Striatal Cholinergic Interneurons in a Knock-in Mouse Model of L-DOPA-Responsive Dystonia.

Striatal cholinergic dysfunction is a common phenotype associated with various forms of dystonia in which anti-cholinergic drugs have some therapeutic benefits. However, the underlying substrate of striatal cholinergic defects in dystonia remain poorly understood. In this study, we used a recently developed knock-in mouse model of dopamine-responsive dystonia (DRD) with strong symptomatic responses to anti-cholinergic drugs, to assess changes in the prevalence and morphology of striatal cholinergic interneurons (ChIs) in a model of generalized dystonia. Unbiased stereological neuronal counts and Sholl analysis were used to address these issues. To determine the potential effect of aging on the number of ChIs, both young (3 months old) and aged (15 months old) mice were used. For purpose of comparisons with ChIs, the number of GABAergic parvalbumin (PV)-immunoreactive striatal interneurons was also quantified in young mice. Overall, no significant change in the prevalence of ChIs and PV-immunoreactive cells was found throughout various functional regions of the striatum in young DRD mice. Similar results were found for ChIs in aged animals. Subtle changes in the extent and complexity of the dendritic tree of ChIs were found in middle and caudal regions of the striatum in DRD mice. Additional immunohistochemical data also suggested lack of significant change in the expression of striatal cholinergic M1 and M4 muscarinic receptors immunoreactivity in DRD mice. Thus, together with our previous data from a knock-in mouse model of DYT-1 dystonia (Song et al., 2013), our data further suggest that the dysregulation of striatal cholinergic transmission in dystonia is not associated with major neuroplastic changes in the morphology or prevalence of striatal ChIs. Highlights -There is no significant change in the number of striatal ChIs in young and aged mice model of DRD-There is no significant change in the prevalence of striatal GABAergic PV-containing interneurons in the striatum of young mice models of DRD-Subtle morphological changes in the dendritic arborization of striatal ChIs are found in the middle and caudal tiers of the striatum in young mice models of DRD-The levels of both M1 and M4 muscarinic receptors immunoreactivity are not significantly changed in the striatum of DRD mice-Major changes in the prevalence and morphology of striatal ChIs are unlikely to underlie striatal cholinergic dysfunction in DRD.

Engineered D2R Variants Reveal the Balanced and Biased Contributions of G-Protein and ß-Arrestin to Dopamine-Dependent Functions.

The dopamine D2 receptor (D2R), like many G-protein-coupled receptors, signals through G-protein- and ß-arrestin-dependent pathways. Preferential activation of one of these pathways is termed functional selectivity or biased signaling and is a promising therapeutic strategy. Though biased signaling through D2Rs has been demonstrated, acquiring the mechanistic details of biased D2R/G-protein and D2R/ß-arrestin signaling in vivo has been challenging because of the lack of techniques that specifically target these interactions in discrete cell populations. To address this question, we employed a cell type-specific viral expression approach to restore D2R variants that preferentially engage either G-protein or ß-arrestin signaling in 'indirect pathway' medium spiny neurons (iMSNs), because of their central role in dopamine circuitry. We found that the effect of haloperidol antagonism on D2R metabolic signaling events is largely mediated by acute blockade of D2R/G-protein signaling. We show that a D2R-driven behavior, nestlet shredding, is similarly driven by D2R/G-protein signaling. On the other hand, D2R-driven locomotion and rearing require coordinated D2R/G-protein and D2R/ß-arrestin signaling. The acute locomotor response to amphetamine and cocaine similarly depend on both G-protein and ß-arrestin D2R signaling. Surprisingly, another psychotropic drug, phencyclidine, displayed a selective D2R/ß-arrestin potentiation of locomotion. These findings highlight how D2R mostly relies upon balanced G-protein and ß-arrestin signaling in iMSNs. However, the response to haloperidol and phencyclidine indicates that normal D2R signaling homeostasis can be dramatically altered, indicating that targeting a specific D2R signal transduction pathway could allow for more precise modulation of dopamine circuit function.

Parkinsonism without dopamine neuron degeneration in aged l-dopa-responsive dystonia knockin mice.

