the effects exerted on the tectal motor center may be better served with a selective dopaminergic modulation.

EZH2 Influences mdDA Neuronal Differentiation, Maintenance and Survival.

Over the last decade several components have been identified to be differentially expressed in subsets of mesodiencephalic dopaminergic (mdDA) neurons. These differences in molecular profile have been implied to be involved in the selective degeneration of the SNc neurons in Parkinson’s disease. The emergence and maintenance of individual subsets is dependent on different transcriptional programs already present during development. In addition to the influence of transcription factors, recent studies have led to the hypothesis that modifications of histones might also influence the developmental program of neurons. In this study we focus on the histone methyltransferase EZH2 and its role in the development and maintenance of mdDA neurons. We generated two different conditional knock out (cKO) mice; an En1Cre driven cKO, for deletion of Ezh2 in mdDA progenitors and a Pitx3Cre driven cKO, to study the effect of post-mitotic deletion of Ezh2 on mdDA neurons maturation and neuronal survival. During development Ezh2 was found to be important for the generation of the proper amount of TH+ neurons. The loss of neurons primarily affected a rostrolateral population, which is also reflected in the analysis of the subset marks, Ahd2 and Cck. In contrast to early genetic ablation, post-mitotic deletion of Ezh2 did not lead to major developmental defects at E14.5. However, in 6 months old animals Cck was found ectopically in the rostral domain of mdDA neurons and Ahd2 expression was reduced in more mediocaudal positioned cells. In addition, Pitx3Cre driven deletion of Ezh2 led to a progressive loss of TH+ cells in the VTA and these animals display reduced climbing behavior. Together, our data demonstrates that Ezh2 is important for the generation of mdDA neurons during development and that during adult stages Ezh2 is important for the preservation of proper neuronal subset identity and survival.

Lewy body-like alpha-synuclein inclusions trigger reactive microgliosis prior to nigral degeneration

BackgroundConverging evidence suggests a role for microglia-mediated neuroinflammation in Parkinson’s disease (PD). Animal models of PD can serve as a platform to investigate the role of neuroinflammation in degeneration in PD. However, due to features of the previously available PD models, interpretations of the role of neuroinflammation as a contributor to or a consequence of neurodegeneration have remained elusive. In the present study, we investigated the temporal relationship of neuroinflammation in a model of synucleinopathy following intrastriatal injection of pre-formed alpha-synuclein fibrils (α-syn PFFS).MethodsMale Fischer 344 rats (N = 114) received unilateral intrastriatal injections of α-syn PFFs, PBS, or rat serum albumin with cohorts euthanized at monthly intervals up to 6 months. Quantification of dopamine neurons, total neurons, phosphorylated α-syn (pS129) aggregates, major histocompatibility complex-II (MHC-II) antigen-presenting microglia, and ionized calcium-binding adaptor molecule-1 (Iba-1) immunoreactive microglial soma size was performed in the substantia nigra. In addition, the cortex and striatum were also examined for the presence of pS129 aggregates and MHC-II antigen-presenting microglia to compare the temporal patterns of pSyn accumulation and reactive microgliosis.ResultsIntrastriatal injection of α-syn PFFs to rats resulted in widespread accumulation of phosphorylated α-syn inclusions in several areas that innervate the striatum followed by significant loss (~ 35%) of substantia nigra pars compacta dopamine neurons within 5–6 months. The peak magnitudes of α-syn inclusion formation, MHC-II expression, and reactive microglial morphology were all observed in the SN 2 months following injection and 3 months prior to nigral dopamine neuron loss. Surprisingly, MHC-II immunoreactivity in α-syn PFF injected rats was relatively limited during the later interval of degeneration. Moreover, we observed a significant correlation between substantia nigra pSyn inclusion load and number of microglia expressing MHC-II. In addition, we observed a similar relationship between α-syn inclusion load and number of microglia expressing MHC-II in cortical regions, but not in the striatum.ConclusionsOur results demonstrate that increases in microglia displaying a reactive morphology and MHC-II expression occur in the substantia nigra in close association with peak numbers of pSyn inclusions, months prior to nigral dopamine neuron degeneration, and suggest that reactive microglia may contribute to vulnerability of SNc neurons to degeneration. The rat α-syn PFF model provides an opportunity to examine the innate immune response to accumulation of pathological α-syn in the context of normal levels of endogenous α-syn and provides insight into the earliest neuroinflammatory events in PD.

