Lack of GSK3ß activation and modulation of synaptic plasticity by dopamine in 5-HT1A-receptor KO mice.

Psychiatric disorders are associated with excitation-inhibition (E-I) balance impairment in the prefrontal cortex. However, how the E-I balance is regulated is poorly known. The E-I balance of neuronal networks is linked to the action of numerous neuromodulators such as dopamine and 5-HT. We investigated the role of D2-receptors in tuning the E-I balance in a mouse model of anxiety, the 5-HT1A-receptor KO mice. We focused on synaptic plasticity of excitation and inhibition on layer 5 pyramidal neurons. We show that D2-receptor activation decreases the excitation and favors HFS-induced LTD of excitatory synapses via the activation of GSK3ß. This effect is absent in 5-HT1A-receptor KO mice. Our data show that the fine control of excitatory transmission by GSK3ß requires recruitment of D2-receptors and depends on the presence of 5-HT1A-receptors. In psychiatric disorders in which the number of 5-HT1A-receptors decreased, therapies should reconsider how serotonin and dopamine receptors interact and control neuronal network activity.

5-HT(1A) receptors direct the orientation of plasticity in layer 5 pyramidal neurons of the mouse prefrontal cortex.

Several psychiatric disorders involving the prefrontal cortex (PFC) are associated with a dysfunction of 5-HT(1A) receptors (5-HT(1A)R). These receptors, located on interneurons and pyramidal neurons, may influence neuronal excitability through a regulation of the balance between excitation (E) and inhibition (I). Patch-clamp recordings in mouse cortical slices were performed to determine the modulatory role of 5-HT(1A)R on the excitability and the synaptic plasticity of layer 5 pyramidal neurons (L5PyNs) of the PFC. This was done by a comparison of postsynaptic currents evoked by electrical stimulation in layer 2/3 of 5-HT(1A)R-KO and wild-type (WT) mice. We observed that the E-I balance was significantly changed from 20% E-80% I in WT mice to 23% E-77% I in 5-HT(1A)R-KO mice, demonstrating that 5-HT(1A)Rs contribute to the control of the balance between excitation and inhibition. Furthermore, we show that interfering with 5-HT(1A)R reduced the magnitude of the long term potentiation of excitation (eLTP) (induced by high frequency stimulation). In addition, we show that 5-HT(1A)Rs determine the orientation of the synaptic plasticity towards LTP or LTD or no plasticity through the modulation of NMDAR-mediated currents. Our data point out to a unique role of 5-HT(1A) postsynaptic receptors in PFC to adapt the functional plasticity of L5PyNs towards LTP, LTD or no plasticity. This brings a new way to intervene on neuronal networks of the PFC in anxiety disorders and schizophrenia.

Serotonergic modulation of LTP at excitatory and inhibitory synapses in the developing rat visual cortex.

The stability and efficacy of neuronal circuits are achieved through a detailed balance between pyramidal cell and interneuron activities. Interestingly, the neocortical excitatory-inhibitory (E-I) balance is actively maintained at the soma of Layer 5 pyramidal neurons which receive 20% of excitation and 80% of inhibition after dendritic integration, and this is not affected by changes in synaptic strength. To infer the role of serotonergic neuromodulation on the activity-dependent maintenance of the E-I balance, we performed continuous voltage clamp measurements of stimulation-locked conductance dynamics in Layer 5 pyramidal neurons before and after long-term potentiation (LTP) induction, together with chronic or acute manipulation of serotonin function. When a theta-burst stimulation was applied in Layer 2/3 of 5-HT depleted cortical slices (after in vivo treatment with the tryptophan hydroxylase inhibitor p-chlorophenylalanine (pCPA)), or after in vitro perfusion of the potent 5-HT1A receptor antagonist WAY-100,635, we observed a persistent shift of the ratio between excitation and inhibition toward more inhibition. This was due to a strong LTP of inhibition co-aligned with a weak LTP of excitation, whereas the same protocol caused a similar potentiation of excitatory and inhibitory inputs when applied in control slices. In contrast, neither excitatory nor inhibitory postsynaptic currents were potentiated when LTP protocols were delivered in the presence of either the selective serotonin reuptake inhibitor citalopram or the 5-HT1A receptor agonist 8-OH-DPAT. This is the first demonstration that serotonergic neuromodulation is crucial for the maintenance of the neocortical E-I balance during high-frequency regimes.

