Ablation of fast-spiking interneurons in the dorsal striatum, recapitulating abnormalities seen post-mortem in Tourette syndrome, produces anxiety and elevated grooming
Introduction
Tic disorders affect 5% of the population and produce significant morbidity (Du et al., 2010). Gilles de la Tourette syndrome (TS), characterized by both vocal and motor tics, represents part of this continuum. Tics are commonly comorbid with other forms of neuropsychiatric pathology, including obsessive–compulsive disorder (OCD) and attention deficit disorder (ADHD); indeed, 90% of patients diagnosed with TS have at least one additional diagnosis (Du et al., 2010, Hirschtritt et al., 2015). Existing pharmacotherapies are of limited efficacy (Bloch, 2008). Tics fluctuate over time and are exacerbated by stress (Leckman, 2002, Du et al., 2010, Kurlan, 2010) and by acute psychostimulant challenge (Denys et al., 2013). Individuals with TS often have deficits in procedural learning (Marsh et al., 2004), sensorimotor gating (Swerdlow et al., 2001, Castellan Baldan et al., 2014), and fine motor control (Bloch et al., 2006), though these are not part of contemporary diagnostic criteria.
Convergent data implicate abnormalities of the basal ganglia-thalamo-cortical circuitry in TS (Leckman et al., 2010, Williams et al., 2013b), although it is increasingly clear that dysfunction in other brain circuitries is also involved (Leckman et al., 2010, Neuner et al., 2013, Williams et al., 2013a). The striatum is the main input nucleus of the basal ganglia. Its principal cells, the medium spiny neurons (MSNs), receive glutamatergic input from the cortex and thalamus and dopaminergic modulation from the substantia nigra. MSNs comprise >90% of the neurons in the rodent striatum; their activity is modulated by several different populations of interneuron.
Parvalbumin-expressing fast-spiking interneurons (FSIs) constitute about 1% of striatal neurons. PV interneurons integrate glutamatergic inputs from the cortex and form strong GABAergic synapses on the somata of nearby MSNs, forming a potent feedforward inhibitory circuit (Mallet et al., 2005, Tepper et al., 2010). A single FSI can exert powerful inhibitory control on the activity of a large number of nearby MSNs (Koos and Tepper, 1999). This feed-forward inhibition is thought to have an important role in orchestrating striatal information processing (Berke, 2011). FSIs coordinate MSN firing in the theta-band range in certain behavioral states (Berke et al., 2004) and fire in concert during action selection (Gage et al., 2010).
Post-mortem examination of brains from individuals with a history of severe TS has revealed abnormalities in striatal FSIs (Kalanithi et al., 2005, Kataoka et al., 2010). However, such observations cannot elucidate the causal role of this deficit: whether it is causal, compensatory, epiphenomenal, or a consequence of years of treatment. Several lines of evidence suggest a casual role. Transient pharmacological inhibition of FSIs produces movement abnormalities (Gittis et al., 2011), but it is unclear what would happen with more chronic disruption of FSI activity, which is implied by the absence of these cells in post-mortem material. Reduced FSIs are also seen in the dtsz dystonic hamster, a spontaneously occurring mutant with movement abnormalities (Gernert et al., 2000), and in the SAPAP3 knockout mouse, which exhibits elevated, repetitive grooming (Burguiere et al., 2013); but these deficits occur in the context of widespread abnormalities, some of which may be developmental, and thus their causal role in the development of abnormal behaviors is no more clear than it is patients.
We sought to more directly address the question of whether chronic disruption of striatal FSIs, in an otherwise normal brain, is sufficient to produce TS-relevant phenomenology. We have recently described a strategy for the targeted ablation of defined interneuronal populations (Xu et al., 2015a). This permits the temporally, spatially, and cell type-specific recapitulation of the neuropathological changes seen post-mortem in TS in an otherwise normal adult brain. Here we applied this strategy to striatal FSIs and examined the consequences of their ablation.
Importantly, demonstration of a causal connection between a FSI deficit and behavioral pathology does not require recapitulation of all aspects of TS phenomenology. It is increasingly recognized that many neuropsychiatric diagnoses do not represent unitary natural kinds (Insel et al., 2010) and are causally heterogeneous, and that modeling trandiagnostic endophenotypes is a more realistic prospect than modeling diagnostic entities in all of their complexity (Nestler and Hyman, 2010). Striatal FSI deficiency has been associated with severe TS (Kalanithi et al., 2005, Kataoka et al., 2010), but it is not known whether this finding is specific to severe, treatment-refractory disease, to TS or tic disorders more generally, or to a range of behavioral pathology that extends beyond the boundaries of current diagnostic entities. We therefore tested a range of behaviors that capture different aspects of the symptomatology of TS and related disorders.
Section snippets
Experimental procedures
All experiments were performed in accordance with the NIH Guide for the Use of Laboratory Animals and were approved by the Yale University Institutional Animal Care and Use Committee. Mice were housed in a temperature- and climate-controlled facility on a 12-h light/dark schedule.
Targeted ablation of PV-expressing interneurons in the adult mouse brain
We injected the dorsal striatum of adult male PV-Cre transgenic mice with virus iDTR-A46 on one side and the negative control virus iDTR-C46 contralaterally (Fig. 1A; see Experimental procedures). Mice were injected with either DT (15 ng/g body weight intraperitoneal daily × 3 days) or saline 2 weeks after surgery and then sacrificed 1 week later. EGFP expression was visible broadly throughout the striatum on both sides, demarcating viral spread; FLAG immunoreactivity, which reflects sDTR expression,
Discussion
These results establish the sufficiency of PV interneuron disruption in the dorsal striatum, which has been documented in severe TS (Kalanithi et al., 2005, Kataoka et al., 2010), to produce repetitive grooming, after two different acute stressors. This suggests that the interneuronal deficit observed in patients is causally related to tics and other movement abnormalities, and not a compensatory change, an epiphenomenon, or a consequence of treatment. Our manipulation is unlikely to precisely
Acknowledgements
The authors gratefully thank Stacey Wilber and Jessica André for assistance with mouse colony maintenance and genotyping and Flora Vaccarino, Mounira Banasr, Marina Picciotto, and Ralph DiLeone for invaluable discussions during the development of the iDTR ablation system. This work has been supported by NIH grants R01MH091861 (CP) and K08MH081190 (CP), the Tourette Syndrome Association (CP, MX), the Allison Family Foundation (CP), and the State of Connecticut through its support of the Ribicoff
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Current address: Brown University, Providence, RI, United States.