Impaired striatal GABA transmission in experimental autoimmune encephalomyelitis
Research highlights
► Spontaneous striatal GABAergic currents are permanently reduced in EAE starting from disease onset. ► Neuronal networks treated in vitro with proinflammatory cytokines display similar alterations. ► PV striatal interneurons reduction in EAE may explain GABAergic alterations.
Introduction
Synaptic alterations are receiving increasing attention as early correlates of primarily neurodegenerative diseases but also of neuroinflammatory disorders. Inflammatory cytokines (Stellwagen et al., 2005, Lai et al., 2006, Stellwagen and Malenka, 2006, Cumiskey et al., 2007, Mizuno et al., 2008, Centonze et al., 2009), resident immune cells such as microglia (Centonze et al., 2009), and brain infiltrating T lymphocytes (Lewitus et al., 2007, Centonze et al., 2009), have been found to alter glutamate transmission, providing support to the emerging concept that glutamate-dependent excitotoxic damage plays a fundamental role in neuronal degeneration accompanying inflammatory disorders, such as multiple sclerosis (MS) (Srinivasan et al., 2005, Cianfoni et al., 2007, Newcombe et al., 2008), and its mouse model, experimental autoimmune encephalomyelitis (EAE) (Wallström et al., 1996, Bolton and Paul, 1997, Pitt et al., 2000, Smith et al., 2000, Matute et al., 2001, Centonze et al., 2009).
GABA is the main inhibitory neurotransmitter in the central nervous system (CNS), whose activity balances that of glutamate in neurons. To date, glutamate transmission has been studied in neurophysiological investigations in EAE mice (Centonze et al., 2009), while the possible changes of GABA signaling in this model of MS have been inferred on the basis of biochemical (Gottesfeld et al., 1976), molecular (Wang et al., 2008), and morphological studies (Ziehn et al., 2010), but never addressed through direct recordings of synaptic activity.
The striatum is a sub-cortical gray matter structure whose activity is finely regulated by both glutamate and GABA inputs (Fisone et al., 2007, Tepper et al., 2007). It is highly sensitive to the neurodegenerative process associated with MS (Henry et al., 2008, Tao et al., 2009, Ceccarelli et al., 2010), and undergoes complex alterations of glutamate transmission and dendritic damage during EAE (Centonze et al., 2009). Importantly, these synaptic alterations occur in the absence of overt demyelinating lesions, and even before the appearance of the EAE-associated neurological deficits, indicating that they are not a mere consequence of axonal damage (Centonze et al., 2009).
Not only glutamate, but also GABAergic synaptic activity can be studied in the striatum by means of whole-cell patch clamp recordings from single neurons in slices. Striatal principal neurons (also known as medium spiny projection neurons, MSNs), in fact, receive GABAergic inputs from axon collateral of MSNs themselves and from GABAergic interneurons (Koos and Tepper, 1999, Tunstall et al., 2002, Guzman et al., 2003, Plenz, 2003, Tepper et al., 2004, Koos et al., 2004, Gustafson et al., 2006), which play a crucial role in limiting the excitatory drive originating from cortico-striatal glutamatergic inputs. Accordingly, blockade of GABAergic inhibition significantly elevates basal activity of MSNs in vivo (Nisenbaum and Berger, 1992), which is driven by glutamate released from cortico-striatal terminals (Wilson and Kawaguchi, 1996, Stern et al., 1998).
Given the complexity of the adult tissue, some of the intrinsic electrical features of a network of neurons can be analyzed in vitro, by taking advantage of using Multi Electrode Arrays (MEA) device. Indeed, primary neuronal cultures retain many of the properties found in their in vivo context (Wagenaar et al., 2006) and have advantages in terms of electrical recording and long term pharmacological manipulation. Neuronal network models display a spontaneous electrical activity that is governed by the balance between excitation and inhibition (Mazzoni et al., 2007).
Local immune response, derived from infiltrating blood derived cells – i.e. T helper 1 (Th1) lymphocytes, is characterized by a broad spectrum of pro-inflammatory cytokines that, acting on oligodendrocytes, astrocytes, resident microglia and neurons, elicit CNS derangement. IFN-γ, TNF-α and IL1-β represent key molecules of adaptive immunity and are secreted by Th1 cells infiltrating CNS of both EAE and MS active lesions. In addition, these pro-inflammatory molecules can also alter neuronal functioning. Indeed, TNF-α enhances synaptic efficacy of cultured neurons (Beattie et al., 2002); IL1-β increases neuronal excitability (Zhang et al., 2008) and IFN-γ causes long term modifications of synaptic activity and neuronal damages (Vikman et al., 2001, Mizuno et al., 2008). Thus, aim of the present investigation is to study GABA transmission in EAE and in Th1 pro-inflammatory treated neuronal cultures, to assess whether such molecules can trigger alterations of synaptic inhibition occurring in these models, and to explore further synaptic mechanisms possibly contributing to neuronal damage occurring during neuro-inflammation.
Section snippets
Materials and methods
All efforts were made to minimize animal suffering and to reduce the number of mice used, in accordance with the European Communities Council Directive of 24 November, 1986 (86/609/EEC).
Neurophysiological properties of GABA transmission in EAE
Spontaneous inhibitory postsynaptic currents (sIPSCs) were measured as an indicator of the physiological activity of GABA signaling in the striatum. When recorded from putative MSNs, sIPSCs of control mice ranged between 5 and 60 pA of amplitude and between 0.5 and 3 Hz of frequency (Fig. 1A–C), and could be entirely blocked following the application of bicuculline, selective antagonist of GABA-A receptors (n = 12) (not shown).
In EAE, striatal sIPSC frequency and amplitude were indistinguishable
Discussion
Previous studies have postulated impaired GABA transmission in MS and in EAE. GABA is reduced in the cerebrospinal fluid of MS subjects (Qureshi and Baig, 1988), and [3H] GABA uptake (Gottesfeld et al., 1976), and protein and mRNA expression of the GABA transporter-1 (GAT-1) are dramatically reduced in the spinal cord of EAE mice (Wang et al., 2008). Furthermore, potentiation of GABA signaling significantly ameliorates EAE clinical course, through a mechanism likely involving a direct
Acknowledgments
We thank Vladimiro Batocchi for helpful technical assistance. This investigation was supported by the Italian National Ministero dell’Università e della Ricerca to D.C.; by the Italian National Ministero della Salute to D.C., by Fondazione Italiana Sclerosi Multipla (FISM) to R.F., G.M. and L.M. (L.M. was supported by FISM grant number 2009/R/18), and by BMW-Italy to G.M.
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S.R. and L.M. contributed equally to this work.