Blockade of different muscarinic receptor subtypes changes the equilibrium between excitation and inhibition in rat visual cortex.

We have shown that cortical acetylcholine modulates the balance between excitation and inhibition evoked in layer 5 pyramidal neurons of rat visual cortex [Lucas-Meunier E, Monier C, Amar M, Baux G, Frégnac Y, Fossier P (2009) Cereb Cortex 19:2411-2427]. Our aim is now to establish a functional basis for the role of the different types of muscarinic receptors (MRs) on glutamate fibers and on GABAergic interneurons and to analyse their contribution to the modulation of excitation-inhibition balance in the rat visual cortex. To ascertain that there was a basis for our functional study, we first checked for the presence of the various MR subtypes by single cell RT-PCR and immunolabeling experiments. Then, recording the composite responses in layer 5 pyramidal neurons to layer 1-2 stimulation (which also recruits cholinergic fibers) in the presence of specific antagonists of the different types of MR allowed us to determine their modulatory role. We show that the specific blockade of the widely distributed M1R (with the mamba toxin, MT7) induced a significant increase in the excitatory conductance without modifying the inhibitory conductance, pointing to a localization of M1R on glutamatergic neurons where their activation would decrease the release of glutamate. From our functional results, M2/M4Rs appear to be located on glutamatergic neurons afferent to the recorded layer 5 pyramidal neuron and they decrease glutamate release. The extended distribution of M4Rs in the cortex compared to the restricted distribution of M2R (layers 3-5) is in favour of a major role as a modulator of M4R. The selective antagonist of M3Rs, 4-DAMP, decreased the inhibitory conductance, showing that activated M3Rs increase the release of GABA and thus are located on GABAergic interneurons. The activation of the different types of MRs located either on glutamatergic neurons or on GABAergic interneurons converges to reinforce the dominance of inhibitory inputs thus decreasing the excitability of layer 5 pyramidal neurons.

Roles of nitric oxide in the homeostatic control of the excitation-inhibition balance in rat visual cortical networks.

The level of excitability of cortical neurons depends on the balance between their excitatory and inhibitory inputs (excitation/inhibition [E/I] balance). In the cortex, the E/I balance received by a neuron is dynamically maintained through a coordinated regulation of the strength of these inputs, described in term of homeostatic plasticity. Using a method allowing the determination of the E/I balance in rat cortical layer 5 pyramidal neurons (L5-PNs, the main output stage of the cortex), while keeping the interactions between excitatory and inhibitory networks functional, we examined the effects of high or low frequency of stimulation (HFS or LFS) protocols in layer 4 (in order to mimic thalamo-cortical entries) on the E-I level of the neuronal network. We previously showed that the E/I balance of L5-PNs remains stable due to a dual potentiation or dual depression of E and I after HFS or LFS protocols. Here, using a specific neuronal nitric oxide synthase (nNOS) inhibitor, we show that the related potentiation or depression of E and I (underlying homeostatic plasticity processes) required nNOS activation. We also show that application of an unspecific blocker of nitric oxide synthase (NOS) or a nitric oxide (NO) scavenger induces an increase of the E/I balance suggesting a role for a tonic NO synthesis in the regulation of the network activity. It is concluded that, in the cortex, a phasic NO effect (due to activation of nNOS) is required for the induction of homeostatic plasticity processes whereas a tonic NO signal is involved in the regulation of a set-point value for the E/I balance.

Impaired GABAergic transmission disrupts normal homeostatic plasticity in rat cortical networks.

In the cortex, homeostatic plasticity appears to be a key process for maintaining neuronal network activity in a functional range. This phenomenon depends on close interactions between excitatory and inhibitory circuits. We previously showed that application of a high frequency of stimulation (HFS) protocol in layer 2/3 induces parallel potentiation of excitatory and inhibitory inputs on layer 5 pyramidal neurons, leading to an unchanged excitation/inhibition (E/I) balance. These coordinated long-term potentiations of excitation and inhibition correspond to homeostatic plasticity of the neuronal networks. We showed here, on the rat visual cortex, that blockade (with gabazine) or overactivation (with 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) of GABA(A) receptors enhanced the E/I balance and prevented the potentiation of excitatory and inhibitory inputs after an HFS protocol. These impairements of the GABAergic transmission led to a long-term depression-like effect after an HFS protocol. We also observed that the blockade of inhibition reduced excitation (by 60%), and conversely, the blockade of excitation decreased inhibition (by 90%). These results support the idea that inhibitory interneurons are critical for recurrent interactions underlying homeostatic plasticity in cortical networks.

