The Dual Role of the GABAA Receptor in Peripheral Inflammation And

International Journal of Molecular Sciences

Article The Dual Role of the GABAA Receptor in Peripheral Inﬂammation and Neuroinﬂammation: A Study in Hyperammonemic Rats

Michele Malaguarnera 1,2 , Tiziano Balzano 1,3 , Mari Carmen Castro 1, Marta Llansola 1,* and Vicente Felipo 1

1 Laboratory of Neurobiology, Centro de Investigación Príncipe Felipe, 46012 Valencia, Spain; [email protected] (M.M.); [email protected] (T.B.); [email protected] (M.C.C.); [email protected] (V.F.) 2 Department of Psychobiology, Facultad de Psicología, Universitat de Valencia, 46010 Valencia, Spain 3 HM CINAC (Centro Integral de Neurociencias Abarca Campal), Hospital Universitario HM Puerta del Sur, HM Hospitales, 28938 Madrid, Spain * Correspondence: [email protected]

Abstract: Cognitive and motor impairment in minimal hepatic encephalopathy (MHE) are mediated by neuroinflammation, which is induced by hyperammonemia and peripheral inflammation. GABAergic neurotransmission in the cerebellum is altered in rats with chronic hyperammonemia. The mechanisms by which hyperammonemia induces neuroinflammation remain unknown. We

hypothesized that GABAA receptors can modulate cerebellar neuroinflammation. The GABAA antagonist bicuculline was administrated daily (i.p.) for four weeks in control and hyperammonemic rats. Its effects on peripheral inflammation and on neuroinflammation as well as glutamate and GABA neurotransmission in the cerebellum were assessed. In hyperammonemic rats, bicuculline Citation: Malaguarnera, M.; Balzano, decreases IL-6 and TNFα and increases IL-10 in the plasma, reduces astrocyte activation, induces T.; Castro, M.C.; Llansola, M.; Felipo, the microglia M2 phenotype, and reduces IL-1β and TNFα in the cerebellum. However, in control V. The Dual Role of the GABAA rats, bicuculline increases IL-6 and decreases IL-10 plasma levels and induces microglial activation. Receptor in Peripheral Inflammation Bicuculline restores the membrane expression of some glutamate and GABA transporters restoring and Neuroinflammation: A Study in the extracellular levels of GABA in hyperammonemic rats. Blocking GABAA receptors improves Hyperammonemic Rats. Int. J. Mol. peripheral inflammation and cerebellar neuroinflammation, restoring neurotransmission in hyperam- Sci. 2021, 22, 6772. https://doi.org/ monemic rats, whereas it induces inflammation and neuroinflammation in controls. This suggests a 10.3390/ijms22136772 complex interaction between GABAergic and immune systems. The modulation of GABAA receptors

Academic Editor: Fabrizio Michetti could be a suitable target for improving neuroinﬂammation in MHE.

Received: 28 May 2021 Keywords: bicuculline; cerebellum; astrocyte activation; microglia phenotype; cytokines; GABA and Accepted: 21 June 2021 glutamate transporters; hepatic encephalopathy Published: 24 June 2021

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- Patients with liver cirrhosis may develop covert or minimal hepatic encephalopathy iations. (MHE) characterized by mild cognitive impairment, attention deficits, and psychomotor slowing, which impair quality of life, reduce life span, and increase the risks of accidents, falls, and hospitalization. MHE affects several million people and is a serious health, social, and economic problem [1]. Copyright: © 2021 by the authors. Hyperammonemia and peripheral inflammation play synergistic roles in inducing Licensee MDPI, Basel, Switzerland. cognitive and motor alterations in MHE [2–4]. This article is an open access article Chronic hyperammonemia per se is enough to induce neuroinflammation with the distributed under the terms and activation of microglia increasing inflammatory markers in the brain associated with the conditions of the Creative Commons impairment of cognitive function. Reducing neuroinflammation with ibuprofen restored Attribution (CC BY) license (https:// the learning of a discrimination task in the Y maze in hyperammonemic rats. In addition, creativecommons.org/licenses/by/ chronic hyperammonemia also induces peripheral inflammation, increasing TNFα and 4.0/).

Int. J. Mol. Sci. 2021, 22, 6772. https://doi.org/10.3390/ijms22136772 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 6772 2 of 18

decreasing IL-10 levels in the plasma [5]. Reducing peripheral inflammation with anti- TNFα reduces cerebellar neuroinflammation by mechanisms involving the inhibition of the activation of TNFR1-NFkB and associated signaling pathways [6]. Hyperammonemic rats show increased GABAergic tone in the cerebellum. Chronic treatment with bicuculline, a GABAA receptor antagonist, restores GABAergic tone, the function of the glutamate-nitric oxide-cGMP pathway in the cerebellum, and learning of the discrimination task in the Y maze modulated by this pathway [7]. The same effects are induced by treatment with pregnenolone sulphate, a negative allosteric modulator of the GABAA receptor, which also improves the motor incoordination caused by increased extracellular GABA in the cerebellum [8]. In addition, an antagonist of allopregnanolone, GR3027, which also antagonizes the GABAA receptor, restores motor incoordination in rats with portacaval anastomosis and in hyperammonemic rats. Decreasing neuroinflammation reverses the increase of GABAergic tone in the cerebellum and restores impaired motor coordination in rats with hyperammonemia or with porta-cava anastomosis, suggesting that neuroinflammation alters GABA neurotransmission in the cerebellum of these rats [9–11]. The presence of GABAA receptors in lymphocytes led to the study of its role in the immune system. Recently, the anti-inflammatory effects of the activation of these receptors was reported [12–14]. GABA acts as an anti-inflammatory agent in rheumatoid arthritis, downregulating the mechanisms that lead to the production of pro-inflammatory agents such as IL-1β [15]. The activation of GABAA receptors also ameliorates inflammatory damage in models of multiple sclerosis [13]. GABA acts as an anti-inflammatory agent, decreasing LPS-induced activation of astrocytes and microglia and inhibiting proinflammatory pathways and the increase in proinflammatory cytokines, partially through the activation of GABAA receptors in microglia and astrocytes [16]. The protective role of GABAA activation in LPS-induced inflammation, neuroinflammation, and neurologic alterations was also widely studied [17–20]. In contrast, some reports suggested that GABA can induce pro-inflammatory cytokine production in pathological conditions. Carmans et al. [17] showed that exogenous systemic administration of GABA increases IL-6 and TNFα mRNA in the central nervous system. Sallam et al. [18] reported that the intracerebral administration of bicuculline inhibited the increase in serum IL-6 and TNFα induced by LPS in rats. Increased GABA levels and the subsequent activation of GABAA receptors induce the activation of astrocytes [21]. Our previous work showed the anti-inflammatory effects of systemic administration of bicuculline in the hippocampus of hyperammonemic rats associated with the improvement of spatial learning and anxiety [22]. We proposed that there is an interplay between neuroinflammation and GABAergic and glutamatergic neurotransmission in the induction of cognitive and motor alterations in rats with MHE [23]. It is necessary to note that the effect of systemic modulation of GABAA could be the result of the modulation of GABAA receptors in both peripheral immune cells and cells in the brain. The aim of this work was to analyze the effects of blocking GABAA receptors on peripheral inflammatory and neuroinflammatory markers in the cerebellum of control and hyperammonemic rats as well as its effects on GABA and glutamate neurotransmission. We used bicuculline as GABAA receptor antagonist, as our previous reports showed effects of this compound on cerebellar-dependent learning and signal transduction pathways associated with glutamate NMDA receptors in hyperammonemic rats and we know the effective dose.

2. Results We checked whether the systemic administration of bicuculline affected hyperammonemia. Bicuculline administration did not modify the blood ammonia level in hyperammonemic rats, which ranged between 100 and 150 µM, whereas, in control rats, ammonia Int. J. Mol. Sci. 2021, 22, 6772 3 of 18

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 19 levels in blood were around 50 µM. Two-way ANOVA with repeated measures was performed (F (3, 58) = 6.234, p < 0.001). No signiﬁcant time effect was found; the p-value between control and hyperammonemic curves was p < 0.001 and was p < 0.05 between hyperammonemiaof hyperammonemic with bicuculline andrats, controls, whereas, with in nocontrol signiﬁcant rats, plasmatic effect of bicuculline levels of IL-10 were de- on the ammoniacreased levels by in hyperammonemicbicuculline chronic rats administration (Figure1a). (Figure 1f,g).