Dopamine Transporter Activation Reduces Kv2.1 Activation Potential and Cluster Size

Functional interactions between transmembrane proteins fine‐tune neuronal excitability in response to changing homeostatic demands. Dysregulation of neuronal excitability is thought to impose a significant energetic challenge on neurons, and it is implicated as a contributing factor in the demise of substantia nigra (SNc) dopamine neurons in Parkinson's Disease (PD). The objective of this study is to examine whether or not a novel interaction discovered by our lab between the dopamine transporter (DAT) and Kv2.1 has the potential to modulate the excitability (and thusly vulnerability) of SNc dopamine neurons. This is of particular interest because DAT can be targeted by numerous FDA‐approved compounds (bupropion, amphetamine, methylphenidate), eliminating a significant hurdle should the interaction reveal neuroprotective potential. DAT efficiently reuptakes dopamine along with co‐substrates Na+ and Cl− to recycle the released monoamine. This process triggers multiple concurrent events such as the induction of an inward depolarizing current as well as an increase in intracellular Ca2+, both of which impact neuronal activity. In this study, we have identified a novel functional interaction between DAT and the delayed‐rectifier K+ channel Kv2.1. Kv2.1 repolarizes dopamine neurons and excitotoxic stimuli elicit a negative shift in its activation potential such that neuronal excitability is decreased. This shift in activation voltage is coupled to a loss of channel phosphorylation as well as a dispersal of membrane‐bound Kv2.1 clusters. Examining DAT and Kv2.1 distribution in SNc and ventral tegmental area (VTA) dopamine neurons revealed increased colocalization of the two proteins in the axon hillock compared to the soma of SNc DA neurons, but there was no difference in sub‐cellular colocalization in the less vulnerable VTA dopamine neurons, suggesting a heightened modulatory potential of this interaction in the SNc. Functionally, we found that DAT activation induces a negative shift in Kv2.1's activation potential (VEH = 5.61 ± 2.8 mV; METH = 2.675 ± 2.6 mV; n=8, p=0.0292) as well as the canonical decrease in Kv2.1 cluster size (VEH = 0.2871 ± 0.05 μm3, n = 2541 clusters; AMPH = 0.1418 ± 0.03 μm3, n = 1486 clusters; Cyto‐D = 0.1123 ± 0.04 μm3, n = 419 clusters), suggesting that DAT can be targeted to modulate excitability through its downstream effects on Kv2.1. Additionally, we found DAT activation weakens the interaction between the two proteins measured via FRET microscopy (VEH = 12.48 ± 2.48 % n=10; AMPH = 6.55 ± 1.76% n=14; p=.0484), further suggesting that the axon hillock of SNc dopamine neurons represents a uniquely promising site to target this interaction. Ongoing experiments utilizing high‐speed in vitro imaging coupled with single neuron recordings and ex vivo two‐photon microscopy aim to identify the functional significance of the DAT/Kv2.1 interaction in the axon hillock of SNc dopamine neurons.Support or Funding InformationNINDS Interdisciplinary T32 in Movement Disorders &amp; Neurorestoration (T32‐NS082168), R01DA026947‐08S1/NIDA (HK), 1S10OD020026‐01/NIH (HK)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Quercetin on Neurodegenerative and Compensatory Processes in the Nigrostriatal System in a Model of the Preclinical Stage of Parkinson’s Disease in Rats

Data obtained over the last ten years on the involvement of molecular chaperones of the heat shock protein 70 (HSP70) family in the pathogenesis of Parkinson’s disease (PD) do not provide an answer to the question of whether a decrease in the expression of stress-inducible Hsp70 in the brain is one of the causes of progression of neurodegeneration in PD. The present study used the HSP expression inhibitor quercetin in a model of proteasomal dysfunction in the nigrostriatal system in rats created by microinjection of the proteasome inhibitor lactacystin into the compact zone of the substantia nigra (SNc). This model reproduces subthreshold levels of nigrostriatal system degradation and neurochemical changes present in the preclinical stage of PD. We provide the first demonstration that preventive administration of quercetin prevents LC-induced Hsp70 expression in SNc neurons and leads to a 1.5-fold increase in the degeneration of dopaminergic neurons in the SNc and a 2.7-fold increase in the number of dopaminergic neuron axons in the striatum, with depletion of compensation processes and HSP70 reserves in SNc neurons. Signs of motor dysfunction typical of the clinical stage of PD were seen. The study results confirm the important role of Hsp70 in the mechanisms of protection of the nigrostriatal system in proteasomal dysfunction typical of the preclinical stage of PD. These data may provide scientific grounds for developing new techniques for the early treatment of PD based on Hsp70 preparations.

