Weighted gene co-expression network analysis reveals immune evasion related genes in Echinococcus granulosus sensu stricto.

Cystic echinococcosis (CE) is a zoonotic disease caused by the tapeworm Echinococcus granulosus sensu lato (s.l). In the intermediate host, this disease is characterized by the growth of cysts in viscera such as liver and lungs, inside of which the parasite develops to the next infective stage known as protoscoleces. There are records that the infected viscera affect the development and morphology of E. granulosus s.l. protoscolex in hosts such as buffalo or humans. However, the molecular mechanisms that drive these differences remains unknown. Weighted gene co-expression network analysis (WGCNA) using a set of RNAseq data obtained from E. granulosus sensu stricto (s.s.) protoscoleces found in liver and lung cysts reveals 34 modules in protoscoleces of liver origin, of which 12 have differential co-expression from protoscoleces of lung origin. Three of these twelve modules contain hub genes related to immune evasion: tegument antigen, tegumental protein, ubiquitin hydrolase isozyme L3, COP9 signalosome complex subunit 3, tetraspanin CD9 antigen, and the methyl-CpG-binding protein Mbd2. Also, two of the twelve modules contain only hypothetical proteins with unknown orthology, which means that there are a group of unknown function proteins co-expressed inside the protoscolex of liver CE cyst origin. This is the first evidence of gene expression differences in protoscoleces from CE cysts found in different viscera, with co-expression networks that are exclusive to protoscoleces from liver CE cyst samples. This should be considered in the control strategies of CE, as intermediate hosts can harbor CE cysts in liver, lungs, or both organs simultaneously.

Pharmacological Signature and Target Specificity of Inhibitory Circuits Formed by Martinotti Cells in the Mouse Barrel Cortex.

In the neocortex, fast synaptic inhibition orchestrates both spontaneous and sensory-evoked activity. GABAergic interneurons (INs) inhibit pyramidal neurons (PNs) directly, modulating their output activity and thus contributing to balance cortical networks. Moreover, several IN subtypes also inhibit other INs, forming specific disinhibitory circuits, which play crucial roles in several cognitive functions. Here, we studied a subpopulation of somatostatin-positive INs, the Martinotti cells (MCs) in layer 2/3 of the mouse barrel cortex (both sexes). MCs inhibit the distal portion of PN apical dendrites, thus controlling dendrite electrogenesis and synaptic integration. Yet, it is poorly understood whether MCs inhibit other elements of the cortical circuits, and the connectivity properties with non-PN targets are unknown. We found that MCs have a strong preference for PN dendrites, but they also considerably connect with parvalbumin-positive, vasoactive intestinal peptide-expressing, and layer 1 (L1) INs. Remarkably, GABAergic synapses from MCs exhibited clear cell type-specific short-term plasticity. Moreover, whereas the biophysical properties of MC-PN synapses were consistent with distal dendritic inhibition, MC-IN synapses exhibited characteristics of fast perisomatic inhibition. Finally, MC-PN connections used α5-containing GABAA receptors (GABAARs), but this subunit was not expressed by the other INs targeted by MCs. We reveal a specialized connectivity blueprint of MCs within different elements of superficial cortical layers. In addition, our results identify α5-GABAARs as the molecular fingerprint of MC-PN dendritic inhibition. This is of critical importance, given the role of α5-GABAARs in cognitive performance and their involvement in several brain diseases.SIGNIFICANCE STATEMENT Martinotti cells (MCs) are a prominent, broad subclass of somatostatin-expressing GABAergic interneurons, specialized in controlling distal dendrites of pyramidal neurons (PNs) and taking part in several cognitive functions. Here we characterize the connectivity pattern of MCs with other interneurons in the superficial layers (L1 and L2/3) of the mouse barrel cortex. We found that the connectivity pattern of MCs with PNs as well as parvalbumin, vasoactive intestinal peptide, and L1 interneurons exhibit target-specific plasticity and biophysical properties. The specificity of α5-GABAARs at MC-PN synapses and the lack or functional expression of this subunit by other cell types define the molecular identity of MC-PN connections and the exclusive involvement of this inhibitory circuits in α5-dependent cognitive tasks.

Biochemical, genomic and structural characteristics of the Acr3 pump in Exiguobacterium strains isolated from arsenic-rich Salar de Huasco sediments.

