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We examine some of the biological and physiological properties of the avian α6 neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, α6 mRNA is abundantly expressed in the developing... more
We examine some of the biological and physiological properties of the avian α6 neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, α6 mRNA is abundantly expressed in the developing chick neuroretina, where it coexists with other nicotinic receptor subunit mRNAs such as α3, β2 and β4. In contrast, α6 mRNA is absent from the optic tectum and from the peripheral ganglia. Despite numerous efforts, the α6 subunit has long failed the critical test of functional reconstitution. Here we use patch‐clamp techniques and confocal laser microscopy to measure ACh‐activated currents and nicotine‐elicited Ca2+ transients in human BOSC 23 cells transfected with chick α6 in combination with other chick nAChR neuronal subunits. Heterologously expressed α6 and β4 subunits form functional heteromeric nAChRs, which are permeable to Ca2+ ions and blocked by the nicotinic antagonist methyllycaconitine (10 μm). Likewise, ACh elicits measurable currents in cells transfected with α6 and β2. Hill analysis of the dose–response curves in cells transfected with α3, β4 and α6 cDNAs, suggests the assembly of functional α3β4α6 receptor, with an apparent affinity for ACh threefold lower than α3β4. Our results indicate that α6‐containing nAChRs assemble in heterologous expression systems and are probably present in retinal cells.
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Glycinergic synapses are implicated in the coordination of reflex responses, sensory signal processing and pain sensation. Their activity is pre- and postsynaptically regulated, although mechanisms are poorly understood. Using patch-clamp... more
Glycinergic synapses are implicated in the coordination of reflex responses, sensory signal processing and pain sensation. Their activity is pre- and postsynaptically regulated, although mechanisms are poorly understood. Using patch-clamp recording and Ca2+ imaging in hypoglossal motoneurones from rat and mouse brainstem slices, we address here the role of cytoplasmic Ca2+ (Cai) in glycinergic synapse modulation. Ca2+ influx through voltage-gated or NMDA receptor channels caused powerful transient inhibition of glycinergic IPSCs. This effect was accompanied by an increase in both the failure rate and paired-pulse ratio, as well as a decrease in the frequency of mIPSCs, suggesting a presynaptic mechanism of depression. Inhibition was reduced by the cannabinoid receptor antagonist SR141716A and occluded by the agonist WIN55,212-2, indicating involvement of endocannabinoid retrograde signalling. Conversely, in the presence of SR141716A, glycinergic IPSCs were potentiated postsynaptically by glutamate or NMDA, displaying a Ca2+-dependent increase in amplitude and decay prolongation. Both presynaptic inhibition and postsynaptic potentiation were completely prevented by strong Cai buffering (20 mm BAPTA). Our findings demonstrate two independent mechanisms by which Ca2+ modulates glycinergic synaptic transmission: (i) presynaptic inhibition of glycine release and (ii) postsynaptic potentiation of GlyR-mediated responses. This dual Ca2+-induced regulation might be important for feedback control eof neurotransmission in a variety of glycinergic networks in mammalian nervous systems.
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Combinations of cDNAs encoding mouse and chick nicotinic acetylcholine receptor (nAChR) subunits were transiently transfected into human BOSC 23 cells, and the expressed receptors were studied by simultaneously recording transmembrane... more
Combinations of cDNAs encoding mouse and chick nicotinic acetylcholine receptor (nAChR) subunits were transiently transfected into human BOSC 23 cells, and the expressed receptors were studied by simultaneously recording transmembrane currents and fluorescence transients using the whole-cell patch-clamp technique, and confocal microscopy with the Ca2+ indicator dye fluo-3.The fractional Ca2+ current, Pf, of nAChRs was evaluated as the normalized ratio of nicotine-evoked fluorescence transient over total charge entering the cell (F/Q ratio). Mouse fetal muscle nAChR channels had a Pf,αβγδ value of 2.1 %. The substitution of the γ subunit with the ɛ subunit resulted in a 2-fold increase in Pf (4.2 %). The difference in Ca2+ permeability was confirmed by determination of Ca2+/Cs+ permeability ratios.Among the chick neuronal nAChRs tested, Pf,α3β4 was 4.6 %, while Pf,α4β4 and Pf,α4β2 were 3.0 % and 2.9 %, respectively.The amplitude of the current elicited by the activation of α3β4 nAChRs increased as the external Ca2+ concentration was raised from 2 to 110 mm, whereas currents flowing through all other nAChRs tested were reduced to various extents.Our findings indicate that the adult-type muscle nAChR (αβɛδ) is more permeable to Ca2+ than the fetal-type (αβγδ), while ganglionic-like α3β4 nAChR is more permeable to Ca2+ than the examined α4-containing nAChRs. The functional significance is discussed.
