Glutamine triggers long-lasting increase in striatal network activity in vitro

    Glutamine triggers long-lasting increase in striatal network activity in vitro Wiebke Fleischer, Stephan Theiss, Alfons Schnitzler, Olga Sergeeva PII: DOI: Reference:

S0014-4886(17)30003-1 doi:10.1016/j.expneurol.2017.01.003 YEXNR 12458

To appear in:

Experimental Neurology

Received date: Revised date: Accepted date:

18 August 2016 5 December 2016 4 January 2017

Please cite this article as: Fleischer, Wiebke, Theiss, Stephan, Schnitzler, Alfons, Sergeeva, Olga, Glutamine triggers long-lasting increase in striatal network activity in vitro, Experimental Neurology (2017), doi:10.1016/j.expneurol.2017.01.003

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1. TITLE: Glutamine triggers long-lasting increase in striatal network activity in vitro

T

2. AUTHOR NAMES AND AFFILIATIONS

SC R

IP

Wiebke Fleischer1,2 ([email protected]) Stephan Theiss1,3 Alfons Schnitzler1 Olga Sergeeva1,2 1

MA

NU

Institute of Clinical Neuroscience and Medical Psychology, Medical Faculty, Heinrich-Heine-University, Duesseldorf, Germany 2 Institute of Neuro- und Sensory Physiology, Medical Faculty, Heinrich-HeineUniversity, Duesseldorf, Germany 3 Result Medical GmbH, Duesseldorf, Germany

CE P

TE

D

3. CORRESPONDING AUTHOR Wiebke Fleischer1 ([email protected]) Institute of Clinical Neuroscience and Medical Psychology, Medical Faculty, Heinrich-Heine-University, Duesseldorf, Germany Building 23.02.03.29 Moorenstr. 5 40225 Duesseldorf  +49 211 81-15456

AC

4. CONFLICT OF INTEREST None declared 5. ABSTRACT Accumulation of ammonium and glutamine in blood and brain is a key factor in hepatic encephalopathy (HE) – a neuropsychiatric syndrome characterized by various cognitive and motor deficits. MRI imaging identified abnormalities notably in the basal ganglia of HE patients, including its major input station, the striatum. While neurotoxic effects of ammonia have been extensively studied, glutamine is primarily perceived as “detoxified” form of ammonia. We applied ammonium and glutamine to striatal and cortical cells from newborn rats cultured on microelectrode arrays. Glutamine, but not ammonium significantly increased spontaneous spike rate with a long-lasting excitation outlasting washout. This effect was more prominent in striatal than in cortical cultures. Calcium imaging revealed that glutamine application caused a rise in intracellular calcium that depended both on system A amino acid transport and activation of ionotropic glutamate receptors. This pointed to downstream glutamate release that was triggered by intracellular glutamine. Using an 1

ACCEPTED MANUSCRIPT

3.

D

4.

NU

2.

6. HIGHLIGHTS Glutamine rapidly boosts neuronal network activity in vitro, ammonium has only minor effects Glutamine is taken up by neurons where it triggers sustained glutamate release Striatal networks are more susceptible to glutamine-induced excitation than cortical networks Cultured striatal neurons possess the synaptic machinery for vesicular glutamate release

MA

1.

SC R

IP

T

enzymatic assay kit we confirmed glutamine-provoked glutamate release from striatal cells. Real-time PCR and immunocytochemistry demonstrated the presence of vesicular glutamate transporters (VGLUT1 and 2) necessary for synaptic glutamate release in striatal neurons. We conclude that extracellular glutamine is taken up by neurons, triggers synaptic release of glutamate which is then taken up by astrocytes and again converted to glutamine. This feedback-loop causes a sustained long-lasting excitation of network activity. Thus, apart from ammonia also its “detoxified” form glutamine might be responsible for the neuropsychiatric symptoms in HE.

TE

CE P

AC

1. 2. 3. 4. 5. 6. 7. 8.

7. KEY WORDS microelectrode array hepatic encephalopathy neuronal networks network activity ammonium striatum glutamine tripartite synapse

8. ABBREVIATIONS APV: (2R)-amino-5-phosphonovaleric acid CNQX: 6-cyano-7-nitroquinoxaline-2,3-dione CSF: cerebrospinal fluid DAPI: 4,6-diamidino-2-phenylindole div: days in vitro GAD: glutamate decarboxylase HE: hepatic encephalopathy MEA: multielectrode array MeAiB: α-(methylamino) isobutyric acid MRI: Magnetic Resonance Imaging MSO: methionine sulfoximine NMDA: N-Methyl-D-aspartic acid RT-PCR: real-time quantitative polymerase chain reaction 2

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

TFB-TBOA: (3S)-3-[[3-[[4-(Trifluoromethyl)benzoyl]amino]phenyl]methoxy]L-aspartic acid TMPH: Tetramethylpiperidin-4-yl heptanoate hydrochloride TTX: tetrodotoxin VGAT: vesicular GABA transporter VGLUT: vesicular glutamate transporter

3

ACCEPTED MANUSCRIPT

NU

SC R

IP

T

9. INTRODUCTION Hepatic encephalopathy (HE) is a neuropsychiatric syndrome characterized by cognitive impairment, altered states of consciousness and motor disturbances. In more advanced stages HE may lead to coma and death. HE is caused by liver failure, and the accumulation of harmful metabolites that are normally eliminated by the liver is thought to provoke the observed neurological symptoms. Although the pathophysiological mechanisms are not completely understood, it is widely agreed upon that ammonia is a key factor in the pathogenesis. Injection of high doses of ammonium salts in rats leads to convulsions, coma and ultimately to death (Marcaida et al., 1992; Rangroo Thrane et al., 2013).

TE

D

MA

Ammonia is transported along the bloodstream into the brain where it is taken up by astrocytes and intracellularly condensed with one glutamate molecule to form glutamine. This reaction is thought to act as detoxification and results in astroglial cell swelling leading to brain edema and increased intracranial pressure (Thumburu et al., 2012). Astrocyte swelling was considered a key factor in HE pathology, but recent studies suggest it may only play a secondary role in early stages of HE (Cauli et al., 2014; Oeltzschner et al., 2016).

AC

CE P

Furthermore, ammonia is thought to interact with glutamatergic neurotransmission. A decrease in glutamate transporter expression associated with reduced glutamate re-uptake was observed under exposure to ammonia (Bender and Norenberg, 1996; Chan and Butterworth, 1999; Desjardins et al., 1999; Suarez et al., 2000). Ammonia also interferes with neuronal ionotropic and metabotropic glutamate receptor function (Felipo and Butterworth, 2002; Llansola et al., 2013; Llansola et al., 2007). Blockade of NMDA receptors for instance prevents animal death after ammonium acetate injection (Felipo et al., 1998; Marcaida et al., 1992). A NMDA-receptor dependent increase in network activity of cortical cells on MEAs (multielectrode arrays) was observed when NH4Cl was applied for 24 hours, but no significant changes in network activity were observed 2, 5 or 30 minutes after application (Schwarz et al., 2012). Dynnik et al reported that high doses of ammonium shift intracellular Ca2+ levels of neurons to higher values and induces strong burst firing in hippocampal cultures by activation of ionotropic glutamate receptors (Dynnik et al., 2015). Seemingly contradictory, hyperammonemia was also found to enhance inhibitory GABAergic neurotransmission (Basile, 2002; Jones and Basile, 1998; Sergeeva, 2013). In accordance with the idea of increased “GABAergic tone”, symptoms in HE animal models with liver failure were ameliorated by administration of GABAA receptor antagonists (Bassett et al., 1987; Gammal et al., 1990; Jones et al., 1990). Current studies showed increased GABA levels 4

ACCEPTED MANUSCRIPT only in some brain regions while levels decreased in others (Cauli et al., 2009; Oeltzschner et al., 2015).

SC R

IP

T

Although ammonia is perceived as the main mediator of brain dysfunction in HE, there is evidence that pathogenic effects of ammonia are indeed attributable to its metabolite glutamine: methionine sulfoximine – an inhibitor of glutamine synthesis – raises the median lethal dose (LD50) in mice acutely intoxicated with ammonia (Warren and Schenker, 1964). Albrecht and Norenberg postulated that glutamine acts as “Trojan Horse” that actually mediates ammonia toxicity (Albrecht and Norenberg, 2006).

MA

NU

Glutamine is precursor of both neurotransmitters glutamate and GABA and is pivotal for maintaining epileptiform activity and neurotransmission at excitatory terminals (Kanamori and Ross, 2013; Tani et al., 2007; Tani et al., 2014; Tani et al., 2010).

CE P

TE

D

Glutamine administration by gavage in rats increases extracellular GABA levels in the striatum while glutamate levels remain unchanged (Wang et al., 2007). However, glutamine externally applied to hippocampal slices left GABA release unchanged but strongly increased glutamate release (Szerb and O'Regan, 1984, 1986). In both studies, the balance between GABA and glutamate release was distorted.