Arsenic is a highly toxic metalloid of major concern for public safety. However, microorganisms have several resistance mechanisms, particularly the expression of arsenic pumps is a critical component for bacterial ability to expel it and decrease intracellular toxicity. In this study, we aimed to characterize the biochemical, structural, and genomic characteristics of the Acr3 pump among a group of Exiguobacterium strains isolated from different sites of the arsenic-rich Salar de Huasco (SH) ecosystem. We also determined whether the differences in As(III) resistance levels presented by the strains could be attributed to changes in the sequence or structure of this protein. In this context, we found that based on acr3 sequences the strains isolated from the SH grouped together phylogenetically, even though clustering based on gene sequence identity did not reflect the strain's geographical origin. Furthermore, we determined the genetic context of the acr3 sequences and found that there are two versions of the organization of acr3 gene clusters, that do not reflect the strain's origin nor arsenic resistance level. We also contribute to the knowledge regarding structure of the Acr3 protein and its possible implications on the functionality of the pump, finding that although important and conserved components of this family of proteins are present, there are several changes in the amino acidic sequences that may affect the interactions among amino acids in the 3D model, which in fact are evidenced as changes in the structure and residues contacts. Finally, we demonstrated through heterologous expression that the Exiguobacterium Acr3 pump does indeed improve the organisms As resistance level, as evidenced in the complemented E. coli strains. The understanding of arsenic detoxification processes in prokaryotes has vast biotechnological potential and it can also provide a lot of information to understand the processes of evolutionary adaptation.

Transcriptome analysis of Echinococcus granulosus sensu stricto protoscoleces reveals differences in immune modulation gene expression between cysts found in cattle and sheep.

Cystic Echinococcosis (CE), a zoonotic parasitic disease, is caused by the cestode Echinococcus granulosus sensu lato. CE inflicts severe damage in cattle, sheep, and human hosts worldwide. Fertile CE cysts are characterized by the presence of viable protoscoleces. These parasite forms are studied with minimal contamination with host molecules. Hosts, cattle and sheep, show differences in their CE cyst fertility. The effect of the host in protoscolex transcriptome is not known. We genotyped and performed transcriptomic analysis on sheep protoscoleces obtained from liver and lung CE cysts. The transcriptomic data of Echinococcus granulosus sensu stricto protoscoleces from 6 lung CE cysts and 6 liver CE cysts were Collected. For host comparison analysis, 4 raw data files belonging to Echinococcus granulosus sensu stricto protoscoleces from cattle liver CE cysts were obtained from the NCBI SRA database. Principal component and differential expression analysis did not reveal any statistical differences between protoscoleces obtained from liver or lung cysts, either within the same sheep or different sheep hosts. Conversely, there are significant differences between cattle and sheep protoscolex samples. We found differential expression of immune-related genes. In cattle, 7 genes were upregulated in protoscoleces from liver cysts. In sheep, 3 genes were upregulated in protoscoleces from liver and lung CE cysts. Noteworthy, are the differential expression of antigen B, tegument antigen, and arginase-2 in samples obtained from sheep CE cysts, and basigin in samples from cattle CE cysts. These findings suggest that the host species is an important factor involved in the differential expression of immune related genes, which in turn is possibly related to the fertility of Echinococcus granulosus sensu stricto cysts.

Activation of regulator ArcA in the presence of hypochlorite in Salmonella enterica serovar Typhimurium.

Oxidative stress is the main mechanism behind efficient disinfectants, causing damage in bacterial macromolecules. Importantly, bacteria activate resistance mechanisms in response to damage generated by oxidative stress. Strategies allowing pathogens to survive oxidative stress are highly conserved among microorganisms. Many of these strategies entail genomic responses triggered by signals transduced through Two Component Systems (TCS). Recently, we demonstrated that the TCS ArcAB (specifically ArcA) participates in bacterial responses to hypochlorite, regulating the uptake of this toxic compound and being involved in resistance and survival inside neutrophils, where hypochlorous acid abounds. Here, we demonstrated that ArcA is required in the response to oxidative stress generated by hypochlorite, independent of its cognate sensor ArcB or the Asp54 of ArcA, the only phosphorylable residue in ArcA, which is required to function as a gene regulator. Our results suggest that ArcA could have additional functions to respond to oxidative stress, independent of its regulatory activity, which might require interaction with other unknown relevant proteins.