Figure 1. EffectFigure of bicuculline 1. Effect of on bicuculline ammonia in on blood ammonia and peripheral in blood andmarkers peripheral of inflammation. markers of(a) inflammation. Ammonia concentration in the blood.(a )( Ammoniab) Time courseconcentration of IL-6 levels in the in blood.the plasma. (b) Time (c) TNF courseα levels of IL-6 in the levels plasma in the after plasma. five weeks (c) TNF of hyperammo-α nemia, expressedlevels inas thepercentage plasma of after controls. five weeks (d) Time of hyperammonemia, course of plasmatic expressed IL-10 levels. as percentage(e) Time course of controls. of TGF-β levels in plasma. Cytokine(d) Time levelscourse are of expresse plasmaticd as IL-10 a percentage levels. (e of) Timecontrols. course (f,g of) The TGF- timeβ levels course in of plasma. change Cytokinein IL-10 and TGF-β plasmatic levelslevels in are hypera expressedmmonemic as a percentagerats without of (f controls.) and with ( fbicuculline,g) The time treatment course of(g). change Values in are IL-10 the mean and ± SEM of 18–22 rats/groupTGF-β forplasmatic ammonia, levels 5–7 inrats hyperammonemic for IL6, 6–9 rats ratsfor TNF withoutα, 7–10 (f) rats and for with IL-10, bicuculline and 6–10 treatment rats for TGF- (g). β. Values significantly different from the control rats are indicated by asterisks. * p < 0.05; ** p < 0.01; *** p < 0.001. Values significantly Values are the mean ± SEM of 18–22 rats/group for ammonia, 5–7 rats for IL6, 6–9 rats for TNFα, different from hyperammonemic rats are indicated by “a”. a, p < 0.05, and aa, p < 0.01. CV, control vehicle; CB, control 7–10 rats for IL-10, and 6–10 rats for TGF-β. Values significantly different from the control rats treated with bicuculline; HA, hyperammonemic rats with vehicle; HB, hyperammonemic rats treated with bicuculline. are indicated by asterisks. * p < 0.05; ** p < 0.01; *** p < 0.001. Values significantly different from hyperammonemic ratsAs are peripheral indicated byinflammation “a”. a, p < 0.05, induces and aa, neuroinflammationp < 0.01. CV, control in vehicle; the cerebellum CB, of hyper- control treated withammonemic bicuculline; rats, HA, we hyperammonemic evaluated the rats effect with of vehicle; bicuculline HB, hyperammonemic on cerebellar neuroinflammation. rats treated with bicuculline.The activation of microglia was assessed by immunohistochemistry, with the microglial marker Iba1 used to analyze the morphology by measuring the perimeter of stained cells. As previous studies of our group reported peripheral inflammation in hyperammone- In hyperammonemic rats, microglia displayed a more amoeboid morphology with shorter mic rats with a transitory increase in the levels of IL-6 early after start of the hyperammone- prolongations. Bicuculline-treated rats, both control and hyperammonemic, also showed mic diet and sustained increase in plasma TNFα, we analyzed the effect of bicuculline microglia with an amoeboid form (Figure 2a). These results were confirmed by perimeter systemic administration on these parameters. measurements. A decrease in perimeter indicated a change in microglia morphology after Hyperammonemic rats showed increased levels of IL-6 (151 ± 15% of control, p < 0.05) in the plasma atactivation. 11 days of The hyperammonemia microglial perimeter but not was thereafter. significantly Bicuculline shorter prevented in hyperammonemic the (189 ± early increase in5 IL-6µm, inp < the 0.01) plasma than ofin hyperammonemiccontrol rats (221 ± rats8 µm). (69 Bicuculline± 9% of controls, did notp

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treatment (11 days of hyperammonemia), suggesting an effect of the chronic administration of bicuculline rather than an acute effect. At five weeks of hyperammonemia, TNFα levels were increased by 217 ± 44% compared to controls (p < 0.05), and bicuculline chronic administration significantly decreased TNFα levels in hyperammonemic rats, returning to control levels (91 ± 23% of controls, p < 0.05 compared with hyperammonemia without bicuculline). F (3, 15) = 5.559 and p < 0.01 were the values for two-way ANOVA (Figure1c). To better assess the effects of bicuculline on inflammatory parameters in the plasma, we also analyzed the levels of some anti-inflammatory cytokines. IL-10 in the plasma of hyperammonemic rats was significantly decreased after three or five weeks of hyperammonemia (59 ± 13 and 47 ± 6% of control, respectively). Two-way ANOVA with repeated measures showed a significant time effect with F (1.4, 61.87) = 11.34, p < 0.001. Bicuculline increased IL-10 after five weeks of hyperammonemia (92 ± 10% of controls, p < 0.05) but not after three weeks (61 ± 12% of controls). In control rats, bicuculline significantly decreased IL-10 in plasma to 61 ± 12 and 72 ± 8% of controls after two or four weeks of chronic administration, respectively, p < 0.05 for both (Figure1d). The plasmatic levels of TGF-β were also significantly increased after three or five weeks of hyperammonemia (164 ± 21 and 165 ± 15% of controls, respectively). Bicuculline decreased the levels of TGF-β in hyperammonemic rats after three (95 ± 13% of controls, p < 0.05) but not five weeks of hyperammonemia (187 ± 33% of controls) (Figure1e). Statistical values in two-way ANOVA with repeated measures were F (1.669, 22.53) = 7.131, p < 0.01 for time effect and F (2, 31) = 4.632, p < 0.05 for group effect. The activation of regulatory T lymphocytes increased the levels of the anti-inflammatory cytokines IL-10 and TGF-β. However, in hyperammonemic rats, IL-10 decreased whereas TGF-β increased, indicating that the changes in levels of these cytokines in hyperammonemic rats were not due to the activation of regulatory T-cells but to the modulation of other cell types or mechanisms modulating the plasmatic levels of these cytokines. However, in hyperammonemic rats treated with bicuculline, the levels of both cytokines increased after five weeks of hyperammonemia, suggesting that chronic bicuculline can activate the regulatory T-cells that increase IL-10 and TGF-β levels in the plasma of hyperammonemic rats, whereas, in control rats, plasmatic levels of IL-10 were decreased by bicuculline chronic administration (Figure1f,g). As peripheral inflammation induces neuroinflammation in the cerebellum of hyperammonemic rats, we evaluated the effect of bicuculline on cerebellar neuroinflammation. The activation of microglia was assessed by immunohistochemistry, with the microglial marker Iba1 used to analyze the morphology by measuring the perimeter of stained cells. In hyperammonemic rats, microglia displayed a more amoeboid morphology with shorter prolongations. Bicuculline-treated rats, both control and hyperammonemic, also showed microglia with an amoeboid form (Figure2a). These results were confirmed by perimeter measurements. A decrease in perimeter indicated a change in microglia morphology after activation. The microglial perimeter was significantly shorter in hyperammonemic (189 ± 5 µm, p < 0.01) than in control rats (221 ± 8 µm). Bicuculline did not reverse the activation of microglia in hyperammonemic rats (192 ± 5 µm, p < 0.01) and significantly decreased the perimeter of microglia in control rats (194 ± 6 µm, p < 0.05), indicating their activation. One-way ANOVA parameters in Figure3B were: F (3, 5) = 4.875, p < 0.01 (Figure2b). The activation of microglia is associated with the induction of the NFkB transcription factor in hyperammonemic rats, demonstrated by the increased nuclear localization of P50 NFkB in cerebellar microglia [6]. The expression of NFkB was significantly increased in microglia of hyperammonemia rats and also in rats treated with bicuculline, both control and hyperammonemic (F (3, 8) = 8.542, p < 0.001). However, the nuclear percentage of P50 NFkB was significantly increased in hyperammonemic rats (62 ± 7%, p < 0.01) compared with controls (25 ± 4% in nucleus), but the treatment with bicuculline significantly decreased the percentage of P50 NFkB in the nucleus in hyperammonemic rats (35 ± 7%, p < 0.05), while, in control rats, it significantly increased nuclear P50 NFkB (62 ± 7%, p < 0.01). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 19

Int. J. Mol. Sci. 2021, 22, 6772 with controls (25 ± 4% in nucleus), but the treatment with bicuculline5 of significantly 18 decreased the percentage of P50 NFkB in the nucleus in hyperammonemic rats (35 ± 7%, p < 0.05), while, in control rats, it significantly increased nuclear P50 NFkB (62 ± 7%, p < 0.01). One-way ANOVA parameters were: F (3, 7) = 7.515, p < 0.001. These results suggest that, One-way ANOVAalthough parameters the microglia were: F (3,morphology 7) = 7.515, wasp < 0.001.not changed These resultsby bicuculline, suggest it that, decreased NFkB although the microgliaactivation morphology in hyperammonemic was not changed rats (Figure by bicuculline, 2c–e). We assessed it decreased whether NFkB this was due to a activation in hyperammonemicchange in the microglia rats (Figure phenotype2c–e). We to anti-i assessednflammatory whether thisor regulato was duery toM2. In order to a change in the microgliaassess this phenotypepossibility, towe anti-inflammatory quantified the content or regulatory of the M2 M2.marker In order YM-1. to The content of assess this possibility,YM-1 was we quantifiedsignificantly the decreased content of(60 the ± 5% M2 of marker controls, YM-1. p < 0.05) The in content hyperammonemic of rats, YM-1 was significantlywhereas decreased it did not (60decrease± 5% ofin controls,hyperammonemicp < 0.05) inrats hyperammonemic treated with bicuculline rats, (101 ± 16% whereas it did notof decreasecontrols, in p hyperammonemic< 0.05 compared with rats treatedhyperammonemia with bicuculline without (101 bicuculline):± 16% of F (3, 36) = controls, p < 0.054.057, compared p < 0.05. with Therefore, hyperammonemia bicuculline without should bicuculline):induce an M2 F (3,phenotype 36) = 4.057, in the cerebellar p < 0.05. Therefore,microglia bicuculline of hyperammone should inducemic an rats M2 (Figure phenotype 2f). in the cerebellar microglia of hyperammonemic rats (Figure2f).