Nucleoplasmic reticulum is not essential in nuclear calcium signalling mediated by cyclic ADPribose in primary neurons.

Nuclear calcium regulation is essential for controlling nuclear processes such as gene expression. Recent studies, mostly performed on immortalized or transformed cell lines, reported the presence of a nucleoplasmic reticulum (NR). It has been suggested that NR acts as a storage organelle having an important role in nuclear Ca2+ signalling. However, whether NR is present and necessary in primary neurons for generation of nuclear Ca2+ signalling has never been investigated. Here, we show, by confocal microscopy and by electronic microscopy, that nuclei in intact neurons or isolated nuclei are not endowed with NR. Finally, our experiments performed on isolated nuclei from Aplysia giant neurons show that the nuclear envelope acts as a functional Ca2+ store which can be mobilized by the second messenger cyclic ADPribose to elicit a nucleoplasmic Ca2+ elevation. Our study provides evidence that nuclear Ca2+ signals can be independent of the presence of NR in neurons.

Control of IsAHP in mouse hippocampus CA1 pyramidal neurons by RyR3-mediated calcium-induced calcium release.

In several neuronal preparations, the ryanodine-sensitive calcium store was reported to participate in the generation of slow afterhyperpolarization currents (IsAHP) involved in spike frequency adaptation. We show that calcium release from the ryanodine-sensitive calcium store is a major determinant of the triggering of IsAHP in mouse CA1 pyramidal neurons. Whole-cell patch clamp recordings in hippocampus slices show that the intracellular calcium stores depletion using an inhibitor of the endoplasmic reticulum Ca2+-ATPase (5 microM cyclopiazonic acid), as well as the specific blockade of ryanodine receptors (100 microM ryanodine) both reduced the IsAHP by about 70%. Immunohistology, using an anti-RyR3 specific antibody, indicates that RyR3 expression is particularly enriched in the CA1 apical dendrites (considered as the most important site for sAHP generation). We show that our anti-RyR3 antibody acts as a functional RyR3 antagonist and induced a reduction in IsAHP by about 70%. The additional ryanodine application (100 micro M) did not further affect IsAHP, thus excluding RyR2 in IsAHP activation. Our results argue in favor of a specialized function of RyR3 in CA1 pyramidal cells in triggering IsAHP due to their localization in the apical dendrite.

Cholinergic modulation of the cortical neuronal network.

Acetylcholine (ACh) is an important neurotransmitter of the CNS that binds both nicotinic and muscarinic receptors to exert its action. However, the mechanisms underlying the effects of cholinergic receptors have still not been completely elucidated. Central cholinergic neurons, mainly located in basal forebrain, send their projections to different structures including the cortex. The cortical innervation is diffuse and roughly topographic, which has prompted some authors to suspect a modulating role of ACh on the activity of the cortical network rather than a direct synaptic role. The cholinergic system is implicated in functional, behavioural and pathological states including cognitive function, nicotine addiction, Alzheimer's disease, Tourette's syndrome, epilepsies and schizophrenia. As these processes depend on the activation of glutamatergic and GABAergic systems, the cholinergic terminals must exert their effects via the modulation of excitatory and/or inhibitory neurotransmission. However, the understanding of cholinergic modulation is complex because it is the result of a mixture of positive and negative modulation, implying that there are various types, or even subtypes, of cholinergic receptors. In this review, we summarize the current knowledge on central cholinergic systems (projections and receptors) and then aim to focus on the implications for ACh in the modulation of cortical neuronal activity.

Ryanodine-, IP3- and NAADP-dependent calcium stores control acetylcholine release.

Injections of inositol trisphosphate (IP3) or nicotinamide adenine dinucleotide phosphate (NAADP) into the presynaptic neurone of an identified cholinergic synapse in the buccal ganglion of Aplysia californica increased the amplitude of the inhibitory postsynaptic current evoked by a presynaptic action potential. This suggests that Ca2+ release from various Ca2+ stores can modulate acetylcholine (ACh) release. Specific blockade of the calcium-induced calcium release (CICR) mechanism with ryanodine, or of IP3-induced calcium release with heparin, abolished the effects of IP3, but not the effects of NAADP, suggesting the presence of an intracellular Ca2+ pool independent of those containing ryanodine receptors (RyR) or IP3 receptors. To reinforce electrophysiological observations, intracellular [Ca2+]i changes were measured using the fluorescent dye rhod-2. Injections of cyclic ADP-ribose (an activator of RyR), IP3 or NAADP into the presynaptic neurone induced transient increases in the free intracellular Ca2+ concentration. RyR- and IP3-induced increases were prevented by application of respective selective antagonists but not NAADP-induced increases. Our results show that RyR-dependent, IP3-dependent, and NAADP-dependent Ca2+ stores are present in the same presynaptic terminal but are differently involved in the regulation of the presynaptic Ca2+ concentration that triggers transmitter release.