The Transcription Factor ArcA Modulates Salmonella's Metabolism in Response to Neutrophil Hypochlorous Acid-Mediated Stress.

Salmonella Typhimurium, a bacterial pathogen with high metabolic plasticity, can adapt to different environmental conditions; these traits enhance its virulence by enabling bacterial survival. Neutrophils play important roles in the innate immune response, including the production of microbicidal reactive oxygen species (ROS). In addition, the myeloperoxidase in neutrophils catalyzes the formation of hypochlorous acid (HOCl), a highly toxic molecule that reacts with essential biomolecules, causing oxidative damage including lipid peroxidation and protein carbonylation. The bacterial response regulator ArcA regulates adaptive responses to oxygen levels and influences the survival of Salmonella inside phagocytic cells. Here, we demonstrate by whole transcriptomic analyses that ArcA regulates genes related to various metabolic pathways, enabling bacterial survival during HOCl-stress in vitro. Also, inside neutrophils, ArcA controls the transcription of several metabolic pathways by downregulating the expression of genes related to fatty acid degradation, lysine degradation, and arginine, proline, pyruvate, and propanoate metabolism. ArcA also upregulates genes encoding components of the oxidative pathway. These results underscore the importance of ArcA in ATP generation inside the neutrophil phagosome and its participation in bacterial metabolic adaptations during HOCl stress.

Arsenic Response of Three Altiplanic Exiguobacterium Strains With Different Tolerance Levels Against the Metalloid Species: A Proteomics Study.

Exiguobacterium is a polyextremophile bacterial genus with a physiology that allows it to develop in different adverse environments. The Salar de Huasco is one of these environments due to its altitude, atmospheric pressure, solar radiation, temperature variations, pH, salinity, and the presence of toxic compounds such as arsenic. However, the physiological and/or molecular mechanisms that enable them to prosper in these environments have not yet been described. Our research group has isolated several strains of Exiguobacterium genus from different sites of Salar de Huasco, which show different resistance levels to As(III) and As(V). In this work, we compare the protein expression patterns of the three strains in response to arsenic by a proteomic approach; strains were grown in absence of the metalloid and in presence of As(III) and As(V) sublethal concentrations and the protein separation was carried out in 2D electrophoresis gels (2D-GE). In total, 999 spots were detected, between 77 and 173 of which showed significant changes for As(III) among the three strains, and between 90 and 143 for As(V), respectively, compared to the corresponding control condition. Twenty-seven of those were identified by mass spectrometry (MS). Among these identified proteins, the ArsA [ATPase from the As(III) efflux pump] was found to be up-regulated in response to both arsenic conditions in the three strains, as well as the Co-enzyme A disulfide reductase (Cdr) in the two more resistant strains. Interestingly, in this genus the gene that codifies for Cdr is found within the genic context of the ars operon. We suggest that this protein could be restoring antioxidants molecules, necessary for the As(V) reduction. Additionally, among the proteins that change their expression against As, we found several with functions relevant to stress response, e.g., Hpf, LuxS, GLpX, GlnE, and Fur. This study allowed us to shed light into the physiology necessary for these bacteria to be able to tolerate the toxicity and stress generated by the presence of arsenic in their niche.

Correction: The ArcAB two-component regulatory system promotes resistance to reactive oxygen species and systemic infection by Salmonella Typhimurium.

[This corrects the article DOI: 10.1371/journal.pone.0203497.].

The ArcAB two-component regulatory system promotes resistance to reactive oxygen species and systemic infection by Salmonella Typhimurium.

Salmonella enterica Serovar Typhimurium (S. Typhimurium) is an intracellular bacterium that overcomes host immune system barriers for successful infection. The bacterium colonizes the proximal small intestine, penetrates the epithelial layer, and is engulfed by macrophages and neutrophils. Intracellularly, S. Typhimurium encounters highly toxic reactive oxygen species including hydrogen peroxide and hypochlorous acid. The molecular mechanisms of Salmonella resistance to intracellular oxidative stress is not completely understood. The ArcAB two-component system is a global regulatory system that responds to oxygen. In this work, we show that the ArcA response regulator participates in Salmonella adaptation to changing oxygen levels and is also involved in promoting intracellular survival in macrophages and neutrophils, enabling S. Typhimurium to successfully establish a systemic infection.

