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Inhibition of nitric oxide synthase increases synaptophysin mRNA expression in the hippocampal formation of rats
Neuroscience Letters 421 (2007) 72–76
Inhibition of nitric oxide synthase increases synaptophysin mRNA expression in the hippocampal formation of rats Sˆamia R.L. Joca b , Francisco S. Guimar˜aes c , Elaine Del-Bel a,∗ a
b
Department of MEF-Physiology, School of Odontology, Campus USP, Ribeir˜ao Preto, SP 14040-904, Brazil Department of Physics and Chemistry, Laboratory of Pharmacology, School of Pharmaceutical Sciences, Campus USP, Ribeir˜ao Preto, SP, Brazil c Department of Pharmacology, School of Medicine, Campus USP, Ribeir˜ ao Preto, SP 14049-900, Brazil Received 2 April 2007; received in revised form 15 May 2007; accepted 16 May 2007
Abstract Synaptophysin is a protein involved in the biogenesis of synaptic vesicles and budding. It has been used as an important tool to investigate plastic effects on synaptic transmission. Nitric oxide (NO) can influence plastic changes in specific brain regions related to cognition and emotion. Experimental evidence suggests that NO and synaptophysin are co-localized in several brain regions and that NO may change synaptophysin expression. Therefore, the aim of the present work was to investigate if inhibition of NO formation would change synaptophysin mRNA expression in the hippocampal formation. Male Wistar rats received single or repeated (once a day for 4 days) i.p. injections of saline or l-nitro-arginine (lNOARG, 40 mg/kg), a non-selective inhibitor of nitric oxide synthase (NOS). Twenty-four hours after the last injection the animals were sacrificed and their brains removed for ‘in situ’ hybridization study using 35 S-labeled oligonucleotide probe complementary to synaptophysin mRNA. The results were analyzed by computerized densitometry. Acute administration of l-NOARG induced a significant (p < 0.05, ANOVA) increase in synaptophysin mRNA expression in the dentate gyrus, CA1 and CA3. The effect disappeared after repeated drug administration. No change was found in the striatum, cingulated cortex, substantia nigra or nucleus accumbens. These results reinforce the proposal that nitric oxide is involved in plastic events in the hippocampus. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Synaptophysin; In situ hybridization; Nitric oxide hippocampus
Nitric oxide (NO) is a unique neurotransmitter synthesized from l-arginine by nitric oxide synthase (NOS) in response to Ca2+ -influx induced by activation of NMDA receptors by glutamate (for review see [24,31,39]). NO diffuses rapidly and influences NO-responsive target cells over a large area. It can module neuronal function in several ways, the most common being through activation of soluble guanylate cyclase (sGC) and nitrosylation of proteins and enzymes [31]. As a result, NO modulates neuronal excitability and neurotransmitter release. At high concentrations it can also be neurotoxic [31]. As a consequence, numerous physiological and pathophysiological conditions, including stress-induced disorders, have been related to NO-mediated neurotransmission [20,25,27]. However, the precise mechanisms of several NO-mediated effects remain not completely understood.
∗
Corresponding author. Tel.: +55 16 3602 4047. E-mail address: [email protected] (E. Del-Bel).
