Altered synaptic connectivity and brain function in mice lacking microglial adapter protein Iba1

Growing evidence indicates that microglia impact brain function by regulating synaptic pruning and formation as well as synaptic transmission and plasticity. Iba1 (ionized Ca+2-binding adapter protein 1), encoded by the Allograft inflammatory factor 1 (Aif1) gene, is an actin-interacting protein in microglia. Although Iba1 has long been used as a cellular marker for microglia, its functional role remains unknown. Here, we used global, Iba1-deficient (Aif1−/−) mice to characterize microglial activity, synaptic function, and behavior. Microglial imaging in acute hippocampal slices and fixed tissues from juvenile mice revealed that Aif1−/− microglia display reductions in ATP-induced motility and ramification, respectively. Biochemical assays further demonstrated that Aif1−/− brain tissues exhibit an altered expression of microglial-enriched proteins associated with synaptic pruning. Consistent with these changes, juvenile Aif1−/− mice displayed deficits in the excitatory synapse number and synaptic drive assessed by neuronal labeling and whole-cell patch-clamp recording in acute hippocampal slices. Unexpectedly, microglial synaptic engulfment capacity was diminished in juvenile Aif1−/− mice. During early postnatal development, when synapse formation is a predominant event in the hippocampus, the excitatory synapse number was still reduced in Aif1−/− mice. Together, these findings support an overall role of Iba1 in excitatory synaptic growth in juvenile mice. Lastly, postnatal synaptic deficits persisted in adulthood and correlated with significant behavioral changes in adult Aif1−/− mice, which exhibited impairments in object recognition memory and social interaction. These results suggest that Iba1 critically contributes to microglial activity underlying essential neuroglia developmental processes that may deeply influence behavior.

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Altered synaptic connectivity and brain function in mice lacking microglial adapter protein Iba1

10.1101/2021.04.23.441023 ◽

2021 ◽

Author(s):

Pablo J. Lituma ◽

Evan Woo ◽

Bruce F. O’Hara ◽

Pablo E. Castillo ◽

Nicholas E. S. Sibinga ◽

...

Keyword(s):

Synaptic Transmission ◽

Neurodegenerative Disorders ◽

Brain Function ◽

Hippocampal Slices ◽

Autism Spectrum ◽

Excitatory Synapse ◽

Adapter Protein ◽

Acute Hippocampal Slices ◽

And Behavior ◽

Synaptic Pruning

AbstractGrowing evidence indicates that microglia impact brain function by regulating synaptic pruning and formation, as well as synaptic transmission and plasticity. Iba1 (Ionized Ca+2-binding adapter protein 1), encoded by the Allograft inflammatory factor 1 (Aif1) gene, is an actin-interacting protein in microglia. Although Iba1 has long been used as a cellular marker for microglia, its functional role remains unknown. Here, we used global Iba1-deficient (Aif1-/-) mice to characterize microglial activity, synaptic function and behavior. Microglial imaging in acute hippocampal slices and fixed tissues from juvenile mice revealed that Aif1-/- microglia display reductions in ATP-induced motility and ramification, respectively. Biochemical assays further demonstrated that Aif1-/- brain tissues exhibit an altered expression of microglial-enriched proteins associated with synaptic pruning. Consistent with these changes, juvenile Aif1-/- mice displayed deficits in excitatory synapse number and synaptic transmission assessed by neuronal labeling and whole-cell patch-clamp recording in acute hippocampal slices. Unexpectedly, microglial synaptic engulfment capacity was diminished in juvenile Aif1-/- mice. During early postnatal development when synapse formation is a predominant event in the hippocampus, excitatory synapse number was still reduced in Aif1-/- mice. Together these findings support an overall role of Iba1 in excitatory synaptic growth in juvenile mice. Lastly, postnatal synaptic deficits persisted in the adulthood and correlated with significant behavioral changes in adult Aif1-/- mice, which exhibited impairments in object recognition memory and social interaction. These results suggest that Iba1 critically contributes to microglial activity underlying essential neuro-glia developmental processes that may deeply influence behavior.SignificanceAbnormal microglia-neuron interaction is increasingly implicated in neurodevelopmental and neuropsychiatric conditions such as autism spectrum disorders and schizophrenia, as well as in neurodegenerative disorders such as Alzheimer’s disease. This study demonstrates that deletion of the microglia-specific protein Iba1, which has long been utilized as a selective microglial marker but whose role has remained unidentified, results in microglial structural and functional impairments that significantly impact synaptic development and behavior. These findings not only highlight the importance of microglia in brain function but may also suggest that modifying microglial function could provide a therapeutic strategy for treatment of neurodevelopmental, neuropsychiatric and neurodegenerative disorders.