The transcription factor SlyA from Salmonella Typhimurium regulates genes in response to hydrogen peroxide and sodium hypochlorite.

Salmonella Typhimurium is an intracellular pathogen that is capable of generating systemic fever in a murine model. Over the course of the infection, Salmonella faces different kinds of stressors, including harmful reactive oxygen species (ROS). Various defence mechanisms enable Salmonella to successfully complete the infective process in the presence of such stressors. The transcriptional factor SlyA is involved in the oxidative stress response and invasion of murine macrophages. We evaluated the role of SlyA in response to H2O2 and NaOCl and found an increase of slyA expression upon exposure to these toxics. However, the SlyA target genes and the molecular mechanisms by which they influence the infective process are unknown. We hypothesised that SlyA regulates the expression of genes required for ROS resistance, metabolism, or virulence under oxidative stress conditions. Transcriptional profiling in wild type and ΔslyA strains confirmed that SlyA regulates the expression of several genes involved in virulence [sopD (STM14_3550), sopE2 (STM14_2244), hilA (STM14_3475)] and central metabolism [kgtP (STM14_3252), fruK (STM14_2722), glpA (STM14_2819)] in response to H2O2 and NaOCl. These findings were corroborated by functional assay and transcriptional fusion assays using GFP. DNA-protein interaction assays showed that SlyA regulates these genes through direct interaction with their promoter regions.

Multiscale single-cell analysis reveals unique phenotypes of raphe 5-HT neurons projecting to the forebrain.

Serotonergic neurons of the raphe nuclei exhibit anatomical, neurochemical and elecrophysiological heterogeneity that likely underpins their specific role in multiple behaviors. However, the precise organization of serotonin (5-HT) neurons to orchestrate 5-HT release patterns throughout the brain is not well understood. We compared the electrophysiological and neurochemical properties of dorsal and median raphe 5-HT neurons projecting to the medial prefrontal cortex (mPFC), amygdala (BLA) and dorsal hippocampus (dHP), combining retrograde tract tracing with brain slice electrophysiology and single-cell RT-PCR in Pet1-EGFP mice. Our results show that 5-HT neurons projecting to the dHP and the mPFC and the BLA form largely non-overlapping populations and that BLA-projecting neurons have characteristic excitability and membrane properties. In addition, using an unbiased clustering method that correlates anatomical, molecular and electrophysiological phenotypes, we find that 5-HT neurons with projections to the mPFC and the dHP segregate from those projecting to the BLA. Single-cell gene profiling showed a restricted expression of the peptide galanin in the population of 5-HT neurons projecting to the mPFC. Finally, cluster analysis allowed identifying an atypical subtype of 5-HT neuron with low excitability, long firing delays and preferential expression of the vesicular glutamate transporter type 3. Overall, these findings allow to define correlated anatomical and physiological identities of serotonin raphe neurons that help understanding how discrete raphe cells subpopulations account for the heterogeneous activities of the midbrain serotonergic system.

Hydrogen peroxide and hypochlorous acid influx through the major S. Typhimurium porin OmpD is affected by substitution of key residues of the channel.

OmpD is the major Salmonella enterica serovar Typhimurium (S. Typhimurium) porin and mediates hydrogen peroxide (H2O2) influx. The results described herein extend this finding to hypochlorous acid (HOCl), another reactive oxygen species that is also part of the oxidative burst generated by the phagosome. S. Typhimurium cells lacking OmpD show decreased HOCl influx, and OmpD-reconstituted proteoliposomes show an increase in the uptake of the toxic compound. To understand this physiologically relevant process, we investigated the role of key OmpD residues in H2O2 and NaOCl transport. Using a theoretical approach, residue K16 was defined as a major contributor to the channel electrostatic properties, and E111 was shown to directly participate in the size-exclusion limit of the channel. Together, we provide theoretical, genetic, and biochemical evidence that OmpD mediates H2O2 and NaOCl uptake, and that key residues of the channel are implicated in this process.

Molecular and functional characterization of GAD67-expressing, newborn granule cells in mouse dentate gyrus.