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.05.026
The NOS enzyme is co-localized in the hippocampus with the 38-kDa calcium-binding glycoprotein synaptophysin [4]. Synaptophysin is a synaptic vesicle protein proposed to regulate exocytosis of synaptic vesicles [2]. Its expression has been linked to synaptic remodeling [16,17,19] and is considered a reliable index of synaptic density [44]. It has been used to evaluate synaptogenesis or synapse removal induced by chronic drug treatment. Stress enhances NO levels and NOS expression in several brain regions ([27] for review) and may also regulate synaptophysin expression [42,49], although it is not known if these two processes are related. Moreover, it has been reported that NO induces tyrosine nitration of synaptophysin [43] and that excessive production of NO by activated microglia blocks the axonal transport of synaptophysin, what may cause axonal and synaptic dysfunction [38]. Altered synaptophysin levels have been described in pathological brain conditions such as Alzheimer’s [41] and Parkinson’s diseases [51], schizophrenia [15,16] and bipolar disorder [45]. In addition, decreased synaptophysin levels corre-
S.R.L. Joca et al. / Neuroscience Letters 421 (2007) 72–76
lates positively with impaired learning and memory capacity in animal models [5,37]. These results suggest, therefore, that the plastic and cognitive alterations found in some neuropsychiatric disorders may be related to changes in synaptophysin levels. Considering that NO has been related to affective, neurodegenerative and emotional disorders [10,20,25,27] and it can modify synaptophysin levels, the objective of the present study was to investigate the effects of inhibition of nitric oxide formation on synaptophysin mRNA expression in brain regions related to the aforementioned disorders such as the hippocampus, nucleus accumbens, striatum, substantia nigra and cingulated cortex. Male Wistar rats (200–250 g) with free access to food and water throughout the experiment and kept in a temperature controlled (23 ± 1 ◦ C) room with a 12 h light/dark cycle (lights on at 06:30 a.m.) were used. They received acute (single) or repeated (once a day for 4 days) i.p. injections of saline or lw-Nitro-l-arginine (l-NOARG, Sigma, 40 mg/kg). The dose of l-NOARG was chosen based on previous studies indicating that it can induce catalepsy [10,11] and modify anxiety-like behavior [8]. Moreover, this dose is able to inhibit NO production in the central nervous system (CNS) [36]. Also, tolerance to repeated treatment with this dose of l-NOARG occurs for the cataleptic effect after 4 days of treatment [9–11]. Twenty-four hour after the last injection the rats were sacrificed by decapitation. Their brains were removed and snap frozen in liquid nitrogen and stored at −70 ◦ C. Twelve micrometer brain sections were cut on a cryostat (Leica) at −15 ◦ C and thawed mounted on sylane immersed slides. Sections were dried and quickly warmed to room temperature (RT), immersed in fresh 4% paraformaldehyde buffered in 0.1 M sodium phosphate (pH 7.4) for 5 min and rinsed twice in 0.1 M phosphate buffered saline pH 7.4 (PBS). Sections were then treated with 0.25% acetic anhydride for 10 min at RT in 0.1 M triethanolamine hydrochloride/0.9% NaCl (pH 8.0). After treatment, sections were dehydrated through 70, 80, 90 and 100% ethanol (3 min each), defatted in chloroform for 5 min, partially rehydrated with absolute and 90% ethanol (5 min each), allowed to dry, and used immediately. A 39mer oligodeoxynucleotide probe complementary to the human synaptophysin mRNA kindly donated by Dr. K. Pearson, University of Sheffield, U.K. and complementary to synaptophysin mRNA sequence (5 -TAG CCT TGC TGC CCA TAG TCG CCC TGA GGC CCG TAG CCA-3 ) was used [15,16]. Prior to use, the oligonucleotide 3 was tail labeled with 35 S (Amersham) using terminal deoxynucleotidyl transferase from a commercially available kit (NEN Dupont) which adds an average of 10 bases per probe molecule. The hybridization procedure was similar to that previously described [21,34]. Dried sections were exposed to Hyperfilm (Amersham), stored at −70 ◦ C and developed after 10 days. RNAase (20 mg/ml, 1 h at room temperature) treated slides were used as experimental controls. The oligonucleotide probe specifically targets the synaptophysin transcript, as shown by northern blotting, sense strand hybridization, and ribonuclease controls [15,16]. Optical density (OD) measure of autoradiographic images of the dorsal hippocampal (made in the cell body, the apical and the basal synaptic layers of CA1, CA2, CA3 and dentate
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gyrus), striatum, nucleus accumbens and cingulated cortex was performed using a computerized system (ImagePro, USA). The modified optical density was obtained by subtracting the original value from 255 (maximum brightness) and converting the product to logarithm. To correct for any background difference this value was subtracted from those obtained from the corpus callosum [34]. The results express the means between both brain sides. The measurements were performed blindly to the group conditions and the results were analyzed by ANOVA followed by the Duncan test for multiple comparisons. Since no differences were found between animals treated acutely or repeatedly with saline, they were joined together in a control group. The significance level was set at p < 0.05. Synaptophysin mRNA expression in the hippocampus can be seen in Figs. 1 and 2. This distribution was similar to that previously described [15,16,28]. Acute administration of l-NOARG significantly increased synaptophysin mRNA expression 24 h later in the dentate gyrus (F2,12 = 4.42, p = 0.036, Duncan test, p < 0.05) and CA1 (F2,12 = 7.67, p < 0.01, Duncan test, p < 0.05) as compared to control animals.