Research Interests: Biophysics, Chemistry, Kinetics, Calcium, Fluorescent Dyes and Reagents, and 15 moreMedicine, Biological Sciences, Cell line, Humans, Mice, Nicotine, Animals, Calcium channels, Nicotinic Acetylcholine Receptors, Neurons, Chickens, Fetus, Patch Clamp, aniline Compounds, and Medical and Health Sciences
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The mature mammalian brain connectome emerges during development via the extension and pruning of neuronal connections. Glial cells have been identified as key players in the phagocytic elimination of neuronal synapses and projections.... more
The mature mammalian brain connectome emerges during development via the extension and pruning of neuronal connections. Glial cells have been identified as key players in the phagocytic elimination of neuronal synapses and projections. Recently, phosphatidylserine has been identified as neuronal “eat‐me” signal that guides elimination of unnecessary input sources, but the associated transduction systems involved in such pruning are yet to be described. Here, we identified Xk‐related protein 8 (Xkr8), a phospholipid scramblase, as a key factor for the pruning of axons in the developing mammalian brain. We found that mouse Xkr8 is highly expressed immediately after birth and required for phosphatidylserine exposure in the hippocampus. Mice lacking Xkr8 showed excess excitatory nerve terminals, increased density of cortico‐cortical and cortico‐spinal projections, aberrant electrophysiological profiles of hippocampal neurons, and global brain hyperconnectivity. These data identify phospholipid scrambling by Xkr8 as a central process in the labeling and discrimination of developing neuronal projections for pruning in the mammalian brain.
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... 2001) and the progression to AIDS in HIV-infected patients, is questioned (Faure et al. 2000; McDermott et al. 2000; Vidal et al. 2005). The first evidence of a direct neuroprotective function of CX3CL1 appeared in a paper published... more
... 2001) and the progression to AIDS in HIV-infected patients, is questioned (Faure et al. 2000; McDermott et al. 2000; Vidal et al. 2005). The first evidence of a direct neuroprotective function of CX3CL1 appeared in a paper published by Meucci et al. ...
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Research Interests: Chronic Pain, Biomarkers, Medicine, Multidisciplinary, In Vitro, and 15 moreMice, Animals, Microglia, Male, Central Nervous System, Neurons, Long Term Depression, Dentate Gyrus, Neurological, Activation, Encephalitis, Nature Communications, Dopaminergic Neurons, Excitatory Postsynaptic Potentials, and Neuralgia
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Complement signaling is thought to serve as an opsonization signal to promote the phagocytosis of synapses by microglia. However, while its role in synaptic remodeling has been demonstrated in the retino-thalamic system, it remains... more
Complement signaling is thought to serve as an opsonization signal to promote the phagocytosis of synapses by microglia. However, while its role in synaptic remodeling has been demonstrated in the retino-thalamic system, it remains unclear whether complement signaling mediates synaptic pruning in the brain more generally. Here we found that mice lacking the Complement receptor 3, the major microglia complement receptor, failed to show a deficit in either synaptic pruning or axon elimination in the developing mouse cortex. Instead, mice lacking Complement receptor 3 exhibited a deficit in the perinatal elimination of neurons in the cortex, a deficit that is associated with increased cortical thickness and enhanced functional connectivity in these regions in adulthood. These data demonstrate a role for complement in promoting neuronal elimination in the developing cortex.
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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... more
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...