Dentate gyrus granule cells (GCs) have been suggested to synthesize both GABA and glutamate immediately after birth and under pathological conditions in the adult. Expression of the GABA synthesizing enzyme GAD67 by GCs during the first few weeks of postnatal development may then allow for transient GABA synthesis and synaptic release from these cells. Here, using the GAD67-EGFP transgenic strain G42, we explored the phenotype of GAD67-expressing GCs in the mouse dentate gyrus. We report a transient, GAD67-driven EGFP expression in differentiating GCs throughout ontogenesis. EGFP expression correlates with the expression of GAD and molecular markers of GABA release and uptake in 2-4 weeks post-mitotic GCs. These rather immature cells are able to fire action potentials (APs) and are synaptically integrated in the hippocampal network. Yet they show physiological properties that differentiate them from mature GCs. Finally, GAD67-expressing GCs express a specific complement of GABAA receptor subunits as well as distinctive features of synaptic and tonic GABA signaling. Our results reveal that GAD67 expression in dentate gyrus GCs is a transient marker of late differentiation that persists throughout life and the G42 strain may be used to visualize newborn GCs at a specific, well-defined differentiation stage.

Input-specific learning rules at excitatory synapses onto hippocampal parvalbumin-expressing interneurons.

Hippocampal parvalbumin-expressing interneurons (PV INs) provide fast and reliable GABAergic signalling to principal cells and orchestrate hippocampal ensemble activities. Precise coordination of principal cell activity by PV INs relies in part on the efficacy of excitatory afferents that recruit them in the hippocampal network. Feed-forward (FF) inputs in particular from Schaffer collaterals influence spike timing precision in CA1 principal cells whereas local feedback (FB) inputs may contribute to pacemaker activities. Although PV INs have been shown to undergo activity-dependent long term plasticity, how both inputs are modulated during principal cell firing is unknown. Here we show that FF and FB synapses onto PV INs are endowed with distinct postsynaptic glutamate receptors which set opposing long-term plasticity rules. Inward-rectifying AMPA receptors (AMPARs) expressed at both FF and FB inputs mediate a form of anti-Hebbian long term potentiation (LTP), relying on coincident membrane hyperpolarization and synaptic activation. In contrast, FF inputs are largely devoid of NMDA receptors (NMDARs) which are more abundant at FB afferents and confer on them an additional form of LTP with Hebbian properties. Both forms of LTP are expressed with no apparent change in presynaptic function. The specific endowment of FF and FB inputs with distinct coincidence detectors allow them to be differentially tuned upon high frequency afferent activity. Thus, high frequency (>20 Hz) stimulation specifically potentiates FB, but not FF afferents. We propose that these differential, input-specific learning rules may allow PV INs to adapt to changes in hippocampal activity while preserving their precisely timed, clockwork operation.

Presynaptic but not postsynaptic GABA signaling at unitary mossy fiber synapses.

Dentate gyrus granule cells have been suggested to corelease GABA and glutamate both in juvenile animals and under pathological conditions in adults. Although mossy fiber terminals (MFTs) are known to express glutamic acid decarboxylase (GAD) in early postnatal development, the functional role of GABA synthesis in MFTs remains controversial, and direct evidence for synaptic GABA release from MFTs is missing. Here, using GAD67-GFP transgenic mice, we show that GAD67 is expressed only in a population of immature granule cells in juvenile animals. We demonstrate that GABA can be released from these cells and modulate mossy fiber excitability through activation of GABAB autoreceptors. However, unitary postsynaptic currents generated by individual, GAD67-expressing granule cells are purely glutamatergic in all postsynaptic cell types tested. Thus GAD67 expression does not endow dentate gyrus granule cells with a full GABAergic phenotype and GABA primarily instructs the pre- rather than the postsynaptic element.

The neuronal K-Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines.

The K-Cl cotransporter KCC2 plays an essential role in neuronal chloride homeostasis, and thereby influences the efficacy and polarity of GABA signaling. Although KCC2 is expressed throughout the somatodendritic membrane, it is remarkably enriched in dendritic spines, which host most glutamatergic synapses in cortical neurons. KCC2 has been shown to influence spine morphogenesis and functional maturation in developing neurons, but its function in mature dendritic spines remains unknown. Here, we report that suppressing KCC2 expression decreases the efficacy of excitatory synapses in mature hippocampal neurons. This effect correlates with a reduced postsynaptic aggregation of GluR1-containing AMPA receptors and is mimicked by a dominant negative mutant of KCC2 interaction with cytoskeleton but not by pharmacological suppression of KCC2 function. Single-particle tracking experiments reveal that suppressing KCC2 increases lateral diffusion of the mobile fraction of AMPA receptor subunit GluR1 in spines but not in adjacent dendritic shafts. Increased diffusion was also observed for transmembrane but not membrane-anchored recombinant neuronal cell adhesion molecules. We suggest that KCC2, likely through interactions with the actin cytoskeleton, hinders transmembrane protein diffusion, and thereby contributes to their confinement within dendritic spines.

