Dopamine in Motor Cortex Is Necessary for Skill Learning and Synaptic Plasticity

Preliminary evidence indicates that dopamine given by mouth facilitates the learning of motor skills and improves the recovery of movement after stroke. The mechanism of these phenomena is unknown. Here, we describe a mechanism by demonstrating in rat that dopaminergic terminals and receptors in primary motor cortex (M1) enable motor skill learning and enhance M1 synaptic plasticity. Elimination of dopaminergic terminals in M1 specifically impaired motor skill acquisition, which was restored upon DA substitution. Execution of a previously acquired skill was unaffected. Reversible blockade of M1 D1 and D2 receptors temporarily impaired skill acquisition but not execution, and reduced long-term potentiation (LTP) within M1, a form of synaptic plasticity critically involved in skill learning. These findings identify a behavioral and functional role of dopaminergic signaling in M1. DA in M1 optimizes the learning of a novel motor skill

Motor skill learning depends on protein synthesis in the dorsal striatum after training

Functional imaging studies in humans and electrophysiological data in animals suggest that corticostriatal circuits undergo plastic modifications during motor skill learning. In motor cortex and hippocampus circuit plasticity can be prevented by protein synthesis inhibition (PSI) which can interfere with certain forms learning. Here, the hypothesis was tested that inducing PSI in the dorsal striatum by bilateral intrastriatal injection of anisomycin (ANI) in rats interferes with learning a precision forelimb reaching task. Injecting ANI shortly after training on days 1 and 2 during 4days of daily practice (n=14) led to a significant impairment of motor skill learning as compared with vehicle-injected controls (n=15, P=0.033). ANI did not affect the animals' motivation as measured by intertrial latencies. Also, ANI did not affect reaching performance once learning was completed and performance reached a plateau. These findings demonstrate that PSI in the dorsal striatum after training impairs the acquisition of a novel motor skill. The results support the notion that plasticity in basal ganglia circuits, mediated by protein synthesis, contributes to motor skill learnin