AC

MRI hyperintensities in the basal ganglia are a common finding in cirrhotic patients with and without HE (Rose et al., 1999; Zeron et al., 2011). In functional imaging studies basal ganglia of HE patients seem to be less affected by the overall decrease in cerebral blood flow and glucose metabolism than most cortical areas but are more metabolically susceptible to changes in blood ammonia (Ahl et al., 2004; Iversen et al., 2009; Kato et al., 2000; Keiding et al., 2006; Lockwood et al., 1991). Significant correlations were found between neuropsychologic performance of HE patients and altered blood flow or glutamate/glutamine-content in the basal ganglia (Catafau et al., 2000; Weissenborn et al., 2007). Here, we investigated the spontaneous neuronal network activity of both striatal and cortical cells cultured on microelectrode arrays that were exposed to pathophysiological doses of ammonium (NH4Cl) or glutamine. The aim of this study was to characterize acute ammonium- and glutamine-induced changes in neuronal network activity and to characterize modes of action. Furthermore, since brain regions are differentially affected in HE, we tried to reproduce such region specificity in a small in vitro environment by comparing cortical with striatal networks. 5

ACCEPTED MANUSCRIPT 10. MATERIAL AND METHODS 1. Cell culture

CE P

TE

D

MA

NU

SC R

IP

T

Organ removal of newborn rats was performed according to the EU directive 2010/63/EU for animal experiments. Primary dissociated cultures of cortex and striatum were prepared from newborn rats according to protocols described elsewhere (Sergeeva et al., 2007). Briefly, animals were anaesthetized and decapitated, skull and meninges were removed. Coronal slices containing the corpus striatum were cut with a scalpel and transferred into a dish with PBS. Striata and the dorsal part of the cortex were isolated and moved into separate dishes where they were cut into small pieces. Brain tissue was triturated after trypsinization (10 minutes) and washed in Dulbecco's modified Eagle's medium (DMEM 42430) enriched with fetal calf serum (10 %), and insulin (25 µg/ml). After mild centrifugation the cells were resuspended in a density of 1 x 106 cells/ml (cortex) or 10 x 106 cells/ml (striatum). Then, 50 µl of the dissociated cells were plated onto the center of poly-D-lysine-coated MEAs (Multi Channel Systems, Reutlingen) or cover slips in 24-wells and cultured in a humidified incubator with 5 % CO2, 95 % humidified air at 37 °C. On the following day serum-free B27-supplemented neurobasal medium (Invitrogen, Karlsruhe, Germany) with 50 U/ml streptomycin/penicillin and 0.5 mM glutamine was added to a final volume of 1.5 ml (MEAs) or 1 ml (cover slips). 2. MEA recordings and analysis

AC

Extracellular potentials were recorded on MEAs with a square grid of 60 planar Ti/TiN-microelectrodes (30 µm diameter, 200 µm spacing; Multi Channel Systems, Reutlingen, Germany). Signals from all 60 electrodes were simultaneously sampled at 25 kHz, visualized and stored using the standard software MCRack. Spike detection was performed off-line with the integrated “Spike Sorter”-tool with a cut-off threshold defined by -5 standard deviations above noise during a “silent phase” with no spike activity. Spontaneous spike rate (spikes per minute) was averaged over all electrodes of the MEA. The burst detection was performed subsequent to the spike detection with the software SpAnNer (RESULT Medizinische Analyseverfahren, Duesseldorf, Germany) and relied on an entropy based algorithm. A minimum entropy of 5 was required for a sequence of at least 3 spikes to constitute a burst. Recordings were performed at 37° C in magnesium-free recording solution with pH 7.4 (150 mM NaCl, 3.75 mM KCl, 2 mM CaCl2, 10 mM HEPES hemisodium salt, and 10 mM glucose). Neurobasal medium on the MEAs was replaced by recording solution and measurements were started after a 30 min long adaptation phase within a MEA2100-2x60 system (Multi Channel Systems, Reutlingen, Germany). 6

ACCEPTED MANUSCRIPT

D

MA

NU

SC R

IP

T

We added substances directly onto the MEA and started the recordings immediately after the addition or washout of the substance. Thus, exposure time and recording time are not significantly different. To investigate long-term effects of substances as presented in part 1 to 3 of the RESULTS section we established a protocol composed of 35 consecutive one-minute long recordings: five control recordings followed by 10 recordings under substance and 20 recordings after washout of the substance. To describe MEA long-term measurements in a quantitative fashion and to test for significance between different conditions, we calculated mean values for all one-minute long recordings of one MEA within a certain condition (“control”, “substance” or “washout”). To exclude possible artefacts caused by manual manipulation we discarded the first two recordings of each measurements series that followed liquid exchanges. Thus, a “control” mean was calculated from five measurements of each MEA recording series, the “substance” mean from eight measurements and the “washout” mean from 18 measurements. For the NH4Cl measurement series we separately analyzed an additional six minute long “transition” phase of minutes 3 to 8 directly after ammonium washout, while the actual washout phase was only twelve minutes long and started nine minutes after washout.

AC

CE P

TE

Because of variances between different MEAs we used relative values to describe certain substance effects. The relative spike rate for instance was defined as the ratio of spontaneous spike rate measured under substance to the basal value that was determined in the preceding control measurement. In longterm measurements values were normalized to the mean value of the five control measurements. Other parameters of network activity were the average burst duration and the ratio of spikes in bursts, which states the proportion of all detected action potentials that were fired within bursts of action potentials. Data are presented as mean  standard deviation (SD). Statistical significance of differences between paired groups of parametric values (such as “burst duration”) was determined using the paired Student’s t-test. For paired comparison of non-normal variables (such as “relative spike rate”) we used the Wilcoxon matched-pairs signed rank test. To test for significance of unpaired groups of values we used either the unpaired Student’s t-test for parametric values or the Mann Whitney test for nonparametric values. The significance level was set to 0.05 (* p≤0.05; ** p≤0.01; *** p≤0.001). 3. Fluo-4 calcium imaging Cultured adherent cells were loaded with 3 µM cell permeant Fluo-4, AM (Invitrogen, Karlsruhe, Germany) for 1-2 hours, washed with and incubated in the same recording solution that we used in MEA experiments. Fluorescence of Fluo-4 was excited by a monochromator (Polychrome IV; TILL Photonics, Gräfelfing, Germany) at 488 nm wavelength. Emission light was measured at 7

ACCEPTED MANUSCRIPT

SC R

IP

T

535 nm. Image series were captured at 0.5 or 1 Hz with an exposure time of 15 ms by a cooled 12-bit IMAGO CCD camera and digitized by a computer running the TILLvisION Multi-Color Ratio Imaging System (TILL Photonics). Imaging protocols were programmed and executed with the software TILLvisION and images were symmetrically binned 4x (to 160 x 120 pixels) to increase the signal-to-noise ratio. Individual cells were defined as regions of interest and intensity changes in fluorescence were analyzed. Neurons were identified by their strong rise in intracellular calcium after NMDA (50 µM) application at the end of a measurement series. 4. Real-time PCR

Gene

AC

CE P

TE

D

MA

NU

Total cellular mRNA was isolated from striata or cortex of Wistar rats of different ages or cultures using an mRNA isolation kit (Pharmacia Biotech) according to the manufacturer´s protocol. For the reverse-transcription 8 µl of eluted mRNA was added to 7 µl of reagents mixture prepared according to the protocol of the “first strand cDNA synthesis kit” (Pharmacia Biotech). After incubation for 2 hours at 37° C the reverse transcription reaction was stopped by freezing at -20° C. The reverse-transcription generated approximately 50 to 190 ng cDNA per µl. The PCR was performed in a StepOnePlus Real-Time PCR system using the SYBR green master mix kit according to the supplier’s manual (Applied Biosystems, Darmstadt, Germany). Absolute cDNA concentrations from different samples were not determined but adjusted in preceding RT-PCR experiments according to their individual -actin expression level. All reactions were normalized on the respective -actin expression. We used the following primers:

-actin VGLUT1 VGLUT2 GAD65 GAD67 VGAT synaptophysin

Forward primer (5’-3’)

Reverse primer (5’-3’)

CGTGAAAAGATGACCCAGATCATGTT ACCCATCGGAGGCCAGATCG TTGAAATCAGCAAGGTTGGCATGTT AAACACAAGTGGAAGCTGAA ATGGGTGTGCTGCTCCAGT ATTCAGGGCATGTTCGTGCT GGCTGAATTCTTTGTCACCGT

GCTCATTGCCGATAGTGAT GCCACTCCTCCCGCGTCTT GGCRATRTCCAAGTGGTT CCTTGTCTCCCGTGTCATA ACAGTGCCCTTTGCTTTGC CACGCGTTAGCTATGGCCA GCCATCTTCACATCGGACA

The reactions were performed in optical tubes capped with MicroAmp optical caps. Four replicates of each DNA sample were used to minimize errors. The reactions were incubated for 10 min at 95 °C followed by 40 cycles of 15 s at 95° C and 1 min at 60° C. The PCR reactions were subjected to a heat dissociation protocol following the final cycle of the PCR. The reactions were 8

ACCEPTED MANUSCRIPT

SC R

IP

T

heat denaturated over a 35° C temperature gradient at 0.03° C/s from 60 to 95° C. Measurements were only evaluated when each PCR product showed a single peak in the denaturation curves. Semiquantitative analysis of expression relative to the -actin endogenous control was performed according to the “2Ct “(-fold) method as described previously (Sergeeva et al., 2003). Onesample t-tests were used to test for significant differences between expression levels in different cell types (striatal versus cortical levels). Accuracy of PCR products was verified by DNA sequencing. 5. Immunocytochemistry