The NO way to increase muscular utrophin expression?

Duchenne muscular dystrophy (DMD), a severe X-linked recessive disorder that results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. An approach to the search for a treatment is to compensate for dystrophin loss by utrophin, another cytoskeletal protein. During development, in normal as in dystrophic embryos, utrophin is found at the membrane surface of immature skeletal fibres and is progressively replaced by dystrophin. Thus, it is possible to consider utrophin as a 'foetal homologue' of dystrophin. In a previous work, we studied the effect of L-arginine, the substrate of nitric oxide synthetase (NOS), on utrophin expression at the muscle membrane. Using a novel antibody, we confirm here that the immunocytochemical staining was indeed due to an increase in utrophin at the sarcolemma. The result is observed not only on mdx (an animal model of DMD) myotubes in culture but also in mdx mice treated with L-arginine. In addition, we show here the utrophin increase in muscle extracts of mdx mice treated with L-arginine, after electrophoretic separation and western-blotting using this novel antibody, and thus extending the electrophoretic results previously obtained on myotube cultures to muscles of treated mice.

Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level.

Monitoring calcium fluxes in real time could help to understand the development, the plasticity, and the functioning of the central nervous system. In jellyfish, the chemiluminescent calcium binding aequorin protein is associated with the green fluorescent protein and a green bioluminescent signal is emitted upon Ca(2+) stimulation. We decided to use this chemiluminescence resonance energy transfer between the two molecules. Calcium-sensitive bioluminescent reporter genes have been constructed by fusing green fluorescent protein and aequorin, resulting in much more light being emitted. Chemiluminescent and fluorescent activities of these fusion proteins have been assessed in mammalian cells. Cytosolic Ca(2+) increases were imaged at the single-cell level with a cooled intensified charge-coupled device camera. This bifunctional reporter gene should allow the investigation of calcium activities in neuronal networks and in specific subcellular compartments in transgenic animals.

Nitric oxide and l-arginine cause an accumulation of utrophin at the sarcolemma: a possible compensation for dystrophin loss in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD), a severe X-linked recessive disorder which results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. An approach to treatment is to compensate for dystrophin loss with utrophin, another cytoskeletal protein with over 80% homology with dystrophin. Utrophin is expressed, at the neuromuscular junction, in normal and DMD muscles and there is evidence that it may perform the same cellular functions as dystrophin. So, the identification of molecules or drugs that could up-regulate utrophin is a very important goal for therapy. We show that in adult normal and mdx mice (an animal model of Duchenne myopathy) treated with l-arginine, the substrate of nitric oxide synthase (NOS), a pool of utrophin localized at the membrane appeared and increased, respectively. In normal and mdx myotubes in culture, l-arginine, nitric oxide (NO), or hydroxyurea increased utrophin levels and enhanced its membrane localization. This effect did not occur with d-arginine, showing the involvement of NOS in this process. The NO-induced increase in utrophin was prevented by oxadiazolo-quinoxalin-1-one, an inhibitor of a soluble guanylate cyclase implicated in NO effects. These results open the way to a potential treatment for Duchenne and Becker dystrophies.

Nitric oxide transforms serotonin into an inactive form and this affects neuromodulation.

Nitric oxide is a highly reactive molecule, diffusible and therefore ubiquitous in the central nervous system. Consequently, nitric oxide or nitric oxide-derived nitrogen oxides must enter into contact with neuromodulators and they can modify these molecules, especially monoamines, and thus change their regulatory action on synaptic transmission. We tested this possibility on a well-known, identified cholinergic synapse of Aplysia buccal ganglion, in which we have found that evoked acetylcholine release was decreased by extracellularly applied serotonin. We show that this modulatory effect of serotonin was largely reduced not only in the presence of 3-morpholinosydnonimine, a nitric oxide donor, but also when endogenous nitric oxide synthase was activated. We have shown that this decrease in the serotonin effect is due to the formation of chemical derivatives of serotonin, mainly a symmetric serotonin dimer, 4-nitroso-serotonin and 4-nitro-serotonin, which are ineffective in reproducing the modulatory effect of serotonin. Serotonin is involved in the regulation of several central functions, such as sleep-wake activity or mood. The consequences of chemical modifications of serotonin by nitric oxide must be taken into account in physiological as well as pathological situations. In addition, our results highlight the importance of the physiological implications of interactions between free radicals and neuromediators in the nervous system.

