Microglial diversity along the hippocampal longitudinal axis impacts synaptic plasticity in adult male mice under homeostatic conditions.

The hippocampus is a plastic brain area that shows functional segregation along its longitudinal axis, reflected by a higher level of long-term potentiation (LTP) in the CA1 region of the dorsal hippocampus (DH) compared to the ventral hippocampus (VH), but the mechanisms underlying this difference remain elusive. Numerous studies have highlighted the importance of microglia-neuronal communication in modulating synaptic transmission and hippocampal plasticity, although its role in physiological contexts is still largely unknown. We characterized in depth the features of microglia in the two hippocampal poles and investigated their contribution to CA1 plasticity under physiological conditions. We unveiled the influence of microglia in differentially modulating the amplitude of LTP in the DH and VH, showing that minocycline or PLX5622 treatment reduced LTP amplitude in the DH, while increasing it in the VH. This was recapitulated in Cx3cr1 knockout mice, indicating that microglia have a key role in setting the conditions for plasticity processes in a region-specific manner, and that the CX3CL1-CX3CR1 pathway is a key element in determining the basal level of CA1 LTP in the two regions. The observed LTP differences at the two poles were associated with transcriptional changes in the expression of genes encoding for Il-1, Tnf-α, Il-6, and Bdnf, essential players of neuronal plasticity. Furthermore, microglia in the CA1 SR region showed an increase in soma and a more extensive arborization, an increased prevalence of immature lysosomes accompanied by an elevation in mRNA expression of phagocytic markers Mertk and Cd68 and a surge in the expression of microglial outward K+ currents in the VH compared to DH, suggesting a distinct basal phenotypic state of microglia across the two hippocampal poles. Overall, we characterized the molecular, morphological, ultrastructural, and functional profile of microglia at the two poles, suggesting that modifications in hippocampal subregions related to different microglial statuses can contribute to dissect the phenotypical aspects of many diseases in which microglia are known to be involved.

UPR activation specifically modulates glutamate neurotransmission in the cerebellum of a mouse model of autism.

An increasing number of rare mutations linked to autism spectrum disorders have been reported in genes encoding for proteins involved in synapse formation and maintenance, such as the post-synaptic cell adhesion proteins neuroligins. Most of the autism-linked mutations in the neuroligin genes map on the extracellular protein domain. The autism-linked substitution R451C in Neuroligin3 (NLGN3) induces a local misfolding of the extracellular domain, causing defective trafficking and retention of the mutant protein in the endoplasmic reticulum (ER). The activation of the unfolded protein response (UPR), due to misfolded proteins accumulating in the ER, has been implicated in pathological and physiological conditions of the nervous system. It was previously shown that the over-expression of R451C NLGN3 in a cellular system leads to the activation of the UPR. Here, we have investigated whether this protective cellular response is detectable in the knock-in mouse model of autism endogenously expressing R451C NLGN3. Our data showed up-regulation of UPR markers uniquely in the cerebellum of the R451C mice compared to WT littermates, at both embryonic and adult stages, but not in other brain regions. Miniature excitatory currents in the Purkinje cells of the R451C mice showed higher frequency than in the WT, which was rescued inhibiting the PERK branch of UPR. Taken together, our data indicate that the R451C mutation in neuroligin3 elicits UPR in vivo, which appears to trigger alterations of synaptic function in the cerebellum of a mouse model expressing the R451C autism-linked mutation.

Fluoxetine effects on molecular, cellular and behavioral endophenotypes of depression are driven by the living environment.

Selective serotonin reuptake inhibitors (SSRIs) represent the most common treatment for major depression. However, their efficacy is variable and incomplete. In order to elucidate the cause of such incomplete efficacy, we explored the hypothesis positing that SSRIs may not affect mood per se but, by enhancing neural plasticity, render the individual more susceptible to the influence of the environment. Consequently, SSRI administration in a favorable environment promotes a reduction of symptoms, whereas in a stressful environment leads to a worse prognosis. To test such hypothesis, we exposed C57BL/6 mice to chronic stress in order to induce a depression-like phenotype and, subsequently, to fluoxetine treatment (21 days), while being exposed to either an enriched or a stressful condition. We measured the most commonly investigated molecular, cellular and behavioral endophenotypes of depression and SSRI outcome, including depression-like behavior, neurogenesis, brain-derived neurotrophic factor levels, hypothalamic-pituitary-adrenal axis activity and long-term potentiation. Results showed that, in line with our hypothesis, the endophenotypes investigated were affected by the treatment according to the quality of the living environment. In particular, mice treated with fluoxetine in an enriched condition overall improved their depression-like phenotype compared with controls, whereas those treated in a stressful condition showed a distinct worsening. Our findings suggest that the effects of SSRI on the depression- like phenotype is not determined by the drug per se but is induced by the drug and driven by the environment. These findings may be helpful to explain variable effects of SSRI found in clinical practice and to device strategies aimed at enhancing their efficacy by means of controlling environmental conditions.

