Giulia Ponterio

Centrality of striatal cholinergic transmission in Basal Ganglia function

Work over the past two decades revealed a previously unexpected role for striatal cholinergic interneurons in the context of basal ganglia function. The recognition that these interneurons are essential in synaptic plasticity and motor learning represents a significant step ahead in deciphering how the striatum processes cortical inputs, and why pathological circumstances cause motor dysfunction. Loss of the reciprocal modulation between dopaminergic inputs and the intrinsic cholinergic innervation within the striatum appears to be the trigger for pathophysiological changes occurring in basal ganglia disorders. Accordingly, there is now compelling evidence showing profound changes in cholinergic markers in these disorders, in particular Parkinsons disease and dystonia. Based on converging experimental and clinical evidence, we provide an overview of the role of striatal cholinergic transmission in physiological and pathological conditions, in the context of the pathogenesis of movement disorders.

Cholinergic dysfunction alters synaptic integration between thalamostriatal and corticostriatal inputs in DYT1 dystonia

Projections from thalamic intralaminar nuclei convey sensory signals to striatal cholinergic interneurons. These neurons respond with a pause in their pacemaking activity, enabling synaptic integration with cortical inputs to medium spiny neurons (MSNs), thus playing a crucial role in motor function. In mice with the DYT1 dystonia mutation, stimulation of thalamostriatal axons, mimicking a response to salient events, evoked a shortened pause and triggered an abnormal spiking activity in interneurons. This altered pattern caused a significant rearrangement of the temporal sequence of synaptic activity mediated by M1 and M2 muscarinic receptors in MSNs, consisting of an increase in postsynaptic currents and a decrease of presynaptic inhibition, respectively. Consistent with a major role of acetylcholine, either lowering cholinergic tone or antagonizing postsynaptic M1 muscarinic receptors normalized synaptic activity. Our data demonstrate an abnormal time window for synaptic integration between thalamostriatal and corticostriatal inputs, which might alter the action selection process, thereby predisposing DYT1 gene mutation carriers to develop dystonic movements.

Developmental Profile of the Aberrant Dopamine D2 Receptor Response in Striatal Cholinergic Interneurons in DYT1 Dystonia

Background DYT1 dystonia, a severe form of genetically determined human dystonia, exhibits reduced penetrance among carriers and begins usually during adolescence. The reasons for such age dependence and variability remain unclear. Methods and Results We characterized the alterations in D2 dopamine receptor (D2R) signalling in striatal cholinergic interneurons at different ages in mice overexpressing human mutant torsinA (hMT). An abnormal excitatory response to the D2R agonist quinpirole was recorded at postnatal day 14, consisting of a membrane depolarization coupled to an increase in spiking frequency, and persisted unchanged at 3 and 9 months in hMT mice, compared to mice expressing wild-type human torsinA and non-transgenic mice. This response was blocked by the D2R antagonist sulpiride and depended upon G-proteins, as it was prevented by intrapipette GDP-β-S. Patch-clamp recordings from dissociated interneurons revealed a significant increase in the Cav2.2-mediated current fraction at all ages examined. Consistently, chelation of intracellular calcium abolished the paradoxical response to quinpirole. Finally, no gross morphological changes were observed during development. Conclusions These results suggest that an imbalanced striatal dopaminergic/cholinergic signaling occurs early in DYT1 dystonia and persists along development, representing a susceptibility factor for symptom generation.

Anticholinergic drugs rescue synaptic plasticity in DYT1 dystonia: Role of M1 muscarinic receptors

Broad‐spectrum muscarinic receptor antagonists have represented the first available treatment for different movement disorders such as dystonia. However, the specificity of these drugs and their mechanism of action is not entirely clear. We performed a systematic analysis of the effects of anticholinergic drugs on short‐ and long‐term plasticity recorded from striatal medium spiny neurons from DYT1 dystonia knock‐in (Tor1a+/Δgag) mice heterozygous for ΔE‐torsinA and their controls (Tor1a+/+ mice). Antagonists were chosen that had previously been proposed to be selective for muscarinic receptor subtypes and included pirenzepine, trihexyphenydil, biperiden, orphenadrine, and a novel selective M1 antagonist, VU0255035. Tor1a+/Δgag mice exhibited a significant impairment of corticostriatal synaptic plasticity. Anticholinergics had no significant effects on intrinsic membrane properties and on short‐term plasticity of striatal neurons. However, they exhibited a differential ability to restore the corticostriatal plasticity deficits. A complete rescue of both long‐term depression (LTD) and synaptic depotentiation (SD) was obtained by applying the M1‐preferring antagonists pirenzepine and trihexyphenidyl as well as VU0255035. Conversely, the nonselective antagonist orphenadrine produced only a partial rescue of synaptic plasticity, whereas biperiden and ethopropazine failed to restore plasticity. The selectivity for M1 receptors was further demonstrated by their ability to counteract the M1‐dependent potentiation of N‐methyl‐d‐aspartate (NMDA) current recorded from striatal neurons. Our study demonstrates that selective M1 muscarinic receptor antagonism offsets synaptic plasticity deficits in the striatum of mice with the DYT1 dystonia mutation, providing a potential mechanistic rationale for the development of improved antimuscarinic therapies for this movement disorder.

