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Guanosine Anxiolytic-Like Effect Involves Adenosinergic and Glutamatergic Neurotransmitter Systems

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Abstract

Accumulating evidences indicate that endogenous modulators of excitatory synapses in the mammalian brain are potential targets for treating neuropsychiatric disorders. Indeed, glutamatergic and adenosinergic neurotransmissions were recently highlighted as potential targets for developing innovative anxiolytic drugs. Accordingly, it has been shown that guanine-based purines are able to modulate both adenosinergic and glutamatergic systems in mammalian central nervous system. Here, we aimed to investigate the potential anxiolytic-like effects of guanosine and its effects on the adenosinergic and glutamatergic systems. Acute/systemic guanosine administration (7.5 mg/kg) induced robust anxiolytic-like effects in three classical anxiety-related paradigms (elevated plus maze, light/dark box, and round open field tasks). These guanosine effects were correlated with an enhancement of adenosine and a decrement of glutamate levels in the cerebrospinal fluid. Additionally, pre-administration of caffeine (10 mg/kg), an unspecific adenosine receptors’ antagonist, completely abolished the behavioral and partially prevented the neuromodulatory effects exerted by guanosine. Although the hippocampal glutamate uptake was not modulated by guanosine (both ex vivo and in vitro protocols), the synaptosomal K+-stimulated glutamate release in vitro was decreased by guanosine (100 μM) and by the specific adenosine A1 receptor agonist, 2-chloro-N 6-cyclopentyladenosine (CCPA, 100 nM). Moreover, the specific adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM) fully reversed the inhibitory guanosine effect in the glutamate release. The pharmacological modulation of A2a receptors has shown no effect in any of the evaluated parameters. In summary, the guanosine anxiolytic-like effects seem closely related to the modulation of adenosinergic (A1 receptors) and glutamatergic systems.

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Acknowledgments

This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Instituto Nacional de Ciência e Tecnologia (INCT) para Excitotoxicidade e Neuroproteção, Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS), and Financiadora de Estudos e Projetos (FINEP) research grant “Rede Instituto Brasileiro de Neurociências (IBN-Net)”, #01.06.0842-00.

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Authors and Affiliations

  1. Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600–anexo, 90035-003, Porto Alegre, RS, Brazil

    Roberto Farina Almeida, Daniel Diniz Comasseto, Denise Barbosa Ramos, Gisele Hansel, Eduardo R. Zimmer, Samanta Oliveira Loureiro, Marcelo Ganzella & Diogo Onofre Souza

  2. Neurobiology Department, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Marcelo Ganzella

  3. Brain Institute of Rio Grande do Sul (BraIns), Pontifical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, Brazil

    Eduardo R. Zimmer

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  1. Roberto Farina Almeida
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Correspondence to Diogo Onofre Souza.

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Material Supplementary 1

CAF per se did not affect the anxiety-related behavior assessed by the EPM task. The percentage of time spent in the open arms (A), the total distance travelled in open arms (cm) (B) and the total distance travelled (cm) (C) were evaluated in the EPM task 60 minutes after i.p. administration of CAF 10 mg/kg. The number of transitions (D) and the time spent in the light compartment (E) were evaluated in the light/dark task 60 minutes after i.p. CAF 10 mg/kg administration. The total distance travelled (F), the time spent in the center zone (G) and the distance travelled in center zone (H) were evaluated in the round open field task 60 minutes after i.p. CAF 10 mg/kg. Data are reported as the mean ± S.E.M. and were analyzed by unpaired Student’s t test. *p < 0.05 and **p < 0.01, compared to the saline group (n = 5 animals/group). (GIF 22 kb)

High Resolution Image (TIF 18454 kb)

Material Supplementary 2

CSF purine levels 60 minutes after i.p. saline administration: a comparison between two different protocols to preserve CSF purines. CSF purine levels 60 minutes after i.p. injection of saline. To avoid purines degradation, 2 different conditions for CSF sampling were evaluated: i) Method I – CSF samples maintained in ice for approximately 1 h and then stored in -80 °C, ii) Method II – CSF samples frozen immediately after collection in dry ice and then stored at −80 °C. By freezing the samples in dry ice immediately after the centrifugation, there was a robust nucleoside preservation. CSF levels of GMP were undetectable. Data are expressed as the mean ± SD and were analyzed by unpaired Student’s t test (n = 7 per group). *p < 0.05 compared to the Method I group. (GIF 14 kb)

High Resolution Image (TIF 1265 kb)

Material Supplementary 3

Analysis of hippocampal synaptosomal preparations. Different stages of our in vitro synaptosomal preparation homogenate (H), supernatant (S1) and synaptosomal preparation (SP) were characterized by evaluating different proteins (SNAP 25, VAMP, β Tub III, EAAC1, PSD 95, GFAP, GLT-1, NR1, GLUA1 and MAO A) by quantitative Western blot analysis as described in the Materials and Methods section; n = 3 animals. (GIF 39 kb)

High Resolution Image (TIF 117 kb)

Material Supplementary 4

DPCPX dose response curve In vitro K+-stimulated GLU release was evaluated after 1 minute of synaptosomal depolarization as described in the Materials and Methods section. The different DPCPX doses (25 nM, 50 nM, 100 nM, 250 nM) were tested. Data are reported as the mean ± S.E.M. and were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 5 animals/group). (GIF 11 kb)

High Resolution Image (TIF 6677 kb)

Material Supplementary 5

CAF modulates CSF levels of XAN and UA. CSF levels of INO and GUO (A); HIPO, XANT and UA (B) were measured 60 minutes after i.p. saline or 7.5 mg/kg GUO administration. Data are reported as the mean ± S.E.M. and were analyzed by unpaired Student’s t test. CSF levels of INO and GUO (C); HIPO, XANT and UA (D) were measured 60 minutes after i.p. saline or 7.5 mg/kg GUO administration, which was preceded by 15 minutes of i.p. saline or 10 mg/kg CAF pre-administration. Data are reported as the mean ± S.E.M., and differences among groups were determined by two-way ANOVA followed by Bonferroni’s post hoc test when applicable. *p < 0.05 and **p < 0.01, compared to the saline group (n = 10–12 animals/group). (GIF 40 kb)

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Almeida, R.F., Comasseto, D.D., Ramos, D.B. et al. Guanosine Anxiolytic-Like Effect Involves Adenosinergic and Glutamatergic Neurotransmitter Systems. Mol Neurobiol 54, 423–436 (2017). https://doi.org/10.1007/s12035-015-9660-x

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