MicroRNA-335-5p modulates spatial memory and hippocampal synaptic plasticity.

MicroRNAs are endogenous, noncoding RNAs crucial for the post-transcriptional regulation of gene expression. In this study, we investigated the role of miR-335-5p in spatial learning and synaptic plasticity. To this end we first showed spatial learning induced down-regulation of miR-335-5p. Next we found impairment in long-term memory and reduction in hippocampal long-term potentiation by exogenous administration of the miRNA. These findings demonstrate that miR-335-5p is a key coordinator of the intracellular pathways that mediate experience-dependent changes in the brain.

ATP release during cell swelling activates a Ca2+-dependent Cl- current by autocrine mechanism in mouse hippocampal microglia.

Microglia cells, resident immune cells of the brain, survey brain parenchyma by dynamically extending and retracting their processes. Cl- channels, activated in the cellular response to stretch/swelling, take part in several functions deeply connected with microglia physiology, including cell shape changes, proliferation, differentiation and migration. However, the molecular identity and functional properties of these Cl- channels are largely unknown. We investigated the properties of swelling-activated currents in microglial from acute hippocampal slices of Cx3cr1 +/GFP mice by whole-cell patch-clamp and imaging techniques. The exposure of cells to a mild hypotonic medium, caused an outward rectifying current, developing in 5-10 minutes and reverting upon stimulus washout. This current, required for microglia ability to extend processes towards a damage signal, was carried mainly by Cl- ions and dependent on intracellular Ca2+. Moreover, it involved swelling-induced ATP release. We identified a purine-dependent mechanism, likely constituting an amplification pathway of current activation: under hypotonic conditions, ATP release triggered the Ca2+-dependent activation of anionic channels by autocrine purine receptors stimulation. Our study on native microglia describes for the first time the functional properties of stretch/swelling-activated currents, representing a key element in microglia ability to monitor the brain parenchyma.

KCa3.1 inhibition switches the phenotype of glioma-infiltrating microglia/macrophages.

Among the strategies adopted by glioma to successfully invade the brain parenchyma is turning the infiltrating microglia/macrophages (M/MΦ) into allies, by shifting them toward an anti-inflammatory, pro-tumor phenotype. Both glioma and infiltrating M/MΦ cells express the Ca(2+)-activated K(+) channel (KCa3.1), and the inhibition of KCa3.1 activity on glioma cells reduces tumor infiltration in the healthy brain parenchyma. We wondered whether KCa3.1 inhibition could prevent the acquisition of a pro-tumor phenotype by M/MΦ cells, thus contributing to reduce glioma development. With this aim, we studied microglia cultured in glioma-conditioned medium or treated with IL-4, as well as M/MΦ cells acutely isolated from glioma-bearing mice and from human glioma biopsies. Under these different conditions, M/MΦ were always polarized toward an anti-inflammatory state, and preventing KCa3.1 activation by 1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), we observed a switch toward a pro-inflammatory, antitumor phenotype. We identified FAK and PI3K/AKT as the molecular mechanisms involved in this phenotype switch, activated in sequence after KCa3.1. Anti-inflammatory M/MΦ have higher expression levels of KCa3.1 mRNA (kcnn4) that are reduced by KCa3.1 inhibition. In line with these findings, TRAM-34 treatment, in vivo, significantly reduced the size of tumors in glioma-bearing mice. Our data indicate that KCa3.1 channels are involved in the inhibitory effects exerted by the glioma microenvironment on infiltrating M/MΦ, suggesting a possible role as therapeutic targets in glioma.

Ca2(+)-dependence of arachidonic acid redistribution among phospholipids of cultured mouse keratinocytes.