Fig. 1. Autoradiograms of sections of rat brain hybridized with a 35 S-labelled oligonucleotide probe complementary to synaptophysin mRNA after single (Nrg-acute) or repeated (once a day injection for 4 days, Nrg-chronic) administration of l-NOARG (40 mg/kg).
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Fig. 2. Increased synaptophysin mRNA expression in the dentate gyrus, CA1, CA2 and CA3 hippocampal regions of rats treated with single (Nrg-ac) or repeated (Nrg-chr, once a day injection for 4 days) l-NOARG (40 mg/kg). Animals (n = 4–6/group) were sacrificed 24 h after the last injection. In situ hybridization was performed using a 35 S-labelled oligonucleotide probe complementary to synaptophysin mRNA. To correct for any background difference the modified optical density obtained in each region was subtracted from that obtained in the corpus callosum. Results are expressed as mean + S.E.M. p < 0.05 compared to (*) controls or (+) Nrg-chr group (ANOVA followed by the Duncan test, p < 0.05).
It also induced a significant increase in synaptophysin mRNA expression when compared to chronically l-NOARG treated animals in CA3 (F2,11 = 3.43, p = 0.06, Duncan test, p < 0.05) and CA1 (Duncan test, p < 0.05). There was no difference between controls and chronically l-NOARG treated animals. Also, no effect was found in the striatum, nucleus accumbens and cingulated cortex (Duncan test, p > 0.05, Table 1). NO is proposed to trigger biochemical responses during synaptic activity and/or modify neurotransmitter secretion in presynaptic terminals [31,39]. It can act as a retrograde neuronal messenger released from the postsynaptic membrane, diffusing across the synaptic cleft to the presynaptic membrane to induce long-term potentiation and depression, or to affect metabolism by regulating cGMP levels [24]. l-NOARG is a non-selective NOS inhibitor that produces a time-dependent irreversible inhibition of the purified brain NOS in vitro [36], which may explain its long-lasting and apparently irreversible inhibition of rat-brain NOS following systemic administration in vivo [14]. The hippocampus contains both the neuronal and endothelial NOS isoforms [13,48] although more recent evidence suggests that only the nNOS isoform is present in hippocampal neurons [3]. This region has been related to several effects observed after systemic administration of NOS inhibitors, including antidepressant, nociceptive and impaired learning effects [18,25,26]. The mechanisms of these effects
have not been completely elucidated, but may involve hippocampal plastic changes. Synaptophysin expression has been linked to synaptic remodeling [15,19] and several pieces of evidence suggest that NO can interfere with synaptophysin function and/or expression. During synaptic remodeling in the adult CNS, NO acts as a signal for synaptic detachment and inhibits synapse formation by cGMPdependent and probably S-nitrosylation-mediated mechanisms [40]. Moreover, NO and cGMP act in the presynaptic neurons during long-lasting potentiation [1] and contribute to activitydependent regulation of synaptic vesicle cycling [29,46]. NO can also interfere with synaptic function by producing peroxynitrite, the product of the radical–radical reaction between NO and superoxide anion. Peroxynitrite is a potent oxidant involved in tissue damage in neurodegenerative disorders [32] and is responsible for the nitration of several proteins, including synaptophysin [12,43]. This effect may have pathophysiological consequences since peroxynitrite formation and subsequent tyrosine nitration of synaptophysin is found in the hippocampus of rats chronically treated with intracerebroventricular infusion of beta-amyloid protein [43]. As this treatment also resulted in impairment of nicotine-evoked acetylcholine release, the authors suggested that tyrosine nitration of synaptophysin may be related to the beta-amyloid-induced impairment of acetylcholine release often related to Alzheimer’s disease.