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Nicotinic Acetylcholine Receptor α7 and α4β2 Subtypes Differentially Control GABAergic Input to CA1 Neurons in Rat Hippocampus

Journal of Neurophysiology ◽

10.1152/jn.2001.86.6.3043 ◽

2001 ◽

Vol 86 (6) ◽

pp. 3043-3055 ◽

Cited By ~ 174

Author(s):

Manickavasagom Alkondon ◽

Edson X. Albuquerque

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Pyramidal Neurons ◽

Hippocampal Slices ◽

Synaptic Function ◽

Gabaergic Inhibition ◽

Patch Clamp Recording ◽

Net Charge ◽

Nicotinic Acetylcholine ◽

Α7 Nachr

The hippocampus, a limbic brain region involved in the encoding and retrieval of memory, has a well-defined structural network assembled from excitatory principal neurons and inhibitory interneurons. Because the GABAergic interneurons form synapses onto both pyramidal neurons and interneurons, the activation of nicotinic acetylcholine receptors (nAChRs) present on certain interneurons could induce either inhibition or disinhibition in the hippocampal circuitry. To understand the role of nAChRs in controlling synaptic transmission in the hippocampus, we evaluated the magnitude of nAChR-modulated GABAergic postsynaptic currents (PSCs) in pyramidal neurons and various interneurons of the CA1 region. Using whole cell patch-clamp recording and post hoc identification of neuronal types in rat hippocampal slices, we show that brief (12-s) nAChR activation by ACh (1 mM) or choline (10 mM) enhances the frequency of GABAergic PSCs in both pyramidal neurons and CA1 interneurons. The magnitude of α7 nAChR-mediated GABAergic inhibition, as assessed by the net charge of choline-induced PSCs, was highest in stratum lacunosum moleculare interneurons followed by pyramidal neurons and s. radiatum interneurons. In contrast, the magnitude of α4β2 nAChR-mediated GABAergic inhibition, as assessed by the difference between the net charge of PSCs induced by ACh and choline, was highest in pyramidal neurons followed by s. lacunosum moleculare and s. radiatum interneurons. The present results suggest that cholinergic cues transmitted via specific subtypes of nAChRs modify the synaptic function in the hippocampus by inducing a differential degree of GABAergic inhibition in the target neurons.

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Brain lactic acidosis and synaptic function

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y90-026 ◽

1990 ◽

Vol 68 (2) ◽

pp. 164-169 ◽

Cited By ~ 23

Author(s):

Wolfgang Walz ◽

Diane E. Harold

Keyword(s):