Alterungsprozess von cerebellaren Körner-Neuronen in vitro

Aging is slow and cumulative process and is experimentally difficult to access. Neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease are considered to be at high risk with increasing age. The molecular mechanisms by which they occur are not completely understood. Aim of the present study was to construct and investigate an in vitro model of neuronal aging. To this purpose we used rat primary cerebellar granule neurons which are a good characterized in vitro model for various paradigms of neuronal cell death, e.g. potassium deprivation or glutamate excitotoxicity. We prepared CGN to a purity of 95% and kept them in culture for either 7 days or up to 60 days. To validate our in vitro model for aging, we tested whether known molecular changes associated with aging occur over time. Moreover, by using DNA microarray analysis we examined how aging in vitro influences the pattern of gene expression. In the end, we studied mechanisms of cell death signaling cascades in young vs. old CGN. Old CGN had increased protein carbonylation and ubiquitination. Proteasomal activity was significantly lower in old CGN even though protein expression of proteasomal subunits did not change over the time. Morphological changes in astrocytes included increase in size, accumulation of autofluorescent, undegradable pigment lipofuscine, and senescence-associated beta -galactosidase activity. Aging in vitro caused increased expression of genes involved in stress response. Down-regulated genes were mostly responsible for control of synaptic function. Young neurons favored apoptosis over necrosis in contrast to old neurons. DIV 50 neurons were much less vulnerable as compared to DIV 7 neurons to classical apoptotic paradigms such as potassium deprivation, or staurosporine treatment. Old CGN had up-regulated anti-apoptotic proteins Bcl-xL and lifeguard, and down-regulated caspase-3 and Bax. On the other hand, old neurons exhibited enhanced sensitivity to glutamate excitotoxicity. Aging in vitro caused ATP depletion, failure of intracellular Ca2+ homeostasis, increase in calpain activity, and changes in ERK signaling. Increased calpain activity caused cleavage of calcium pump NCX3 and blocked pumping excessive Ca2+ outside of the cell. This made old neurons extremely sensitive glutamate insult. When calpains were inhibited over longer period of time by regular application of calpeptin, sensitivity to excitotoxic stimulus was significantly reduced. These data show that cerebellar granule neurons can successfully be cultured over period of 60 days in vitro and may represent a valuable tool for modeling aging and neurodegenerative diseases.Der Alterungsprozess ist ein langsamer und kumulativer Vorgang, der experimentell nur schwer erfassen ist. Neurodegenerative Erkrankungen wie Alzheimer oder Parkinson sind Erkrankungen, die im Alter eintreten können. Der molekulare Mechanismus dieser Erkrankungen ist nicht vollständig aufgeklärt. Das Ziel dieser Studie war es ein in-vitro Modell zu entwickeln und daran den Alterungsprozess auf neuronaler Ebene zu untersuchen. Dazu wurden primäre zerebellare Körnerzellen (CGN = cerebellar granule neurons) der Ratte verwendet. Diese haben sich als sehr gute in-vitro Modelle für diverse Variationen von neuronalem Zelltod, wie z.B. Kalium-Deprivation oder Glutamat-Exotoxizität erwiesen. Wir reinigten die CGN zu 95% auf und kultivierten die aufgereinigten Zellen im Nährmedium zwischen 7 und 60 Tage. Zur Validierung unseres in-vitro Alterungsmodels führten wir für den Alterungsprozess evaluierte molekulare Testreihen durch. Darüber hinaus untersuchten wir mit DNA microarray Analyse in wieweit in-vitro-Alterung der Zellen die Genexpression beeinflusst bzw. verändert. Zudem untersuchten wir die Mechanismen der Zelltod-Signalkaskade in jungen CGN im Vergleich zu gealterten CGN. Alte CGN haben eine erhöhte Protein-Karbonylierung und -Ubiquitinierung. Die proteasomale Aktivität war signifikant niedriger in alten CGN obwohl die Proteinexpression der proteasomalen Untereinheiten sich über die Zeit nicht veränderte. Morphologische Veränderungen in Astrozyten schlossen Vergrößerung, Akkumulation der Autofluoreszenz, nicht abbaubare Lipofuscin Pigment, und mit Vergreisung assoziierte Beta-Galaktosidase Aktivität ein. Der in vitro Alterungsprozess verursachte eine Expression von Genen die erwiesenermaßen in der Stressantwort eine Rolle spielen. Wohingegen die Expression von Genen, die meist für die Kontrolle der synaptischen Funktion verantwortlich sind, herabgesetzt waren. Junge Neurone starben im Gegensatz zu den gealterten Zellen eher durch Apoptose als durch Nekrose. DIV 50 Neurone waren im Vergleich zu DIV 7 Neurone weniger anfällig für klassische Apoptose-Arten wie Kaliumdeprivation oder Gabe von Staurosporinen. Alte CGN hatten einen erhöhten Spiegel der anti-apoptotischen Proteine Bcl-xl und Lifeguard, und einen abgesetzten Spiegel der Apoptose-induzierenden Protease Caspase-3 und dem Protein Bax. Andererseits zeigten die alten Neurone eine erhöhte Erregbarkeit durch Glutamat. Der in-vitro Alterungsprozess verursachte zudem eine ATP-Verarmung, eine Versagen der intrazellulären Ca2+-Homöostase, eine Erhöhung der Calpain-Aktivität und Veränderungen in ERK-regulierten Signalkaskaden.Die Erhöhung der Calpain-Aktivität verursachte eine Spaltung der Kalziumpumpe NCX3 und blockierte damit die übermäßige Abgabe von Ca2+ nach außen. Dies führte dazu, dass gealterte Neuronen extrem sensitiv für auf Glutamat-Insulte waren. Wenn Calpain über eine längere Zeit durch die Gabe von Calpeptin inhibiert wurde, war die Sensitivität auf exozytotische Stimuli herabgesetzt. Diese Daten zeigen, dass zerebellare Körnerzellen erfolgreich über eine Zeitdauer von 60 Tagen in vitro kultiviert werden können und daher ein nützliches Modell für den Alterungsprozess und neurodegenerative Krankheiten darstellt

Data from: Dopamine promotes motor cortex plasticity and motor skill learning via PLC activation

Dopaminergic neurons in the ventral tegmental area, the major midbrain nucleus projecting to the motor cortex, play a key role in motor skill learning and motor cortex synaptic plasticity. Dopamine D1 and D2 receptor antagonists exert parallel effects in the motor system: they impair motor skill learning and reduce long-term potentiation. Traditionally, D1 and D2 receptor modulate adenylyl cyclase activity and cyclic adenosine monophosphate accumulation in opposite directions via different G-proteins and bidirectionally modulate protein kinase A (PKA), leading to distinct physiological and behavioral effects. Here we show that D1 and D2 receptor activity influences motor skill acquisition and long term synaptic potentiation via phospholipase C (PLC) activation in rat primary motor cortex. Learning a new forelimb reaching task is severely impaired in the presence of PLC, but not PKA-inhibitor. Similarly, long term potentiation in motor cortex, a mechanism involved in motor skill learning, is reduced when PLC is inhibited but remains unaffected by the PKA inhibitor. Skill learning deficits and reduced synaptic plasticity caused by dopamine antagonists are prevented by co-administration of a PLC agonist. These results provide evidence for a role of intracellular PLC signaling in motor skill learning and associated cortical synaptic plasticity, challenging the traditional view of bidirectional modulation of PKA by D1 and D2 receptors. These findings reveal a novel and important action of dopamine in motor cortex that might be a future target for selective therapeutic interventions to support learning and recovery of movement resulting from injury and disease

PLC activation prevents DA antagonist-induced learning deficits.

<p>(a) Co-administration of the PLC agonist m-3M3FBS and D1 receptor antagonist SCH 23390 (red) prevents SCH 23390 (orange) induced deficit in the success rate (left) compared to vehicle injected control (black). Intertrial latencies remained unaffected regardless of drug or drug combination injected (right). (b) D2 receptor antagonist raclopride (yellow) impaired skill acquisition. This impairment was prevented by PLC agonist co-administration (red). PLC agonist alone (green) had no effect on learning as compared to controls (black). Intertrial latencies remain unaffected by all treatments (right). Error bars indicate SEM. Arrows indicate days of local injections.</p

Activation of intracellular PLC pathway rescues the LTP impairment caused by DA receptor block.