Table 1 Synaptophysin mRNA expression in the striatum, nucleus accumbens, substantia nigra and cingulated cortex of rats treated with single (Nrg-ac) or repeated (Nrg-chr) administration of saline (controls) or l-NOARG (40 mg/kg)
Control (n = 6) Nrg-ac (n = 5) Nrg-chr (n = 4)
Striatum
Accumbens
Substantia nigra
Cingulated cortex
0.134 (±0.017) 0.128 (±0.007) 0.141 (±0.020)
0.244 (±0.020) 0.241 (±0.015) 0.246 (±0.010)
0.340 (±0.016) 0.395 (±0.022) 0.330 (±0.027)
0.440 (±0.020) 0.460 (±(0.010) 0.409 (±0.037)
Animals (n = 4–6/group) were sacrificed 24 h after the last injection. In situ hybridization was performed using a 35 S-labelled oligonucleotide probe complementary to synaptophysin mRNA. To correct for any background difference the modified optical density obtained in each region was subtracted from that obtained in the corpus callosum. Results are expressed as mean ± S.E.M. No effect was found among groups.
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Another condition related to increased NO production and reduced synaptophysin expression is stress exposure [27,42,49]. For example, acute or chronic exposure to restraint stress decreases synaptophysin expression in the hippocampus [42,49]. On the other hand, chronic treatment with drugs that attenuate behavioral deficits induced by restraint exposure, such as antidepressants, increases synaptophysin mRNA expression in the hippocampus [33] and accelerates the recovery of hippocampal synaptophysin expression after chronic stress [49]. Plastic effects induced by antidepressant drugs in the hippocampus have been linked to their antidepressant effects [7,22]. It is possible that a reduction in NO production contributes to these plastic effects, since it appears be one of the common actions of these drugs [47]. In agreement with this proposal, NOS inhibitors also induce antidepressant-like effects after systemic or intra-hippocampal injection in the forced swimming model [23,25]. These effects appear after acute or sub-chronic administration, but their mechanisms are still unknown. The present results suggest that synaptic plastic modifications mediated by changes in synaptophysin expression could be involved. This could be an indirect effect due to changes in BDNF expression [30]. BDNF enhances synaptophysin expression in cultured hippocampal neurons [50] and NO is able to decrease BDNF secretion in cultured hippocampal cells [6]. In the present study, however, the acute effect of l-NOARG disappeared after 4 days of repeated treatment. Changes in synaptophysin expression depending on length of exposure to stress regimens have been previously reported. For example, intermittent exposure of rats to 6 h of restraint stress for 14 days did not produce significant changes in synaptophysin immunoreactivity [35]. Also, tolerance to some effects of NOS inhibitors has been reported before [10,11]. For example, the cataleptic effect of these drugs observed in rodents suffers tolerance after 4 days of chronic treatment. These animals also develop cross-tolerance for haloperidol [9]. The mechanisms of this rapid tolerance are still unknown. Our data, however, showing no effect on striatal synaptophysin mRNA expression of either acute or chronic NOS inhibition, suggest that it does not involve changes in this expression. Although the other investigated regions also contain NOS positive cells, the present results suggest that local NO effects are probably not mediated by synaptophysin expression changes. In conclusion, acute, but not repeated, systemic administration of l-NOARG in rats induced a significant and region specific increase in synaptophysin mRNA expression in the dentate gyrus, CA1, CA2 and CA3. This change could be related to effects involving plastic modifications of this region such as the antidepressant-like effect observed after systemic or intrahippocampal injection of NOS inhibitors.
