Neurochemistry International 128 (2019) 175–185 Contents lists available at ScienceDirect Neurochemistry International journal homepage: www.elsevier.com/locate/neuint Modulation of extrasynaptic GABAergic receptor activity influences T glutamate release and neuronal survival following excitotoxic damage to mouse spinal cord neurons ∗ Graciela L. Mazzonea, , Andrea Nistrib a Instituto de Investigaciones en Medicina Traslacional (IIMT), CONICET-Universidad Austral, Derqui-Pilar, Buenos Aires, Argentina b Neuroscience Dept., International School for Advanced Studies (SISSA), Trieste, Italy ARTICLE INFO ABSTRACT Keywords: Excitotoxic levels of released glutamate trigger a cascade of deleterious cellular events leading to delayed Neuroprotection neuronal death. This phenomenon implies extensive dysregulation in the balance between network excitation Kainic acid and inhibition. Our hypothesis was that enhancing network inhibition should prevent excitotoxicity and provide Spinal cord injury neuroprotection. To test this notion, we used mouse organotypic spinal slice cultures and explored if ex- Extrasynaptic GABA receptor citotoxicity caused by the potent glutamate analogue kainate was blocked by pharmacological increase in Excitotoxicity GABAA receptor activity. To this end we monitored (with a biosensor) real-time glutamate release following 1 h kainate application and quantified neuronal survival 24 h later. Glutamate release evoked by kainate was strongly decreased by the allosteric GABAA modulator midazolam (10 nM) or the GABA agonist THIP (10 μM), leading to neuroprotection. On the contrary, much higher glutamate release was induced by the GABA an- tagonist bicuculline (20 μM) that inhibits synaptic and extrasynaptic GABAA receptors. Gabazine (20 μM), an antagonist of synaptic GABAA receptors, had no effect on glutamate release or neuroprotection. No effect was observed with the glycine antagonist strychnine or the glycine agonist L-alanine. These findings indicate that enhancement of GABA receptor activity was an effective tool to counteract excitotoxic death in spinal networks. In view of the potent activity by THIP, preferentially acting on extrasynaptic GABAA receptors, the present data imply a significant role for extrasynapticA GABA receptors in sparing spinal cord neurons from injury. 1. Introduction harm to white matter which is, on the other hand, damaged by transient oxygen-glucose deprivation (Kuzhandaivel et al., 2011; Mazzone et al., In the spinal cord massive release of the excitatory transmitter 2010; Sámano and Nistri, 2019; Taccola et al., 2008). In particular, glutamate evoked by a lesion generates excitotoxicity, namely a pa- using spinal cord organotypic cultures, we were able to estimate da- thophysiological process that largely expands the initial damage mage induced by kainate by measuring real-time release of endogenous through free radical production and metabolic dysfunction (Ahuja glutamate (via a specific biosensor), and evaluating cell losses et al., 2017). To achieve neuroprotection against this process, previous (Mazzone and Nistri, 2011a). studies have focused on blocking excitatory glutamatergic transmission This model prompted us to investigate if enhancing inhibition may with drugs like riluzole or glutamate receptor antagonists (Ramer et al., effectively contrast excitotoxicity. Indeed, clues to the potential use- 2014; Ulndreaj et al., 2017), yet the result has not been satisfactory fulness of enhancing inhibition were provided by the observation that (Cifra et al., 2012b; Sámano and Nistri, 2019). To identify novel po- general anaesthetics like methoxyflurane and propofol could be ex- tential targets for neuroprotection, our former studies had devised a perimentally neuroprotective, though with different degree of effec- model of neuronal excitotoxicity by applying the glutamate analogue tiveness and cellular mechanisms (Bajrektarevic and Nistri, 2016; Kaur kainate to in vitro spinal networks to destroy neurons with minimal et al., 2016; Shabbir et al., 2015). Nonetheless, translating the Abbreviations: ANOVA, analysis of variance; AU, arbitrary unit; BIC, bicuculline; DAPI, 4′,6-diamidino-2-phenylindole; DIV, days in vitro; DME/HIGH, Dulbecco's modified Eagle's medium high glucose; FCS, fetal calf serum; KA, kainate; GABA, gama‐aminobutyric acid; GBZ, gabazine; MDZ, midazolam; NGF,nervegrowth factor; NeuN, neuronal specific nuclear protein; PBS, phosphate-buffered saline; S100β, astroglial calcium-binding protein S100β; SCI, spinal cord injury;SEM, standard error of the mean; STRY, strychnine; THIP, 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol ∗ Corresponding author. IIMT-CONICET-Universidad Austral, Av. Perón 1500 5° piso, B1629 AHJ, Derqui (Pilar), Buenos