Mouse keratinocytes cultured in a medium containing less than 0.1 mM Ca2+ (low Ca2+) incorporated [1-14C]arachidonic acid (AA) into phospholipids by kinetics including; (i) a rapid labelling of phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer) and both acid-stable and alkenylacyl forms of phosphatidylcholine (PtdCho); and (ii) a slow but long-lasting radiolabel incorporation into both acid-stable and alkenylacyl forms of phosphatidylethanolamine (PtdEtn), partly associated with a net radioactivity loss from acid stable-PtdCho. Under low Ca2+ conditions no radioactivity transfer apparently occurred between PtdIns and other phospholipid classes. When cells were prelabelled for 24 h with [1-14C]AA and reincubated in label-free medium containing 1.2 mM Ca2+ (normal Ca2+), an early and extensive loss of radioactivity from PtdIns was observed, reasonably in connection with Ca2+ stimulation of phosphoinositide turnover. Cell shift to normal Ca2+ did not result in an increased synthesis of labelled eicosanoids, but was consistent with an increase of radioactivity incorporation into diacylglycerol (DAG) and with a complex pattern of [1-14C]AA redistribution, eventually leading to a marked radioactivity incorporation into acid stable-PtdEtn (but not into alkenylacyl-PtdEtn) and to a labelling decrease of acid stable-PtdCho. The possible mechanisms driving AA recycling after cell shift to normal Ca2+ are discussed.

Acetylcholine-activated inward current induces cytosolic Ca2+ mobilization in mouse C2C12 myotubes.

We examined the spatiotemporal pattern of intracellular Ca2+ liberation in mouse myotubes by means of fluorescence imaging of cytosolic free Ca2+ together with the simultaneous recording of membrane whole-cell currents. Acetylcholine (ACh) applications to C2C12 myotubes equilibrated in Ca(2+)-free medium and voltage clamped at -50 mV evoked localized fluorescence transients of variable amplitude with less than 0.5 s delay. Under the same experimental conditions, fluorescence transients were elicited by ACh also in mouse primary myotubes. Ca2+ transients were inhibited in myotubes clamped at depolarized potentials (-10 mV to +50 mV), or equilibrated in a Na+,Ca(2+)-free medium as well as in cells loaded with heparin, or with inositol (1,4,5) trisphosphate (InsP3). To investigate whether InsP3 could induce Ca2+ mobilization, [Ca2+]i determinations were carried out in myotubes loaded with InsP3 through the whole-cell patch-clamp recording pipette or by extracellular application in permeabilized cells. InsP3 diffusion into the myoplasm caused Ca2+ spikes with 5 +/- 1 s (mean +/- SEM) delay from the rupture of the membrane patch. Spikes were followed by sustained increases in fluorescence or by damped oscillations. In permeabilized myotubes, InsP3 induced the release of sequestered 45Ca2+ with a half-maximally effective concentration (EC50) of 0.28 +/- 0.05 microM, and Hill coefficient of 0.79 +/- 0.09. It is concluded that the ACh-activated inward current in mouse myotubes is coupled to cytosolic Ca2+ mobilization from internal InsP3-sensitive pools.

Signaling pathways activated by chemokine receptor CXCR2 and AMPA-type glutamate receptors and involvement in granule cells survival.

We show that treatment of cerebellar granules with interleukin-8 (IL-8), growth-related gene product beta (GRObeta) or AMPA induced activation of PI3-K/Akt and of ERK pathways, the latter being independent of PI3-K and dependent on PTX-sensitive G proteins. We also show that AMPA-mediated neuron survival was abolished both by ERK kinase inhibitor PD98059 and AMPA-Rs blocker CNQX, and that chemokine-mediated survival was blocked by the PI3-K inhibitors LY294002 and wortmannin. We conclude that the neurotrophic effects of AMPA need the contemporary activation of ERKs and stimulation of AMPA-Rs, and that PI3-K/Akt activation is a determinant pathway for the IL-8/GRObeta anti-apoptotic activity.

Chemokine receptor CXCR2 regulates the functional properties of AMPA-type glutamate receptor GluR1 in HEK cells.