Figure 2. FigureEffect of 2. bicucullineEffect of bicuculline on microglia on phen microgliaotype phenotypein white matter in white of the matter cerebellum. of the cerebellum.Microglia was Microglia marked with anti- Iba1. Representativewas marked images with anti-Iba1.of microglial Representative morphology (a images) are shown. of microglial The perimeter morphology of microglia (a) are was shown. quantified The as a measure of microglialperimeter activation of microglia (b). The was expression quantified of as NFkB a measure p50 subunit of microglial in microglia activation is shown (b). in The (c) expressionand its quantification of in (d). The NFkBnuclear p50 localization subunit in of microglia the p50 issubunit shown of in NFkB (c) and was its quantified quantification from in immunofluorescence (d). The nuclear localization (e). Microglia M2 marker YM-1of the was p50 quantified subunit of by NFkB Western was quantifiedblot and expressed from immunofluorescence as a percentage of ( econtrols). Microglia (f). Data M2 markerare the YM-1mean ± SEM of 4–5 rats for microglia analysis, 3–4 rats for NFkB p50 analysis, and 8–10 rats per group for YM-1 quantification. Values was quantified by Western blot and expressed as a percentage of controls (f). Data are the mean significantly different from control rats are indicated by asterisks. *p < 0.05, **p < 0.01, ***p < 0.001. Values significantly ± different fromSEM HV of rats 4–5 are rats indicated for microglia by a. a analysis,p < 0.05, aa 3–4, p rats< 0.01 for. And NFkB significantly p50 analysis, different and 8–10from ratsCB are per indicated group by b. bb p < 0.01. CV,for YM-1control quantification. vehicle; CB, control Values treated significantly with bicu differentculline; HV, from hyperammonemic control rats are indicated rats with byvehicle; asterisks. HB, hyperammonemic* ratsp < treated 0.05, ** withp < 0.01,bicuculline. *** p < 0.001. Values significantly different from HV rats are indicated by a. a p < 0.05, aa, p < 0.01. And significantly different from CB are indicated by b. bb p < 0.01. CV, control vehicle; CB, controlWe also treated analyzed with bicuculline; the astrocyte HV, activation hyperammonemic in the cerebellum rats with vehicle; through HB, immunohisto- hyperammonemicchemistry rats treated with with GFAP. bicuculline. In Figure 3a, an increase of GFAP staining in cerebellum of hyperammonemic rats is shown, whereas bicuculline treatment reversed this increase. Quanti- We also analyzedficationthe of the astrocyte GFAP-stained activation area in (Figure the cerebellum 3b) showed through that it was immunohisto- significantly increased in chemistry with GFAP.hyperammonemic In Figure3a, rats an increase(26 ± 0.6 µm of GFAP2, p < 0.001) staining compared in cerebellum to controls of hyper-(22 ± 0.4 µm2) but not ammonemic ratsin is control shown, or whereas hyperammonemic bicuculline treatmentrats treated reversed with bicuculline this increase. (23 ± Quantifi- 0.6 and 23 ± 0.6 µm2, cation of the GFAP-stainedrespectively, area p < (Figure 0.01 compared3b) showed with that hyperammonemia it was significantly without increased bicuculline) in (Welch hyperammonemic rats (26 ± 0.6 µm2, p < 0.001) compared to controls (22 ± 0.4 µm2) but not in control or hyperammonemic rats treated with bicuculline (23 ± 0.6 and 23 ± 0.6 µm2, respectively, p < 0.01 compared with hyperammonemia without bicuculline) (Welch ANOVA

test: F (3.0, 336.6) = 11.17, p < 0.0001), indicating that bicuculline reversed astrocyte activation in the cerebellum of hyperammonemic rats. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 19

Int. J. Mol. Sci. 2021, 22, 6772 ANOVA test: F (3.0, 336.6) = 11.17, p < 0.0001), indicating that bicuculline reversed6 of 18 astrocyte activation in the cerebellum of hyperammonemic rats.

Figure 3. Effect of bicuculline on astrocyte activation in white matter of the cerebellum. Astrocytes Figure 3. Effect of bicuculline on astrocyte activation in white matter of the cerebellum. Astrocytes were stained with anti-GFAP. Representative images are shown in (a). The GFAP-stained area was were stained with anti-GFAP. Representative images are shown in (a). The GFAP-stained area was ± quantifiedquantified (b ().b). Data Data are are the the mean mean SEM± SEM of of four four rats rats per per group. group. Values Values significantly significantly different different from from controlcontrol rats rats are are indicated indicated by by asterisks. asterisks. *** ***p p< < 0.001. 0.001.Values Values significantly significantly different different from from hyperam- hyperammo- monemicnemic rats rats are are indicated indicated by by “aa”, pp << 0.01.0.01.CV, CV, control control vehicle; vehicle; CB, CB, control control treated treated with with bicuculline; bicuculline; HV,HV, hyperammonemic hyperammonemic rats rats with with vehicle; vehicle; HB, HB, hyperammonemic hyperammonemic rats treatedrats treated with bicuculline.with bicuculline.

In order to understand whether bicuculline can restore the increase of pro-inflammatory In order to understand whether bicuculline can restore the increase of pro-inflamma- cytokines in the cerebellum of hyperammonemic rats, we analyzed the content of IL-1β tory cytokines in the cerebellum of hyperammonemic rats, we analyzed the content of IL- and of TNFα. 1β and of TNFα. IL-1β levels were assessed by immunohistochemistry in the white matter of the cerebellum (FigureIL-1β 4levelsa). Quantification were assessed of cells by expressingimmunohistochemistry IL-1 β showed in that the the white number matter of cells of ex-the cer- pressingebellum IL-1 (Figureβ was 4a). significantly Quantification increased of cells in hyperammonemic expressing IL-1 ratsβ showed (1005 ± that29 cells/mm the number2, of pcells< 0.0001) expressing compared IL-1 withβ was controls significantly (807 ± 31increased cells/mm in2 ).hyperammonemic Bicuculline treatment rats signifi- (1005 ± 29 cantlycells/mm2, reversed p < IL-10.0001)β expression compared in with hyperammonemic controls (807 rats± 31 (794 cells/mm2).± 41 cells/mm Bicuculline2, p < 0.001,treatment comparedsignificantly with reversed hyperammonemia IL-1β expression without in bicuculline), hyperammonemi confirmingc rats the(794 results ± 41 cells/mm2, obtained p < by0.001, Western compared blot (Figure with 4hyperammonemiab). The parameters without for the Brown–Forsythe bicuculline), confirming ANOVA testthe results were ob- Ftained (3.0, 178.7) by Western = 17.54, blotp < 0.0001.(Figure To 4b). confirm The parameters expression offor IL-1 theβ Brown–Forsythein microglia or astrocytes, ANOVA test wewere performed F (3.0, 178.7) a double = 17.54, immunofluorescence p < 0.0001. To confirm of IL-1β expressionwith GFAP of or IL-1 Iba1β ininhyperammone- microglia or astro- miccytes, rats. we IL-1 performedβ was expressed a double in immunofluorescence both astrocytes and in of microglial IL-1β with cells GFAP (Figure or4 Iba1c,d). Thein hyper- totalammonemic content of rats. IL-1 βIL-1assessedβ was byexpressed Western in blot both was astrocytes significantly and increased in microglial in the cells cerebel- (Figure ± lum4c,d). of The hyperammonemic total content of rats IL-1 (160β assessed14%of by controls, Westernp blot< 0.01). was Bicucullinesignificantly treatment increased in did not alter the IL-1β content in control rats but significantly reversed the increase in the cerebellum of hyperammonemic rats (160 ± 14% of controls, p < 0.01). Bicuculline treat- hyperammonemic rats (97 ± 9% of controls, p < 0.01 compared with hyperammonemia ment did not alter the IL-1β content in control rats but significantly reversed the increase without bicuculline) (F (3, 46) = 8.695), p < 0.0001) (Figure4e). in hyperammonemicThe expression of TNFrats α(97was ± 9% also of assessed controls, by p Western< 0.01 compared blot and immunohistochem-with hyperammonemia istrywithout in white bicuculline) matter of(F cerebellum.(3, 46) = 8.695), The p < number 0.0001) of(Figure cells expressing4e). TNFα increased in hyperammonemic rats treated with bicuculline or not (141 ± 7 cells/mm2 in HA, p < 0.05 and 154 ± 9 cells/mm2 in HA + BIC, p < 0.01) compared with control rats (113 ± 8 cells/mm2). In addition, bicuculline treatment also increased TNFα expression in control rats (139 ± 5 cells/mm2, p < 0.05) (F (3, 145) = 4.792, p < 0.01) (Figure5a,b). However, transmembrane TNFα (26 kd isoform) content, analyzed by Western blotting, was significantly increased in hyperammonemic rats (141 ± 10% of controls, p < 0.05), but bicuculline treatment decreased its content to control values (84 ± 10% of controls, p < 0.01, compared with hyperammonemia without bicuculline). The content of transmembrane TNFα (26 KD) was not altered in control rats treated with bicuculline (99 ± 11% of controls) (F (3, 37) = 5.378, p < 0.01) (Figure5c). Membrane expression of TNFR1 increased in the cerebellum of hyperammonemic rats, but bicuculline did not restore it (Figure5d) ( 188 ± 26%