Lactic Acid ◽

Synaptic Transmission ◽

Hippocampal Slices ◽

Sodium Lactate ◽

Extracellular Ph ◽

Synaptic Function ◽

Excitatory Postsynaptic Potential ◽

Irreversible Damage ◽

Acid Release ◽

Neuronal Transmission

Measurements of the presynaptic fiber volley (PSFV), the population excitatory postsynaptic potential (EPSP), and the extracellular pH in the dendritic CA1 layer of rat hippocampal slices were used to evaluate the effects of lactacidosis on central synaptic transmission. Replacement of NaCl with sodium lactate (up to 30 mM) was found not to affect the PSFV; however, the EPSP was reversibly suppressed. Sodium citrate, with added CaCl2 to adjust for Ca2+ chelation, had the same effect as sodium lactate. Addition of lactic acid influenced the PSFV only when, at a concentration of 30 mM, the extracellular pH dropped to 6.6 or lower. With lactic acid concentrations of up to 20 mM, which produced pH levels of 6.8 in the slice, effects on the EPSP were reversible. However, 30 mM lactic acid suppressed both the PSFV and EPSP irreversibly. These results show that synaptic transmission is much more susceptible to lactacidosis than presynaptic axonal transmission. They also show that high levels of lactate, albeit causing suppression of synaptic transmission, do not cause irreversible damage. However, acidosis associated with lactic acid release may damage synaptic transmission irreversibly.Key words: acidosis, hippocampal slice, ischemia, lactate, lactic acid, neuronal transmission, synapse.

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Identification of Neuronal Pentraxins as Synaptic Binding Partners of C1q and the Involvement of NP1 in Synaptic Pruning in Adult Mice

Frontiers in Immunology ◽

10.3389/fimmu.2020.599771 ◽

2021 ◽

Vol 11 ◽

Author(s):

Réka Á. Kovács ◽

Henrietta Vadászi ◽

Éva Bulyáki ◽

György Török ◽

Vilmos Tóth ◽

...

Keyword(s):

Molecular Mechanisms ◽

Synapse Formation ◽

Potential Interaction ◽

Synaptic Function ◽

Classical Pathway ◽

Microglial Phagocytosis ◽

Classical Complement Pathway ◽

Adult Mice ◽

Synaptic Pruning

Elements of the immune system particularly that of innate immunity, play important roles beyond their traditional tasks in host defense, including manifold roles in the nervous system. Complement-mediated synaptic pruning is essential in the developing and healthy functioning brain and becomes aberrant in neurodegenerative disorders. C1q, component of the classical complement pathway, plays a central role in tagging synapses for elimination; however, the underlying molecular mechanisms and interaction partners are mostly unknown. Neuronal pentraxins (NPs) are involved in synapse formation and plasticity, moreover, NP1 contributes to cell death and neurodegeneration under adverse conditions. Here, we investigated the potential interaction between C1q and NPs, and its role in microglial phagocytosis of synapses in adult mice. We verified in vitro that NPs interact with C1q, as well as activate the complement system. Flow cytometry, immunostaining and co-immunoprecipitation showed that synapse-bound C1q colocalizes and interacts with NPs. High-resolution confocal microscopy revealed that microglia-surrounded C1q-tagged synapses are NP1 positive. We have also observed the synaptic occurrence of C4 suggesting that activation of the classical pathway cannot be ruled out in synaptic plasticity in healthy adult animals. In summary, our results indicate that NPs play a regulatory role in the synaptic function of C1q. Whether this role can be intensified upon pathological conditions, such as in Alzheimer’s disease, is to be disclosed.

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A novel and simple fluorescence method for the measurement of presynaptic vesicular zinc release in acute hippocampal slices with a fluorescence plate reader

Brain Research Bulletin ◽

10.1016/j.brainresbull.2007.06.001 ◽

2007 ◽

Vol 74 (1-3) ◽

pp. 183-187 ◽

Cited By ~ 6

Author(s):

Zsolt L. Datki ◽

Ákos Hunya ◽

Botond Penke

Keyword(s):

Hippocampal Slices ◽

Fluorescence Method ◽

Plate Reader ◽

Zinc Release ◽

Acute Hippocampal Slices ◽

Fluorescence Plate Reader

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Abstract WMP106: Reversal of Tonic Inhibition Contributes to Recovery of Synaptic Function After Transient Focal Cerebral Ischemia

Stroke ◽

10.1161/str.47.suppl_1.wmp106 ◽

2016 ◽

Vol 47 (suppl_1) ◽

Author(s):

James E Orfila ◽

Robert M Dietz ◽

Himmat Grewal ◽

Takeru Shimizu ◽

Frank F Strnad ◽

...