BACKGROUND: Recent neuroimaging studies implicate nigrostriatal degeneration as a critical factor in producing late-onset parkinsonism in patients with l-dopa-responsive dystonia-causing mutations. However, postmortem anatomical studies do not reveal neurodegeneration in l-dopa-responsive dystonia patients. These contrasting findings make it unclear how parkinsonism develops in l-dopa-responsive dystonia mutation carriers. METHODS: We prospectively assessed motor dysfunction, responses to dopaminergic challenge, and dopamine neuron degeneration with aging in a validated knockin mouse model bearing a l-dopa-responsive dystonia-causing mutation found in humans. RESULTS: As l-dopa-responsive dystonia mice aged, dystonic movements waned while locomotor activity decreased and initiation of movements slowed. Despite the age-related reduction in movement, there was no evidence for degeneration of midbrain dopamine neurons. Presynaptically mediated dopaminergic responses did not change with age in l-dopa-responsive dystonia mice, but responses to D1 dopamine receptor agonists decreased with age. CONCLUSIONS: We have demonstrated for the first time the co-occurrence of dystonia and Parkinson's-like features (mainly consisting of hypokinesia) in a genetic mouse model. In this model we show that these features evolve without dopaminergic neurodegeneration, suggesting that postsynaptic plasticity, rather than presynaptic degeneration, may contribute to the development of parkinsonism in patients with l-dopa-responsive dystonia. © 2017 International Parkinson and Movement Disorder Society.

A commentary on the utility of a new L-DOPA-responsive dystonia mouse model.

In a recent issue of Brain, we reported on the generation and characterization of a mouse model of the rare disease L-DOPA-responsive dystonia (DRD). Here, we discuss the utility of these mice for understanding broader disease processes and treatment strategies. Using specific experimental designs that either work "forward" from genetic etiology or "backward" from the symptomatic presentation, we discuss how our data and future work can be used to understand broader themes.

A new knock-in mouse model of l-DOPA-responsive dystonia.

Abnormal dopamine neurotransmission is associated with many different genetic and acquired dystonic disorders. For instance, mutations in genes critical for the synthesis of dopamine, including GCH1 and TH cause l-DOPA-responsive dystonia. Despite evidence that implicates abnormal dopamine neurotransmission in dystonia, the precise nature of the pre- and postsynaptic defects that result in dystonia are not known. To better understand these defects, we generated a knock-in mouse model of l-DOPA-responsive dystonia (DRD) mice that recapitulates the human p.381Q>K TH mutation (c.1141C>A). Mice homozygous for this mutation displayed the core features of the human disorder, including reduced TH activity, dystonia that worsened throughout the course of the active phase, and improvement in the dystonia in response to both l-DOPA and trihexyphenidyl. Although the gross anatomy of the nigrostriatal dopaminergic neurons was normal in DRD mice, the microstructure of striatal synapses was affected whereby the ratio of axo-spinous to axo-dendritic corticostriatal synaptic contacts was reduced. Microinjection of l-DOPA directly into the striatum ameliorated the dystonic movements but cerebellar microinjections of l-DOPA had no effect. Surprisingly, the striatal dopamine concentration was reduced to ∼1% of normal, a concentration more typically associated with akinesia, suggesting that (mal)adaptive postsynaptic responses may also play a role in the development of dystonia. Administration of D1- or D2-like dopamine receptor agonists to enhance dopamine signalling reduced the dystonic movements, whereas administration of D1- or D2-like dopamine receptor antagonists to further reduce dopamine signalling worsened the dystonia, suggesting that both receptors mediate the abnormal movements. Further, D1-dopamine receptors were supersensitive; adenylate cyclase activity, locomotor activity and stereotypy were exaggerated in DRD mice in response to the D1-dopamine receptor agonist SKF 81297. D2-dopamine receptors exhibited a change in the valence in DRD mice with an increase in adenylate cyclase activity and blunted behavioural responses after challenge with the D2-dopamine receptor agonist quinpirole. Together, our findings suggest that the development of dystonia may depend on a reduction in dopamine in combination with specific abnormal receptor responses.

Delaying the onset of treadmill exercise following peripheral nerve injury has different effects on axon regeneration and motoneuron synaptic plasticity.

Transection of a peripheral nerve results in withdrawal of synapses from motoneurons. Some of the withdrawn synapses are restored spontaneously, but those containing the vesicular glutamate transporter 1 (VGLUT1), and arising mainly from primary afferent neurons, are withdrawn permanently. If animals are exercised immediately after nerve injury, regeneration of the damaged axons is enhanced and no withdrawal of synapses from injured motoneurons can be detected. We investigated whether delaying the onset of exercise until after synapse withdrawal had occurred would yield similar results. In Lewis rats, the right sciatic nerve was cut and repaired. Reinnervation of the soleus muscle was monitored until a direct muscle (M) response was observed to stimulation of the tibial nerve. At that time, rats began 2 wk of daily treadmill exercise using an interval training protocol. Both M responses and electrically-evoked H reflexes were monitored weekly for an additional seven wk. Contacts made by structures containing VGLUT1 or glutamic acid decarboxylase (GAD67) with motoneurons were studied from confocal images of retrogradely labeled cells. Timing of full muscle reinnervation was similar in both delayed and immediately exercised rats. H reflex amplitude in delayed exercised rats was only half that found in immediately exercised animals. Unlike immediately exercised animals, motoneuron contacts containing VGLUT1 in delayed exercised rats were reduced significantly, relative to intact rats. The therapeutic window for application of exercise as a treatment to promote restoration of synaptic inputs onto motoneurons following peripheral nerve injury is different from that for promoting axon regeneration in the periphery.

