GABAergic signaling and connectivity on Purkinje cells are impaired in experimental autoimmune encephalomyelitis

Abstract

A significant proportion of multiple sclerosis (MS) patients have functionally relevant cerebellar deficits, which significantly contribute to disability. Although clinical and experimental studies have been conducted to understand the pathophysiology of cerebellar dysfunction in MS, no electrophysiological and morphological studies have investigated potential alterations of synaptic connections of cerebellar Purkinje cells (PC). For this reason we analyzed cerebellar PC GABAergic connectivity in mice with MOG_(35–55)-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We observed a strong reduction in the frequency of the spontaneous inhibitory post-synaptic currents (IPSCs) recorded from PCs during the symptomatic phase of the disease, and in presence of prominent microglia activation not only in the white matter (WM) but also in the molecular layer (ML). The massive GABAergic innervation on PCs from basket and stellate cells was reduced and associated to a decrease of the number of these inhibitory interneurons. On the contrary no significant loss of the PCs could be detected. Incubation of interleukin-1beta (IL-1β) was sufficient to mimic the electrophysiological alterations observed in EAE mice.

We thus suggest that microglia and pro-inflammatory cytokines, together with a degeneration of basket and stellate cells and their synaptic terminals, contribute to impair GABAergic transmission on PCs during EAE. Our results support a growing body of evidence that GABAergic signaling is compromised in EAE and in MS, and show a selective susceptibility to neuronal and synaptic degeneration of cerebellar inhibitory interneurons.

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.