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Gabaa Receptors ◽

Western Blot Analysis ◽

Hippocampal Slices ◽

Long Term Potentiation ◽

The United States ◽

Synaptic Function ◽

Gaba Transporter ◽

Adult Mice

Introduction: Ischemic stroke is the fourth leading cause of death in the United States and is increasingly being recognized as a disease that occurs in people of all ages, not just the elderly. Studies suggest that the immature developing brain may have a greater degree of plasticity compared to the adult, thereby enhancing functional recovery to a greater extent during development. Hypothesis: Pediatric mice exhibit greater recovery from hippocampal synaptic function following experimental stroke than adults. Methods: Extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared at, 7 or 30 days after recovery from middle cerebral artery occlusion (MCAO). A behavioral fear conditioning paradigm was done to evaluate contextual memory in both pediatric and adult mice 30 days after MCAO. α5 GABAA receptors and GABA transporter expression was evaluated by western blot analysis. Results: In adult mice following MCAO, hippocampal long-term potentiation (LTP), defined as an increase in synaptic strength, remained impaired for at least 30 days in the ipsilateral and contralateral, non-injured hemisphere. However, in pediatric mice following MCAO, LTP was only impaired in the ipsilateral side 7 days after MCAO and showed full recovery of synaptic function at 30 days. Behavioral data confirmed these data, showing that only adult mice displayed memory deficits 30 days after MCAO. L655,708 (100nM), an inverse agonist selective for α5 GABAA receptors, rescued LTP in acute slices in both pediatric and adult mice at all-time points tested. Western blot analysis failed to identify any changes in α5 GABAA receptors or GABA transporter (GAT1 or GAT3) levels after MCAO. Conclusion: The present study demonstrates that transient focal ischemia causes functional impairment in the hippocampus and that recovery of behavioral and synaptic function is more robust in the young brain. In addition, inhibition of tonic GABA activity rescues synaptic function, indicating that targeting of excessive GABA activity may provide a therapeutic approach to improve cognitive recovery after stroke.

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GPR68 deletion impairs hippocampal long-term potentiation and passive avoidance behavior

Molecular Brain ◽

10.1186/s13041-020-00672-8 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Yuanyuan Xu ◽

Mike T. Lin ◽

Xiang-ming Zha

Keyword(s):

Passive Avoidance ◽

Pyramidal Neurons ◽

Granule Cells ◽

Hippocampal Slices ◽

Synaptic Cleft ◽

Long Term Potentiation ◽

Synaptic Function ◽

Metabotropic Receptor ◽

Term Potentiation

Abstract Increased neural activities reduced pH at the synaptic cleft and interstitial spaces. Recent studies have shown that protons function as a neurotransmitter. However, it remains unclear whether protons signal through a metabotropic receptor to regulate synaptic function. Here, we showed that GPR68, a proton-sensitive GPCR, exhibited wide expression in the hippocampus, with higher expression observed in CA3 pyramidal neurons and dentate granule cells. In organotypic hippocampal slice neurons, ectopically expressed GPR68-GFP was present in dendrites, dendritic spines, and axons. Recordings in hippocampal slices isolated from GPR68−/− mice showed a reduced fiber volley at the Schaffer collateral-CA1 synapses, a reduced long-term potentiation (LTP), but unaltered paired-pulse ratio. In a step-through passive avoidance test, GPR68−/− mice exhibited reduced avoidance to the dark chamber. These findings showed that GPR68 contributes to hippocampal LTP and aversive fear memory.

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Sleep-wake cycles drive daily dynamics of synaptic phosphorylation

Science ◽

10.1126/science.aav3617 ◽

2019 ◽

Vol 366 (6462) ◽

pp. eaav3617 ◽

Cited By ~ 39

Author(s):

Franziska Brüning ◽

Sara B. Noya ◽

Tanja Bange ◽

Stella Koutsouli ◽

Jan D. Rudolph ◽

...