AC

CE P

TE

D

MA

NU

Cell cultures on cover slips were fixed in 4 % paraformaldehyde in PBS, pH 7.4, and stored at -20 °C in PBS with 20 % sucrose if the cover slips were not immediately used. The cultures were permeabilized in PBS with 0.25 % Triton X-100 (PBS-T) for 15 min and then covered with Image-iT FX signal enhancer (Invitrogen, Karlsruhe, Germany) for 30 min to reduce unspecific background staining of the secondary antibodies. Subsequently, cultures were incubated with 10 % goat serum in PBS-T for 30 min at room temperature. Primary antibodies were diluted in PBS: VGLUT1 (Millipore #AB5905, Schwalbach, Germany; 1:10,000), synaptophysin (Sigma-Aldrich #S5768, Munich, Germany; 1:200), VGAT (Millipore #AB5062P; 1:1,000), MAP2 (Sigma-Aldrich, Munich, Germany; 1:500). The antibody solution was applied to the cultures overnight at 4 °C. After washing, cultures were incubated with Alexa Fluor 488-labeled goatanti guinea pig IgG (1:1,000; VGLUT1), Alexa Fluor 594-labeled goat-anti rabbit IgG (1:1,000; VGAT, synaptophysin) or with Alexa Fluor 350-labeled goat-anti-mouse IgG (1:200; MAP2; all from Molecular Probes, Eugene, Oregon, USA) overnight at 4 °C. Negative controls without primary antibodies were performed to estimate the specificity of the second antibodies. Finally, cell nuclei labeling was performed with 500 ng/ml DAPI (Sigma-Aldrich). Images were taken with an AxioCam MRm camera fixed to a ZEISS Observer.D1 microscope using the software AxioVision (Carl Zeiss Microscopy, Jena, Germany). 6. Glutamate measurement Glutamine (1 mM) alone or in combination with TFB-TBOA (1 µM) was applied to cell cultures on cover slips in 24-multiwells for 10 min at 37°C in 500 µl recording solution. Then, the recording solution was collected into individual reaction tubes, which were centrifuged at 14,000 rpm for 2 min to remove cell debris. The supernatant was transferred into fresh reaction tubes and frozen at -20 °C until further use. L-glutamate in the medium was measured using the AmplexTM Red Glutamic Acid/Glutamate Oxidase Assay Kit (Molecular Probes, Inc. Eugene, Oregon, USA) according to the manufacturer's protocol. In brief, 50 µl of the recording 9

ACCEPTED MANUSCRIPT

SC R

IP

T

solution were mixed with 20 µl reaction mixture and allowed to react for 10 min at 37°C in the dark. The highly fluorescent product resorufin was detected using absorption and emission filters set to 540 ± 15 nm and 584 ± 16 nm, respectively, with a Fluoroskan Ascent FL fluorometer (Thermo Electron Corporation, Dreieich, Germany). Fluorescence intensity was normalized to values obtained from supernatant of cell cultures where no substance had been added. To account for possible contaminations of glutamine with glutamate we performed the AmplexTM Red Glutamic Acid/Glutamate Oxidase Assay with glutamine solutions of varying concentrations. Fluorescence intensity was compared to that of a glutamate standard curve to obtain absolute concentrations.

NU

7. Drugs

CE P

11. RESULTS

TE

D

MA

Drugs used in the present study: (2R)-amino-5-phosphonovaleric acid (APV), ammonium chloride (NH4Cl), glutamine, α-(methylamino) isobutyric acid (MeAiB), N-Methyl-D-aspartic acid (NMDA), scopolamine hydrobromide, 2,2,6,6-Tetramethylpiperidin-4-yl heptanoate hydrochloride (TMPH), and glutamate were purchased from Sigma-Aldrich/Fluka (Munich, Germany), (3S)3-[[3-[[4-(Trifluoromethyl)benzoyl]amino]phenyl]methoxy]-L-aspartic acid (TFB-TBOA) from Tocris (Bristol, UK) and 6-cyano-7-nitroquinoxaline-2,3dione (CNQX) from Biotrend (Cologne, Germany), and Bafilomycin A1 from Wako Chemicals (Neuss, Germany)

1. Glutamine strongly excites striatal and cortical network activity in a long-lasting fashion while ammonium exerts

AC

only minor effects Isolated cells from striatum or cortex of newborn rats were cultured on MEAs. Both cell types developed into neuronal networks exhibiting spontaneous arraywide spike bursts. We investigated effects of ammonium chloride on cortical and striatal network activity according to the protocol given in the Materials and Methods section. We used an ammonium dose of 5 mM because it was used in several other former in vitro studies (Chan et al., 2000; Dynnik et al., 2015; Schwarz et al., 2012). However, blood, CSF or extracellular brain levels of ammonia/ammonium are significantly lower: Microdialysis in animal models of liver failure revealed extracellular brain ammonia levels of 500 µM to 1.2 mM (Rose et al., 2007; Zwirner et al., 2010). Fig. 1A shows two seconds of raw data from four MEA electrodes before, during and after application of NH4Cl (5 mM) in an exemplary measurement with a striatal culture. The activity pattern was hardly affected by addition of 10

ACCEPTED MANUSCRIPT

T

ammonium. Quantitative analysis with eleven MEAs covered either with striatal or cortical cells confirmed that NH4Cl (5 mM) elicited minor effects on network activity (fig. 2). Burst rate of striatal cells was significantly reduced while burst rate of cortical cells significantly increased, but overall the changes induced by ammonium were rather small. See left-hand side of table 1 for the exact values.

SC R

IP

From the graphs depicted in fig. 2, we identified a (~4 minute long) transition phase directly after washout with increased spike and burst rate and a diminished ratio of spikes within bursts, but these changes did not reach statistical significance for the average of all experiments.

MA

NU

Brain glutamine is mainly located in astrocytes. Extracellular concentrations in animal models or patients with liver failure have been reported in the range from 300 µM to 9.5 mM (Bjerring et al., 2008; Kanamori and Ross, 2004; Michalak et al., 1996; Rose et al., 2007). In this study, we used glutamine concentrations of 1-2 mM which is a conservative value for slightly elevated extracellular levels.

CE P

TE

D

Fig. 1B shows two seconds of raw data from an exemplary recording of a striatal culture. Note that the burst rate clearly increased under glutamine and that the activity remained on a high level ten minutes after glutamine wash-out. This long-lasting activation by glutamine was also evident in the quantitative analysis of six striatal and nine cortical cultures as depicted in fig. 2 (right-hand side).

AC

All four investigated parameters of network activity were significantly altered by addition of 2 mM glutamine both in striatal and in cortical cultures, but the increase in spike rate was far more prominent in striatal cultures (p = 0.002). Glutamine raised the lower spike activity of striatal cultures onto a level comparable with that of cortical cultures: During the five-minute long control phase an average of 46,000 action potentials were recorded from cortical cultures, but only 17,000 from striatal cultures. During the first five minutes of glutamine application a cortical culture fired 51,000 action potentials and a striatal culture more than 54,000. Burst rate increased fivefold in cortical and sevenfold in striatal cultures. Both cell types reacted to glutamine application with a comparable pattern change: the average burst duration became shorter and the ratio of spikes that were not organized in bursts increased. Interestingly, all investigated parameters of network activity remained on a comparable level after wash-out of glutamine and did not return to baseline values during the twenty minutes long washout phase, neither in striatal nor in cortical cultures. Commercially available glutamine has been found to contain low doses of glutamate. With the Amplex Red Glutamic Acid/Glutamate Oxidase enzymatic assay we determined the following concentrations of glutamate in our glutamine 11

ACCEPTED MANUSCRIPT

IP

T

solutions: 280 nM glutamate in 1 mM glutamine, 2.4 µM in 5 mM and 4.9 µM in 10 mM. This data confirmed a previously reported 0.05 % glutamate contamination in glutamine solutions (Sands and Barish, 1989). We tested comparable doses of glutamate in MEA experiments with striatal cells. Fig. 3A shows that glutamate concentrations up to 2 µM did not increase the spontaneous spike rate like 1 mM glutamine. Thus, contaminating glutamate was not responsible for the glutamine-mediated excitation.

NU

SC R

Clearly, the effects of glutamine on network activity of both cell types were much stronger than those elicited by ammonium, and glutamine provoked a stronger response in striatal than in cortical cultures. We therefore examined the effect of glutamine on striatal cultures in more detail. 2. Glutamine-induced excitation depends on active transport

MA

into neurons

AC

CE P

TE

D

To test if glutamine acted directly via ion channels or involved energyconsuming processes, we checked for a temperature-dependency in the glutamine-induced excitation of striatal network activity. We incubated four striatal cultures on MEAs with 1 mM glutamine for 10 min at room temperature and measured an increase of spike rate to only 134 % ± 14 % (p = 0.03; fig. 3B). When we measured the same MEAs at 37° C the base level of activity was one third higher and glutamine increased spike rate to 235 % ± 38 % (p = 0.03). Spike rate did not relapse to base level during the 20 minutes long washout phase. This temperature dependency makes the involvement of energy-consuming processes such as active transport across the membrane very likely. Glutamine is transported into neurons against its concentration gradient via Na+dependent amino acid transport system A. The N-methylated amino acid analogue MeAiB is a selective substrate of amino acid system A transport, but only large doses (250-500 mM) competitively inhibit the transport of natural substrates like glutamine (Kanamori and Ross, 2005; Mackenzie and Erickson, 2004). In calcium imaging experiments at room temperature, we observed a glutamineinduced intracellular calcium rise in cultured striatal neurons loaded with Fluo-4, AM (fig. 4). This rise did not depend on neuronal spike activity since coapplication with TTX (1 µM) did not inhibit the calcium rise. Perfusion with glutamine (1 mM) caused a maximum increase of fluorescence intensity to 161 ± 18 % of base level (n = 3; p = 0.03). In combination with MeAiB (25 mM), glutamine raised fluorescence intensity to only 110 ± 5 % (n = 3; p = 0.03, fig. 4). This shows that the rise in intracellular calcium actually depended on glutamine uptake. Since the imaging experiments were performed 12

ACCEPTED MANUSCRIPT at room temperature we assume that the calcium rise triggered by glutamine was considerably smaller than it would have been at 37 °C. 3. Glutamine triggers glutamate release and downstream

T

activation of ionotropic glutamate receptors

MA

NU

SC R

IP

No rise in intracellular calcium was observed when we co-applied glutamine with APV (100 µM) and CNQX (20 µM), antagonists of NMDA- and AMPA/kainate receptor, resp. (102 ± 5 %; n = 3; p = 0.03 to glutamine rise; fig. 4). This shows that the glutamine effect on intracellular calcium depended on calcium influx through ionotropic glutamate receptors and not on recruitment of intracellular calcium stores. But since extracellular glutamine was ineffective when its neuronal uptake was blocked by MeAiB, we assume that intracellular glutamine triggered downstream glutamate release which in turn activated receptors at postsynaptic neurons.