A conotoxin from Conus textile with unusual posttranslational modifications reduces presynaptic Ca2+ influx.

Cone snails are gastropod mollusks of the genus Conus that live in tropical marine habitats. They are predators that paralyze their prey by injection of venom containing a plethora of small, conformationally constrained peptides (conotoxins). We report the identification, characterization, and structure of a gamma-carboxyglutamic acid-containing peptide, conotoxin epsilon-TxIX, isolated from the venom of the molluscivorous cone snail, Conus textile. The disulfide bonding pattern of the four cysteine residues, an unparalleled degree of posttranslational processing including bromination, hydroxylation, and glycosylation define a family of conotoxins that may target presynaptic Ca2+ channels or act on G protein-coupled presynaptic receptors via another mechanism. This conotoxin selectively reduces neurotransmitter release at an Aplysia cholinergic synapse by reducing the presynaptic influx of Ca2+ in a slow and reversible fashion. The three-dimensional structure, determined by two-dimensional 1H NMR spectroscopy, identifies an electronegative patch created by the side chains of two gamma-carboxyglutamic acid residues that extend outward from a cavernous cleft. The glycosylated threonine and hydroxylated proline enclose a localized hydrophobic region centered on the brominated tryptophan residue within the constrained intercysteine region.

Calcium transients and neurotransmitter release at an identified synapse.

It is widely accepted that the modulation of the presynaptic Ca2+ influx is one of the main mechanisms by which neurotransmitter release can be controlled. The well-identified cholinergic synapse in the buccal ganglion of Aplysia has been used to study the modulations that affect presynaptic Ca2+ transients and to relate this to quantal evoked neurotransmitter release. Three types of Ca2+ channel (L, N and P) are present in the presynaptic neurone at this synapse. Influxes of Ca2+ through N- and P-type channels trigger the release of ACh with only N-type Ca2+ channels being regulated by presynaptic neuromodulator receptors. In addition, presynaptic Ca2+ stores, via complex mechanisms of Ca2+ uptake and Ca2+ release, control the Ca2+ concentration that triggers this evoked ACh release.

Regulatory roles of complexins in neurotransmitter release from mature presynaptic nerve terminals.

Complexins are presynaptic proteins whose functional roles in synaptic transmission are still unclear. In cultured rat hippocampal neurons, complexins are distributed throughout the cell bodies, dendrites and axons, whereas synaptotagmin I and synaptobrevin/VAMP-2, essential proteins for neurotransmitter release, accumulated in the synaptic-releasing sites as early as 1 week in culture. With a maturation of synapses in vitro, complexins also accumulated in the synaptic release sites and co-localized with synaptotagmin I and synaptobrevin/VAMP-2 after 3-4 weeks in culture. Complexins I and II were expressed in more than 90 and 70% of the cultured neurons, respectively; however, they were largely distributed in different populations of synaptic terminals. In the developing rat brain, complexins were distributed in neuronal cell bodies in the early stage of postnatal development, but gradually accumulated in the synapse-enriched regions with development. In mature presynaptic neurons of Aplysia buccal ganglia, injection of anticomplexin II antibody caused a stimulation of neurotransmitter release. Injection of recombinant complexin II and alphaSNAP caused depression and facilitation of neurotransmitter release from nerve terminals, respectively. The effect of complexin was reversed by a subsequent injection of recombinant alphaSNAP, and vice versa. These results suggest that complexins are not essential but have some regulatory roles in neurotransmitter release from presynaptic terminals of mature neurons.

Control of the calcium concentration involved in acetylcholine release and its facilitation: an additional role for synaptic vesicles?