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of controls, p < 0.05 for hyperammonemic rats and 208 ± 27% of controls, p < 0.01 for hyperammonemic rats treated with bicuculline). In addition, bicuculline also increased the Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 19 membrane expression of TNFR1 in control rats (172 ± 26% of controls) (F (3, 43) = 4.956, p < 0.01) (Figure5d).

Figure 4. EffectFigure of 4.bicucullineEffect of bicucullineon the content on the of contentIL1β in ofwhite IL1 βmatterin white of the matter cerebellu of them. cerebellum. Immunohistochemistry Immunohis- was performed usingtochemistry antibody against was performed IL-1β. Representative using antibody images against of IL-1 IL-1ββ. Representativestaining in white images matter of of IL-1 theβ cerebellumstaining in are shown (a). The numberwhite of matter cells ofexpressing the cerebellum IL-1β arewas shown quantified. (a). The Data number are the of cellsmean expressing ± SEM of IL-13–4β ratswas per quantified. group (b). Double immunofluorescenceData are the of meanIL-1β± andSEM Iba1 of 3–4is shown rats per in group(c) and (b of). DoubleIL-1β and immunofluorescence GFAP in (d), confirming of IL-1 βtheand expression Iba1 is of IL-1β in both microgliashown and in (c astr) andocytes. of IL-1 Theβ and content GFAP of in IL-1 (d),β confirmingwas also analyzed the expression by Western of IL-1 blotβ in in both the microgliatotal cerebellum. and Values are mean ± astrocytes.SEM of 12–14 The samples content per of IL-1groupβ was (e). Values also analyzed significantly by Western different blot from in the control total cerebellum.rats are indicated Values by asterisks: * p < 0.05, **are p < mean 0.01, ±****SEM p < 0.0001. of 12–14 Values samples significantly per group different (e). Values from significantly hyperammonemic different rats from are control indicated rats by “aa”, p < 0.01, “aaa”, p < 0.001. CV, control vehicle; CB, control treated with bicuculline; HV, hyperammonemic rats with vehicle; are indicated by asterisks: * p < 0.05, ** p < 0.01, **** p < 0.0001. Values significantly different from HB, hyperammonemic rats treated with bicuculline. hyperammonemic rats are indicated by “aa”, p < 0.01, “aaa”, p < 0.001. CV, control vehicle; CB, control treated with bicuculline;The expression HV, hyperammonemic of TNFα was also rats withassessed vehicle; by HB, Western hyperammonemic blot and immunohistochem- rats treated with bicuculline.istry in white matter of cerebellum. The number of cells expressing TNFα increased in 2 Previously,hyperammonemic we reported an association rats treated between with bi neuroinflammationcuculline or not (141 and ± the 7 cells/mm membrane in HA, p < 0.05 2 2 expression of glutamateand 154 ± and9 cells/mm GABA transportersin HA + BIC, in p < the 0.01) cerebella compared of hyperammonemic with control rats (113 rats. ± 8 cells/mm ). In hyperammonemicIn addition, rats, membrane bicuculline expression treatment of also glutamate increased transporters TNFα expression was significantly in control rats (139 ± 5 2 decreased (66 cells/mm± 8%, p <, p 0.05 < 0.05) of controls(F (3, 145) for = GLT-14.792, andp < 0.01) 48 ± (Figure7% of 5a,b). controls, However,p < 0.01 transmembrane for GLAST) (FigureTNFα6 a,b)(26 kd according isoform) to content, previous analyzed results. by Bicuculline Western blotting, restored was membrane significantly increased expression of GLASTin hyperammonemic in hyperammonemic rats (141 rats ± (107 10%± of14% controls, of controls, p < 0.05),p < 0.01 but with bicuculline respect treatment de- to hyperammonemiacreased withoutits content bicuculline) to control butvalues not (84 those ± 10% of GLT-1 of controls, (63 ± p10% < 0.01, ofcontrols, compared with hyper- p < 0.01) (Figureammonemia6a,b). Statistical without analysis bicuculline). with one-way The content ANOVA of transmembrane showed the following TNFα (26 KD) was not parameters: GLT-1,altered F (3,in control 47) = 5.468, rats treatedp < 0.01, with and bicuculline GLAST, F (3,(99 24)± 11% = 7.854, of controls)p < 0.001. (F (3, In 37) = 5.378, p < addition, GABA0.01) transporters (Figure 5c). were Membrane also altered. expression Membrane of expressionTNFR1 increased of GABA in transporterthe cerebellum of hyper- GAT-1 was significantlyammonemic decreased rats, but (66 bicuculline± 12% of did controls, not restorep < 0.05) it (Figure in hyperammonemic 5d) (188 ± 26% of controls, p < rats (Figure6c).0.05 Moreover, for hyperammonemic GAT-3 was significantly rats and 208 increased ± 27% of in controls, hyperammonemic p < 0.01 forrats hyperammonemic (161 ± 34% of controls,rats treatedp < with 0.05) bicuculline). (Figure6d); thisIn addition, was related bicuculline to a reversal also ofincreased its function, the membrane ex- indicating thatpression there was of a TNFR1 release in of control GABA rats through (172 this± 26% transporter of controls) instead (F (3, of43) an = 4.956, uptake. p < 0.01) (Figure Bicuculline effectively5d). decreased the membrane expression of GAT-3 (53 ± 12% of controls, p < 0.001) (Figure6d) but did not reverse the decrease of membrane expression of GAT-1

Int. J. Mol. Sci. 2021, 22, 6772 8 of 18

in hyperammonemic rats (51 ± 13% of controls, p < 0.01) (F (3, 38) = 7.598, p < 0.001). In addition, bicuculline also decreased membrane expression of GAT-1 in control rats (51 ± 6% of controls, p < 0.01) (F (3, 38) = 6.969, p < 0.001) (Figure6c). Both alterations led to increased extracellular GABA (59 ± 13 nM, p < 0.05, compared with 14.4 ± 6 nM in control rats) (Figure6e). The extracellular concentration of GABA was decreased by bicuculline in hyperammonemic rats (17 ± 7% nM, p < 0.05 compared with hyperammonemia without bicuculline) (F (3, 21) = 4.329, p < 0.05) (Figure6e), surely due to a reversal of the increase of GAT-3 membrane expression, as GAT-1 remained decreased in hyperammonemic rats Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 19 treated with bicuculline. An increase in extracellular GABA, but not a signiﬁcant one, was observed in control rats treated with bicuculline.