Regional specificity of synaptic plasticity deficits in a knock-in mouse model of DYT1 dystonia

DYT1 dystonia is a movement disorder caused by a deletion in the C-terminal of the protein torsinA. It is unclear how torsinA mutation might disrupt cellular processes encoding motor activity, and whether this impairment occurs in specific brain regions. Here, we report a selective impairment of corticostriatal synaptic plasticity in knock-in mice heterozygous for Δ-torsinA (Tor1a(+/Δgag) mice) as compared to controls (Tor1a(+/+) mice). In striatal spiny neurons from Tor1a(+/Δgag) mice, high-frequency stimulation failed to induce long-term depression (LTD), whereas long-term potentiation (LTP) exhibited increased amplitude. Of interest, blockade of D2 dopamine receptors (D2Rs) increased LTP in Tor1a(+/+) mice to a level comparable to that measured in Tor1a(+/Δgag) mice and normalized the levels of potentiation across mouse groups. A low-frequency stimulation (LFS) protocol was unable to depotentiate corticostriatal synapses in Tor1a(+/Δgag) mice. Muscarinic M1 acetylcholine receptor (mAChR) blockade rescued plasticity deficits. Additionally, we found an abnormal responsiveness of cholinergic interneurons to D2R activation, consisting in an excitatory response rather than the expected inhibition, further confirming an imbalance between dopaminergic and cholinergic signaling in the striatum. Conversely, synaptic activity and plasticity in the CA1 hippocampal region were unaltered in Tor1a(+/Δgag) mice. Importantly, the M1 mAChR-dependent enhancement of hippocampal LTP was unaffected in both genotypes. Similarly, both basic properties of dopaminergic nigral neurons and their responses to D2R activation were normal. These results provide evidence for a regional specificity of the electrophysiological abnormalities observed and demonstrate the reproducibility of such alterations in distinct models of DYT1 dystonia.

Activation of 5-HT6 receptors inhibits corticostriatal glutamatergic transmission

We investigated the effect of 5-HT6 receptor subtype activation on glutamatergic transmission by means of whole-cell patch-clamp electrophysiological recordings from medium spiny neurons of the striatum and layer V pyramidal neurons of the prefrontal cortex. To this aim, we took advantage of a novel ligand, ST1936, showing nM affinity and agonist activity at the 5-HT6 receptor subtype. Our data show that 5-HT6 receptor activation by ST1936 reduces the frequency of spontaneous excitatory postsynaptic currents, with an IC50 of 1.3 μM. Moreover, 5-HT6 receptor activation also reduced the amplitude of spontaneous excitatory postsynaptic currents recorded from medium spiny neurons, suggesting a mechanism of action involving postsynaptic 5-HT6 receptors, as further confirmed by the paired-pulse analysis on evoked excitatory postsynaptic currents and by recordings of miniature glutamatergic events. The inhibitory effect of ST1936 on glutamatergic transmission was prevented by the selective 5-HT6 receptor antagonist SB258585 and mimicked by a different agonist, WAY-181187. Conversely, in the cortex ST1936 reduced the frequency, but not the amplitude, of spontaneous excitatory postsynaptic currents suggesting a presynaptic or indirect effect of the 5-HT6 receptor.

Optogenetic stimulation reveals distinct modulatory properties of thalamostriatal vs corticostriatal glutamatergic inputs to fast-spiking interneurons

Parvalbumin-containing fast-spiking interneurons (FSIs) exert a powerful feed-forward GABAergic inhibition on striatal medium spiny neurons (MSNs), playing a critical role in timing striatal output. However, how glutamatergic inputs modulate their firing activity is still unexplored. Here, by means of a combined optogenetic and electrophysiological approach, we provide evidence for a differential modulation of cortico- vs thalamo-striatal synaptic inputs to FSIs in transgenic mice carrying light-gated ion channels channelrhodopsin-2 (ChR2) in glutamatergic fibers. Corticostriatal synapses show a postsynaptic facilitation, whereas thalamostriatal synapses present a postsynaptic depression. Moreover, thalamostriatal synapses exhibit more prominent AMPA-mediated currents than corticostriatal synapses, and an increased release probability. Furthermore, during current-evoked firing activity, simultaneous corticostriatal stimulation increases bursting activity. Conversely, thalamostriatal fiber activation shifts the canonical burst-pause activity to a more prolonged, regular firing pattern. However, this change in firing pattern was accompanied by a significant rise in the frequency of membrane potential oscillations. Notably, the responses to thalamic stimulation were fully abolished by blocking metabotropic glutamate 1 (mGlu1) receptor subtype, whereas both acetylcholine and dopamine receptor antagonists were ineffective. Our findings demonstrate that cortical and thalamic glutamatergic input differently modulate FSIs firing activity through specific intrinsic and synaptic properties, exerting a powerful influence on striatal outputs.

