Impaired striatal GABA transmission in experimental autoimmune encephalomyelitis

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Abstract

Synaptic dysfunction triggers neuronal damage in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). While excessive glutamate signaling has been reported in the striatum of EAE, it is still uncertain whether GABA synapses are altered. Electrophysiological recordings showed a reduction of spontaneous GABAergic synaptic currents (sIPSCs) recorded from striatal projection neurons of mice with MOG(35−55)-induced EAE. GABAergic sIPSC deficits started in the acute phase of the disease (20–25 days post immunization, dpi), and were exacerbated at later time-points (35, 50, 70 and 90 dpi). Of note, in slices they were independent of microglial activation and of release of TNF-α. Indeed, sIPSC inhibition likely involved synaptic inputs arising from GABAergic interneurons, because EAE preferentially reduced sIPSCs of high amplitude, and was associated with a selective loss of striatal parvalbumin (PV)-positive GABAergic interneurons, which contact striatal projection neurons in their somatic region, giving rise to more efficient synaptic inhibition. Furthermore, we found also that the chronic persistence of pro-inflammatory cytokines were able, per se, to produce profound alterations of electrophysiological network properties, that were reverted by GABA administration.

The results of the present investigation indicate defective GABA transmission in MS models depending from alteration of PV cells number and, in part, deriving from the effects of a chronic inflammation, and suggest that pharmacological agents potentiating GABA signaling might be considered to limit neuronal damage in MS patients.

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.

References (66)

  • L.K. Friedman et al.

    Early exposure of cultured hippocampal neurons to excitatory amino acids protects from later excitotoxicity

    Int. J. Dev. Neurosci.

    (2010)
  • Y. Kawaguchi et al.

    Striatal interneurones: chemical, physiological and morphological characterization

    Trends Neurosci.

    (1995)
  • A.Y. Lai et al.

    Interleukin-1 beta modulates AMPA receptor expression and phosphorylation in hippocampal neurons

    J. Neuroimmunol.

    (2006)
  • C. Matute et al.

    The link between excitotoxic oligodendroglial death and demyelinating diseases

    Trends Neurosci.

    (2001)
  • L. Muzio et al.

    Cxcl10 enhances blood cells migration in the sub-ventricular zone of mice affected by experimental autoimmune encephalomyelitis

    Mol. Cell. Neurosci.

    (2010)
  • E.S. Nisenbaum et al.

    Functionally distinct subpopulations of striatal neurons are differentially regulated by GABAergic and dopaminergic inputs-I. In vivo analysis

    Neuroscience

    (1992)
  • D. Plenz

    When inhibition goes incognito: feedback interaction between spiny projection neurons in striatal function

    Trends Neurosci.

    (2003)
  • G.A. Qureshi et al.

    Quantitation of free amino acids in biological samples by high-performance liquid chromatography. Application of the method in evaluating amino acid levels in cerebrospinal fluid and plasma of patients with multiple sclerosis

    J. Chromatogr.

    (1988)
  • S. Rossi et al.

    Exercise attenuates the clinical, synaptic and dendritic abnormalities of experimental autoimmune encephalomyelitis

    Neurobiol. Dis.

    (2009)
  • G. Tao et al.

    Deep gray matter atrophy in multiple sclerosis: a tensor based morphometry

    J. Neurol. Sci.

    (2009)
  • J.M. Tepper et al.

    Basal ganglia macrocircuits

    Prog. Brain Res.

    (2007)
  • J.M. Tepper et al.

    GABAergic microcircuits in the neostriatum

    Trends Neurosci.

    (2004)
  • K. Vikman et al.

    Interferon-g-induced changes in synaptic activity and AMPA receptor clustering in hippocampal cultures

    Brain Res.

    (2001)
  • T. Vyshkina et al.

    Genetic variants of Complex I in multiple sclerosis

    J. Neurol. Sci.

    (2005)
  • E. Wallström et al.

    Memantine abrogates neurological deficits, but not CNS inflammation, in Lewis rat experimental autoimmune encephalomyelitis

    J. Neurol. Sci.

    (1996)
  • Z.Q. Xiong et al.

    Fleeting activation of ionotropic glutamate receptors sensitizes cortical neurons to complement attack

    Neuron

    (2002)
  • R. Zhang et al.

    Inhibition of NMDA-induced outward currents by interleukin-1beta in hippocampal neurons

    Biochem. Biophys. Res. Commun.

    (2008)
  • B. Zhu et al.

    Dendritic and synaptic pathology in experimental autoimmune encephalomyelitis

    Am. J. Pathol.

    (2003)
  • M.O. Ziehn et al.

    Hippocampal CA1 atrophy and synaptic loss during experimental autoimmune encephalomyelitis, EAE

    Lab. Invest.

    (2010)
  • E.C. Beattie et al.

    Control of synaptic strength by glial TNFalpha

    Science

    (2002)
  • R. Bhat et al.

    Inhibitory role for GABA in autoimmune inflammation

    Proc. Natl. Acad. Sci. USA

    (2010)
  • A. Blankenship et al.

    Mechanisms underlying spontaneous patterned activity in developing neural circuits

    Nat. Rev. Neurosci.

    (2010)
  • C. Bolton et al.

    MK-801 limits neurovascular dysfunction during experimental allergic encephalomyelitis

    J. Pharmacol. Exp. Ther.

    (1997)
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    S.R. and L.M. contributed equally to this work.

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