Figure 5Figure. Effect 5.of Effectbicuculline of bicuculline on the content on the of contentTNFα and of TNFmembraneα and membraneexpression of expression TNFR1 in ofthe TNFR1 cerebellum. in the Immuno- histochemistrycerebellum. was performed Immunohistochemistry using the antibody was performed against TNF usingα. Representative the antibody against images TNF of TNFα. Representativeα staining in the white matter ofimages the cerebellum of TNFα arestaining shown in ( thea). The white number matter of cells of the expressing cerebellum TNF areα was shown quantified. in (a). Data The numberare the mean of ± SEM of three ratscells per expressing group (b). TNF Theα contentwas quantified. of TNFα was Data also are analyzed the mean by Western± SEM ofblot three in the rats total per cerebellum group (b). (c The), along with the membrane expression of TNFR1 (d). Values are the mean ± SEM of 10–12 samples per group. Values significantly content of TNFα was also analyzed by Western blot in the total cerebellum (c), along with the different from control rats are indicated by asterisks: * p < 0.05, ** p < 0.01. Values significantly different from hyperammonemic ratsmembrane are indicated expression by “aa”, of p TNFR1< 0.01. CV, (d). control Values vehicle; are the meanCB, control± SEM treated of 10–12 with samples bicuculline; per group.HV, hyperammonemic Values rats withsignificantly vehicle; HB, hyperammon different fromemic control rats treated rats are with indicated bicuculline. by asterisks: * p < 0.05, ** p < 0.01. Values significantly different from hyperammonemic rats are indicated by “aa”, p < 0.01. CV, control vehicle; CB, control treated withPreviously, bicuculline; HV,we reported hyperammonemic an association rats with between vehicle; neuroinflammation HB, hyperammonemic and the mem- rats treated with bicuculline.brane expression of glutamate and GABA transporters in the cerebella of hyperammonemic rats. In hyperammonemic rats, membrane expression of glutamate transporters was As increasedsignificantly extracellular decreased GABA (66 and ± activation8%, p < 0.05 of of GABA controlsA receptors for GLT-1 induce and 48motor ± 7% of controls, p incoordination, we< 0.01 confirmed for GLAST) that (Figure blocking 6a,b) GABA accordingA receptors to previous with results. bicuculline Bicuculline adminis- restored mem- tration improvedbrane motor expression coordination of GLAST in hyperammonemic in hyperammonemic rats. rats As (107 shown ± 14% inof Figurecontrols,7, p < 0.01 with hyperammonemicrespect rats showed to hyperammonemia higher number without of slips (1.7bicuculline)± 0.2 slips, butp not< 0.01) those than of GLT-1 control (63 ± 10% of rats (1.1 ± 0.1 slips).controls, The numberp < 0.01) of (Figure slips was 6a,b). significantly Statistical analysis decreased with in hyperammonemicone-way ANOVA showed the rats treated withfollowing bicuculline parameters: (0.8 ± 0.2, GLT-1,p < 0.01, F (3, compared47) = 5.468, with p < 0.01, hyperammonemic and GLAST, F (3, rats 24) = 7.854, p < without bicuculline)0.001. (F In (3, addition, 76) = 4.116, GABAp < 0.01).transporters As indicated were also by thealtered. increase Membrane in extracellular expression of GABA GABA in the cerebellum,transporter some GAT-1 motor was significantly incoordination decreased (but not(66 ± a 12% significant of controls, effect) p < 0.05) was in hyperam- observed in controlmonemic rats treated rats (Figure with bicuculline 6c). Moreover, (Figure GAT-37). was significantly increased in hyperammo- A schematicnemic summary rats (161 of the ± 34% main of findingscontrols, inp < control 0.05) (Figure (a) and 6d); hyperammonemic this was related to rats a reversal of its (b) is shown in Figurefunction,8. indicating that there was a release of GABA through this transporter instead of an uptake. Bicuculline effectively decreased the membrane expression of GAT-3 (53 ± 12% of controls, p < 0.001) (Figure 6d) but did not reverse the decrease of membrane expression of GAT-1 in hyperammonemic rats (51 ± 13% of controls, p < 0.01) (F (3, 38) = 7.598, p < 0.001). In addition, bicuculline also decreased membrane expression of GAT-1 in control rats (51 ± 6% of controls, p < 0.01) (F (3, 38) = 6.969, p < 0.001) (Figure 6c). Both alterations led to increased extracellular GABA (59 ± 13 nM, p < 0.05, compared with 14.4 ± 6 nM in control rats) (Figure 6e). The extracellular concentration of GABA was decreased by bicuculline in hyperammonemic rats (17 ± 7% nM, p < 0.05 compared with hyperammonemia without bicuculline) (F (3, 21) = 4.329, p < 0.05) (Figure 6e), surely due to a reversal of the

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increase of GAT-3 membrane expression, as GAT-1 remained decreased in hyperammo- Int. J. Mol. Sci. 2021, 22, 6772 nemic rats treated with bicuculline. An increase in extracellular GABA, but not9 a of signifi- 18 cant one, was observed in control rats treated with bicuculline.

FigureFigure 6. Effect ofof bicuculline bicuculline on on membrane membrane expression expressi ofon glutamate of glutamate and GABA and GABA transporters transporters and on and onextracellular extracellular GABA GABA content. content. Membrane Membrane expression expression of glutamate of glutamate transporters transporters GLT-1 (a) and GLT-1 GLAST (a) and GLAST(b) and of(b GABA) and of transporters GABA transporters GAT-1 (c) and GAT-1 GAT-3 (c ()d )and in the GAT-3 cerebellum (d) in was the analyzedcerebellum using was the analyzed BS3 usingcrosslinker the BS3 procedure, crosslinker as describedprocedure, in as the described Methods in section. the Methods Representative section. images Representative are shown. images The are shown.extracellular The extracellular concentration concentration of GABA was of measured GABA was by HPLC-MSmeasured inby samples HPLC-MS obtained in samples by in vivoobtained microdialysis in the cerebellum (e). Values are expressed as percentage of control rats and are the Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWby in vivo microdialysis in the cerebellum (e). Values are expressed as percentage of control10 rats of 19 andmean are± theSEM mean of 8–12 ± SEM rats of per 8–12 group. ratsValues per group. signiﬁcantly Values differentsignificantly from different control rats from are control indicated rats are indicatedby asterisks: by asterisks: *p < 0.05, ***pp << 0.05, 0.01 ** and p < from 0.01 hyperammonemicand from hyperammonemic rats by “a”: rats a, p by< 0.05; “a”: aa,a, pp << 0.05; 0.01; aa, p

As increased extracellular GABA and activation of GABAA receptors induce motor incoordination, we confirmed that blocking GABAA receptors with bicuculline administration improved motor coordination in hyperammonemic rats. As shown in Figure 7, hyperammonemic rats showed higher number of slips (1.7 ± 0.2 slips, p < 0.01) than control rats (1.1 ± 0.1 slips). The number of slips was significantly decreased in hyperammonemic

rats treated(Slips) with bicuculline (0.8 ± 0.2, p < 0.01, compared with hyperammonemic rats without bicuculline) (F (3, 76) = 4.116, p < 0.01). As indicated by the increase in extracellular GABA in the cerebellum, some motor incoordination (but not a significant effect) was ob- servedincoordination Motor in control rats treated with bicuculline (Figure 7).

Figure 7. Effects of bicuculline on motor coordination with the beam walking test. Motor coordination Figurewas assessed 7. Effects with of thebicuculline beam walking on motor test ascoordination described in with the Methods the beam section. walking The test. graph Motor shows coordina- tionMean was±SEM assessed of 12 ratswith per the group. beam Valueswalking signiﬁcantly test as described different in from the Methods control rats section. are indicated The graph by shows Mean±SEMasterisks and of from12 rats hyperammonemic per group. Values rats by significantly “a”, * p < 0.05; different aa, p < 0.01. from control rats are indicated by asterisks and from hyperammonemic rats by “a”, * p < 0.05; aa, p < 0.01.

A schematic summary of the main findings in control (a) and hyperammonemic rats (b) is shown in Figure 8.

(a)

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(Slips) Motor incoordination Motor

Figure 7. Effects of bicuculline on motor coordination with the beam walking test. Motor coordination was assessed with the beam walking test as described in the Methods section. The graph shows Mean±SEM of 12 rats per group. Values significantly different from control rats are indicated by Int. J. Mol. Sci. 2021, 22, 6772 asterisks and from hyperammonemic rats by “a”, * p < 0.05; aa, p < 0.01. 10 of 18

A schematic summary of the main findings in control (a) and hyperammonemic rats (b) is shown in Figure 8.