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Microglia, the brain's resident macrophages, actively contribute to the homeostasis of cerebral parenchyma by sensing neuronal activity and supporting synaptic remodeling and plasticity. While several studies demonstrated different... more
Microglia, the brain's resident macrophages, actively contribute to the homeostasis of cerebral parenchyma by sensing neuronal activity and supporting synaptic remodeling and plasticity. While several studies demonstrated different roles for astrocytes in sleep, the contribution of microglia in the regulation of sleep/wake cycle and in the modulation of synaptic activity in the different day phases has not been deeply investigated. Using light as a zeitgeber cue, we studied the effects of microglial depletion with the colony stimulating factor‐1 receptor antagonist PLX5622 on the sleep/wake cycle and on hippocampal synaptic transmission in male mice. Our data demonstrate that almost complete microglial depletion increases the duration of NREM sleep and reduces the hippocampal excitatory neurotransmission. The fractalkine receptor CX3CR1 plays a relevant role in these effects, because cx3cr1GFP/GFP mice recapitulate what found in PLX5622‐treated mice. Furthermore, during the ligh...
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Glioma patients commonly suffer from epileptic seizures. However, the mechanisms of glioma-associated epilepsy are far to be completely understood. Using glioma-neurons co-cultures, we found that tumor cells are able to deeply influence... more
Glioma patients commonly suffer from epileptic seizures. However, the mechanisms of glioma-associated epilepsy are far to be completely understood. Using glioma-neurons co-cultures, we found that tumor cells are able to deeply influence neuronal chloride homeostasis, by depolarizing the reversal potential of γ-aminobutyric acid (GABA)-evoked currents (EGABA). EGABA depolarizing shift is due to zinc-dependent reduction of neuronal KCC2 activity and requires glutamate release from glioma cells. Consistently, intracellular zinc loading rapidly depolarizes EGABA in mouse hippocampal neurons, through the Src/Trk pathway and this effect is promptly reverted upon zinc chelation. This study provides a possible molecular mechanism linking glioma invasion to excitation/inhibition imbalance and epileptic seizures, through the zinc–mediated disruption of neuronal chloride homeostasis.
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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... more
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 ...
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The mRNA levels of NKCC1, an inwardly directed Na + , K + -2Cl − cotransporter that facilitates the accumulation of intracellular Cl − , and of KCC2, an outwardly directed K + -Cl − cotransporter that extrudes Cl − , were studied in... more
The mRNA levels of NKCC1, an inwardly directed Na + , K + -2Cl − cotransporter that facilitates the accumulation of intracellular Cl − , and of KCC2, an outwardly directed K + -Cl − cotransporter that extrudes Cl − , were studied in surgically resected brain specimens from drug-resistant temporal lobe (TL) epilepsy (TLE) patients. Quantitative RT-PCR analyses of the mRNAs extracted from the human TLE-associated brain regions revealed an up-regulation of NKCC1 mRNA and a down-regulation of KCC2 mRNA in the hippocampal subiculum, compared with the hippocampus proper or the TL neocortex, suggesting an abnormal transcription of Cl − transporters in the TLE subiculum. In parallel experiments, cell membranes isolated from the same TLE-associated brain regions were injected into Xenopus oocytes that rapidly incorporated human GABA A receptors into their surface membrane. The GABA currents elicited in oocytes injected with membranes from the subiculum had a more depolarized reversal potenti...
Research Interests: Neuroscience, Biology, Medicine, Multidisciplinary, Hippocampus, and 15 moreTemporal Lobe, Humans, Animals, Drug Resistance, Temporal Lobe Epilepsy, Subiculum, Xenopus laevis, Neocortex, Oocytes, Gene Expression Regulation, Hippocampal formation, Excitatory Postsynaptic Potentials, Cell Membrane, GABAA receptor, and Bumetanide
The properties of γ-aminobutyric acid (GABA) type A receptors (GABA A receptors) microtransplanted from the human epileptic brain to the plasma membrane of Xenopus oocytes were compared with those recorded directly from neurons, or glial... more
The properties of γ-aminobutyric acid (GABA) type A receptors (GABA A receptors) microtransplanted from the human epileptic brain to the plasma membrane of Xenopus oocytes were compared with those recorded directly from neurons, or glial cells, in human brains slices. Cell membranes isolated from brain specimens, surgically obtained from six patients afflicted with drug-resistant temporal lobe epilepsy (TLE) were injected into frog oocytes. Within a few hours, these oocytes acquired GABA A receptors that generated GABA currents with an unusual run-down, which was inhibited by orthovanadate and okadaic acid. In contrast, receptors derived from membranes of a nonepileptic hippocampal uncus, membranes from mouse brain, or recombinant rat α1β2γ2-GABA receptors exhibited a much less pronounced GABA-current run-down. Moreover, the GABA A receptors of pyramidal neurons in temporal neocortex slices from the same six epileptic patients exhibited a stronger run-down than the receptors of rat ...
