Microglial Cytokines Mediate Plasticity Induced by 10 Hz Repetitive Magnetic Stimulation.

Microglia, the resident immune cells of the CNS, sense the activity of neurons and regulate physiological brain functions. They have been implicated in the pathology of brain diseases associated with alterations in neural excitability and plasticity. However, experimental and therapeutic approaches that modulate microglia function in a brain region-specific manner have not been established. In this study, we tested for the effects of repetitive transcranial magnetic stimulation (rTMS), a clinically used noninvasive brain stimulation technique, on microglia-mediated synaptic plasticity; 10 Hz electromagnetic stimulation triggered a release of plasticity-promoting cytokines from microglia in mouse organotypic brain tissue cultures of both sexes, while no significant changes in microglial morphology or microglia dynamics were observed. Indeed, substitution of tumor necrosis factor α (TNFα) and interleukin 6 (IL6) preserved synaptic plasticity induced by 10 Hz stimulation in the absence of microglia. Consistent with these findings, in vivo depletion of microglia abolished rTMS-induced changes in neurotransmission in the mPFC of anesthetized mice of both sexes. We conclude that rTMS affects neural excitability and plasticity by modulating the release of cytokines from microglia.SIGNIFICANCE STATEMENT Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive brain stimulation technique that induces cortical plasticity. Despite its wide use in neuroscience and clinical practice (e.g., depression treatment), the cellular and molecular mechanisms of rTMS-mediated plasticity remain not well understood. Herein, we report an important role of microglia and plasticity-promoting cytokines in synaptic plasticity induced by 10 Hz rTMS in organotypic slice cultures and anesthetized mice, thereby identifying microglia-mediated synaptic adaptation as a target of rTMS-based interventions.

The Amyloid Precursor Protein Regulates Synaptic Transmission at Medial Perforant Path Synapses.

The perforant path provides the primary cortical excitatory input to the hippocampus. Because of its important role in information processing and coding, entorhinal projections to the dentate gyrus have been studied in considerable detail. Nevertheless, synaptic transmission between individual connected pairs of entorhinal stellate cells and dentate granule cells remains to be characterized. Here, we have used mouse organotypic entorhino-hippocampal tissue cultures of either sex, in which the entorhinal cortex (EC) to dentate granule cell (GC; EC-GC) projection is present, and EC-GC pairs can be studied using whole-cell patch-clamp recordings. By using cultures of wild-type mice, the properties of EC-GC synapses formed by afferents from the lateral and medial entorhinal cortex were compared, and differences in short-term plasticity were identified. As the perforant path is severely affected in Alzheimer's disease, we used tissue cultures of amyloid precursor protein (APP)-deficient mice to examine the role of APP at this synapse. APP deficiency altered excitatory neurotransmission at medial perforant path synapses, which was accompanied by transcriptomic and ultrastructural changes. Moreover, presynaptic but not postsynaptic APP deletion through the local injection of Cre-expressing adeno-associated viruses in conditional APPflox/flox tissue cultures increased the neurotransmission efficacy at perforant path synapses. In summary, these data suggest a physiological role for presynaptic APP at medial perforant path synapses that may be adversely affected under altered APP processing conditions.SIGNIFICANCE STATEMENT The hippocampus receives input from the entorhinal cortex via the perforant path. These projections to hippocampal dentate granule cells are of utmost importance for learning and memory formation. Although there is detailed knowledge about perforant path projections, the functional synaptic properties at the level of individual connected pairs of neurons are not well understood. In this study, we investigated the role of APP in mediating functional properties and transmission rules in individually connected neurons using paired whole-cell patch-clamp recordings and genetic tools in organotypic tissue cultures. Our results show that presynaptic APP expression limits excitatory neurotransmission via the perforant path, which could be compromised in pathologic conditions such as Alzheimer's disease.

Microglia modulate TNFα-mediated synaptic plasticity.

The pro-inflammatory cytokine tumor necrosis factor α (TNFα) tunes the capacity of neurons to express synaptic plasticity. It remains, however, unclear how TNFα mediates synaptic positive (=change) and negative (=stability) feedback mechanisms. We assessed effects of TNFα on microglia activation and synaptic transmission onto CA1 pyramidal neurons of mouse organotypic entorhino-hippocampal tissue cultures. TNFα mediated changes in excitatory and inhibitory neurotransmission in a concentration-dependent manner, where low concentration strengthened glutamatergic neurotransmission via synaptic accumulation of GluA1-only-containing AMPA receptors and higher concentration increased inhibition. The latter induced the synaptic accumulation of GluA1-only-containing AMPA receptors as well. However, activated, pro-inflammatory microglia mediated a homeostatic adjustment of excitatory synapses, that is, an initial increase in excitatory synaptic strength at 3 h returned to baseline within 24 h, while inhibitory neurotransmission increased. In microglia-depleted tissue cultures, synaptic strengthening triggered by high levels of TNFα persisted and the impact of TNFα on inhibitory neurotransmission was still observed and dependent on its concentration. These findings underscore the essential role of microglia in TNFα-mediated synaptic plasticity. They suggest that pro-inflammatory microglia mediate synaptic homeostasis, that is, negative feedback mechanisms, which may affect the ability of neurons to express further plasticity, thereby emphasizing the importance of microglia as gatekeepers of synaptic change and stability.

Denervated mouse CA1 pyramidal neurons express homeostatic synaptic plasticity following entorhinal cortex lesion.