BDNF is required for the induction of a presynaptic component of the functional conversion of silent synapses.

Long-term potentiation (LTP) has received attention because of its proposed role in learning and memory. Despite substantial effort the pre- or postsynaptic expression site of LTP remains unsettled. It has been proposed that LTP is expressed postsynaptically through the functional conversion of "silent synapses." We had shown that Schaffer collateral (SC) silent and "functional synapses," which lack and express AMPA receptors, respectively exhibit distinct transmitter release properties. Therefore the functional conversion of silent synapses with LTP should be associated with presynaptic modifications. We now show that the pairing-induced LTP at SC synapses is mediated by combined pre- and postsynaptic modifications involving the postsynaptic emergence of an AMPA response coupled with an enhanced glutamate release. BDNF replicates the changes associated with this LTP by activating TrkBRs, suggesting that the neurotrophin is required for the coordinated changes on both sides of the synaptic cleft.

Distinct transmitter release properties determine differences in short-term plasticity at functional and silent synapses.

Recent evidence suggests that functional and silent synapses are not only postsynaptically different but also presynaptically distinct. The presynaptic differences may be of functional importance in memory formation because a proposed mechanism for long-term potentiation is the conversion of silent synapses into functional ones. However, there is little direct experimentally evidence of these differences. We have investigated the transmitter release properties of functional and silent Schaffer collateral synapses and show that on the average functional synapses displayed a lower percentage of failures and higher excitatory postsynaptic current (EPSC) amplitudes than silent synapses at +60 mV. Moreover, functional but not silent synapses show paired-pulse facilitation (PPF) at +60 mV and thus presynaptic short-term plasticity will be distinct in the two types of synapse. We examined whether intraterminal endoplasmic reticulum Ca2+ stores influenced the release properties of these synapses. Ryanodine (100 microM) and thapsigargin (1 microM) increased the percentage of failures and decreased both the EPSC amplitude and PPF in functional synapses. Caffeine (10 mM) had the opposite effects. In contrast, silent synapses were insensitive to both ryanodine and caffeine. Hence we have identified differences in the release properties of functional and silent synapses, suggesting that synaptic terminals of functional synapses express regulatory molecular mechanisms that are absent in silent synapses.

Selective muscarinic regulation of functional glutamatergic Schaffer collateral synapses in rat CA1 pyramidal neurons.

Analysis of the cholinergic regulation of glutamatergic neurotransmission is an essential step in understanding the hippocampus because it can influence forms of synaptic plasticity that are thought to underlie learning and memory. We studied in vitro the cholinergic regulation of excitatory postsynaptic currents (EPSCs) evoked in rat CA1 pyramidal neurons by Schaffer collateral (SC) stimulation. Using "minimal" stimulation, which activates one or very few synapses, the cholinergic agonist carbamylcholine (CCh) increased the failure rate of functional more (36 %) than of silent synapses (7 %), without changes in the EPSC amplitude. These effects of CCh were insensitive to manipulations that increased the probability of release, such as paired pulse facilitation, increases in temperature and increases in the extracellular Ca(2+) : Mg(2+) ratio. Using "conventional" stimulation, which activates a large number of synapses, CCh inhibited more the pharmacologically isolated non-NMDA (86 %) than the NMDA (47 %) EPSC. The changes in failure rate, EPSC variance and the increased paired pulse facilitation that paralleled the inhibition imply that CCh decreased release probability. Muscarine had similar effects. The inhibition by both CCh and by muscarine was prevented by atropine. We conclude that CCh reduces the non-NMDA component of SC EPSCs by selectively inhibiting transmitter release at functional synapses via activation of muscarinic receptors. The results suggest that SCs have two types of terminals, one in functional synapses, selectively sensitive to regulation through activation of muscarinic receptors, and the other in silent synapses less sensitive to that regulation. The specific inhibition of functional synapses would favour activity-dependent plastic phenomena through NMDA receptors at silent synapses without the activation of non-NMDA receptors and functional synapses.