Laura Maggi

Synaptic pruning by microglia is necessary for normal brain development.

A good brain needs a good vacuum cleaner. Microglia are highly motile phagocytic cells that infiltrate and take up residence in the developing brain, where they are thought to provide a surveillance and scavenging function. However, although microglia have been shown to engulf and clear damaged cellular debris after brain insult, it remains less clear what role microglia play in the uninjured brain. Here, we show that microglia actively engulf synaptic material and play a major role in synaptic pruning during postnatal development in mice. These findings link microglia surveillance to synaptic maturation and suggest that deficits in microglia function may contribute to synaptic abnormalities seen in some neurodevelopmental disorders.

SDF‐1α‐mediated modulation of synaptic transmission in rat cerebellum

The functional expression of the seven‐transmembrane domain G protein‐coupled chemokine receptor CXCR‐4/fusin in rat nerve cell was demonstrated by staining with a polyclonal anti‐CXCR‐4 Ab, and by evaluating the calcium responses to the physiological agonist stromal‐derived cell factor‐1α (SDF‐1α) in both cerebellar granule cells in culture and Purkinje neurons (PNs) in cerebellar slices. Cerebellar glial, granule and Purkinje cells showed a pronounced staining for CXCR‐4. Furthermore, cultured granule cells exhibited Ca2+ transients elicited by the application of SDF‐1α, both in cell bodies and in neuronal processes. Whole‐cell patch‐clamped PNs in cerebellar slices responded to SDF‐1α application by a slow inward current followed by an increase of both intracellular Ca2+ level and spontaneous synaptic activity. In particular, the SDF‐1α‐induced slow inward current was considerably reduced by ionotropic glutamate receptor blockers, but developed fully in a medium in which synaptic transmission was inhibited, indicating that this current might be, at least in part, mediated by extrasynaptic glutamate, possibly released from the surrounding glial and/or nerve cells. Taken together, these findings indicate a functional involvement of CXCR‐4 in the modulation of synaptic transmission, adding another member to the repertoire of the chemokine receptors exerting a neuromodulatory role in the cerebellum.

Chemokine Fractalkine/CX3CL1 Negatively Modulates Active Glutamatergic Synapses in Rat Hippocampal Neurons

We examined the effects of the chemokine fractalkine (CX3CL1) on EPSCs evoked by electrical stimulation of Schaffer collaterals in patch-clamped CA1 pyramidal neurons from rat hippocampal slices. Acute application of CX3CL1 caused a sustained reduction of EPSC amplitude, with partial recovery after washout. CX3CL1-induced EPSC depression is postsynaptic in nature, because paired-pulse ratio was maintained, amplitude distribution of spontaneous excitatory postsynaptic currents shifted to lower values, and whole-cell current responses to AMPA were reversibly inhibited. EPSC depression by CX3CL1 is mediated by CX3CL1 receptor (CX3CR1), because CX3CL1 was unable to influence EPSC amplitude in CA1 pyramidal neurons from CX3CR1 knock-out mice. CX3CL1-induced depression of both EPSC and AMPA current was not observed in the absence of afferent fiber stimulation or AMPA receptor activation, respectively, indicating the requirement of sustained receptor activity for its development. Findings obtained from hippocampal slices, cultured hippocampal neurons, and transfected human embryonic kidney cells indicate that a Ca2+-, cAMP-, and phosphatase-dependent process is likely to modulate CX3CL1 effects because of the following: (1) CX3CL1-induced depression was antagonized by intracellular BAPTA, 8Br-cAMP, phosphatase inhibitors, and pertussis toxin (PTX); (2) CX3CL1 inhibited forskolin-induced cAMP formation sensitive to PTX; and (3) CX3CL1 inhibited forskolin-induced Ser845 GluR1 phosphorylation, which was sensitive to PTX and dependent on Ca2+ and phosphatase activity. Together, these findings indicate that CX3CL1 negatively modulates AMPA receptor function at active glutamatergic synapses through cell-signaling pathways by influencing the balance between kinase and phosphatase activity.