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(a)

(b)

Figure 8. Scheme showing the effects of blocking GABAA receptors with bicuculline on peripheral and cerebellar neuroin- Figure 8. Schemeflammation showing and neurotransmission. the effects of blocking(a) Control rats. GABA ChronicA receptors treatment with with bicuculline bicuculline induced a on peripheral peripheral pro-inflam- and cerebellar neu- roinflammationmatory and environment neurotransmission. shown by an increase (a) Control in IL-6 rats.and a decr Chronicease in treatmentIL-10. In the cerebellum, with bicuculline bicuculline inducedalso induced a peripheral pro- neuroinflammation, increasing TNFα levels and inducing NFkB in activated microglia. In addition, GABA neurotrans- inflammatory environmentmission was also shownimpaired by by andecreasing increase the inGAT-1 IL-6 membrane and a decrease expression, in which IL-10. led Into a the weak cerebellum, increase in extracellular bicuculline also induced neuroinflammation,GABA and increasing motor incoordination. TNFα levels (b) Hyperammonemic and inducing rats. NFkB In hyperammonemic in activated microglia.rats, bicuculline In prevented addition, the GABAincrease neurotransmis- of the pro-inflammatory cytokines IL-6 and TNFα, increased IL-10, and decreased TGF-β levels in plasma, improving sion was also impairedperipheral inflammation. by decreasing In the the cerebell GAT-1um, membranebicuculline induced expression, a change whichin the ph ledenotype to a of weak microglia increase to a more in extracellularanti- GABA and motor incoordination.inflammatory state, (b) M2, Hyperammonemic decreasing NFkB nuclear rats. localization, In hyperammonemic but did not affect rats,the increased bicuculline membrane prevented expression the of increase of the TNFR1. Bicuculline prevented astrocyteα activation and decreased the content of the pro-inflammatoryβ cytokines TNFα pro-inflammatoryand IL-1 cytokinesβ. Bicuculline IL-6 restored and TNF the membrane, increased expression IL-10, of andGLAST decreased and decreased TGF- the membranelevels in expression plasma, of improvingGAT-3 peripheral inflammation.without In the any cerebellum, effect on GLT1 bicuculline and GAT1 tran inducedsporters. a Even change so, extracellular in the phenotype levels of GABA of microgliawere also returned to a more to control anti-inflammatory levels by bicuculline. This suggests that bicuculline should mainly affect peripheral inflammation and astrocyte activation, state, M2, decreasingpossibly NFkBthrough nuclear a direct effect localization, on GABAA butreceptor dids in not peripheral affect the immune increased cells and membrane in astrocytes. expression of TNFR1. Bicuculline prevented astrocyte activation and decreased the content of the pro-inflammatory cytokines TNFα and IL-1β. Bicuculline restored the membrane expression of3. GLASTDiscussion and decreased the membrane expression of GAT-3 without any effect on GLT1 and GAT1 transporters. Even so, extracellularWe analyzed levels the effects of GABA of blocking were GABA also returnedA receptors to with control the chronic levels systemic by bicuculline. ad- This ministration of bicuculline on inflammatory markers in blood and in the cerebellum, both suggests that bicuculline should mainlyin control affect and peripheral in hyperammonemic inflammation rats, the and last astrocyte showing peripheral activation, inflammation possibly through and a direct effect on GABAA receptors in peripheralneuroinflammation. immune cells and in astrocytes. Hyperammonemic rats showed increased extracellular GABA in the cerebellum, which increased the activation of GABA receptors, both GABAA and GABAB. In this work, we analyzed only the effect of blocking GABAA, but effects of GABAB receptors activation on inflammation are also reported in the literature [24–26]. Therefore, future research can address the role of GABAB receptors on inflammation and neuroinflammation in hyperammonemic rats. It is widely reported that GABAA activation has anti-inflammatory effects in some pathological situations [16,27–30]. We found that the chronic inhibition of the GABAA re-

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3. Discussion

We analyzed the effects of blocking GABAA receptors with the chronic systemic administration of bicuculline on inflammatory markers in blood and in the cerebellum, both in control and in hyperammonemic rats, the last showing peripheral inflammation and neuroinflammation. Hyperammonemic rats showed increased extracellular GABA in the cerebellum, which increased the activation of GABA receptors, both GABAA and GABAB. In this work, we analyzed only the effect of blocking GABAA, but effects of GABAB receptors Int. J. Mol. Sci. 2021, 22, x FOR PEERactivation REVIEW on inflammation are also reported in the literature [24–26]. Therefore, future 12 of 19 research can address the role of GABAB receptors on inflammation and neuroinflammation in hyperammonemic rats. It is widely reported that GABAA activation has anti-inflammatory effects in some pathologicalceptor in control situations rats [induced16,27–30 ].slight We foundperipheral that the inflammation, chronic inhibition increasing of the IL-6 GABA andA decreas- receptoring IL-10. in controlIn addition, rats induced bicuculline slight also peripheral induced inflammation, microglial activation, increasing IL-6NFkB and nuclear de- local- creasingization, IL-10. and an In increase addition, in bicuculline the memb alsorane induced expression microglial of TNFR1 activation, and NFkBin TNF nuclearα in the white localization,matter of the and cerebellum an increase from in the control membrane rats expression(Figure 9). of This TNFR1 suggests and in that, TNF αinin physiological the white matter of the cerebellum from control rats (Figure9). This suggests that, in phys- conditions, not only in inflammatory pathologies but also the GABA system modulate the iological conditions, not only in inflammatory pathologies but also the GABA system modulateimmune thesystem, immune as was system, shown as was in shown[31], where in [31 ],an where increase an increase in TNF inα TNFin naïveα in naïve macrophages macrophagesexposed to picrotoxin exposed to picrotoxinwas found. was found.

FigureFigure 9. 9.Scheme Scheme showing showing the experimentalthe experimental design. design. However, bicuculline improved inflammatory parameters in hyperammonemic rats, decreasingHowever, IL-6 and bicuculline TNFα and improved increasing inflammatory IL-10 levels in parameters the plasma. Ain similarhyperammonemic effect of rats, intracisternaldecreasing administrationIL-6 and TNF ofα and bicuculline increasing was found IL-10 by levels Sallam in etthe al. plasma. [18], which A showedsimilar effect of aintracisternal decrease in serum administration levels of IL-6 of and bicuculline TNFα in ratswas injected found by intraventricularly Sallam et al. [18], with which LPS showed aftera decrease bicuculline in serum injection. levels These of dataIL-6 suggestand TNF thatα thein rats effect injected of bicuculline intraventricularly on peripheral with LPS inflammationafter bicuculline could injection. be due to itsThes effecte data on brain suggest receptors. that the effect of bicuculline on peripheral inflammationThe inflammatory could effectsbe due of to GABA its effect and on anti-inflammatory brain receptors. effects of GABAA receptor inhibition were also observed previously by other authors in hepatic injury [32], endotox- The inflammatory effects of GABA and anti-inflammatory effects of GABAA receptor emia [18], or autoimmune encephalomyelitis [17]. Different mechanisms for the opposed inhibition were also observed previously by other authors in hepatic injury [32], endotox- function of GABA on different inflammatory aspects were proposed. Carmans et al. [17] suggestedemia [18], that or autoimmune endogenous GABA encephalomyelitis improves inflammation, [17]. Different whereas mechanisms exogenous for GABA the opposed enhancesfunction it,of inGABA a model on ofdifferent autoimmune inflammatory encephalomyelitis. aspects were Lee proposed. et al. [33] reported Carmans an et al. [17] enhancementsuggested that of inflammatoryendogenous hypersensitivityGABA improves by inflammation, GABA through whereas peripheral exogenous GABAA GABA receptorenhances activation. it, in a model It was suggestedof autoimmune that this enceph dual actionalomyelitis. of GABA ALeereceptors et al. [33] was reported due to an en- differenthancement subunit of inflammatory composition in hypersensiti stress-inducedvity gut by inflammation GABA through [34]. peripheral GABAA receptor Carmansactivation. et al.It was [17] showedsuggested that that systemic this dual GABA action induced of GABA proliferationA receptors of T cells was and due to dif- increasedferent subunit IFNγ, whereascomposition it decreased in stress-induced IL-4 levels in gut splenocytes, inflammation inducing [34]. a Th1 response. We can suggest that bicuculline can inhibit the GABA induction of Th1, decreasing the ratio Carmans et al. [17] showed that systemic GABA induced proliferation of T cells and Th1/Th2. Th2 production of IL-10 and TGFβ may induce M2 phenotype in macrophages, contributingincreased IFN to theγ, whereas decrease init TNFdecreasedα production. IL-4 levels in splenocytes, inducing a Th1 response. We Oncan the suggest other hand, that thebicuculline activation can of regulatory inhibit the T-lymphocytes GABA induction (T-regs) of also Th1, increased decreasing the bothratio IL-10 Th1/Th2. and TGF- Th2β [ 35production,36]. Our results of IL-10 showed and a TGF clearβ increase may induce in TGF- M2β when phenotype IL-10 was in macrophages, contributing to the decrease in TNFα production. On the other hand, the activation of regulatory T-lymphocytes (T-regs) also increased both IL-10 and TGF-β [35,36]. Our results showed a clear increase in TGF-β when IL-10 was decreased in hyperammonemic rats, suggesting that the increase in TGF-β can be induced from a different source than the activation of T-regs. A possible explanation for this situation is that, since liver damage is an important source of TGF-β [37], and hyperammonemia is thought to impair liver function [38,39], this can contribute to the increase in TGF-β in plasma. In hyperammonemic rats, both cytokines increased after four weeks of chronic treatment with bicuculline, suggesting that bicuculline could activate T-regs in these rats, thereby inducing an increase in both IL-10 and TGF-β. Augmented levels of these anti-inflammatory cytokines may lead to the decrease in TNFα. The increase in IL- 10 and the decrease in TNFα serum levels by bicuculline were also reported by Hsu et al. [32]. These authors showed a beneficial effect of bicuculline treatment on hepatic injury in a model with sepsis and suggested that this effect should be mediated by modulation of cytokine production, which agrees with the results presented here.