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Research Interests: Neuroscience, Cognitive Science, Fear, Magnetic Resonance Imaging, Classical Conditioning, and 15 moreBrain, Mice, Functional Magnetic Resonance Imaging, Animals, Male, Amygdala, Neurons, Arousal, Biological neural network, Neuron, Neural pathways, Action Potentials, Neurosciences, Forebrain, and In Vitro Techniques
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In this paper we show for the first time that: i) astrocytes are required for the neuroprotective activity of CX3CL1 against excitotoxicity; ii) inhibition of the glutamate transporter 1 (GLT-1) prejudices CX3CL1-mediated neuroprotection;... more
In this paper we show for the first time that: i) astrocytes are required for the neuroprotective activity of CX3CL1 against excitotoxicity; ii) inhibition of the glutamate transporter 1 (GLT-1) prejudices CX3CL1-mediated neuroprotection; iii) CX3CL1 increases GLT-1 activity on astrocytes. The modulation of GLT-1 activity induced by CX3CL1 on astrocytes requires the presence and the activity of A1 adenosine receptor (A1R), being blocked by the specific antagonist DPCPX and absent in A1R(-/-) astrocytes. These data introduce the astrocytes as active players in CX3CL1-mediated signaling between microglia and neurons, identifying GLT-1 as a key mediator of the neuroprotective activity of CX3CL1.
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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... more
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.
Research Interests: Chemistry, Biology, Cell Biology, Glutamate, Humans, and 15 moreEC, Animals, Central Nervous System, AMPA, Correlation coefficient, Ec, Cerebellar Cortex, Binding Site, Glutamic Acid, Gene Expression Regulation, AMPA receptors, Glutamate Receptor, Functional Properties, Excitatory Postsynaptic Potentials, and binding sites
The chemokine fractalkine (CX3CL1) is constitutively expressed by central neurons, regulating microglial responses including chemotaxis, activation, and toxicity. Through the activation of its own specific receptor, CX3CR1, CX3CL1 exerts... more
The chemokine fractalkine (CX3CL1) is constitutively expressed by central neurons, regulating microglial responses including chemotaxis, activation, and toxicity. Through the activation of its own specific receptor, CX3CR1, CX3CL1 exerts both neuroprotection against glutamate (Glu) toxicity and neuromodulation of the glutamatergic synaptic transmission in hippocampal neurons. Using cultured hippocampal neuronal cell preparations, obtained from CX3CR1−/− (CX3CR1GFP/GFP) mice, we report that these same effects are mimicked by exposing neurons to a medium conditioned with CX3CL1-treated mouse microglial cell line BV2 (BV2-st medium). Furthermore, CX3CL1-induced neuroprotection from Glu toxicity is mediated through the adenosine receptor 1 (AR1), being blocked by neuronal cell preparations treatment with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a specific inhibitor of AR1, and mimicked by both adenosine and the specific AR1 agonist 2-chloro-N6-cyclopentyladenosine. Similarly, experim...
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The activation of ion channels is crucial during cell movement, including glioblastoma cell invasion in the brain parenchyma. In this context, we describe for the first time the contribution of intermediate conductance Ca2+-activated K... more
The activation of ion channels is crucial during cell movement, including glioblastoma cell invasion in the brain parenchyma. In this context, we describe for the first time the contribution of intermediate conductance Ca2+-activated K (IKCa) channel activity in the chemotactic response of human glioblastoma cell lines, primary cultures, and freshly dissociated tissues to CXC chemokine ligand 12 (CXCL12), a chemokine whose expression in glioblastoma has been correlated with its invasive capacity. We show that blockade of the IKCachannel with its specific inhibitor 1-[(2-chlorophenyl) diphenylmethyl]-1 H-pyrazole (TRAM-34) or IKCachannel silencing by short hairpin RNA (shRNA) completely abolished CXCL12-induced cell migration. We further demonstrate that this is not a general mechanism in glioblastoma cell migration since epidermal growth factor (EGF), which also activates IKCachannels in the glioblastoma-derived cell line GL15, stimulate cell chemotaxis even if the IKCachannels have...