CX3CR1 Deficiency Alters Hippocampal-Dependent Plasticity Phenomena Blunting the Effects of Enriched Environment

In recent years several evidence demonstrated that some features of hippocampal biology, like neurogenesis, synaptic transmission, learning, and memory performances are deeply modulated by social, motor, and sensorial experiences. Fractalkine/CX3CL1 is a transmembrane chemokine abundantly expressed in the brain by neurons, where it modulates glutamatergic transmission and long-term plasticity processes regulating the intercellular communication between glia and neurons, being its specific receptor CX3CR1 expressed by microglia. In this paper we investigated the role of CX3CL1/CX3CR1 signaling on experience-dependent hippocampal plasticity processes. At this aim wt and CX3CR1GFP/GFP mice were exposed to long-lasting-enriched environment (EE) and the effects on hippocampal functions were studied by electrophysiological recordings of long-term potentiation of synaptic activity, behavioral tests of learning and memory in the Morris water maze paradigm and analysis of neurogenesis in the subgranular zone of the dentate gyrus (DG). We found that CX3CR1 deficiency increases hippocampal plasticity and spatial memory, blunting the potentiating effects of EE. In contrast, exposure to EE increased the number and migration of neural progenitors in the DG of both wt and CX3CR1GFP/GFP mice. These data indicate that CX3CL1/CX3CR1-mediated signaling is crucial for a normal experience-dependent modulation of hippocampal functions.

Dark microglia: A new phenotype predominantly associated with pathological states.

The past decade has witnessed a revolution in our understanding of microglia. These immune cells were shown to actively remodel neuronal circuits, leading to propose new pathogenic mechanisms. To study microglial implication in the loss of synapses, the best pathological correlate of cognitive decline across chronic stress, aging, and diseases, we recently conducted ultrastructural analyses. Our work uncovered the existence of a new microglial phenotype that is rarely present under steady state conditions, in hippocampus, cerebral cortex, amygdala, and hypothalamus, but becomes abundant during chronic stress, aging, fractalkine signaling deficiency (CX3CR1 knockout mice), and Alzheimers disease pathology (APP‐PS1 mice). Even though these cells display ultrastructural features of microglia, they are strikingly distinct from the other phenotypes described so far at the ultrastructural level. They exhibit several signs of oxidative stress, including a condensed, electron‐dense cytoplasm and nucleoplasm making them as “dark” as mitochondria, accompanied by a pronounced remodeling of their nuclear chromatin. Dark microglia appear to be much more active than the normal microglia, reaching for synaptic clefts, while extensively encircling axon terminals and dendritic spines with their highly ramified and thin processes. They stain for the myeloid cell markers IBA1 and GFP (in CX3CR1‐GFP mice), and strongly express CD11b and microglia‐specific 4D4 in their processes encircling synaptic elements, and TREM2 when they associate with amyloid plaques. Overall, these findings suggest that dark microglia, a new phenotype that we identified based on their unique properties, could play a significant role in the pathological remodeling of neuronal circuits, especially at synapses. GLIA 2016;64:826–839

Fractalkine receptor deficiency impairs microglial and neuronal responsiveness to chronic stress

Chronic stress is one of the most relevant triggering factors for major depression. Microglial cells are highly sensitive to stress and, more generally, to environmental challenges. However, the role of these brain immune cells in mediating the effects of stress is still unclear. Fractalkine signaling - which comprises the chemokine CX3CL1, mainly expressed by neurons, and its receptor CX3CR1, almost exclusively present on microglia in the healthy brain - has been reported to critically regulate microglial activity. Here, we investigated whether interfering with microglial function by deleting the Cx3cr1 gene affects the brains response to chronic stress. To this purpose, we housed Cx3cr1 knockout and wild-type adult mice in either control or stressful environments for 2weeks, and investigated the consequences on microglial phenotype and interactions with synapses, synaptic transmission, behavioral response and corticosterone levels. Our results show that hampering neuron-microglia communication via the CX3CR1-CX3CL1 pathway prevents the effects of chronic unpredictable stress on microglial function, short- and long-term neuronal plasticity and depressive-like behavior. Overall, the present findings suggest that microglia-regulated mechanisms may underlie the differential susceptibility to stress and consequently the vulnerability to diseases triggered by the experience of stressful events, such as major depression.