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decreased in hyperammonemic rats, suggesting that the increase in TGF-β can be induced from a different source than the activation of T-regs. A possible explanation for this situation is that, since liver damage is an important source of TGF-β [37], and hyperammonemia is thought to impair liver function [38,39], this can contribute to the increase in TGF-β in plasma. In hyperammonemic rats, both cytokines increased after four weeks of chronic treatment with bicuculline, suggesting that bicuculline could activate T-regs in these rats, thereby inducing an increase in both IL-10 and TGF-β. Augmented levels of these anti- inflammatory cytokines may lead to the decrease in TNFα. The increase in IL-10 and the decrease in TNFα serum levels by bicuculline were also reported by Hsu et al. [32]. These authors showed a beneficial effect of bicuculline treatment on hepatic injury in a model with sepsis and suggested that this effect should be mediated by modulation of cytokine production, which agrees with the results presented here. In the cerebellum, the chronic administration of bicuculline decreased astrocyte activation, induced polarization of microglia to an anti-inflammatory phenotype M2, and decreased IL-1β content (Figure8b). The effect of bicuculline on peripheral cytokines could contribute to the decrease in cerebellar neuroinflammation, as neuroinflammation is due to peripheral inflammation. Blocking peripheral TNFα signaling with infliximab prevented neuroinflammation, preventing microglia and astrocyte activation and the increase in IL-1β [5,40]. However, we cannot ignore that a direct effect of bicuculline on cerebellar receptors can also lead to inhibition of astrocyte activation as well as to the decrease in IL-1β and the change in microglia phenotype in hyperammonemic rats. A direct effect of GABA on astrocyte morphology increasing branching and GFAP expression was reported [21]. Therefore, we can suppose that the observed effect of bicuculline on astrocyte activation can be mediated, at least partially, by direct decrease of GABA signaling. Bicuculline does not restore TNFα content in the white matter, but its levels were decreased in the total cerebella of hyperammonemic rats treated with bicuculline. This discrepancy can be explained by the decrease in peripheral TNFα or in the content of TNFα in other cerebellar layers but not of TNFα expression in white matter, as the whole cerebellum, including endogenous capillaries, was analyzed by Western blot. A complex role of GABAA in macrophage polarization was also reported [31]. These authors showed that inflammation induced by LPS also modulated the GABA system in macrophages, showing a bidirectional modulation between GABA signaling and immune cells function. Our results also suggest an effect of GABAA on microglia phenotype and a possible effect of microglia on GABA system, as bicuculline activated microglia in control rats and increased GABA extracellular levels by decreased membrane expression of GAT-1, whereas, in hyperammonemic rats showing neuroinflammation, bicuculline induced M2 phenotype in microglia and decreased GABA extracellular levels, decreasing membrane expression of GAT-3 and astrocyte activation. Bicuculline restored the membrane expression of GLAST and GAT3 but not of GLT-1 and GAT1. This suggests that the membrane expression of GLAST and GAT3 should be modulated by astrocyte activation and IL1β increase, whereas the membrane expression of GAT1 and GLT1 should be only modulated by TNFα or directly modulated by GABAA inhibition by bicuculline with a small neuroinflammation effect. This observation is in accordance with the effect of sulforaphane in the cerebellum of hyperammonemic rats, since sulforaphane also inhibited the increase in membrane expression of GAT3, the activation of astrocytes, and induced a change in the microglia phenotype from M1 to M2, leading to an improvement in motor coordination. Sulforaphane also improved learning ability in the Y maze, as was previously reported for bicuculline chronic treatment [7]. This suggests that future research should analyze some possible anti-oxidant effects of bicuculline as well as the effect of bicuculline on the Nrf2 signaling pathway, as sulforaphane exerts its effect through this pathway. Kochiyama et al. [41] showed the modulation of Nrf2 by GABAA receptors. It was shown that the activation of TNFR1 induced NFkB nuclear translocation in hyperammonemic rats [6]. Bicuculline did not prevent the membrane expression of TNFR1 but it prevented NFkB nuclear localization, indicating that its effect Int. J. Mol. Sci. 2021, 22, 6772 13 of 18

could be mediated by the inhibition of a downstream step. Ferrari et al. [42] reported that Nrf2 activation prevented NFkB activation by TNFα [42]. Therefore, this may also be the possible mechanism of inhibition of NFkB by bicuculline. Our results also show that decreasing the peripheral levels of TNFα in chronic hyperammonemia did not prevent all the effects on cerebellar neuroinflammation and neurotransmission but should be necessary to prevent the initial increase of TNFα in plasma, as, in this work, bicuculline treatment started after one week of hyperammonemia, when cerebellar neuroinflammation was already observed [5], whereas, in our previous works, Infliximab treatment started before hyperammonemic diet [5,40]. Our results suggest that bicuculline-induced peripheral inflammation, microglial activation in the cerebellum, and a decrease in the membrane expression of GAT-1, leading to a small increase in extracellular GABA, were not sufficient to lead to significant impairment of motor coordination in control rats (Figure8a). However, in hyperammonemic rats, which also showed astrocyte activation and decrease in GLAST and GLT1 and increase in GAT-3 membrane expression, extracellular GABA levels were higher, leading to motor coordination impairment (Figure8b). We want to point out that bicuculline treatment, by blocking GABAA receptors, can directly improve motor incoordination in hyperammonemic rats, but our results show that, in addition, bicuculline decreased extracellular GABA levels via modulation of the membrane expression of GABA transporters.

4. Materials and Methods Study design: chronic hyperammonemia in rats and treatment with bicuculline. Male Wistar rats (120–140 g, Charles River Laboratories, Barcelona, Spain) were made hyperammonemic by feeding them an ammonium-containing diet (ammonium acetate 30%), as in [43]. Animals were distributed into four groups: control with vehicle (CV); control treated with bicuculline (CB); hyperammonemic rats (HA); hyperammonemic rats treated with bicuculline (HB). Bicuculline ((+)-Bicuculline, Sigma-Aldrich, Saint Louis, MI, USA) was injected intraperitoneally, 0.3 mg/kg, once per day. The dose was chosen based on a previous study of the use of bicuculline in hyperammonemic rats [7]. Bicuculline was dissolved in physiological serum (NaCl 0.9%) with 0.3% DMSO, and this solution was used as vehicle. The experiment was repeated four times, and nine animals per group were used in each experiment (a total of 36 rats per group). The experimental design is summarized in Figure9. The experiments were approved by the Comit é de Ética y Experimentación Ani- mal, Prince Felipe Research Center-Consellería de Agricultura, Generalitat Valenciana and were carried out in accordance with the Directive of the European Commission (2010/63/EU) for care and management of experimental animals and complied with the ARRIVE guidelines for animal research. Measurement of ammonia and cytokines in plasma. Blood was collected from the saphe- nous vein on days 11, 25, and 36 after starting the hyperammonemic diet. For plasma preparation, blood was collected in EDTA-containing vials, centrifuged at 6000× g for 5 min, and stored at −80 ◦C. Twenty microliters of blood were immediately used to measure ammonia with the Ammonia Test Kit II for the PocketChemBA system (Arkay, Inc., Kyoto, Japan). TNFα in plasma was measured using an ELISA Kit from eBioscience (San Diego, CA, USA). The content of all other cytokines in plasma was analyzed by Western blot using primary antibodies against IL-10, TGF-β (1:1000) from Abcam (Cambridge, UK), and IL-6 (1:500) from MyBioSource (San Diego, CA, USA). Secondary antibodies were anti-rabbit or anti-mouse (1:4000) IgG, conjugated with alkaline phosphatase (Sigma). The images were captured using a Hewlett Packard (San José, CA, USA) ScanJet 5300C, and the band intensities were quantiﬁed using the AlphaImager 2200 software (AInnotech Corporation, San Francisco, CA, USA). Int. J. Mol. Sci. 2021, 22, 6772 14 of 18