Cerebellar synaptogenesis is compromised in mouse models of DYT1 dystonia.

Early-onset torsion dystonia (DYT1) is an autosomal-dominant movement disorder characterized by sustained muscle contractions and abnormal posturing. It is caused by a three base-pair deletion (ΔGAG) in the gene encoding the AAA(+) protein torsinA, which gives rise to a loss of function mutation responsible of neuronal functional abnormalities. Symptoms typically appear during childhood, suggesting the presence of an early critical period of sensorimotor circuit susceptibility to torsinA dysfunction. Here, we identified in two different DYT1 mouse strains, heterozygous torsinA knockout mice (Tor1a+/-) and human ΔGAG mutant torsinA transgenic mice (hMT), the anatomical abnormalities in the cerebellum, during a critical age for synaptogenesis (postnatal day 14, P14). By means of immunofluorescence, confocal analysis and western blot quantification, we observed a reduced inhibitory input on Purkinje cells (PCs) as well as an unbalanced excitatory innervation; a significant reduction of the parallel fiber (PF) synaptic terminals and an increase of the climbing fiber (CF) inputs. Finally, in support of the in vivo results, we also provide evidence of an impaired PF synaptogenesis in a co-culture system. Of note, these alterations were rescued and in part over-compensated in the adult age in both mouse strains, suggesting that torsinA dysfunction can induce an altered maturation of cerebellar synaptic contacts. Altogether these results indicate that a loss of function of torsinA during cerebellar synaptogenesis induces important developmental alterations, that might contribute to the age-dependent susceptibility to develop dystonia in mutation carriers.

Rhes regulates dopamine D2 receptor transmission in striatal cholinergic interneurons

Ras homolog enriched in striatum (Rhes) is highly expressed in striatal medium spiny neurons (MSNs) of rodents. In the present study, we characterized the expression of Rhes mRNA across species, as well as its functional role in other striatal neuron subtypes. Double in situ hybridization analysis showed that Rhes transcript is selectively localized in striatal cholinergic interneurons (ChIs), but not in GABAergic parvalbumin- or in neuropeptide Y-positive cell populations. Rhes is closely linked to dopamine-dependent signaling. Therefore, we recorded ChIs activity in basal condition and following dopamine receptor activation. Surprisingly, instead of an expected dopamine D2 receptor (D2R)-mediated inhibition, we observed an aberrant excitatory response in ChIs from Rhes knockout mice. Conversely, the effect of D1R agonist on ChIs was less robust in Rhes mutants than in controls. Although Rhes deletion in mutants occurs throughout the striatum, we demonstrate that the D2R response is altered specifically in ChIs, since it was recorded in pharmacological isolation, and prevented either by intrapipette BAPTA or by GDP-β-S. Moreover, we show that blockade of Cav2.2 calcium channels prevented the abnormal D2R response. Finally, we found that the abnormal D2R activation in ChIs was rescued by selective PI3K inhibition thus suggesting that Rhes functionally modulates PI3K/Akt signaling pathway in these neurons. Our findings reveal that, besides its expression in MSNs, Rhes is localized also in striatal ChIs and, most importantly, lack of this G-protein, significantly alters D2R modulation of striatal cholinergic excitability.

Torsin A Localization in the Mouse Cerebellar Synaptic Circuitry

Torsin A (TA) is a ubiquitous protein belonging to the superfamily of proteins called “ATPases associated with a variety of cellular activities” (AAA+ ATPase). To date, a great deal of attention has been focused on neuronal TA since its mutant form causes early-onset (DYT1) torsion dystonia, an inherited movement disorder characterized by sustained muscle contractions and abnormal postures. Interestingly, it has been proposed that TA, by interacting with the cytoskeletal network, may contribute to the control of neurite outgrowth and/or by acting as a chaperone at synapses could affect synaptic vesicle turnover and neurotransmitter release. Accordingly, both its peculiar developmental expression in striatum and cerebellum and evidence from DYT1 knock-in mice suggest that TA may influence dendritic arborization and synaptogenesis in the brain. Therefore, to better understand TA function a detailed description of its localization at synaptic level is required. Here, we characterized by means of rigorous quantitative confocal analysis TA distribution in the mouse cerebellum at postnatal day 14 (P14), when both cerebellar synaptogenesis and TA expression peak. We observed that the protein is broadly distributed both in cerebellar cortex and in the deep cerebellar nuclei (DCN). Of note, Purkinje cells (PC) express high levels of TA also in the spines and axonal terminals. In addition, abundant expression of the protein was found in the main GABA-ergic and glutamatergic inputs of the cerebellar cortex. Finally, TA was observed also in glial cells, a cellular population little explored so far. These results extend our knowledge on TA synaptic localization providing a clue to its potential role in synaptic development.