Direct Dopaminergic Projections from the SNc Modulate Visuomotor Transformation in the Lamprey Tectum

Dopamine neurons in the SNc play a pivotal role inmodulating motor behavior via striatum. Here, weshow that the same dopamine neuron that targets striatum also sends a direct branch to the optic tectum (superior colliculus). Whenever SNc neurons are activated, both targets will therefore be affected. Visual stimuli (looming or bars) activate the dopamine neurons coding saliency and also elicit distinct motor responses mediated via tectum (eye, orienting or evasive), which are modulated by the dopamine input. Whole-cell recordings from tectal projection neurons and interneurons show that dopamine, released by SNc stimulation, increases or decreases the excitability depending on whether they express the dopamine D1 or the D2 receptor. SNc thus exerts its effects on the visuomotor system through a combined effect directly on tectum and also via striatum. This direct SNc modulation will occur regardless of striatum and represents a novel mode of motor control.

Enhanced Sensitivity to Hyperpolarizing Inhibition in Mesoaccumbal Relative to Nigrostriatal Dopamine Neuron Subpopulations.

Midbrain dopamine neurons recorded in vivo pause their firing in response to reward omission and aversive stimuli. While the initiation of pauses typically involves synaptic or modulatory input, intrinsic membrane properties may also enhance or limit hyperpolarization, raising the question of how intrinsic conductances shape pauses in dopamine neurons. Using retrograde labeling and electrophysiological techniques combined with computational modeling, we examined the intrinsic conductances that shape pauses evoked by current injections and synaptic stimulation in subpopulations of dopamine neurons grouped according to their axonal projections to the nucleus accumbens or dorsal striatum in mice. Testing across a range of conditions and pulse durations, we found that mesoaccumbal and nigrostriatal neurons differ substantially in rebound properties with mesoaccumbal neurons displaying significantly longer delays to spiking following hyperpolarization. The underlying mechanism involves an inactivating potassium (IA) current with decay time constants of up to 225 ms, and small-amplitude hyperpolarization-activated currents (IH), characteristics that were most often observed in mesoaccumbal neurons. Pharmacological block of IA completely abolished rebound delays and, importantly, shortened synaptically evoked inhibitory pauses, thereby demonstrating the involvement of A-type potassium channels in prolonging pauses evoked by GABAergic inhibition. Therefore, these results show that mesoaccumbal and nigrostriatal neurons display differential responses to hyperpolarizing inhibitory stimuli that favors a higher sensitivity to inhibition in mesoaccumbal neurons. These findings may explain, in part, observations from in vivo experiments that ventral tegmental area neurons tend to exhibit longer aversive pauses relative to SNc neurons.SIGNIFICANCE STATEMENT Our study examines rebound, postburst, and synaptically evoked inhibitory pauses in subpopulations of midbrain dopamine neurons. We show that pauses in dopamine neuron firing, evoked by either stimulation of GABAergic inputs or hyperpolarizing current injections, are enhanced by a subclass of potassium conductances that are recruited at voltages below spike threshold. Importantly, A-type potassium currents recorded in mesoaccumbal neurons displayed substantially slower inactivation kinetics, which, combined with weaker expression of hyperpolarization-activated currents, lengthened hyperpolarization-induced delays in spiking relative to nigrostriatal neurons. These results suggest that input integration differs among dopamine neurons favoring higher sensitivity to inhibition in mesoaccumbal neurons and may partially explain in vivo observations that ventral tegmental area neurons exhibit longer aversive pauses relative to SNc neurons.

Abstract WP102: Retrograde Neurodegeneration of Substantia Nigra Projections to the Striatum Following Long Term Survival After Ischemic Stroke

Introduction: Stroke survivors suffer from long-term physical, cognitive and affective disabilities. These disabilities lower the quality of life, contribute to social isolation and anhedonia, and clinical disorders such as post-stroke depression (PSD), which disproportionately affects women. Preclinical studies show that lesions of the substantia nigra (pars compacta) impair both motor performance and reward seeking behavior. Neurons from the SNc project to the striatum, a region that is significantly infarcted by middle cerebral artery occlusion (MCAo). Hypothesis: Ischemia induced by MCAo leads to retrograde degeneration of the SNc pathway projecting to the striatum. Methods: Middle-aged Sprague-Dawley female rats (12 months old) were subjected to ischemic stroke using a silicon-coated nylon filament suture to occlude the middle cerebral artery (MCA) which was removed 75min after to allow for reperfusion. After 14 weeks of survival, rats were again anesthetized and Fluorogold (Flg) was injected into the left and right striatum. Four days later, animals were overdosed with anesthetic, perfused with saline and formaldehyde and the brain removed for cryosectioning. In three sections per animal, Flg-labeled cells in the SNc were counted on both hemispheres, using fluorescent illumination. Results: Flg-injections into the striatum retrogradely labeled neurons in the midbrain. There was a 35% decrease (p&lt;0.009) in the number of Flg labeled SNc cells in the ischemic hemisphere (85.5+18.2) as compared to the non-ischemic hemisphere (132.4+22.7). The reduced number of projection neurons in the ischemic hemisphere is consistent with the loss of trophic support from the striatum. Conclusions: SNc neurons are a critical component of reward pathway and motor function, hence degeneration of these neurons could lead to long term motor deficits and depressive symptoms including anhedonia that are common after stroke.