LTP impairment by fractalkine/CX3CL1 in mouse hippocampus is mediated through the activity of adenosine receptor type 3 (A3R)

We have examined how the chemokine fractalkine/CX(3)CL1 influences long-term potentiation (LTP) in CA1 mouse hippocampal slices. Field potentials (fEPSPs) were recorded upon electrical stimulation of Schaffer collaterals. It was found that application of CX(3)CL1 inhibits LTP when present during the critical induction period. LTP impairment (i) failed to occur in CX(3)CR1 deficient mice (CX(3)CR1(GFP/GFP)) and in the presence of okadaic acid (OA); (ii) required the activation of adenosine receptor 3 (A(3)R), since it was prevented in A(3)R-deficient mice or by MRS1523, a selective A(3)R antagonist. Together, these findings indicate that CX(3)CL1 inhibits hippocampal LTP through A(3)R activity.

Homeostatic Control of Synaptic Activity by Endogenous Adenosine is Mediated by Adenosine Kinase

Extracellular adenosine, a key regulator of neuronal excitability, is metabolized by astrocyte-based enzyme adenosine kinase (ADK). We hypothesized that ADK might be an upstream regulator of adenosine-based homeostatic brain functions by simultaneously affecting several downstream pathways. We therefore studied the relationship between ADK expression, levels of extracellular adenosine, synaptic transmission, intrinsic excitability, and brain-derived neurotrophic factor (BDNF)-dependent synaptic actions in transgenic mice underexpressing or overexpressing ADK. We demonstrate that ADK: 1) Critically influences the basal tone of adenosine, evaluated by microelectrode adenosine biosensors, and its release following stimulation; 2) determines the degree of tonic adenosine-dependent synaptic inhibition, which correlates with differential plasticity at hippocampal synapses with low release probability; 3) modulates the age-dependent effects of BDNF on hippocampal synaptic transmission, an action dependent upon co-activation of adenosine A2A receptors; and 4) influences GABAA receptor-mediated currents in CA3 pyramidal neurons. We conclude that ADK provides important upstream regulation of adenosine-based homeostatic function of the brain and that this mechanism is necessary and permissive to synaptic actions of adenosine acting on multiple pathways. These mechanistic studies support previous therapeutic studies and implicate ADK as a promising therapeutic target for upstream control of multiple neuronal signaling pathways crucial for a variety of neurological disorders.

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

&NA; 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.

Enriched environment reduces glioma growth through immune and non-immune mechanisms in mice

Mice exposed to standard (SE) or enriched environment (EE) were transplanted with murine or human glioma cells and differences in tumour development were evaluated. We report that EE exposure affects: (i) tumour size, increasing mice survival; (ii) glioma establishment, proliferation and invasion; (iii) microglia/macrophage (M/Mφ) activation; (iv) natural killer (NK) cell infiltration and activation; and (v) cerebral levels of IL-15 and BDNF. Direct infusion of IL-15 or BDNF in the brain of mice transplanted with glioma significantly reduces tumour growth. We demonstrate that brain infusion of IL-15 increases the frequency of NK cell infiltrating the tumour and that NK cell depletion reduces the efficacy of EE and IL-15 on tumour size and of EE on mice survival. BDNF infusion reduces M/Mφ infiltration and CD68 immunoreactivity in tumour mass and reduces glioma migration inhibiting the small G protein RhoA through the truncated TrkB.T1 receptor. These results suggest alternative approaches for glioma treatment.