<p>(a) Maximum synaptic strength of horizontal layer II/III connections, measured by saturating LTP with multiple attempts of TBS (arrows) in raclopride (D2antag) co-administered with m-3m3fbs (PLCactivator, red) resulted in normal amounts of LTP (left). m-3m3fbs alone did not affect LTP saturation (green). The histogram (right) illustrates LTP saturation for control condition (grey), PLC agonist (green), PLC antagonist (blue), D2 antagonist (yellow). D1 and D2 antagonist co-applied with the PLC agonist (red) illustrates the rescue of D1 and D2 block. (b) FP amplitudes were not affected by co-administration of D2 antagonist and PLC agonist or by PLC agonist alone (left). The histogram (right) illustrates group data of synaptic strength for all the different conditions (color code as in a). Values are calculated relative to the baseline recordings before drug application.</p

Experimental design.

<p>(a) Timeline for experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124986#pone.0124986.g002" target="_blank">Fig 2A</a> using continuous drug release via minipump (red bar) during motor skill training. (b) Timeline for experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124986#pone.0124986.g002" target="_blank">Fig 2B</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124986#pone.0124986.g003" target="_blank">Fig 3A and 3B</a> using acute drug injections on day 2 and 3 of motor skill training (red arrows). (c) Timeline for experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124986#pone.0124986.g002" target="_blank">Fig 2</a> using continuous drug release via minipump (red bar) after successful acquisition of the motor task.</p

PLC inhibitor impairs motor skill acquisition and the potential for M1 synaptic plasticity.

<p>(a) Motor skill acquisition was assessed in rats that received continuous intracortical injection (grey bar) of PLC inhibitor U-73122 (blue), PKA inhibitor H-89 (green) and inactive PLC inhibitor U-73343 (black) into the M1 forelimb area via osmotic minipumps. The success rate and the latency, a measure of motivation, were impaired in the presence of PLC but not PKA inhibitor. Error bars indicate SEM. (b) Temporary application of PLC and PKA inhibitors directly to M1 on training day 2 & 3 (time of steepest learning) reveal the same effect as with continuous application. Arrows indicate local injection days. The inset illustrates the rate of learning from day 1–2, day 3–4, and day 4–5 indicating a lack of improvement from day 2–3. (c) Maximum synaptic strength (LTP saturation) by repeated induction of LTP (multiple arrows) in layer II/III horizontal connections in brain slices. Peak amplitudes were assessed in the M1 forelimb area in ACSF (dark blue) and in the presence of PLC inhibitor U-73122 (light blue). Each FP trace (insets) represents an average of 10 individual responses at times indicated by numbers. (d) Maximum LTP in the presence of PKA inhibitor is not significantly different from control LTP. Arrows indicate multipe LTP attempts. (e) Summary histogram of LTP. Grey: Control; green: PKA inhibition; blue: PLC inhibition.</p

Identification of dopaminergic terminals in M1.

<p>(a) Western blot analysis of M1 cortical tissue injected with vehicle (sham-lesioned) and 6-OHDA in conjunction with desipramine (i.p.) using tyroxine hydroxylase (TH) reactivity indicated reduced TH expression after elimination of dopaminergic terminals. (b) Quantification of protein expression in DA-lesioned (6-OHDA+D) and sham-lesioned hemispheres reveals reduced protein expression after elimination of dopaminergic terminals. (c) Immunofluorescence staining of cortical dopaminergic terminals (TH immunoreactivity) in an exemplary vehicle and DA-lesioned hemisphere (6-OHDA injections into M1) indicated almost no staining in layer I and II/III and reduced staining in deeper layers in the lesioned M1. Similar findings were obtained in the other two animals treated analogously.</p

Functional D1 and D2 receptors in M1 are necessary for optimal motor skill acquisition but not for movement execution.

<p>(a) Blocking D1 receptors with SCH02339 (green) and D2 receptors with raclopride (blue) or sulpiride (orange) on the second and third day (arrows) of motor skill training significantly impaired reaching success compared to vehicle injected animals (black). When antagonists were discontinued, success rate began to increase normally. No significant differences in success rate existed at day 8 between all 4 groups. Inset: exemplary Nissl stain to verify cannula placement. (b) Raclopride injected into M1 (arrows) after the task had been acquired did not affect the performance. Inset: exemplary Nissl stain to verify injection cannula placement. (c,d) To exclude the possibility that the antagonists spread to other brain regions receiving important DA projections thereby causing the observed learning impairment, raclopride was injected into the dorsal striatum (c, blue) and the prefrontal cortex (d, blue) and compared to vehicle injected controls (black). Skill acquisition was not impaired in these animals. Insets: exemplary Nissl stain to verify cannula placement.</p