2,5-Diterbutyl-1,4-benzohydroquinone, a specific blocker of Ca2+-ATPase pumps, increased acetylcholine release from an identified synapse of Aplysia, as well as from Torpedo and mouse caudate nucleus synaptosomes. Because 2,5-diterbutyl-1,4-benzohydroquinone does not change the presynaptic Ca2+ influx, the enhancement of acetylcholine release could be due to an accumulation of Ca2+ in the terminal. This possibility was further checked by studying the effects of 2,5-diterbutyl-1,4-benzohydroquinone on twin pulse facilitation, classically attributed to residual Ca2+. While preventing the fast sequestration of Ca2+ by presynaptic organelles, 2,5-diterbutyl-1,4-benzohydroquinone magnified both twin pulse facilitation observed under low extracellular Ca2+ concentration and twin pulse dysfacilitation observed under high extracellular Ca2+ concentration. Thus, it is concluded that 2,5-diterbutyl-1,4-benzohydroquinone, by preventing Ca2+ buffering near transmitter release sites, modulates acetylcholine release. As 2,5-diterbutyl-1,4-benzohydroquinone was also shown to decrease by 50% the uptake of 45Ca2+ by isolated synaptic vesicles, we propose that synaptic vesicles can control the presynaptic Ca2+ concentration triggering the release of neurotransmitter.

Cyclic ADP-ribose and calcium-induced calcium release regulate neurotransmitter release at a cholinergic synapse of Aplysia.

1. Presynaptic injection of cyclic ADP-ribose (cADPR), a modulator of the ryanodine receptor, increased the postsynaptic response evoked by a presynaptic spike at an identified cholinergic synapse in the buccal ganglion of Aplysia californica. 2. The statistical analysis of long duration postsynaptic responses evoked by square depolarizations of the voltage-clamped presynaptic neurone showed that the number of evoked acetylcholine (ACh) quanta released was increased following cADPR injection. 3. Overloading the presynaptic neurone with cADPR led to a transient increase of ACh release followed by a depression. 4. cADPR injections did not modify the presynaptic Ca2+ current triggering ACh release. 5. Ca2+ imaging with the fluorescent dye rhod-2 showed that cADPR injection rapidly increased the free intracellular Ca2+ concentration indicating that the effects of cADPR on ACh release might be related to Ca2+ release from intracellular stores. 6. Ryanodine and 8-amino-cADPR, a specific antagonist of cADPR, decreased ACh release. 7. ADP-ribosyl cyclase, which cyclizes NAD+ into cADPR, was present in the presynaptic neurone as shown by reverse transcriptase-polymerase chain reaction experiments. 8. Application of NAD+, the substrate of ADP-ribosyl cyclase, increased ACh release and this effect was prevented by both ryanodine and 8-amino-cADPR. 9. These results support the view that Ca(2+)-induced Ca2+ release might be involved in the build-up of the Ca2+ concentration which triggers ACh release, and thus that cADPR might have a role in transmitter release modulation.

Opposite actions of nitric oxide on cholinergic synapses: which pathways?

Nitric oxide (NO) produced opposite effects on acetylcholine (ACh) release in identified neuroneuronal Aplysia synapses depending on the excitatory or the inhibitory nature of the synapse. Extracellular application of the NO donor, SIN-1, depressed the inhibitory postsynaptic currents (IPSCs) and enhanced the excitatory postsynaptic currents (EPSCs) evoked by presynaptic action potentials (1/60 Hz). Application of a membrane-permeant cGMP analog mimicked the effect of SIN-1 suggesting the participation of guanylate cyclase in the NO pathway. The guanylate cyclase inhibitor, methylene blue, blocked the NO-induced enhancement of EPSCs but only reduced the inhibition of IPSCs indicating that an additional mechanism participates to the depression of synaptic transmission by NO. Using nicotinamide, an inhibitor of ADP-ribosylation, we found that the NO-induced depression of ACh release on the inhibitory synapse also involves ADP-ribosylation mechanism(s). Furthermore, application of SIN-1 paired with cGMP-dependent protein kinase (cGMP-PK) inhibitors showed that cGMP-PK could play a role in the potentiating but not in the depressing effect of NO on ACh release. Increasing the frequency of stimulation of the presynaptic neuron from 1/60 Hz to 0.25 or 1 Hz potentiated the EPSCs and reduced the IPSCs. In these conditions, the potentiating effect of NO on the excitatory synapse was reduced, whereas its depressing effect on the inhibitory synapse was unaffected. Moreover the frequency-dependent enhancement of ACh release in the excitatory synapse was greatly reduced by the inhibition of NO synthase. Our results indicate that NO may be involved in different ways of modulation of synaptic transmission depending on the type of the synapse including synaptic plasticity.