AC

CE P

TE

D

To verify glutamine-triggered glutamate release from striatal cultures, we used the same Amplex Red enzymatic assay as mentioned in paragraph 1. After addition of 1 mM glutamine for 10 min to striatal cultures, we observed only a very slight and not significant increase of fluorescence intensity in the extracellular fluid arguing against a glutamine-triggered glutamate release (108 ± 28 %; n = 7; p = 0.69; fig. 5). However, when glutamine was combined with a blocker of glial glutamate uptake (TFB-TBOA; 1 µM), we saw a significant increase in extracellular glutamate to 178 ± 26 % (n = 7; p = 0.016). TFB-TBOA alone did not induce a rise in extracellular glutamate (102 ± 13 %; n = 7; p = 0.93). These results confirm that glutamine caused subsequent glutamate release and also illustrate the efficiency of glutamate removal by glial glutamate transporters. We assume that glutamate is not released randomly along the cell membrane but in close vicinity to astroglial uptake sites, likely synapses. Former microdialysis studies with HE patients and animal models of HE found increased glutamate level in the extracellular fluid (Brusilow et al., 2010; Felipo and Butterworth, 2002; Michalak et al., 1996) in part due to impaired glial glutamate uptake (Chan et al., 2000), while other studies failed to find changes in extracellular glutamate (Hermenegildo et al., 2000; Kanamori and Ross, 2005). Our study shows that in striatal cultures glutamate is released in response to glutamine but extracellular glutamate levels are brought back to normal values by astrocytic re-uptake. Thus neither glutamate measurement in the extracellular fluid nor in the whole brain would enable us to decide whether glutamatergic neurotransmission is impaired or activated. Both variants are static descriptions 13

ACCEPTED MANUSCRIPT that do not account for the dynamics of glutamine/glutamate shuttling and metabolism within the neuronal-glial network system.

SC R

IP

T

Glutamine is a metabolic precursor of both glutamate and the inhibitory neurotransmitter GABA. Extracellular glutamine (0.5 mM) multiplied glutamate release from hippocampal slices while GABA release remained unchanged (Szerb and O'Regan, 1984). Blocking glutamine synthesis with MSO strongly reduced glutamate release, but hardly affected GABA release (Szerb and O'Regan, 1986). Thus, GABA release seemed to be relatively independent on changes in the supply of its precursor glutamine. 4. Spontaneous spike activity of striatal cultures is driven by

NU

synaptically released glutamate

CE P

TE

D

MA

While only 20 % of cortical neurons are thought to be GABAergic (Sahara et al., 2012), 95 % of striatal neurons in the adult brain are GABAergic spiny projection neurons (Kreitzer, 2009). Electric activity of the striatum is thought to be driven by cortical and thalamic input and to convey inhibition to tonically active downstream targets. A small number of spontaneously active cholinergic interneurons are found within the striatum but the existence of glutamatergic neurons was denied until recently, when Perreault et al. described medium spiny neurons with a dual GABA/glutamate phenotype. The number of these neurons that expressed VGLUT along with the GABA-synthesizing enzyme glutamate decarboxylase was especially high in cultured neonatal striatal neurons but a small proportion retained in adult animals (Perreault et al., 2014).

AC

Studies in our laboratories have shown that electric activity in striatal cultures is not due to cholinergic excitation but driven by synaptically released glutamate. After blockade of NMDA- and AMPA-receptors, cultures were silent, while cholinergic antagonists had little effect on network activity (supplemental figure 9). Synaptic drive of striatal network activity was confirmed by incubation with bafilomycin A1 (1 µM), a toxin that dissipates the pH gradient necessary for filling synaptic vesicles with neurotransmitters (Pocock and Nicholls, 1998). Bafilomycin abolished electric activity, both spontaneous and stimulus induced. Biphasic electric stimuli applied via one of the MEA electrodes that usually elicited network-wide spike bursts were ineffective under bafilomycin (supplemental figure 8). Thus, fast glutamate release via synapses and not tonic release, e.g. by astrocytes, drives the synchronous bursting activity of striatal networks. In MEA experiments, the blocker of glial glutamate transport TFB-TBOA doubled spike rate although the addition of TFB-TBOA alone onto striatal cultures had not resulted in a measurable increase of glutamate in the extracellular fluid (supplemental fig. 9). We think that TFB-TBOA application primarily affects the kinetics of glutamate shuttle between neurons and 14

ACCEPTED MANUSCRIPT

T

astrocytes but does not necessarily cause an overall rise of extracellular glutamate over a longer time period. The high impact of GABAergic transmission in striatal cultures, however, was evident by the remarkable prolongation of burst duration after addition of GABAA-receptor antagonists (supplemental fig. 9) (Sergeeva et al., 2007).

IP

5. Striatal neurons express low levels of glutamatergic

SC R

markers

MA

NU

We used semiquantitative RT-PCR with cDNA derived from striata and cortices of newborn rats to compare expression levels of GABAergic and glutamatergic markers. The two isoforms of the GABA-synthesizing enzyme glutamate decarboxylase (GAD65 and GAD67) were used as GABAergic markers along with VGAT, the vesicular GABA transporter. The vesicular glutamate transporters VGLUT1 and VGLUT2 were used us glutamatergic markers. Synaptophysin is a presynaptic protein, present in both excitatory and inhibitory synapses.

CE P

TE

D

GABAergic markers were more strongly expressed in striatal than in cortical tissue (fig. 6A, table 2): While the general presynaptic marker synaptophysin was expressed at comparable levels in both tissue types, the expression of the vesicular GABA transporter VGAT was 4.5 times higher in striatal tissue. The two isoforms of the GABA-synthesizing enzyme glutamate decarboxylase GAD67 and GAD65 were expressed fivefold compared to cortical tissue, respectively.

AC

On the other hand, expression of synaptophysin was lower in striatal cultures than in cortical cultures. This result is in line with our observation that a high cell density was necessary for striatal neurons to develop into functional networks. No significant difference between striatal and cortical cultures was found in the expression levels of GABAergic markers. Glutamatergic markers were only expressed at very low levels in striatal tissue compared to cortical values (table 2; fig. 6B). In cultures, the expression levels of VGLUT1 and VGLUT2 were even lower but this can be explained by an overall lower synaptic density in striatal compared to cortical cultures. Although the expression of vesicular glutamate transporters was very small both in striatal tissue and cultures, it was clearly detectable. 6. Glutamatergic and synapses in striatal cultures do not colocalize with GABAergic synapses We used immunocytochemistry to determine the cellular localization of vesicular glutamate transporters. VGLUT1- and VGLUT2-positive puncta were 15

ACCEPTED MANUSCRIPT

SC R

IP

T

clearly visible in striatal cultures and co-localized with the presynaptic protein synaptophysin (fig. 7A). Glutamatergic synapses were ubiquitous and were equally distributed across the whole network. GABAergic synapses were visualized by using an antibody against VGAT. VGAT positive puncta were also widely present but not co-localized with VGLUT1 (fig. 7B). We found VGAT+- and VGLUT1+-punctae located along fibers of the same neuron but the possibility that the presynaptic part of these synapses belonged to a different cell with a very thin – thus not detectable – axon cannot completely be ruled out. 12.Supplementary material

1. Striatal network activity is synaptically driven.

TE

D

MA

NU

Bafilomycin A1 prevents filling of synaptic vesicles by dissipating the transmembrane pH gradient and hence the driving force for neurotransmitter uptake. After one hour incubation with bafilomycin A1 network bursts in striatal cultures on MEAs had completely ceased. The spontaneous activity was reduced to only 19 % ± 11 % (n = 4; p < 0.001) of base level with profound loss of synchrony between electrodes. Electric stimulation of electrodes that had led to network-wide spike bursts under control conditions had no effect after bafilomycin A1 incubation (supplemental fig. 8). 2. Striatal network activity depends on glutamate.

AC

CE P

To determine short-term effects of substances on neuronal activity of striatal cells, every MEA experiment comprised three recordings – control, substance and washout – each two minutes long and separated by an intermediate delay of at least 15 seconds to eliminate artifacts due to mechanical manipulations. Blocking fast synaptic transmission with a combination of NMDA-, AMPA- and GABAA-receptor antagonists resulted in an almost complete block of spike activity (0.3 % ± 0.1 %; n = 11; p < 0.0001; supplemental fig. 9 A). NMDAreceptor blockade alone with APV reduced electric activity to 22 % ± 23 % (n = 8; p < 0.0001) and AMPA-receptor blockade alone with CNQX to 44 % ± 21 % (n = 10; p < 0.0001) of base level. Although cholinergic neurons are known to exhibit excitatory drive within the striatum, a combination of nicotinic and muscarinic receptor antagonists (TMPH, scopolamine) only marginally attenuated spontaneous spike activity (85 % ± 15 %; n = 9; p = 0.02). Blocking of glial glutamate uptake with TFB-TBOA on the other hand doubled spike rate (206 % ± 85 %; n = 10; p = 0.003). 3. Modulation of network activity by intrinsic GABA Striatal cells express high amounts of GABAergic markers. Astonishingly, application of the GABAA receptor antagonists gabazine did not cause a significant increase of spontaneous spike or burst rate (spike rate: 16

ACCEPTED MANUSCRIPT

SC R

IP

T

120 % ± 63 %; n = 11; p = 0.3; burst rate: 76 % ± 50 %; n = 11; p = 0.15; supplemental fig. 9 B). But the influence of intrinsic GABA was apparent when we looked at other parameters of network activity: The number of spikes within an average burst significantly increased to 182 % ± 56 % (p = 0.0007) under gabazine and the duration of bursts to 188 % ± 59 % (p = 0.0006). Thus, disinhibition does not necessarily cause an increase in overall spike activity but clearly changes the spatiotemporal organization of network activity. Inherent GABA not suppresses but modulates neuronal network activity 4. The excitatory effect of glutamine is not due to disinhibition

TE

D

MA

NU

Disinhibiting the network activity of striatal cultures with application of the GABAA receptor blocker gabazine (n = 7) had only minor effects on spike rate (96 % ± 25 %; p = 0.94) and even decreased burst rate (63 % ± 30 %; p = 0.03) but clearly increased the ratio of spikes that were part of bursts (from 78 % ± 11 % to 90 % ± 6 %; p = 0.02) and the mean burst duration (from 48 ± 6 ms to 100 ± 27 ms; p = 0.003; supplemental fig. 10). We observed opposing effects when we added glutamine to the disinhibited networks: Spike and burst rate significantly increased (spikes: 159 %± 29 %; p = 0.03; bursts: 136 %± 27 %; p = 0.03), but the ratio of spikes (82 %± 10 %; p = 0.03) and the burst duration (58 ± 10 ms; p = 0.009) decreased. This clearly shows that glutamine does not cause disinhibition of the networks.