Acknowledgements We acknowledge the helpful technical support of J.C. de Aguiar and E.L.T. Gomes. This research was supported by grants from FAPESP, CAPES, CNPq and Wellcome Foundation, UK.
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Inhibition of nitric oxide synthase increases synaptophysin mRNA expression in the hippocampal formation of rats Sˆamia R.L. Joca b , Francisco S. Guimar˜aes c , Elaine Del-Bel a,∗ a
b
Department of MEF-Physiology, School of Odontology, Campus USP, Ribeir˜ao Preto, SP 14040-904, Brazil Department of Physics and Chemistry, Laboratory of Pharmacology, School of Pharmaceutical Sciences, Campus USP, Ribeir˜ao Preto, SP, Brazil c Department of Pharmacology, School of Medicine, Campus USP, Ribeir˜ ao Preto, SP 14049-900, Brazil Received 2 April 2007; received in revised form 15 May 2007; accepted 16 May 2007
Abstract Synaptophysin is a protein involved in the biogenesis of synaptic vesicles and budding. It has been used as an important tool to investigate plastic effects on synaptic transmission. Nitric oxide (NO) can influence plastic changes in specific brain regions related to cognition and emotion. Experimental evidence suggests that NO and synaptophysin are co-localized in several brain regions and that NO may change synaptophysin expression. Therefore, the aim of the present work was to investigate if inhibition of NO formation would change synaptophysin mRNA expression in the hippocampal formation. Male Wistar rats received single or repeated (once a day for 4 days) i.p. injections of saline or l-nitro-arginine (lNOARG, 40 mg/kg), a non-selective inhibitor of nitric oxide synthase (NOS). Twenty-four hours after the last injection the animals were sacrificed and their brains removed for ‘in situ’ hybridization study using 35 S-labeled oligonucleotide probe complementary to synaptophysin mRNA. The results were analyzed by computerized densitometry. Acute administration of l-NOARG induced a significant (p < 0.05, ANOVA) increase in synaptophysin mRNA expression in the dentate gyrus, CA1 and CA3. The effect disappeared after repeated drug administration. No change was found in the striatum, cingulated cortex, substantia nigra or nucleus accumbens. These results reinforce the proposal that nitric oxide is involved in plastic events in the hippocampus. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Synaptophysin; In situ hybridization; Nitric oxide hippocampus
Nitric oxide (NO) is a unique neurotransmitter synthesized from l-arginine by nitric oxide synthase (NOS) in response to Ca2+ -influx induced by activation of NMDA receptors by glutamate (for review see [24,31,39]). NO diffuses rapidly and influences NO-responsive target cells over a large area. It can module neuronal function in several ways, the most common being through activation of soluble guanylate cyclase (sGC) and nitrosylation of proteins and enzymes [31]. As a result, NO modulates neuronal excitability and neurotransmitter release. At high concentrations it can also be neurotoxic [31]. As a consequence, numerous physiological and pathophysiological conditions, including stress-induced disorders, have been related to NO-mediated neurotransmission [20,25,27]. However, the precise mechanisms of several NO-mediated effects remain not completely understood.