Experiments were conducted in both HEK cells and cerebellar neurons to investigate whether CXC chemokine receptor 2 (CXCR2) is functionally coupled to GluR1. The co-expression of CXCR2 with GluR1 in HEK cells increased (i) the GluR1 "apparent" affinity for the transmitter; (ii) the GluR1 channel open probability; and (iii) GluR1 binding site cooperativity upon CXCR2 stimulation with CXC chemokine ligand 2 (CXCL2). The affinity of C-terminal-deleted GluR1 for glutamate (Glu) remained stable instead. Furthermore, CXCL2 increased the binding site cooperativity of AMPA receptors in rat cerebellar granule cells; and the amplitude of spontaneous excitatory postsynaptic current (sEPSCs) in Purkinje neurons (PNs). Our findings indicate that the coupling of CXCR2 with GluR1 may modulate glutamatergic synaptic transmission.

CXC chemokines interleukin-8 (IL-8) and growth-related gene product alpha (GROalpha) modulate Purkinje neuron activity in mouse cerebellum.

We give here evidence that Purkinje neurons (PNs) of mouse cerebellar slices studied with patch clamp technique combined with laser confocal microscopy, respond to human IL-8 and GROalpha by (i) a cytosolic Ca2+ transient compatible with inositol (1,4,5) trisphosphate (InsP3) formation; (ii) an enhancement of the neurotransmitter release; and (iii) an impairment of the long-term depression of synaptic strength (LTD). It was also found the expression of IL-8 receptor type 2 in PN and granule cells by immunofluorescence, immunoblotting and RT-PCR analysis. Considered together these findings suggest that IL-8 and GROalpha may play a neuromodulatory role on mouse cerebellum.

Nicotinic cholinergic stimulation promotes survival and reduces motility of cultured rat cerebellar granule cells.

Despite many studies on the functional expression of neuronal nicotinic acetylcholine receptors (nAChRs), an exhaustive description of the long-term effects of nicotine (Nic) stimulation in cerebellar granules is still far to be completed. For this reason, we addressed the experiments stimulating cultured cerebellar granule neurons (CGN) with Nic, focusing on the effects on cell motility and survival. Using electrophysiological and Ca(2+)-fluorescence techniques, we found a subset of rat CGN that responded to Nic by inward whole cell currents and by short-delay Ca(2+) transients. These responses were mediated through both homomeric and heteromeric nAChRs, as assessed by their sensitivity to alpha-bungarotoxin (alpha-BTX), dihydro-beta-erythroidine (DHbetaE), methyllicaconitine (MLA) and 5-hydroxyindole (5OH-indole). Once established the expression of alpha-BTX-sensitive and insensitive nAChRs and their ability to trigger Ca(2+) responses in CGN, we aimed at investigating their possible role on cell survival and motility. We demonstrate that Nic stimulation significantly increases the survival of CGN exposed to the apoptosis-promoting low K(+) medium. This anti-apoptotic effect is likely mediated through alpha7* nAChRs since we found that it was mimicked by choline, was insensitive to DHbetaE and was fully inhibited by alpha-BTX. Furthermore, we report that Nic negatively modulates CGN motility, reducing the basal cell movement through a pored membrane by the activation of alpha-BTX-insensitive nAChRs. We conclude that CGN express various types of nAChRs, which are differently involved in regulating Nic-mediated modulation of cell survival and migration, and we suggest potential regulatory roles for cholinergic receptors during cerebellar development.

Effects of perphenazine on the metabolism of inositol phospholipids in SK-N-BE(2) human neuroblastoma cells.

Administration of myo-[3H]inositol to SK-N-BE(2) human neuroblastoma cells for 24 hr resulted in equilibrium labelling of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2), as well as in retention of a large intracellular pool of free myo-[3H]inositol. Equilibrium labelling was no longer observed when cells were treated for 2 hr with 20 microM perphenazine (PPZ) in label-free medium; under these conditions, myo-[3H]inositol from the retained intracellular pool was incorporated into PI and PIP but not into PIP2. Analysis of water-soluble myo-[3H]inositol derivatives and inositol 1,4,5-trisphosphate mass determination indicated that PPZ did not stimulate phosphoinositide hydrolysis by phospholipase C. These results indicate that PPZ raises PI and PIP levels, whereas it is ineffective in expanding the PIP2 pool. The latter effect is not due to a concomitant synthesis and hydrolysis of this lipid.

Phenothiazines inhibit acetylcholinesterase by concentration-dependent-type kinetics. A study with trifluoperazine and perphenazine.