Research Interests: Physiology, Biology, Cell Migration, Cell Biology, Medicine, and 15 moreHumans, Glioblastoma, Calcium Signaling, American, Chemotaxis, Medical Physiology, Mitogen Activated Protein Kinase, Epidermal Growth Factor, Neoplasm Invasiveness, Chemokines, Recombinant Proteins, Chelating Agents, Protein Kinase Inhibitors, Brain Neoplasms, and Biochemistry and cell biology
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BACKGROUND AND PURPOSE Studies using intermittent access drug self-administration show increased motivation to take and seek cocaine and fentanyl, relative to continuous access. In this study, we examined the effects of intermittent- and... more
BACKGROUND AND PURPOSE Studies using intermittent access drug self-administration show increased motivation to take and seek cocaine and fentanyl, relative to continuous access. In this study, we examined the effects of intermittent- and continuous access self-administration on heroin intake, patterns of self-administration, and cue-induced heroin seeking, after forced or voluntary abstinence, in male and female rats. We also modeled brain levels of heroin and its active metabolites. EXPERIMENTAL APPROACH Rats were trained to self-administer a palatable solution and then heroin (0.075 mg/kg/inf) either continuously (6-h/d; 10 d) or intermittently (6-h/d; 5-min access/30-min; 10 d). Brain levels of heroin and its metabolites were modeled using a pharmacokinetic software. Next, heroin-seeking was assessed after 1 or 21 abstinence days. Between tests, rats underwent either forced or voluntary abstinence. The estrous cycle was measured using a vaginal smear test. KEY RESULTS Intermittent access exacerbated heroin self-administration and was characterized by a burst-like intake, yielding higher brain peaks of heroin and 6-monoacetylmorphine concentrations. Moreover, intermittent access increased cue-induced heroin-seeking during early, but not late abstinence. Heroin-seeking was higher in females after intermittent, but not continuous access and this effect was independent of the estrous cycle. CONCLUSIONS AND IMPLICATIONS Intermittent heroin access in rats resembles critical features of heroin use disorder: a self-administration pattern characterized by repeated large doses of heroin and higher relapse vulnerability during early abstinence. This has significant implications for refining animal models of substance use disorder and for better understanding of the neuroadaptations responsible for this disorder.
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Exposure to aversive events during sensitive developmental periods can affect the preferential coping strategy adopted by individuals later in life, leading to either stress-related psychiatric disorders, including depression, or to... more
Exposure to aversive events during sensitive developmental periods can affect the preferential coping strategy adopted by individuals later in life, leading to either stress-related psychiatric disorders, including depression, or to well-adaptation to future adversity and sources of stress, a behavior phenotype termed “resilience”. We have previously shown that interfering with the development of mother-pups bond with the Repeated Cross Fostering (RCF) stress protocol can induce resilience to depression-like phenotype in adult C57BL/6J female mice. Here, we used patch-clamp recording in midbrain slice combined with both in vivo and ex vivo pharmacology to test our hypothesis of a link between electrophysiological modifications of dopaminergic neurons in the intermediate Ventral Tegmental Area (VTA) of RCF animals and behavioral resilience. We found reduced hyperpolarization-activated (Ih) cation current amplitude and evoked firing in VTA dopaminergic neurons from both young and adult RCF female mice. In vivo, VTA-specific pharmacological manipulation of the Ih current reverted the pro-resilient phenotype in adult early-stressed mice or mimicked behavioral resilience in adult control animals. This is the first evidence showing how pro-resilience behavior induced by early events is linked to a long-lasting reduction of Ih current and excitability in VTA dopaminergic neurons.