Balance between cholinergic and dopaminergic signaling is central to striatal control of movement and cognition. In dystonia, a common disorder of movement, anticholinergic therapy is often beneficial. This observation suggests there is a pathological increase in cholinergic tone, yet direct confirmation is lacking. In DYT1, an early-onset genetic form of dystonia caused by a mutation in the protein torsinA (TorA), the suspected heightened cholinergic tone is commonly attributed to faulty dopamine D2 receptor (D2R) signaling where D2R agonists cause excitation of striatal cholinergic interneurons (ChIs), rather than the normal inhibition of firing observed in wild-type animals, an effect known as “paradoxical excitation”. Here, we provide for the first time direct measurement of elevated striatal extracellular acetylcholine (ACh) in a knock-in mouse model of human DYT1 dystonia (TorAΔE/+ mice), confirming a striatal hypercholinergic state. We hypothesized that this elevated extracellular ACh might cause chronic over-activation of muscarinic acetylcholine receptors (mAChRs) and disrupt normal D2R function due to their shared coupling to Gi/o-proteins. We tested this concept in vitro first using a broad-spectrum mAChR antagonist, and then using a M2/M4 mAChR selective antagonist to specifically target mAChRs expressed by ChIs. Remarkably, we found that mAChR inhibition reverses the D2R–mediated paradoxical excitation of ChIs recorded in slices from TorAΔE/+ mice to a typical inhibitory response. Furthermore, we recapitulated the paradoxical D2R excitation of ChIs in striatal slices from wild type mice within minutes by simply increasing cholinergic tone through pharmacological inhibition of acetylcholinesterase (AChE) or by prolonged agonist activation of mAChRs. Collectively, these results show that enhanced mAChR tone itself is sufficient to rapidly reverse the polarity of D2R regulation of ChI excitability, correcting the previous notion that the D2R mediated paradoxical ChI excitation causes the hypercholinergic state in dystonia. Further, using a combination of genetic and pharmacological approaches, we found evidence that this switch in D2R polarity results from a change in coupling from the preferred Gi/o pathway to non-canonical β-arrestin signaling. These results highlight the need to fully understand how the mutation in TorA leads to pathologically heightened extracellular ACh. Furthermore the discovery of this novel ACh-dopamine interaction and the participation of β-arrestin in regulation of cholinergic interneurons is likely important for other basal ganglia disorders characterized by perturbation of ACh-dopamine balance, including Parkinson and Huntington diseases, l-DOPA-induced dyskinesia and schizophrenia.
DYT1 dystonia is an early-onset, hyperkinetic movement disorder caused by a deletion in the gene TOR1A, which encodes the protein torsinA. Several lines of evidence show that in animal models of DTY1 dystonia, there is impaired basal dopamine (DA) release and enhanced acetylcholine tone. Clinically, anticholinergic drugs are the most effective pharmacological treatment for DYT1 dystonia, but the currently used agents are non-selective muscarinic antagonists and associated with side effects. We used a DYT1 ∆GAG knock-in mouse model (DYT1 KI) to investigate whether nicotine and/or a non-desensitizing nicotinic agonist, AZD1446, would increase DA output in DYT1 dystonia. Using in vivo microdialysis, we found that DYT1 KI mice showed significantly increased DA output and greater sensitivity to nicotine compared to wild type (WT) littermate controls. In contrast, neither systemic injection (0.25–0.75 mg/kg) or intrastriatal infusion (30 μM–1 mM) of AZD1446 had a significant effect on DA efflux in WT or DYT1 KI mice. In vitro, we found that AZD1446 had no effect on the membrane properties of striatal spiny projection neurons (SPNs) and did not alter the spontaneous firing of ChI interneurons in either WT or DYT1 KI mice. We did observe that the firing frequency of dopaminergic neurons was significantly increased by AZD1446 (10 μM), an effect blocked by dihydro-beta-erythroidine (DHβE 3 μM), but the effect was similar in WT and DYT1 KI mice. Our results support the view that DYT1 models are associated with abnormal striatal cholinergic transmission, and that the DYT1 KI animals have enhanced sensitivity to nicotine. We found little effect of AZD1446 in this model, suggesting that other approaches to nicotinic modulation should be explored.
Carnitine is an essential molecule for mitochondrial beta‐oxidation of long‐chain fatty acids and other cellular functions. Several rare, inherited disorders of carnitine metabolism occur in humans, and secondary carnitine deficiency is an important feature in a variety of clinical settings. Many of these conditions can be detected via quantitative analysis of free and esterified carnitine in plasma or urine, which thus offers an effective means for assessing the transport and initial processing of fatty acids. Here, we describe some of the methods most commonly employed for quantification of plasma carnitine and consider some of the advantages and disadvantages of these approaches. © 2019 by John Wiley & Sons, Inc.
Cragg SJ. Cortical control of striatal dopamine transmission via striatal cholinergic interneurons [published online ahead of print August 27, 2016]. Cereb Cortex.Striatal dopamine release is pivotal in many reward-related behaviors and basal ganglia diseases including dystonia and Parkinson's disease. Dopamine release arises from dopaminergic axons innervating the striatum, but the mechanisms regulating this release have been surprisingly difficult to unravel. Clinicians have long understood that a key factor in the regulation of movement is the dopamine-acetylcholine dynamic; this is often conceived as a "seesaw," with anticholinergic medications acting to oppose the effects of striatal dopamine depletion. Although this idea captures the clinical experience, the molecular and cellular bases of dopaminergic-cholinergic interactions have been obscure. Furthermore, the "seesaw" concept does not help to integrate the neocortex into the circuit, which clearly must have a central role in the regulation of voluntary movement.A new study from Kosillo and colleagues 1 sheds new light on these issues using optogenetic activation of channelrhodopsinexpressing corticostriatal axons or intralaminar thalmostriatal axons and measuring striatal dopamine release by fast-scan cyclic voltammetry. They find that both cortical and thalamic stimulation can drive striatal dopamine release. Both pathways are blocked by dihydro-b-erythroidine, an antagonist of nicotinic cholinergic receptors, showing that these effects are mediated by the action of acetylcholine on dopamine terminals (well known to be populated by nicotinic receptors). Interestingly, both the cortical and thalamic pathways required glutamatergic signaling but used receptors of different types: Cortex inputs stopped triggering the release of dopamine when an antagonist of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors antagonist was applied, whereas the thalamic inputs relied on not only AMPA but also a N-Methyl-D-aspartic acid (NMDA) receptor to drive dopamine release. These pathways also differ in their dynamics, with cortical inputs leading to short-latency action potentials, whereas thalamostriatal inputs produce action potentials generated during longer time scales.This important conclusion of this study is that striatal dopamine release is regulated by a disynaptic circuit: Glutamatergic axons from cortex and thalamus act on cholinergic interneurons, and these in turn stimulate nicotinic acetylcholine receptors on dopamine axons to activate the release of dopamine. This provides an answer to the long-standing puzzle of why glutamate is a potent regulator of striatal dopamine release, when few if any glutamate receptors can be found on dopamine terminals. It also explains how nicotinic signaling is essential for the transduction of cortical and thalamic signals that modulate dopamine release. The authors suggest that the key to dopamine release is likely the synchronized activation of networks of cholinergic interneurons, drive...
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