Keyword(s):

Brain Function ◽

Synaptic Function ◽

Synaptic Activity ◽

Regulation Of Transcription ◽

Protein Abundance ◽

Cellular Processes ◽

Cytoskeleton Reorganization ◽

And Function ◽

Daily Changes ◽

Rest Activity

The circadian clock drives daily changes of physiology, including sleep-wake cycles, through regulation of transcription, protein abundance, and function. Circadian phosphorylation controls cellular processes in peripheral organs, but little is known about its role in brain function and synaptic activity. We applied advanced quantitative phosphoproteomics to mouse forebrain synaptoneurosomes isolated across 24 hours, accurately quantifying almost 8000 phosphopeptides. Half of the synaptic phosphoproteins, including numerous kinases, had large-amplitude rhythms peaking at rest-activity and activity-rest transitions. Bioinformatic analyses revealed global temporal control of synaptic function through phosphorylation, including synaptic transmission, cytoskeleton reorganization, and excitatory/inhibitory balance. Sleep deprivation abolished 98% of all phosphorylation cycles in synaptoneurosomes, indicating that sleep-wake cycles rather than circadian signals are main drivers of synaptic phosphorylation, responding to both sleep and wake pressures.

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Adenosine A1 receptor-mediated protection of mouse hippocampal synaptic transmission against oxygen and/or glucose deprivation: a comparative study

Journal of Neurophysiology ◽

10.1152/jn.00813.2018 ◽

2019 ◽

Vol 122 (2) ◽

pp. 721-728 ◽

Cited By ~ 3

Author(s):

Masahito Kawamura ◽

David N. Ruskin ◽

Susan A. Masino

Keyword(s):

Synaptic Transmission ◽

Hippocampal Slices ◽

Glucose Deprivation ◽

Acute Response ◽

Recording Conditions ◽

Receptor Inactivation ◽

Acute Hippocampal Slices ◽

Metabolic Stresses ◽

The Impact

Adenosine receptors are widely expressed in the brain, and adenosine is a key bioactive substance for neuroprotection. In this article, we clarify systematically the role of adenosine A1 receptors during a range of timescales and conditions when a significant amount of adenosine is released. Using acute hippocampal slices obtained from mice that were wild type or null mutant for the adenosine A1 receptor, we quantified and characterized the impact of varying durations of experimental ischemia, hypoxia, and hypoglycemia on synaptic transmission in the CA1 subregion. In normal tissue, these three stressors rapidly and markedly reduced synaptic transmission, and only treatment of sufficient duration led to incomplete recovery. In contrast, inactivation of adenosine A1 receptors delayed and/or lessened the reduction in synaptic transmission during all three stressors and reduced the magnitude of the recovery significantly. We reproduced the responses to hypoxia and hypoglycemia by applying an adenosine A1 receptor antagonist, validating the clear effects of genetic receptor inactivation on synaptic transmission. We found activation of adenosine A1 receptor inhibited hippocampal synaptic transmission during the acute phase of ischemia, hypoxia, or hypoglycemia and caused the recovery from synaptic impairment after these three stressors using genetic mutant. These studies quantify the neuroprotective role of the adenosine A1 receptor during a variety of metabolic stresses within the same recording system. NEW & NOTEWORTHY Deprivation of oxygen and/or glucose causes a rapid adenosine A1 receptor-mediated decrease in synaptic transmission in mouse hippocampus. We quantified adenosine A1 receptor-mediated inhibition during and synaptic recovery after ischemia, hypoxia, and hypoglycemia of varying durations using a genetic mutant and confirmed these findings using pharmacology. Overall, using the same recording conditions, we found the acute response and the neuroprotective ability of the adenosine A1 receptor depended on the type and duration of deprivation event.

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