∗
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The NOS enzyme is co-localized in the hippocampus with the 38-kDa calcium-binding glycoprotein synaptophysin [4]. Synaptophysin is a synaptic vesicle protein proposed to regulate exocytosis of synaptic vesicles [2]. Its expression has been linked to synaptic remodeling [16,17,19] and is considered a reliable index of synaptic density [44]. It has been used to evaluate synaptogenesis or synapse removal induced by chronic drug treatment. Stress enhances NO levels and NOS expression in several brain regions ([27] for review) and may also regulate synaptophysin expression [42,49], although it is not known if these two processes are related. Moreover, it has been reported that NO induces tyrosine nitration of synaptophysin [43] and that excessive production of NO by activated microglia blocks the axonal transport of synaptophysin, what may cause axonal and synaptic dysfunction [38]. Altered synaptophysin levels have been described in pathological brain conditions such as Alzheimer’s [41] and Parkinson’s diseases [51], schizophrenia [15,16] and bipolar disorder [45]. In addition, decreased synaptophysin levels corre-
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lates positively with impaired learning and memory capacity in animal models [5,37]. These results suggest, therefore, that the plastic and cognitive alterations found in some neuropsychiatric disorders may be related to changes in synaptophysin levels. Considering that NO has been related to affective, neurodegenerative and emotional disorders [10,20,25,27] and it can modify synaptophysin levels, the objective of the present study was to investigate the effects of inhibition of nitric oxide formation on synaptophysin mRNA expression in brain regions related to the aforementioned disorders such as the hippocampus, nucleus accumbens, striatum, substantia nigra and cingulated cortex. Male Wistar rats (200–250 g) with free access to food and water throughout the experiment and kept in a temperature controlled (23 ± 1 ◦ C) room with a 12 h light/dark cycle (lights on at 06:30 a.m.) were used. They received acute (single) or repeated (once a day for 4 days) i.p. injections of saline or lw-Nitro-l-arginine (l-NOARG, Sigma, 40 mg/kg). The dose of l-NOARG was chosen based on previous studies indicating that it can induce catalepsy [10,11] and modify anxiety-like behavior [8]. Moreover, this dose is able to inhibit NO production in the central nervous system (CNS) [36]. Also, tolerance to repeated treatment with this dose of l-NOARG occurs for the cataleptic effect after 4 days of treatment [9–11]. Twenty-four hour after the last injection the rats were sacrificed by decapitation. Their brains were removed and snap frozen in liquid nitrogen and stored at −70 ◦ C. Twelve micrometer brain sections were cut on a cryostat (Leica) at −15 ◦ C and thawed mounted on sylane immersed slides. Sections were dried and quickly warmed to room temperature (RT), immersed in fresh 4% paraformaldehyde buffered in 0.1 M sodium phosphate (pH 7.4) for 5 min and rinsed twice in 0.1 M phosphate buffered saline pH 7.4 (PBS). Sections were then treated with 0.25% acetic anhydride for 10 min at RT in 0.1 M triethanolamine hydrochloride/0.9% NaCl (pH 8.0). After treatment, sections were dehydrated through 70, 80, 90 and 100% ethanol (3 min each), defatted in chloroform for 5 min, partially rehydrated with absolute and 90% ethanol (5 min each), allowed to dry, and used immediately. A 39mer oligodeoxynucleotide probe complementary to the human synaptophysin mRNA kindly donated by Dr. K. Pearson, University of Sheffield, U.K. and complementary to synaptophysin mRNA sequence (5 -TAG CCT TGC TGC CCA TAG TCG CCC TGA GGC CCG TAG CCA-3 ) was used [15,16]. Prior to use, the oligonucleotide 3 was tail labeled with 35 S (Amersham) using terminal deoxynucleotidyl transferase from a commercially available kit (NEN Dupont) which adds an average of 10 bases per probe molecule. The hybridization procedure was similar to that previously described [21,34]. Dried sections were exposed to Hyperfilm (Amersham), stored at −70 ◦ C and developed after 10 days. RNAase (20 mg/ml, 1 h at room temperature) treated slides were used as experimental controls. The oligonucleotide probe specifically targets the synaptophysin transcript, as shown by northern blotting, sense strand hybridization, and ribonuclease controls [15,16]. Optical density (OD) measure of autoradiographic images of the dorsal hippocampal (made in the cell body, the apical and the basal synaptic layers of CA1, CA2, CA3 and dentate
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gyrus), striatum, nucleus accumbens and cingulated cortex was performed using a computerized system (ImagePro, USA). The modified optical density was obtained by subtracting the original value from 255 (maximum brightness) and converting the product to logarithm. To correct for any background difference this value was subtracted from those obtained from the corpus callosum [34]. The results express the means between both brain sides. The measurements were performed blindly to the group conditions and the results were analyzed by ANOVA followed by the Duncan test for multiple comparisons. Since no differences were found between animals treated acutely or repeatedly with saline, they were joined together in a control group. The significance level was set at p < 0.05. Synaptophysin mRNA expression in the hippocampus can be seen in Figs. 1 and 2. This distribution was similar to that previously described [15,16,28]. Acute administration of l-NOARG significantly increased synaptophysin mRNA expression 24 h later in the dentate gyrus (F2,12 = 4.42, p = 0.036, Duncan test, p < 0.05) and CA1 (F2,12 = 7.67, p < 0.01, Duncan test, p < 0.05) as compared to control animals.