Brain immunohistochemistry and immunofluorescence. At week 5 of hyperammonemia, the rats were anaesthetized with sodium pentobarbital and transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4). Brains were removed and post-fixed in the same fixative solution for 24 h at 4 ◦C. Paraffin- embedded sections (5 µm) were cut and mounted on coated glass slides. The tissue sections were then processed with the Envision Flex + kit (DAKO, Santa Clara, CA, USA), blocking endogenous peroxidase activity for 5 min, and then incubated with antibodies. Primary antibodies were against Iba-1 (Wako 019-19741; 1:300 for 30 min), Glial Fibrillary Acidic Protein (GFAP) (Dako IR524; ready for use for 20 min), IL-1β (Abcam AB9722; 1:100 dilution for 30 min), and TNFα (Abcam; 1:2000 for 45 min). The reaction was visualized by incubation with Envision Flex plus horseradish peroxidase for 20 min and, finally, diaminobenzidine for 10 min. Sections were counterstained with Mayer’s hematoxylin for 5 min. A double immunofluorescence was performed to confirm IL1b co-localization with microglia (using Iba1, 1:300, Abcam) and astrocytes (using GFAP, Sigma, 1:400). Analysis of microglial activation. Analysis of Iba-1-stained microglia was performed in the cerebellum white matter using ImageJ 1.48v software. Microglial activation was assessed by measuring the cell perimeter in eight randomly selected areas (0.45 mm2) per section, according to [44]. The area of interest was selected. Using Auto Local Threshold and Analyze particle functions in ImageJ, the intensity thresholds and the size filter were applied. To measure the perimeter of microglia, the Bernsen method was used, and a 2000–20,000 size filter was applied. For each rat, at least 30–40 cells were quantified, and the results were converted from pixels to micrometers. Analysis of astrocyte activation. The astrocytic area (µm2) covered by GFAP was measured using the ImageJ software. Using the Auto Local Threshold and the Analyze Particles functions, the intensity thresholds and the size filter were applied. To measure the total cell size, the Bernsen method was used, and a 1500–7000 size filter was applied. For each rat, at least 20 cells from three different sections were counted. Analysis of NF-κB p50 nuclear localization in microglia. An analysis of the expression of the p50 subunit of NF-κB in microglia was performed by double immunofluorescence. Sections from six different animals per group were selected, washed in 0.1 M phosphate buffer, and blocked with normal serum from the same species as the secondary antibody before being incubated overnight with a primary antibody (NF-κB p50, 1:200; Iba1, 1:300) from Abcam, diluted in blocking buffer and secondary fluorescent antibodies (1:400) from Invitrogen (Carlsbad, CA, USA). The nuclei were counterstained with DAPI (Sigma- Aldrich) and the sections cover-slipped. The images were observed under a confocal microscope (TCS-SP2-AOBS, Leica (Wetzlar, Germany) and photographically recorded. The cells positive for NFkB p50 were counted and are expressed as cells/mm2. The p50 subunit may be located in the nuclei or in the cytosol. The nuclear and cytoplasmic intensity of the p50 subunit was analyzed using ImageJ (1.48 v). Nuclei were outlined using the ROI manager function on the DAPI blue channel, and the selection was applied on the green channel (p50) to measure fluorescence. The mean intensity (MI) for each nucleus was measured. For cytoplasmic analysis of the p50 subunit of NF-κB, green channels were used, the cytosol of each cell was manually outlined using a freehand selection of ImageJ, and MI was recorded. Results are expressed as the percentage of cells expressing the p50 subunit of NF-κB in the nucleus. Analysis of protein content in cerebellum by Western blot. Homogenates of cerebellum were subjected to immunoblotting. Primary antibodies were against IL-1β (1:500 dilution (AF-510-NA) from R&D SYSTEMS, Minneapolis, MN, USA), TNFα (1: 500, R&D SYSTEMS), and YM-1 (1:500, Abcam). Secondary antibodies were anti-rabbit, anti-goat, or anti-mouse IgG, 1:4000 dilution (cat. #A8025, A7650, and A3562, respectively), conjugated with alkaline phosphatase from Sigma (St. Louis, MO, USA). The images were captured using ScanJet 5300C (Hewlett-Packard, Amsterdam, the Netherlands) and the band in- Int. J. Mol. Sci. 2021, 22, 6772 15 of 18

tensities quantified using AlphaImager 2200, version 3.1.2 (Alpha Innotech, Watertown, MA, USA). Analysis of membrane surface expression of GABA and glutamate transporters and of TNFR1. Cerebella were dissected, and transversal slices (400 µm) were obtained using a chopper. Slices were added to tubes containing ice-cold standard buffer with or without 2 mM BS3 (crosslinker) (Pierce, Rockford, IL, USA) and incubated for 30 min at 4 ◦C. Cross-linking was terminated by adding 100 mM glycine (10 min, 4 ◦C). The slices were homogenized by sonicating for 20 s. Samples treated with BS3 or not were analyzed by Western blot. The antibodies used were: anti-GLT1 (1:1000, Invitrogen), anti-GLAST (1:4000, Novus Biologicals (Centennial, CO, USA)), anti-GAT1 (1:500, Abcam), anti-GAT3 (1:500, Millipore (Burlington, MA, USA)), and anti-TNFR1 (1:1000, Abcam). The membrane surface expression of each receptor was calculated as the difference between the intensity of the bands without BS3 (total protein) and with BS3 (cytosolic protein), as described by [45]. In vivo cerebellar microdialysis and determination of extracellular GABA. Rats were anesthetized using isoflurane, and a microdialysis guide was implanted in the cerebellum (AP—10.2, ML—1.6, and DV—1.2), as in [46]. After 48 h, a microdialysis probe was implanted in the freely moving rat. Probes were perfused (3 µL/min) with artificial cerebrospinal fluid (in mM): NaCl, 145; KCl, 3.0; CaCl2, 2.26; buffered at pH 7.4 with 2 mM phosphate. After a 2 h stabilization period, samples were collected every 30 min. EDTA was added to the samples at a final concentration of 4 mM and stored at −80 ◦C until analysis of GABA levels. To assess the basal level of extracellular GABA in the cerebellum, five consecutive microdialysis samples were collected from each rat. The extracellular concentration of GABA was measured by HPLC-MS, as in [47]. Motor coordination in the beam-walking test. The beam-walking test consists of a wood strip 20 mm in diameter and 1 m long, located 1 m above the ground. Rats have to cross the beam, and two observers count the number of slips committed by the rats, as described in [8]. The average number of foot faults (slips) observed by two experimenters is recorded as a measure of incoordination. Statistical analysis. Data are expressed as mean ± SEM. All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Prism Software, Inc., San Diego, CA, USA). All data were tested for normality with D’Agostino and Pearson’s tests. When the data did not pass a normality test, Kruskal–Wallis’s test with Dunn’s test for multiple comparisons were performed. If the data showed normal distribution, we used one-way ANOVA with Tukey’s test for multiple comparisons or Welch’s test with Dunnett’s test when the deviations were not equal. To test the progression of cytokine levels in the plasma, two-way ANOVA with repeated measures followed by Bonferroni’s test for multiple comparisons was performed. A confidence level of 95% was considered significant.

5. Conclusions

In summary, we showed here that the inhibition of GABAA receptors with the systemic administration of bicuculline altered peripheral inflammatory markers in control rats, inducing a pro-inflammatory environment, whereas, in hyperammonemic rats with a previous pro-inflammatory environment, bicuculline prevented it, leading to a more anti-inflammatory environment. In the same way, in the cerebellum, bicuculline increased microglial activation in controls, while, in hyperammonemic rats, bicuculline induced a microglial phenotype change to M2 (anti-inflammatory) and inhibited astrocyte activation. This modulation of the immune system is associated with altered membrane expression of the GABA transporters and GABA levels in both control and hyperammonemic rats, supporting the close interplay between GABAergic neurotransmission and immune modulation. Thus, our results bring new data on this interplay. On the other hand, this work also supports the beneficial role of bicuculline in cerebellar neuroinflammation associated with restored GABA neurotransmission and improvement of motor incoordination in hyperammonemic rats. Int. J. Mol. Sci. 2021, 22, 6772 16 of 18

Author Contributions: Conceptualization, V.F. and M.L.; methodology, M.C.C., M.M. and T.B.; software, M.M., T.B. and M.L.; validation, M.L. and M.M.; formal analysis, M.L. and M.M.; investiga- tion, M.L. and M.M.; resources, V.F.; data curation, M.L. and V.F.; writing—original draft preparation, M.L.; writing—review and editing, M.M., M.L. and V.F.; visualization, M.M. and V.F.; supervision, V.F.; project administration, V.F.; funding acquisition, V.F. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Ministerio de Ciencia e Innovación of Spain, grant number SAF2014-51851-R and SAF2017-82917-R and Consellería Educación Generalitat Valenciana, co-funded by the European Regional Development Funds (ERDF), grant numbers PROMETEOII/2014/033 and PROMETEOII/2018/051. Michele Malaguarnera was supported by the Ministry of Econ- omy and Competitiveness of Spain through a Juan de la Cierva postdoctoral fellowship, grant number FJCI-2015-25825. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of CENTRO DE INVESTIGACIÓN PRÍNCIPE FELIPE (protocol code 17-003, 20 April 2017). Informed Consent Statement: Not applicable. Data Availability Statement: Data are contained within the article. The data presented in this study are available in this article: The dual role of the GABAA receptor in peripheral inflammation and neuroinflammation: a study in hyperammonemic rats. Conflicts of Interest: The authors declare no conflict of interest.

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