AC

CE P

13.DISCUSSION Our study showed that network activity of cultured striatal neurons is driven by synaptically released glutamate, although vesicular glutamate transporters are only expressed at low levels. While the majority of striatal neurons is generally perceived to convey downstream inhibition via GABA release, the presence of VGLUT3 in cholinergic interneurons as well as VGLUT1/2 expression in a subtype of GABAergic projection neurons was described in adult mice (Nelson et al., 2014; Perreault et al., 2014). A small VGLUT expression might suffice to support glutamate-driven activity also in the adult animal. We showed that extracellular glutamine is taken up by neurons and boosts striatal network activity in vitro by enhancing neuronal glutamate release. However, this potentiation of glutamatergic drive did not cause measurable increase of glutamate in the extracellular fluid due to rapid and efficient glutamate uptake by astrocytes. This indicates that glutamine-triggered neuronal glutamate release happens in close vicinity of glial uptake sites, likely synapses. Thus, glutamine fuels the metabolic glutamine-glutamate cycle and causes recurrent and sustained excitation of neuronal activity that outlasts washout of extracellular glutamine.

17

ACCEPTED MANUSCRIPT

SC R

IP

T

Although perceived as the main toxic agent in hepatic encephalopathy, ammonium did not elicit comparable effects in our study, neither in striatal nor in cortical networks. The increase in cortical network activity observed in a different study after 24 hour incubation with 5 mM NH4Cl was probably mediated by glial conversion of ammonium to glutamine (Schwarz et al., 2012). Apparently, conversion of ammonia to glutamine is not immediate and depends on the availability of sufficient glutamate molecules that are not bound to other metabolic pathways such as the tricarboxylic acid cycle. We cannot rule out neurotoxic actions of ammonium itself but the immediate effects on neuronal electric activity were clearly weaker than those elicited by even smaller doses of glutamine.

AC

CE P

TE

D

MA

NU

Kam and Nicoll (2007) reported that excitatory synaptic transmission in hippocampal slices and cultures persisted independently from the glutamateglutamine cycle (Kam and Nicoll, 2007). In the contrary, Billups et al. describe in a study using the Calyx of Held Synapse that glutamine transport is necessary to maintain glutamatergic neurotransmission, but only at higher stimulation frequencies (Billups et al., 2013). Blockade of glutamine uptake by MeAiB had no effect on mEPSC amplitude or stimulated EPSCs when the frequency of the presynaptic stimulation was very low (0.1 Hz). Thus, the basic neuronal activity in the investigated system seems to be decisive for the glutamine utilization of neurons for synaptic release of glutamate. The cultures used in our study were seeded in a considerably high density and displayed high levels of neuronal activity with recurrent oscillatory bursts of action potentials imbedding the whole network covered by the electrodes (an area of 2 mm2 and several thousand neurons). In these high-density cultures, an electric signal is not conducted via a defined pathway to a recording site after traversing “one” synapse such as in stimulated slices but is carried to a multitude of randomly connected neurons and constantly regenerated across the whole network. Thus, an effect that has considerably low effect at one synapse can be potentiated along an extensive synaptic circuit. This might explain why we see such strong effects in our cell cultures system elicited by considerably low doses of glutamine. Our study supports the idea that neuronal activity and glutamine metabolization are reciprocally linked: While a high level of electric activity stimulates neuronal glutamine uptake, glutamine uptake – enforced by high levels of extracellular glutamine – also stimulates neuronal activity. An enhanced state of network activity then results in an enhanced response of the network. Astrocytes are able to store high concentrations of glutamine and to meet increased neuronal demands for glutamine even if extracellular concentrations of glutamine are low. This might explain why the enhanced activity of the neuronal networks in our study survived washout of glutamine. Increased burst activity might also result in increased neuronal glutamate release which in turn results in 18

ACCEPTED MANUSCRIPT

T

more glutamate uptake by the astrocytes and more conversion of glutamate to glutamine. In this way more glutamate/glutamine molecules are circulating within the system and allow for a long-lasting excitation of striatal network activity.

CE P

TE

D

MA

NU

SC R

IP

Another aspect of our study is the particular susceptibility of striatal networks to glutamine in comparison to cortical networks. Although glutamine is precursor of both GABA and glutamate and the striatal neurons used in our experiments expressed high levels of GABAergic markers, glutamine increased excitation but not inhibition. We cannot rule out that more GABA was released, but the glutamate-driven excitation clearly outweighed any possible GABA release, thus obscuring a potential increase in “GABAergic tone”. Szerb et al. reported increased glutamate levels in slices treated with glutamine while GABA levels remained constant (Szerb and O'Regan, 1984, 1986). The authors suggested that the enzyme glutamate decarboxylase works rather slowly and represents the rate-limiting step in GABA synthesis. Thus, when glutamine enters the cell, it is readily converted to glutamate but a small turnover number of GAD prevents fast metabolization to GABA. Thus, striatal networks are exposed to high levels of intracellular glutamate which may in part enter the tricarboxylic acid cycle via α-ketoglutarate, or is synaptically released and results in postsynaptic excitation of spike activity and increased energy demands. We also tested the hypothesis if the glutamine-induced excitation of striatal networks was due to disinhibition but addition of glutamine to striatal cells that had already been treated with the GABAA-receptor antagonist gabazine strongly increased spike rate (see supplemental figure 10). This observation contradicts a glutaminetriggered disinhibition.

AC

Cortical networks consist of mainly glutamatergic neurons and express only low levels of GABAergic markers. They exhibited higher spike activity than striatal networks under control conditions and might have a higher glutamine/glutamate metabolization rate per se. Thus, they might be better adapted to changes in glutamine concentration by way of homeostatic mechanisms to maintain a constant level of neuronal activity. In addition, a preferential expression of the amino acid transporter SNAT2 over SNAT1 was reported for glutamatergic neurons. SNAT2 yields a higher Km for glutamine (1.7 mM) than SNAT1 (300 µM) which is predominantly expressed in GABAergic neurons (Bröer, 2007; Jenstad et al., 2009; Mackenzie et al., 2003; Solbu et al., 2010; Yao et al., 2000). More striking effects of glutamine in cortical cultures might have been seen at higher doses. But higher doses of glutamine are problematic due to the contaminating glutamate. A brain region-specific expression of the highly sensitive glutamine transporter subtype might explain why some brain regions are more strongly affected in HE than others. 19

ACCEPTED MANUSCRIPT

TE

D

MA

NU

SC R

IP

T

Since glutamine had a much stronger effect on striatal neuronal function than ammonia, blocking glutamine transport or synthesis may be considered a therapeutic option in HE. Glutamine starving is also a therapeutic goal in oncology, but most tested substances showed such a high toxicity that preclinical studies were discontinued (Wise and Thompson, 2010). The glutamine scavenger phenylbutyrate is already used for treatment of hyperammonemia in clinical practice and diminishes glutamine and ammonia load (Rockey et al., 2014). Interestingly, phenylbutyrate is also discussed for treatment of Huntington’s disease (HD) due to its inhibiting action on the enzyme histone deacetylase. But the beneficial effects of phenylbutyrate in HD might be at least in part due to its function as glutamine scavenger, because elevated levels of extracellular glutamine and perturbations of the glutamateglutamine cycle have been reported for animal models of HD as well (Behrens et al., 2002). Thus, an excess of free glutamine might be a functional link between HE and early stages of HD since both affections show a surprising overlap in clinical features and diagnostics, such as MRI hyperintensities in the basal ganglia, changes in resting connectivity of the cortico-basal gangliathalamic loops, high levels of proinflammatory cytokines, and impaired cognitive function (Carroll et al., 2015).

AC

CE P

14.CONCLUSIONS To sum up: We propose that extracellular glutamine triggers downstream release of glutamate which is taken up by astrocytes, re-converted to glutamine and released into the extracellular space. This continuous circular flow causes a recurrent and long-lasting excitation of striatal network activity in vitro. Free glutamine might not only be a pathological factor in hepatic encephalopathy but also in brain dysfunction observed in polyglutamine diseases. 15.ACKNOWLEDGEMENTS This work was supported by the German Ministry of Education and Research (BMBF: FKZ 031B0010B) and the European Union (EURO-TRANS-BIO project In-HEALTH) and by the German Research Foundation (SFB974 B07). We thank Helmut L. Haas for critical review of the manuscript. 16.FIGURE AND TABLE LEGENDS Table 1: Quantitative analysis of the MEA recordings illustrated in fig. 2. “CS” stands for cell cultures isolated from the corpus striatum and “Cx” for cortical cell cultures. Asterisks indicate significant differences with respect to the individual control recordings or – in the lines labeled with “Cx vs CS” – significant differences between cell types. NH4Cl analysis on the left-hand side comprised eleven measurements with striatal and cortical cultures, respectively. 20

ACCEPTED MANUSCRIPT Glutamine measurements were performed with six striatal and nine cortical cultures.