The first knockin mouse model of episodic ataxia type 2.

Episodic ataxia type 2 (EA2) is an autosomal dominant disorder associated with attacks of ataxia that are typically precipitated by stress, ethanol, caffeine or exercise. EA2 is caused by loss-of-function mutations in the CACNA1A gene, which encodes the α1A subunit of the CaV2.1 voltage-gated Ca(2+) channel. To better understand the pathomechanisms of this disorder in vivo, we created the first genetic animal model of EA2 by engineering a mouse line carrying the EA2-causing c.4486T>G (p.F1406C) missense mutation in the orthologous mouse Cacna1a gene. Mice homozygous for the mutated allele exhibit a ~70% reduction in CaV2.1 current density in Purkinje cells, though surprisingly do not exhibit an overt motor phenotype. Mice hemizygous for the knockin allele (EA2/- mice) did exhibit motor dysfunction measurable by rotarod and pole test. Studies using Cre-flox conditional genetics explored the role of cerebellar Purkinje cells or cerebellar granule cells in the poor motor performance of EA2/- mice and demonstrate that manipulation of either cell type alone did not cause poor motor performance. Thus, it is possible that subtle dysfunction arising from multiple cell types is necessary for the expression of certain ataxia syndromes.

Serotonin 2A receptors differentially contribute to abuse-related effects of cocaine and cocaine-induced nigrostriatal and mesolimbic dopamine overflow in nonhuman primates.

Two of the most commonly used procedures to study the abuse-related effects of drugs in laboratory animals are intravenous drug self-administration and reinstatement of extinguished behavior previously maintained by drug delivery. Intravenous self-administration is widely accepted to model ongoing drug-taking behavior, whereas reinstatement procedures are accepted to model relapse to drug taking following abstinence. Previous studies indicate that 5-HT2A receptor antagonists attenuate the reinstatement of cocaine-maintained behavior but not cocaine self-administration in rodents. Although the abuse-related effects of cocaine have been closely linked to brain dopamine systems, no previous study has determined whether this dissociation is related to differential regulation of dopamine neurotransmission. To elucidate the neuropharmacological and neuroanatomical mechanisms underlying this phenomenon, we evaluated the effects of the selective 5-HT2A receptor antagonist M100907 on intravenous cocaine self-administration and drug- and cue-primed reinstatement in rhesus macaques (Macaca mulatta). In separate subjects, we evaluated the role of 5-HT2A receptors in cocaine-induced dopamine overflow in the nucleus accumbens (n = 4) and the caudate nucleus (n = 5) using in vivo microdialysis. Consistent with previous studies, M100907 (0.3 mg/kg, i.m.) significantly attenuated drug- and cue-induced reinstatement but had no significant effects on cocaine self-administration across a range of maintenance doses. Importantly, M100907 (0.3 mg/kg, i.m.) attenuated cocaine-induced (1.0 mg/kg, i.v.) dopamine overflow in the caudate nucleus but not in the nucleus accumbens. These data suggest that important abuse-related effects of cocaine are mediated by distinct striatal dopamine projection pathways.

Effects of treadmill training on functional recovery following peripheral nerve injury in rats.

Exercise, in the form of moderate daily treadmill training following nerve transection and repair leads to enhanced axon regeneration, but its effect on functional recovery is less well known. Female rats were exercised by walking continuously, at a slow speed (10 m/min), for 1 h/day on a level treadmill, beginning 3 days after unilateral transection and surgical repair of the sciatic nerve, and conducted 5 days/wk for 2 wk. In Trained rats, both direct muscle responses to tibial nerve stimulation and H reflexes in soleus reappeared earlier and increased in amplitude more rapidly over time than in Untrained rats. The efficacy of the restored H reflex was greater in Trained rats than in Untrained controls. The reinnervated tibialis anterior and soleus were coactivated during treadmill locomotion in Untrained rats. In Trained animals, the pattern of activation of soleus, but not tibialis anterior, was not significantly different from that found in Intact rats. The overall length of the hindlimb during level and up- and downslope locomotion was conserved after nerve injury in both groups. This conservation was achieved by changes in limb orientation. Limb length was conserved effectively in all rats during downslope walking but only in Trained rats during level and upslope walking. Moderate daily exercise applied immediately after sciatic nerve transection is sufficient to promote axon regeneration, to restore muscle reflexes, and to improve the ability of rats to cope with different biomechanical demands of slope walking.