The properties of perphenazine (PPZ) and trifluoperazine (TFP) as fluorescent dyes were exploited to calculate their critical micellar concentrations. The relative fluorescence quantum yield of the two amphiphiles was dependent on their concentration, abruptly decreasing above 30-40 microM PPZ and 20-30 microM TFP. Evidence is presented that this phenomenon is driven by the formation of non-fluorescent drug aggregates. The type of inhibition kinetics displayed by PPZ and TFP on human erythrocyte acetylcholinesterase (AChE) was also dependent on drug concentration, turning from non-competitive to a "mixed" inhibition type at concentrations at which PPZ and TFP were demonstrated to undergo micelle formation. Results support the notion that phenothiazines may interact with AChE both as monomers and micellar aggregates, producing different inhibitory effects.

Modulation of the neurotransmitter release in rat cerebellar neurons by GRO beta.

We report here that, in cultured cerebellar granule cells, the CXC chemokine GRObeta stimulates the signaling pathway of the extracellular signal-regulated kinases, and enhances both evoked and spontaneous postsynaptic currents in patch clamped Purkinje neurons from rat cerebellar slices. The GRObeta-induced enhancement of the excitatory post-synaptic currents evoked by stimulating the parallel fibres is blocked by the inhibitor of the extracellular signal-regulated kinases pathway PD98059, which also reduces both basal frequency of spontaneous post-synaptic currents and mean amplitude of evoked excitatory post-synaptic currents. Our results suggest that GRObeta modulates neurotransmitter release in the cerebellum through the activation of the extracellular signal-regulated kinases pathway.

Alpha 5 subunit forms functional alpha 3 beta 4 alpha 5 nAChRs in transfected human cells.

nAChRs heterologously expressed in human cells after transient transfection with alpha 3 beta 4 alpha 5 or alpha 3 beta 4 subunit cDNAs exhibited similar sensitivities to antagonists and comparable functional channel profiles. However, the sum of two Hill equations was required for best fitting the ACh dose-current response curves after co-expression of alpha 5, alpha 3 and beta 4 subunits. One component was comparable to that obtained in alpha 3 beta 4-transfected cells, while the additional component, putatively attributed to an alpha 3 beta 4 alpha 5 nAChR population, showed a Hill coefficient > 2 and a nine-fold greater half-maximal ACh concentration (EC50). These results suggest that the alpha 5 subunit participates in the assembly of alpha 3 beta 4 alpha 5 nAChRs complexes in human cells, adding a new member to the family of neuronal nicotinic receptors.

Phorbol ester modulation of both delta-mutant and subunit-omitted nicotinic receptors expressed in Xenopus oocytes.

The action of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the potent stimulator of protein kinase C (PKC), on acetylcholine-activated currents (I(Ach)) was investigated in voltage clamped Xenopus laevis oocytes injected with RNAs encoding murine embryonic nicotinic acetylcholine receptor (AChR) subunits. Comparable potentiation and acceleration of decay of I(ACh) were observed within minutes of phorbol ester application in oocytes injected with various RNA subunit combinations: (i) alpha beta gamma delta; (ii) alpha beta gamma; (iii) alpha beta delta; and (iv) alpha beta gamma delta(AAA), a mutant of the delta subunit with serine residues 360-361-362 mutated to alanine. Our findings indicate that the effects on I(ACh) induced by PKC stimulation are independent of both gamma and delta subunits and, accordingly, of the presence of PKC phosphorylation sites on delta subunit. It is here suggested a novel PKC-dependent modulatory mechanism of cholinergic receptor which does not involve direct phosphorylation of the AChR and requires phosphorylation of intermediate regulatory protein(s).

Phosphoinositide-derived diacylglycerol conversion to phosphatidic acid is a receptor-dependent and compartmentalized phenomenon in human neuroblastoma.

We report that upon muscarinic stimulation of SK-N-BE(2) human neuroblastoma cells, the extent of phosphoinositide-derived diacylglycerol (DG) conversion to phosphatidic acid (PA), operated by a DG kinase, is dependent on the potency of receptor stimulation and correlates with the reduction of phosphatidylinositol 4,5-bisphosphate mass. Evidence is provided that agonist-evoked Ca2+ mobilisation or protein kinase activation are not key events in triggering receptor-generated DG conversion to PA; furthermore, the phenomenon is compartmentalized, namely it occurs within a topologically restricted area that is poorly accessible to DG artificially generated by cell treatment with bacterial phosphatidylinositol-specific phospholipase C. Possible mechanisms driving regulation of the DG kinase operating in the transduction system investigated are discussed.