TE

D

MA

NU

SC R

IP

T

Figure 1: Glutamine boosts striatal neuronal network activity in vitro. A) Exemplary traces of four MEA electrodes, detecting spontaneous spike activity of a striatal culture after 23 div before, during, and after incubation with 5 mM NH4Cl. Under control conditions, extracellular action potentials were clustered into compact bursts of about 100 to 200 ms duration (upper segment). Several seconds long quiescent periods prevailed between burst firing. This activity pattern was not changed by ammonium application (middle segment). The bottom segment shows the activity pattern ten minutes after a ten-minute long incubation with 5 mM NH4Cl. B) Extracellular activity of a striatal culture after 15 div before, during, and after incubation with 2 mM glutamine. Depicted are two second long original traces from four different MEA electrodes. Under control conditions (upper segment) action potentials were fired in bursts that were followed by silent periods of several seconds. During incubation with 2 mM glutamine (middle segment), the burst frequency significantly increased while silent periods were shortened. This activity pattern persisted ten minutes after washout of glutamine (bottom segment.)

AC

CE P

Figure 2: Diverse actions of ammonium and glutamine on neuronal network function. Effects of ammonium chloride (5 mM NH4Cl) on network activity of striatal (n = 11) and cortical cultures (n = 11) are depicted on the left hand side, glutamine (2 mM) effects on the right hand side (n = 6 striatal cultures; n = 9 cortical cultures). Each row represents a certain aspect of neuronal network activity determined with MEA recordings. (A) The spike rate of both cell cultures types was not altered under application of 5 mM NH4Cl, but a slight increase of spike rate was observed after washout of the substance lasting for about five minutes. (B) This increase in spike rate was mirrored by the slight increase in burst rate after washout. (C) NH4Cl marginally reduced the ratio of spikes that were part of bursts, a clear drop was observed directly after washout in both cortical and striatal cultures. However, this reduction was only temporary and not significant. (D) The effect of ammonium on average burst duration was negligible. E) Glutamine (2 mM) effects on network activity were strikingly different from ammonium effects: Spike rate increased during substance application and – after a short-term decline directly after washout – stayed at a very high level during the whole washout phase lasting for twenty minutes. In striatal networks this effect was particularly pronounced. (F) The activating effect of glutamine was also evident in the burst rate. Here, 21

ACCEPTED MANUSCRIPT

SC R

IP

T

cortical cells showed a strong and long-lasting increase comparable to striatal cells. This is due to the fact that cortical bursts under glutamine became considerably shorter (as seen in H). Thus, the overall number of spikes did not change in cortical networks but the temporal organization of spikes. (G) Glutamine decreased the ratio of spikes that were part of bursts from around 93 % to 64 % in striatal cultures and to only 50 % in cortical cultures. (H) The average cortical burst duration under glutamine application conditions shortened from 330 ms to 30 ms and stayed on this level after washout. Striatal bursts were 160 ms long under control conditions and were only 50 ms long under glutamine and after washout.

AC

CE P

TE

D

MA

NU

Figure 3: Glutamine – not glutamate – excites network activity via a temperature-sensitive mechanism. A) Commercially available glutamine is contaminated with low levels of glutamate. Thus, we tested if low levels of glutamate could be responsible for the observed glutamine-induced excitation of network activity. Glutamate concentrations up to 2 µM – a value comparable to those found in a 5 mM glutamine solution – failed to activate spontaneous spike rate of striatal cultures (n = 7 for each concentration). The same seven cultures were exposed to 1 mM glutamine which caused a significant increase of spike rate. B) Glutamine (1 mM) doubled striatal network spike rate when the cells were incubated at 37 °C (n = 4). At room temperature (~ 20 °C), the spike rate of the same four striatal cultures increased by only 30 % under glutamine (1 mM). This phenomenon points to the involvement of active, energydependent processes rather than passive flow through ion channels as mediator for glutamine-induced excitation. Figure 4: The glutamine-triggered calcium-rise in striatal neurons depends on system A transport and ionotropic glutamate receptors. A) In Fluo-4 imaging experiments, glutamine (1 mM) perfusion induced a significant rise in intracellular calcium of cultured striatal neurons, reflected by an increase of fluorescence intensity to 160 % (n = 3). When glutamine was combined either with the system A transport blocker MeAiB (25 mM) or a combination of the ionotropic glutamate receptor blockers CNQX (20 µM) and APV (50 µM), no significant rise of intracellular calcium was observed. These results indicate that the glutamine-induced calcium rise is mediated by glutamate receptors and depends on cellular glutamine uptake. B) This panel depicts calcium imaging of three consecutive substance applications to one Fluo-4-loaded striatal culture, averaged over 34 neurons. Substances were gradually perfused into the recording chamber to the desired concentration and then rinsed out again. Glutamine (1 mM) caused 22

ACCEPTED MANUSCRIPT

T

an increase in fluorescence intensity to 170 % of control level. This increase was blocked when the ionotropic glutamate receptor antagonists APV (50 µM) and CNQX (20 µM) or transport blocker MeAiB (25 mM) were perfused in combination with glutamine.

MA

NU

SC R

IP

Figure 5: Glutamine causes extracellular glutamate release. We measured the relative glutamate concentration in recording solution covering striatal cultures with the Amplex Red Glutamic Acid/Glutamate Oxidase assay. No significant increase in extracellular glutamate was observed after exposure to 1 mM glutamine for 10 min compared to cultures where no glutamine was administered. However, when glutamine was applied in combination with the glial glutamate transporter blocker TFB-TBOA (1 µM) the glutamate concentration doubled. No rise in fluorescence intensity was observed when TFB-TBOA was applied alone. Thus, glutamine causes glutamate release into the extracellular space which is efficiently taken up by astrocytes.

TE

D

Table 2: mRNA expression of GABAergic and glutamatergic markers in striatal (CS) tissue and cultures in comparison to cortical (Cx) values. “n” refers to the number of investigated cDNA samples. Asterisks indicate significant differences between striatal and cortical expression levels.

AC

CE P

Figure 6: Expression of GABAergic and glutamatergic markers in striatal tissue and cultures compared to cortical values. A) Using RT-PCR, we investigated the expression of GABAergic markers in striatal tissue of newborn rats (left side) and in striatal cultures (right side) in comparison to cortical equivalents (tissue and cultures, resp.). mRNA of the presynaptic marker synaptophysin was detected in comparable amounts in cortical and striatal tissue but the vesicular GABA transporter VGAT and both GABA-synthetizing enzymes GAD67 and GAD65 were significantly higher expressed in striatal tissue. In striatal cultures, all GABAergic markers were expressed in comparable amounts with respect to cortical cultures. The general synaptic marker synaptophysin was expressed at much lower levels than in cortical cultures. We presume that fewer synapses are formed in striatal than in cortical cultures but that most of them present a GABAergic phenotype. B) Both vesicular glutamate transporters VGLUT1 and VGLUT2 were present in striatal tissue and cultures albeit at very low levels compared to cortical values. Figure 7: Glutamatergic synapses in striatal cultures do not co-localize with GABAergic synapses.

23

ACCEPTED MANUSCRIPT

T

A) The microscopic pictures in the top row show immunostainings of a striatal culture after 26 div. VGLUT1-positive punctae (green) co-localize with the presynaptic protein synaptophysin (red). B) No co-localization was observed for VGLUT1 (green) and VGAT (red; bottom row) in a striatal culture after 20 div.

TE

D

MA

NU

SC R

IP

Supplemental figure 8: Striatal network activity is synaptically driven. MEA recordings of the same striatal culture at 13 div: (A) in recording solution and (B) after one-hour incubation with 1 µM bafilomycin A1. Top row: Spike raster plots depict the temporal and spatial distribution of action potentials (bars) detected by different MEA electrodes (vertical axis) over a time period of one minute. Arrows indicate a biphasic electrical pulse applied to the cultures through a single MEA electrode. Under standard conditions (A) this stimulus induced a network-wide burst but had little effect after incubation with bafilomycin (B). Bottom row: Original spike trains of four different MEA electrodes from the measurements depicted in the spike raster plots. In both cases, the top electrode was used for stimulation (note the artifact at time point 5 s). (C) A closer look at the signal of one electrode of the measurement given in (A) after the stimulus (indicated by the arrow). No burst could be elicited after bafilomycin incubation (D).

AC

CE P

Supplemental figure 9: Striatal network activity depends on glutamate. A) Virtually no spike activity was detected after block of fast synaptic transmission with a combination of NMDA-receptor antagonist APV (100 µM), AMPA-receptor antagonist CNQX (20 µM) and GABAAreceptor antagonist gabazine (10 µM). APV and CNQX alone partially inhibited spike activity. A mixture of the nicotinic antagonist TMPH (1 µM) and the muscarinic antagonist scopolamine (1 µM) resulted in a slight decrease of spike activity. The blocker of glial glutamate reuptake TFB-TBOA (1 µM) caused a significant increase of spike rate. B) No significant effect on spike or burst rate was observed after addition of gabazine (10 µM), an antagonist of GABAA receptors. However, gabazine strongly increased the mean number of spikes that constituted a burst and the burst duration. Supplemental figure 10: Glutamine-induced excitation is not due to GABAergic disinhibition. Application of 10 µM gabazine for ten minutes did not result in a substantial increase of spike or burst rate in striatal (n = 7) cultures on MEAs. However, the ratio of spikes that were part of bursts and the average burst duration significantly increased. Additional application of glutamine (2 mM) counteracted this disinhibiting effect: Spike and burst rate increased whereas the ratio of spikes within bursts and the burst duration decreased. 24