Mest/Peg1 Is Essential for the Development and Maintenance of a SNc Neuronal Subset.

Mesodiencephalic dopaminergic (mdDA) neurons originate at the floor plate and floor plate-basal plate boundary of the midbrain ventricular zone. During development mdDA neurons are specified by a unique set of transcription factors and signaling cascades, to form the different molecular subsets of the mdDA neuronal population. In a time series micro-array study performed previously, mesoderm specific transcript (Mest) was found to be one of the most upregulated genes during early mdDA neuronal development. Here, we show that Mest transcript is expressed in the midbrain throughout development and becomes restricted to the substantia nigra (SNc) at late stages. In Mest KO animals mdDA neurons are progressively lost in the adult, mostly affecting the SNc, reflected by a 50% decrease of TH protein and DA release in the striatum and a reduction of climbing behavior. Analysis of Lrp6 KO embryos suggest a subtle opposite phenotype to the Mest KO, hinting toward the possibility that specific loss of mdDA neurons in Mest ablated animals could be due to affected WNT-signaling. Interestingly, the mdDA neuronal region affected by the loss of Mest remains relatively unaffected in Pitx3 mutants, suggesting that both genes are essential for the development and/or maintenance of different mdDA neuronal subsets within the SNc. Overall, the neuroanatomical and phenotypical consequences detected upon the loss of Mest, resemble the loss of SNc neurons and loss of movement control as seen in Parkinson’s Disease (PD), suggesting that the Mest mouse model may be used as a model-system for PD.

Heterogeneity of the midbrain dopamine system: Implications for Parkinson disease.

The midbrain dopaminergic (DA) system is composed primarily of neurons in the substantia nigra pars compacta (SNc, area A10) and ventral tegmental area (VTA, area A9). These include at least 5 neuron subpopulations with distinct patterns of gene expression, electrophysiologic properties, projections to the striatum and cerebral cortex, and response to environmental stimuli. They are involved in several key brain functions, including action selection, motor performance, motivation and reward-based learning, working memory, and cognition. Midbrain DA neurons are activated by alerting signals involved in rapid detection of potentially relevant sensory cues. Most increase their activity in response to reward stimuli and are inhibited by aversive stimuli, thereby coding motivational value for learning reward-oriented behavior. Others are activated by aversive stimuli and code motivational salience of the stimulus. These different types of midbrain DA neurons are anatomically segregated and have specific afferent and efferent connections supporting specific aspects of behavior. Neurons encoding motivational value are located in the VTA and medial SNc and project primarily to the nucleus accumbens (NAc) and ventromedial prefrontal cortex and support Pavlovian learning of actions leading to successful outcomes. Neurons encoding motivational salience are located in the dorsolateral SNc and project to the dorsomedial striatum (primarily the caudate) and dorsolateral prefrontal cortex and modulate goal-oriented behavior. Neurons in the ventral tier of the SNc project to the dorsolateral striatum (putamen) and regulate habit formation and automatic motor behavior. Several features of ventral tier SNc neurons render them susceptible to oxidative stress and the subsequent neurodegeneration underlying Parkinson disease (PD). Involvement of other SNc and VTA neurons may contribute to nonmotor aspects of PD, such as depression, cognitive dysfunction, and impulse dyscontrol, in response to DA agonists. All these topics have been extensively discussed in several reviews.1–18

Transplantation of human mesenchymal stem cells into the cisterna magna and its neuroprotective effects in a parkinsonian animal model

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases in the elderly, and therefore, the demand for effective therapies against PD has greatly increased. Therapeutic applications of stem cells have been considered to be one of the promising approaches in PD therapy. In the present study, the neuroprotective effects of hMSCs were evaluated in a parkinsonian rat model. The animal model was established by injecting 6-hydroxydopamine into the striatum of rats. Two weeks later, cultured hMSCs were transplanted into the cisterna magna. We subsequently identified changes in the expression of inflammatory cytokines, neurotrophic factors, microglial activation, and the survival rate of dopaminergic neurons in SNc. Behavioral recovery was also examined. The results indicated that hMSC transplantation increased the expression of anti-inflammatory cytokines as well as neurotrophic factors, and decreased the number of activated microglia. Compared to the sham-grafted group, relatively large numbers of TH-positive neurons were found in the ipsilateral SNc, and amphetamine-induced asymmetrical rotation was significantly reduced after hMSC transplantation. These findings suggest that hMSCs might be neuroprotective, probably through up-regulation of neurotrophic factors and anti-inflammatory cytokines, and this could have a functional impact in reversing PD symptoms.