GABAergic signaling and connectivity on Purkinje cells are impaired in experimental autoimmune encephalomyelitis
Highlights
► Cerebellar deficits are very frequent in patients with multiple sclerosis (MS). ► Inhibitory synaptic inputs to Purkinje cells show neurophysiological and pathological deficits in EAE, a mouse model of MS. ► The pro-inflammatory cytokine IL-1β also inhibits GABAergic transmission in acute cerebellar slice. ► Cerebellar GABAergic interneurons selectively degenerate in EAE in response to inflammation.
Introduction
Common symptoms of multiple sclerosis (MS) such as gait ataxia, poor coordination of hands, and intention tremors, are usually the result of lesions in the cerebellum. Besides giving a significant contribution to disability, cerebellar deficits seem also relatively refractory to symptomatic therapy and progress even under disease-modifying agents (Waxman, 2005). The pathophysiology of the cerebellar symptoms in MS is complex and only partially understood (Giovannoni and Ebers, 2007). Cerebellar cortex is a major predilection site for demyelination, in particular in patients with primary and secondary progressive MS (Kutzelnigg et al., 2007). In MS patients and in EAE mice, cerebellar deficits have been associated with cerebellar atrophy caused by PCs death as well as degeneration of neurons in olivary nuclei (Kumar and Timperley, 1988, Chin et al., 2009, MacKenzie-Graham et al., 2009). In addition, functional abnormalities in PCs, caused by aberrant expression of surface receptors or ion channels, have been reported both in MS patients and EAE mice. In particular, an atypical repertoire of sodium channels detected in PCs was related to an abnormal bursting activity of PCs (Black et al., 2000, Craner et al., 2003a, Craner et al., 2003b, Renganathan et al., 2003, Saab et al., 2004, Waxman, 2005).
Recently, abnormal expression of metabotropic glutamate receptors (Fazio et al., 2008), cannabinoid CB1 receptors (Cabranes et al., 2006, Centonze et al., 2007), and glutamate transporters (Mitosek-Szewczyk et al., 2008) have been reported in cerebellum during EAE. Altogether these studies suggest that synaptic changes in the cerebellum could contribute to the pathophysiology of MS.
A physiological hallmark of MS and of its animal model EAE is an unbalance between glutamatergic and GABAergic transmission accompanied by synaptic degeneration (Centonze et al., 2009, Centonze et al., 2010, Ziehn et al., 2010, Rossi et al., 2011). These start early before symptoms onset and have been proposed to underlie gray matter dysfunction and also cognitive deficits (Mandolesi et al., 2010). Recently, it has been shown that a decrease in GABAergic signal gives a relevant contribution in the enhancement of neuronal excitability in striatum during EAE, likely representing a further cause of excitotoxic damage together with an increase of glutamatergic transmission (Centonze et al., 2009, Rossi et al., 2011). Moreover, a loss in GABAergic interneurons was observed early in the acute phase of EAE both in hippocampus (Ziehn et al., 2010) and striatum (Rossi et al., 2011), as well as in motor cortex of post-mortem MS patients (Clements et al., 2008). GABA is reduced in the cerebrospinal fluid of MS subjects (Qureshi and Baig, 1988). Furthermore, potentiation of GABA signaling significantly ameliorates EAE clinical course, through a mechanism likely involving a direct neuroprotective effect and an inhibitory action on antigen-presenting cells and the resulting inflammatory response (Bhat et al., 2010).
To date, synaptic transmission and connectivity on cerebellar PCs during MS or EAE have never been investigated. In the present work we studied transmission, neuroinflammation and pathology of GABAergic inhibitory interneurons impinging on cerebellar PCs during EAE.
Section snippets
EAE induction and clinical evaluation
Female C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME, USA) were used for all the experiments. EAE was induced in 6–8 weeks animals as previously described (Centonze et al., 2009, Rossi et al., 2010, Rossi et al., 2011). Mice were injected subcutaneously at the flanks with 200 μg of myelin oligodendrocyte glycoprotein p35–55 (MOG(35–55)) emulsion to induce EAE by active immunization. The emulsion was prepared under sterile conditions using MOG(35–55) (> 85% purity, Espikem, Florence, Italy)
WM lesions and microglia activation in the cerebellum of EAE mice
In EAE mice, brain infiltrating T lymphocytes, resident immune cells such as microglia and inflammatory cytokines have been found to be responsible of altered synaptic transmission in the striatum (Centonze et al., 2009, Rossi et al., 2011). Therefore, we first investigated the presence of infiltrating T lymphocytes and the degree of activation of the microglia/macrophage population by immunohistochemistry and western blot in the cerebellum of mice with EAE and compared the results with those
Discussion
In the present work we have shown that in EAE mice PCs receive a reduced inhibitory tone during the symptomatic phase of the disease. The reduction of GABAergic sIPSC frequency was accompanied by synaptic degenerative processes and loss of stellate and basket cells. Such populations of inhibitory neurons belong to the PV+ interneurons, a class of neurons particularly susceptible to degeneration in MS and EAE (Dutta et al., 2006, Clements et al., 2008, Rossi et al., 2011). Our findings suggest
Acknowledgments
We wish to thank Massimo Tolu and Vladimiro Batocchi for helpful technical assistance. We want also to thank Prof. Piergiorgio Strata for his support. This investigation was supported by the Italian National Ministero della Salute to DC, by Fondazione TERCAS to DC, and by Fondazione Italiana Sclerosi Multipla (FISM) to DC, by a grant from the European Community (AXREGEN: Axonal regeneration, plasticity & stem cells— Grant agreement 21 4003) founding PhD fellowship of NH.
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GM and GG contributed equally to this work.