Fig. 1. Autoradiograms of sections of rat brain hybridized with a 35 S-labelled oligonucleotide probe complementary to synaptophysin mRNA after single (Nrg-acute) or repeated (once a day injection for 4 days, Nrg-chronic) administration of l-NOARG (40 mg/kg).
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Fig. 2. Increased synaptophysin mRNA expression in the dentate gyrus, CA1, CA2 and CA3 hippocampal regions of rats treated with single (Nrg-ac) or repeated (Nrg-chr, once a day injection for 4 days) l-NOARG (40 mg/kg). Animals (n = 4–6/group) were sacrificed 24 h after the last injection. In situ hybridization was performed using a 35 S-labelled oligonucleotide probe complementary to synaptophysin mRNA. To correct for any background difference the modified optical density obtained in each region was subtracted from that obtained in the corpus callosum. Results are expressed as mean + S.E.M. p < 0.05 compared to (*) controls or (+) Nrg-chr group (ANOVA followed by the Duncan test, p < 0.05).
It also induced a significant increase in synaptophysin mRNA expression when compared to chronically l-NOARG treated animals in CA3 (F2,11 = 3.43, p = 0.06, Duncan test, p < 0.05) and CA1 (Duncan test, p < 0.05). There was no difference between controls and chronically l-NOARG treated animals. Also, no effect was found in the striatum, nucleus accumbens and cingulated cortex (Duncan test, p > 0.05, Table 1). NO is proposed to trigger biochemical responses during synaptic activity and/or modify neurotransmitter secretion in presynaptic terminals [31,39]. It can act as a retrograde neuronal messenger released from the postsynaptic membrane, diffusing across the synaptic cleft to the presynaptic membrane to induce long-term potentiation and depression, or to affect metabolism by regulating cGMP levels [24]. l-NOARG is a non-selective NOS inhibitor that produces a time-dependent irreversible inhibition of the purified brain NOS in vitro [36], which may explain its long-lasting and apparently irreversible inhibition of rat-brain NOS following systemic administration in vivo [14]. The hippocampus contains both the neuronal and endothelial NOS isoforms [13,48] although more recent evidence suggests that only the nNOS isoform is present in hippocampal neurons [3]. This region has been related to several effects observed after systemic administration of NOS inhibitors, including antidepressant, nociceptive and impaired learning effects [18,25,26]. The mechanisms of these effects
have not been completely elucidated, but may involve hippocampal plastic changes. Synaptophysin expression has been linked to synaptic remodeling [15,19] and several pieces of evidence suggest that NO can interfere with synaptophysin function and/or expression. During synaptic remodeling in the adult CNS, NO acts as a signal for synaptic detachment and inhibits synapse formation by cGMPdependent and probably S-nitrosylation-mediated mechanisms [40]. Moreover, NO and cGMP act in the presynaptic neurons during long-lasting potentiation [1] and contribute to activitydependent regulation of synaptic vesicle cycling [29,46]. NO can also interfere with synaptic function by producing peroxynitrite, the product of the radical–radical reaction between NO and superoxide anion. Peroxynitrite is a potent oxidant involved in tissue damage in neurodegenerative disorders [32] and is responsible for the nitration of several proteins, including synaptophysin [12,43]. This effect may have pathophysiological consequences since peroxynitrite formation and subsequent tyrosine nitration of synaptophysin is found in the hippocampus of rats chronically treated with intracerebroventricular infusion of beta-amyloid protein [43]. As this treatment also resulted in impairment of nicotine-evoked acetylcholine release, the authors suggested that tyrosine nitration of synaptophysin may be related to the beta-amyloid-induced impairment of acetylcholine release often related to Alzheimer’s disease.