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

17.REFERENCES Ahl, B., Weissenborn, K., van den Hoff, J., Fischer-Wasels, D., Kostler, H., Hecker, H., Burchert, W., 2004. Regional differences in cerebral blood flow and cerebral ammonia metabolism in patients with cirrhosis. Hepatology 40, 73-79. Albrecht, J., Norenberg, M.D., 2006. Glutamine: a Trojan horse in ammonia neurotoxicity. Hepatology 44, 788-794. Basile, A.S., 2002. Direct and indirect enhancement of GABAergic neurotransmission by ammonia: implications for the pathogenesis of hyperammonemic syndromes. Neurochemistry international 41, 115-122. Bassett, M.L., Mullen, K.D., Skolnick, P., Jones, E.A., 1987. Amelioration of hepatic encephalopathy by pharmacologic antagonism of the GABAAbenzodiazepine receptor complex in a rabbit model of fulminant hepatic failure. Gastroenterology 93, 1069-1077. Behrens, P.F., Franz, P., Woodman, B., Lindenberg, K.S., Landwehrmeyer, G.B., 2002. Impaired glutamate transport and glutamate-glutamine cycling: downstream effects of the Huntington mutation. Brain : a journal of neurology 125, 1908-1922. Bender, A.S., Norenberg, M.D., 1996. Effects of ammonia on L-glutamate uptake in cultured astrocytes. Neurochemical research 21, 567-573. Billups, D., Marx, M.C., Mela, I., Billups, B., 2013. Inducible presynaptic glutamine transport supports glutamatergic transmission at the calyx of Held synapse. The Journal of neuroscience : the official journal of the Society for Neuroscience 33, 17429-17434. Bjerring, P.N., Hauerberg, J., Frederiksen, H.J., Jorgensen, L., Hansen, B.A., Tofteng, F., Larsen, F.S., 2008. Cerebral glutamine concentration and lactatepyruvate ratio in patients with acute liver failure. Neurocritical care 9, 3-7. Bröer, S., 2007. SLC38 Family of Transporters for Neutral Amino Acids, in: Lajtha, A., Reith, M.E.A. (Eds.), Handbook of Neurochemistry and Molecular Neurobiology: Neural Membranes and Transport. Springer US, Boston, MA, pp. 327-338. Brusilow, S.W., Koehler, R.C., Traystman, R.J., Cooper, A.J., 2010. Astrocyte glutamine synthetase: importance in hyperammonemic syndromes and potential target for therapy. Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics 7, 452-470. Carroll, J.B., Bates, G.P., Steffan, J., Saft, C., Tabrizi, S.J., 2015. Treating the whole body in Huntington's disease. The Lancet. Neurology 14, 1135-1142. Catafau, A.M., Kulisevsky, J., Berna, L., Pujol, J., Martin, J.C., Otermin, P., Balanzo, J., Carrio, I., 2000. Relationship between cerebral perfusion in frontallimbic-basal ganglia circuits and neuropsychologic impairment in patients with subclinical hepatic encephalopathy. J Nucl Med 41, 405-410. Cauli, O., Llansola, M., Agusti, A., Rodrigo, R., Hernandez-Rabaza, V., Rodrigues, T.B., Lopez-Larrubia, P., Cerdan, S., Felipo, V., 2014. Cerebral oedema is not responsible for motor or cognitive deficits in rats with hepatic 25

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

encephalopathy. Liver international : official journal of the International Association for the Study of the Liver 34, 379-387. Cauli, O., Mansouri, M.T., Agusti, A., Felipo, V., 2009. Hyperammonemia increases GABAergic tone in the cerebellum but decreases it in the rat cortex. Gastroenterology 136, 1359-1367, e1351-1352. Chan, H., Butterworth, R.F., 1999. Evidence for an astrocytic glutamate transporter deficit in hepatic encephalopathy. Neurochemical research 24, 13971401. Chan, H., Hazell, A.S., Desjardins, P., Butterworth, R.F., 2000. Effects of ammonia on glutamate transporter (GLAST) protein and mRNA in cultured rat cortical astrocytes. Neurochemistry international 37, 243-248. Desjardins, P., Rao, K.V., Michalak, A., Rose, C., Butterworth, R.F., 1999. Effect of portacaval anastomosis on glutamine synthetase protein and gene expression in brain, liver and skeletal muscle. Metabolic brain disease 14, 273280. Dynnik, V.V., Kononov, A.V., Sergeev, A.I., Teplov, I.Y., Tankanag, A.V., Zinchenko, V.P., 2015. To Break or to Brake Neuronal Network Accelerated by Ammonium Ions? PloS one 10, e0134145. Felipo, V., Butterworth, R.F., 2002. Neurobiology of ammonia. Progress in neurobiology 67, 259-279. Felipo, V., Hermenegildo, C., Montoliu, C., Llansola, M., Minana, M.D., 1998. Neurotoxicity of ammonia and glutamate: molecular mechanisms and prevention. Neurotoxicology 19, 675-681. Gammal, S.H., Basile, A.S., Geller, D., Skolnick, P., Jones, E.A., 1990. Reversal of the behavioral and electrophysiological abnormalities of an animal model of hepatic encephalopathy by benzodiazepine receptor ligands. Hepatology 11, 371-378. Hermenegildo, C., Monfort, P., Felipo, V., 2000. Activation of N-methyl-Daspartate receptors in rat brain in vivo following acute ammonia intoxication: characterization by in vivo brain microdialysis. Hepatology 31, 709-715. Iversen, P., Sorensen, M., Bak, L.K., Waagepetersen, H.S., Vafaee, M.S., Borghammer, P., Mouridsen, K., Jensen, S.B., Vilstrup, H., Schousboe, A., Ott, P., Gjedde, A., Keiding, S., 2009. Low cerebral oxygen consumption and blood flow in patients with cirrhosis and an acute episode of hepatic encephalopathy. Gastroenterology 136, 863-871. Jenstad, M., Quazi, A.Z., Zilberter, M., Haglerod, C., Berghuis, P., Saddique, N., Goiny, M., Buntup, D., Davanger, S., FM, S.H., Barnes, C.A., McNaughton, B.L., Ottersen, O.P., Storm-Mathisen, J., Harkany, T., Chaudhry, F.A., 2009. System A transporter SAT2 mediates replenishment of dendritic glutamate pools controlling retrograde signaling by glutamate. Cerebral cortex 19, 1092-1106. Jones, E.A., Basile, A.S., 1998. Does ammonia contribute to increased GABAergic neurotransmission in liver failure? Metabolic brain disease 13, 351-360. 26

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Jones, E.A., Basile, A.S., Skolnick, P., 1990. Hepatic encephalopathy, GABAergic neurotransmission and benzodiazepine receptor ligands. Advances in experimental medicine and biology 272, 121-134. Kam, K., Nicoll, R., 2007. Excitatory synaptic transmission persists independently of the glutamate-glutamine cycle. The Journal of neuroscience : the official journal of the Society for Neuroscience 27, 9192-9200. Kanamori, K., Ross, B.D., 2004. Quantitative determination of extracellular glutamine concentration in rat brain, and its elevation in vivo by system A transport inhibitor, alpha-(methylamino)isobutyrate. Journal of neurochemistry 90, 203-210. Kanamori, K., Ross, B.D., 2005. Suppression of glial glutamine release to the extracellular fluid studied in vivo by NMR and microdialysis in hyperammonemic rat brain. Journal of neurochemistry 94, 74-85. Kanamori, K., Ross, B.D., 2013. Electrographic seizures are significantly reduced by in vivo inhibition of neuronal uptake of extracellular glutamine in rat hippocampus. Epilepsy research 107, 20-36. Kato, A., Suzuki, K., Kaneta, H., Obara, H., Fujishima, Y., Sato, S., 2000. Regional differences in cerebral glucose metabolism in cirrhotic patients with subclinical hepatic encephalopathy using positron emission tomography. Hepatology research : the official journal of the Japan Society of Hepatology 17, 237-245. Keiding, S., Sorensen, M., Bender, D., Munk, O.L., Ott, P., Vilstrup, H., 2006. Brain metabolism of 13N-ammonia during acute hepatic encephalopathy in cirrhosis measured by positron emission tomography. Hepatology 43, 42-50. Kreitzer, A.C., 2009. Physiology and pharmacology of striatal neurons. Annual review of neuroscience 32, 127-147. Llansola, M., Montoliu, C., Cauli, O., Hernandez-Rabaza, V., Agusti, A., Cabrera-Pastor, A., Gimenez-Garzo, C., Gonzalez-Usano, A., Felipo, V., 2013. Chronic hyperammonemia, glutamatergic neurotransmission and neurological alterations. Metabolic brain disease 28, 151-154. Llansola, M., Rodrigo, R., Monfort, P., Montoliu, C., Kosenko, E., Cauli, O., Piedrafita, B., El Mlili, N., Felipo, V., 2007. NMDA receptors in hyperammonemia and hepatic encephalopathy. Metabolic brain disease 22, 321335. Lockwood, A.H., Yap, E.W., Rhoades, H.M., Wong, W.H., 1991. Altered cerebral blood flow and glucose metabolism in patients with liver disease and minimal encephalopathy. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 11, 331-336. Mackenzie, B., Erickson, J.D., 2004. Sodium-coupled neutral amino acid (System N/A) transporters of the SLC38 gene family. Pflugers Archiv : European journal of physiology 447, 784-795. 27