Aires, Argentina. E-mail address: [email protected] (G.L. Mazzone). https://doi.org/10.1016/j.neuint.2019.04.018 Received 16 January 2019; Received in revised form 8 April 2019; Accepted 30 April 2019 Available online 30 April 2019 0197-0186/ © 2019 Elsevier Ltd. All rights reserved. G.L. Mazzone and A. Nistri Neurochemistry International 128 (2019) 175–185 laboratory use of general anesthetics to in vivo injury might be difficult because these drugs may further depress compromised functions like respiration and blood pressure control. Because GABA is the principal inhibitory neurotransmitter in the central nervous system (CNS) acting predominantly on GABAA re- ceptors (Nistri and Constanti, 1979; Sivilotti and Nistri, 1991), up or downregulating such receptors strongly affects the maintenance of dynamic neuronal homeostasis (Le Magueresse and Monyer, 2013). GABAA receptors located on the postsynaptic membrane mediate sy- naptic inhibition, while those on the extrasynaptic membrane respond to ambient GABA and confer long-term excitability decrease (Baur et al., 2009; Sigel and Steinmann, 2012). As recently demonstrated in lampreys with complete spinal cord transection, GABA release is also an important factor to promote axonal regeneration mediated by GABAA and GABAB receptors via decreased caspase activation (Romaus- Sanjurjo et al., 2018; Sobrido-Cameán et al., 2018). Thus, potentiating the role of GABA might be a strategy for experimental spinal neuro- protection and is a development of an earlier proposal for the protective role of spinal inhibitory interneurons against motor degeneration (Ramírez-Jarquín et al., 2014). Fig. 1. Mouse spinal cord organotypic culture. Example of 22 DIV slice with To this end, using the in vitro model of excitotoxicity in organotypic two dorsal and ventral ROIs. Inset shows typical neuronal staining NeuN spinal culture (Mazzone et al., 2010), we studied the effects of mid- (green) restricted to the spinal cord tissue region, S100β (red) for glial staining, and DAPI (blue) used for general nuclear staining. azolam (a water soluble benzodiazepine) that binds to the benzodia- zepine binding site on GABAA receptors (Rudolph and Knoflach, 2011) to allosterically potentiate them via increased opening frequency of the fetuses were delivered by caesarian section and were pooled together to intrinsic chloride channel (Bounds and Nelson, 2017). Furthermore, we obtain on average 40 slices. Slices were then embedded in a plasma tested the effect of the GABA analogue THIP (4,5,6,7-tetra- clot, cultured on glass coverslips and introduced into individual tubes hydroisothiazolo-[5,4-c]pyridine-3-ol), an agonist preferentially acting that undergo continuous slow rotation until use (Gahwiler et al., 1997). on extrasynaptic α4βδ-GABAA receptors (Iversen, 2004) that are re- Slices were maintained for 22 days in vitro (DIV) in a medium con- ported to be mainly responsible for controlling neuronal excitability taining Dulbecco's Modified Eagle medium with high glucose (DME/ changes and damage induced by reactive oxygen species in rat spinal HIGH, 82%), sterile water for tissue culture (8%), nerve growth factor cord slices (Ohashi et al., 2016). As a proof of principle for the role of (NGF, 5 ng/ml), fetal calf serum (FCS, 10%; Invitrogen, Carlsbad, CA, GABAA receptors in excitotoxicity, we also examined the effects of ga- USA), at controlled osmolarity (300 mOsm) and pH 7.35, in accordance bazine, an antagonist of synaptic GABAA receptors, and bicuculline that with standard procedures (Spenger et al., 1991; Streit et al., 1991). has a broader antagonistic action (Krall et al., 2015), to find out if they DME/HIGH, penicillin and streptomycin were purchased from Euro- could intensify the effect evoked by kainate. Finally, since glycine is clone (Devon, UK). NGF was obtained from D.B.A. Italia (Segrate, Italy), also an important inhibitory neurotransmitter in spinal networks while the other reagents were purchased from Sigma-Aldrich, Milan, (Aprison and Werman, 1965; Davidoff et al., 1967; Prescott, 2015), we Italy. Fig. 1 shows examples of typical spinal cord organotypic slice explored the effect of L-alanine (a glycine receptor agonist; Curtis et al., cultures with staining for neurons (NeuN positive cells in green) and 1968; Schmieden and Betz, 1995), and strychnine, a glycine receptor astrocytes (S100β positive cells in red) after 22 DIV in two region of antagonist (Curtis, 1969; Davidoff et al., 1969; Du et al., 2015). Since interest (ROIs) indicated by white boxes, namely dorsal and ventral the complex mechanisms regarding GABA receptor pathophysiological ones. role remain incompletely understood, we propose that drug pre- application protocols can provide an insight into mechanisms of action 2.2. Protocols