Table 1 Synaptophysin mRNA expression in the striatum, nucleus accumbens, substantia nigra and cingulated cortex of rats treated with single (Nrg-ac) or repeated (Nrg-chr) administration of saline (controls) or l-NOARG (40 mg/kg)
Control (n = 6) Nrg-ac (n = 5) Nrg-chr (n = 4)
Striatum
Accumbens
Substantia nigra
Cingulated cortex
0.134 (±0.017) 0.128 (±0.007) 0.141 (±0.020)
0.244 (±0.020) 0.241 (±0.015) 0.246 (±0.010)
0.340 (±0.016) 0.395 (±0.022) 0.330 (±0.027)
0.440 (±0.020) 0.460 (±(0.010) 0.409 (±0.037)
Animals (n = 4–6/group) were sacrificed 24 h after the last injection. In situ hybridization was performed using a 35 S-labelled oligonucleotide probe complementary to synaptophysin mRNA. To correct for any background difference the modified optical density obtained in each region was subtracted from that obtained in the corpus callosum. Results are expressed as mean ± S.E.M. No effect was found among groups.
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Another condition related to increased NO production and reduced synaptophysin expression is stress exposure [27,42,49]. For example, acute or chronic exposure to restraint stress decreases synaptophysin expression in the hippocampus [42,49]. On the other hand, chronic treatment with drugs that attenuate behavioral deficits induced by restraint exposure, such as antidepressants, increases synaptophysin mRNA expression in the hippocampus [33] and accelerates the recovery of hippocampal synaptophysin expression after chronic stress [49]. Plastic effects induced by antidepressant drugs in the hippocampus have been linked to their antidepressant effects [7,22]. It is possible that a reduction in NO production contributes to these plastic effects, since it appears be one of the common actions of these drugs [47]. In agreement with this proposal, NOS inhibitors also induce antidepressant-like effects after systemic or intra-hippocampal injection in the forced swimming model [23,25]. These effects appear after acute or sub-chronic administration, but their mechanisms are still unknown. The present results suggest that synaptic plastic modifications mediated by changes in synaptophysin expression could be involved. This could be an indirect effect due to changes in BDNF expression [30]. BDNF enhances synaptophysin expression in cultured hippocampal neurons [50] and NO is able to decrease BDNF secretion in cultured hippocampal cells [6]. In the present study, however, the acute effect of l-NOARG disappeared after 4 days of repeated treatment. Changes in synaptophysin expression depending on length of exposure to stress regimens have been previously reported. For example, intermittent exposure of rats to 6 h of restraint stress for 14 days did not produce significant changes in synaptophysin immunoreactivity [35]. Also, tolerance to some effects of NOS inhibitors has been reported before [10,11]. For example, the cataleptic effect of these drugs observed in rodents suffers tolerance after 4 days of chronic treatment. These animals also develop cross-tolerance for haloperidol [9]. The mechanisms of this rapid tolerance are still unknown. Our data, however, showing no effect on striatal synaptophysin mRNA expression of either acute or chronic NOS inhibition, suggest that it does not involve changes in this expression. Although the other investigated regions also contain NOS positive cells, the present results suggest that local NO effects are probably not mediated by synaptophysin expression changes. In conclusion, acute, but not repeated, systemic administration of l-NOARG in rats induced a significant and region specific increase in synaptophysin mRNA expression in the dentate gyrus, CA1, CA2 and CA3. This change could be related to effects involving plastic modifications of this region such as the antidepressant-like effect observed after systemic or intrahippocampal injection of NOS inhibitors.
Acknowledgements We acknowledge the helpful technical support of J.C. de Aguiar and E.L.T. Gomes. This research was supported by grants from FAPESP, CAPES, CNPq and Wellcome Foundation, UK.
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