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Mackenzie, B., Schafer, M.K., Erickson, J.D., Hediger, M.A., Weihe, E., Varoqui, H., 2003. Functional properties and cellular distribution of the system A glutamine transporter SNAT1 support specialized roles in central neurons. The Journal of biological chemistry 278, 23720-23730. Marcaida, G., Felipo, V., Hermenegildo, C., Minana, M.D., Grisolia, S., 1992. Acute ammonia toxicity is mediated by the NMDA type of glutamate receptors. FEBS letters 296, 67-68. Michalak, A., Rose, C., Butterworth, J., Butterworth, R.F., 1996. Neuroactive amino acids and glutamate (NMDA) receptors in frontal cortex of rats with experimental acute liver failure. Hepatology 24, 908-913. Nelson, A.B., Bussert, T.G., Kreitzer, A.C., Seal, R.P., 2014. Striatal cholinergic neurotransmission requires VGLUT3. The Journal of neuroscience : the official journal of the Society for Neuroscience 34, 8772-8777. Oeltzschner, G., Butz, M., Baumgarten, T.J., Hoogenboom, N., Wittsack, H.J., Schnitzler, A., 2015. Low visual cortex GABA levels in hepatic encephalopathy: links to blood ammonia, critical flicker frequency, and brain osmolytes. Metabolic brain disease 30, 1429-1438. Oeltzschner, G., Butz, M., Wickrath, F., Wittsack, H.J., Schnitzler, A., 2016. Covert hepatic encephalopathy: elevated total glutathione and absence of brain water content changes. Metabolic brain disease 31, 517-527. Perreault, M.L., O'Dowd, B.F., George, S.R., 2014. Dopamine D(1)-D(2) receptor heteromer regulates signaling cascades involved in addiction: potential relevance to adolescent drug susceptibility. Developmental neuroscience 36, 287-296. Pocock, J.M., Nicholls, D.G., 1998. Exocytotic and nonexocytotic modes of glutamate release from cultured cerebellar granule cells during chemical ischaemia. Journal of neurochemistry 70, 806-813. Rangroo Thrane, V., Thrane, A.S., Wang, F., Cotrina, M.L., Smith, N.A., Chen, M., Xu, Q., Kang, N., Fujita, T., Nagelhus, E.A., Nedergaard, M., 2013. Ammonia triggers neuronal disinhibition and seizures by impairing astrocyte potassium buffering. Nature medicine 19, 1643-1648. Rockey, D.C., Vierling, J.M., Mantry, P., Ghabril, M., Brown, R.S., Jr., Alexeeva, O., Zupanets, I.A., Grinevich, V., Baranovsky, A., Dudar, L., Fadieienko, G., Kharchenko, N., Klaryts'ka, I., Morozov, V., Grewal, P., McCashland, T., Reddy, K.G., Reddy, K.R., Syplyviy, V., Bass, N.M., Dickinson, K., Norris, C., Coakley, D., Mokhtarani, M., Scharschmidt, B.F., Group, H.-H.S., 2014. Randomized, double-blind, controlled study of glycerol phenylbutyrate in hepatic encephalopathy. Hepatology 59, 1073-1083. Rose, C., Butterworth, R.F., Zayed, J., Normandin, L., Todd, K., Michalak, A., Spahr, L., Huet, P.M., Pomier-Layrargues, G., 1999. Manganese deposition in basal ganglia structures results from both portal-systemic shunting and liver dysfunction. Gastroenterology 117, 640-644. 28

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Rose, C., Ytrebo, L.M., Davies, N.A., Sen, S., Nedredal, G.I., Belanger, M., Revhaug, A., Jalan, R., 2007. Association of reduced extracellular brain ammonia, lactate, and intracranial pressure in pigs with acute liver failure. Hepatology 46, 1883-1892. Sahara, S., Yanagawa, Y., O'Leary, D.D., Stevens, C.F., 2012. The fraction of cortical GABAergic neurons is constant from near the start of cortical neurogenesis to adulthood. The Journal of neuroscience : the official journal of the Society for Neuroscience 32, 4755-4761. Sands, S.B., Barish, M.E., 1989. NMDA receptor activation by residual glutamate in glutamine preparations: a cautionary note regarding weak NMDA receptor agonists. Brain research 495, 193-197. Schwarz, C.S., Ferrea, S., Quasthoff, K., Walter, J., Gorg, B., Haussinger, D., Schnitzler, A., Hartung, H.P., Dihne, M., 2012. Ammonium chloride influences in vitro-neuronal network activity. Experimental neurology 235, 368-373. Sergeeva, O.A., 2013. GABAergic transmission in hepatic encephalopathy. Archives of biochemistry and biophysics 536, 122-130. Sergeeva, O.A., Chepkova, A.N., Doreulee, N., Eriksson, K.S., Poelchen, W., Monnighoff, I., Heller-Stilb, B., Warskulat, U., Haussinger, D., Haas, H.L., 2003. Taurine-induced long-lasting enhancement of synaptic transmission in mice: role of transporters. The Journal of physiology 550, 911-919. Sergeeva, O.A., Fleischer, W., Chepkova, A.N., Warskulat, U., Haussinger, D., Siebler, M., Haas, H.L., 2007. GABAA-receptor modification in taurine transporter knockout mice causes striatal disinhibition. The Journal of physiology 585, 539-548. Solbu, T.T., Bjorkmo, M., Berghuis, P., Harkany, T., Chaudhry, F.A., 2010. SAT1, A Glutamine Transporter, is Preferentially Expressed in GABAergic Neurons. Frontiers in neuroanatomy 4, 1. Suarez, I., Bodega, G., Fernandez, B., 2000. Modulation of glutamate transporters (GLAST, GLT-1 and EAAC1) in the rat cerebellum following portocaval anastomosis. Brain research 859, 293-302. Szerb, J.C., O'Regan, P.A., 1984. Glutamine enhances glutamate release in preference to gamma-aminobutyrate release in hippocampal slices. Canadian journal of physiology and pharmacology 62, 919-923. Szerb, J.C., O'Regan, P.A., 1986. Possible reasons for the failure of glutamine to influence GABA release in rat hippocampal slices; Effect of nipecotic acid and methionine sulfoximine. Neurochemistry international 8, 389-395. Tani, H., Bandrowski, A.E., Parada, I., Wynn, M., Huguenard, J.R., Prince, D.A., Reimer, R.J., 2007. Modulation of epileptiform activity by glutamine and system A transport in a model of post-traumatic epilepsy. Neurobiology of disease 25, 230-238. Tani, H., Dulla, C.G., Farzampour, Z., Taylor-Weiner, A., Huguenard, J.R., Reimer, R.J., 2014. A local glutamate-glutamine cycle sustains synaptic excitatory transmitter release. Neuron 81, 888-900. 29

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Tani, H., Dulla, C.G., Huguenard, J.R., Reimer, R.J., 2010. Glutamine is required for persistent epileptiform activity in the disinhibited neocortical brain slice. The Journal of neuroscience : the official journal of the Society for Neuroscience 30, 1288-1300. Thumburu, K.K., Taneja, S., Vasishta, R.K., Dhiman, R.K., 2012. Neuropathology of acute liver failure. Neurochemistry international 60, 672-675. Wang, L., Maher, T.J., Wurtman, R.J., 2007. Oral L-glutamine increases GABA levels in striatal tissue and extracellular fluid. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 21, 1227-1232. Warren, K.S., Schenker, S., 1964. Effect of an Inhibitor of Glutamine Synthesis (Methionine Sulfoximine) on Ammonia Toxicity and Metabolism. The Journal of laboratory and clinical medicine 64, 442-449. Weissenborn, K., Ahl, B., Fischer-Wasels, D., van den Hoff, J., Hecker, H., Burchert, W., Kostler, H., 2007. Correlations between magnetic resonance spectroscopy alterations and cerebral ammonia and glucose metabolism in cirrhotic patients with and without hepatic encephalopathy. Gut 56, 1736-1742. Wise, D.R., Thompson, C.B., 2010. Glutamine addiction: a new therapeutic target in cancer. Trends in biochemical sciences 35, 427-433. Yao, D., Mackenzie, B., Ming, H., Varoqui, H., Zhu, H., Hediger, M.A., Erickson, J.D., 2000. A novel system A isoform mediating Na+/neutral amino acid cotransport. The Journal of biological chemistry 275, 22790-22797. Zeron, H.M., Rodriguez, M.R., Montes, S., Castaneda, C.R., 2011. Blood manganese levels in patients with hepatic encephalopathy. Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements 25, 225-229.

30

ACCEPTED MANUSCRIPT Table 1

*

CE P

NU

0.78±0.1 0.93±0.03

* 118±33ms 172±86ms

T IP

1 (n=6) 1 (n=9)

glutamine washout 3.54±1.4* 3.75±1.3* 1.34±0.39* 1.36±0.38

** *** 7.15±4.25* 7.31±3.75 4.99±3.05** 5.33±3.25*

0.93±0.06 0.64±0.12* 0.65±0.11 0.95±0.04 0.50±0.16** 0.49±0.12*

*** * * 107±22ms* 161±31ms 52±6ms*** 53±6ms** 201±91ms 333±135ms 38±24ms*** 32±17ms**

MA

* 119±30ms 180±70ms

control 1 (n=6) 1 (n=9)

SC R

*** 0.82±0.09 0.77±0.17 0.94±0.04 0.88±0.09**

AC 31

washout 0.89±0.26 1.04±0.3

*** 1 (n=11) 0.77±0.26* 0.93±0.29 1 (n=11) 1.32±0.19*** 1.09±0.44

D

CS Cx Cx vs CS relative CS burst Cx rate Cx vs CS ratio CS spikes in Cx bursts Cx vs CS burst CS duration Cx Cx vs CS

TE

relative spike rate

control NH4Cl 1 (n=11) 0.75±0.2** 1 (n=11) 1.03±0.1

**

**

*

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Table 2 CS vs Cx GAD65 GAD67 VGAT synaptophysin VGLUT2 VGLUT1 P0 tissue 6.76±1,4*** 5.02±1.94** 4.66±1.71*** 1.35±0.77 0.12±0.07*** 0.01±0.01 n 5 7 10 12 16 13 culture 1.21±0,1 1.02±0.49 0.72±0.27 0.19±0.07** 0.02±0.01*** 0.04±0.01 n 2 3 5 3 7 6

32

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 1

33

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 2

34

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 3

35

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 4

36

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 5

37

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 6

38

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 7

39

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 8

40

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 9

41

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 10

42

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

8.

T

7.

IP

6.

SC R

5.

HIGHLIGHTS Glutamine rapidly boosts neuronal network activity in vitro, ammonium has only minor effects Glutamine is taken up by neurons where it triggers sustained glutamate release Striatal networks are more susceptible to glutamine-induced excitation than cortical networks Cultured striatal neurons possess the synaptic machinery for vesicular glutamate release

43