Structural, functional, and molecular reorganization of denervated neural networks is often observed in neurological conditions. The loss of input is accompanied by homeostatic synaptic adaptations, which can affect the reorganization process. A major challenge of denervation-induced homeostatic plasticity operating in complex neural networks is the specialization of neuronal inputs. It remains unclear whether neurons respond similarly to the loss of distinct inputs. Here, we used in vitro entorhinal cortex lesion (ECL) and Schaffer collateral lesion (SCL) in mouse organotypic entorhino-hippocampal tissue cultures to study denervation-induced plasticity of CA1 pyramidal neurons. We observed microglia accumulation, presynaptic bouton degeneration, and a reduction in dendritic spine numbers in the denervated layers 3 days after SCL and ECL. Transcriptome analysis of the CA1 region revealed complex changes in differential gene expression following SCL and ECL compared to non-lesioned controls with a specific enrichment of differentially expressed synapse-related genes observed after ECL. Consistent with this finding, denervation-induced homeostatic plasticity of excitatory synapses was observed 3 days after ECL but not after SCL. Chemogenetic silencing of the EC but not CA3 confirmed the pathway-specific induction of homeostatic synaptic plasticity in CA1. Additionally, increased RNA oxidation was observed after SCL and ECL. These results reveal important commonalities and differences between distinct pathway lesions and demonstrate a pathway-specific induction of denervation-induced homeostatic synaptic plasticity.

Timing differences between HFOs and interictal epileptiform discharges generated in vitro by different mechanisms in rat hippocampal slices: A novel approach.

OBJECTIVE: To investigate the effect of generating mechanism on the relationship between interictal-like epileptiform discharges (IEDs) and the underlying High Frequency Oscillations (HFOs; Ripples, R, and Fast Ripples, FR). METHODS: Synchronous spontaneous IEDs were recorded from the CA1 area of hippocampal slices from adult rats, perfused by Mg2+ -free ACSF (n = 41slices/14 animals) or 4-aminopyridine (50 µM, n = 37slices/16 animals); IED filtering revealed Rs and FRs and several metrics were calculated and compared (amplitude, duration, relative onset, time lag, % overlap, peak frequency, peak power, FR/R). RESULTS: Longer IEDs and higher 1st Population Spike (PS) amplitude in Mg2+ -free ACSF (vs 4-AP; P < .001, P < .001) correlated with longer duration and higher amplitude Rs (P < .0001, P = .001) and longer duration FRs (P < .001). In both media, Rs and FRs appeared before IED onset with Rs preceding FRs; R- and FR-IED lag (P = .008, P = .01) as well as R-FR lag (P = .04) were significantly longer in Mg2+ -free ACSF vs in 4-AP. R peak frequency and power were higher in Mg2+ -free ACSF, while no such differences were observed in FRs. Inter-model differences were mostly reflected in Rs, not FRs, suggesting that mechanisms unique to R generation are more active in Mg2+ -free ACSF vs in 4-AP. FRs appeared to contribute equally to IEDs irrespective of generating mechanism. SIGNIFICANCE: Several of the metrics used, particularly those regarding the timing between HFOs and IEDs, appear to correlate with the synchronizing mechanism and we propose that they may be useful when investigating antiepileptic substance effects on neuronal network activity.

Scavenging Tumor Necrosis Factor α Does Not Affect Inhibition of Dentate Granule Cells Following In Vitro Entorhinal Cortex Lesion.

Neurons that lose part of their afferent input remodel their synaptic connections. While cellular and molecular mechanisms of denervation-induced changes in excitatory neurotransmission have been identified, little is known about the signaling pathways that control inhibition in denervated networks. In this study, we used mouse entorhino-hippocampal tissue cultures of both sexes to study the role of the pro-inflammatory cytokine tumor necrosis factor α (TNFα) in denervation-induced plasticity of inhibitory neurotransmission. In line with our previous findings in vitro, an entorhinal cortex lesion triggered a compensatory increase in the excitatory synaptic strength of partially denervated dentate granule cells. Inhibitory synaptic strength was not changed 3 days after the lesion. These functional changes were accompanied by a recruitment of microglia in the denervated hippocampus, and experiments in tissue cultures prepared from TNF-reporter mice [C57BL/6-Tg(TNFa-eGFP)] showed increased TNFα expression in the denervated zone. However, inhibitory neurotransmission was not affected by scavenging TNFα with a soluble TNF receptor. In turn, a decrease in inhibition, i.e., decreased frequencies of miniature inhibitory postsynaptic currents, was observed in denervated dentate granule cells of microglia-depleted tissue cultures. We conclude from these results that activated microglia maintain the inhibition of denervated dentate granule cells and that TNFα is not required for the maintenance of inhibition after denervation.

Denervated mouse dentate granule cells adjust their excitatory but not inhibitory synapses following in vitro entorhinal cortex lesion.

Neurons adjust their synaptic strength in a homeostatic manner following changes in network activity and connectivity. While this form of plasticity has been studied in detail for excitatory synapses, homeostatic plasticity of inhibitory synapses remains not well-understood. In the present study, we employed entorhinal cortex lesion (ECL) of organotypic entorhino-hippocampal tissue cultures to test for homeostatic changes in GABAergic neurotransmission onto partially denervated dentate granule cells. Using single and paired whole-cell patch-clamp recordings, as well as immunostainings for synaptic markers, we find that excitatory synaptic strength is robustly increased 3 days post lesion (dpl), whereas GABAergic neurotransmission is not changed after denervation. Even under conditions of pharmacological inhibition of glutamatergic neurotransmission, which prevents neurons to compensate for the loss of input via excitatory synaptic scaling, down-scaling of GABAergic synapses does not emerge 3 days after denervation. We conclude that granule cells maintain structural and functional properties of GABAergic synapses even in the face of substantial changes in network connectivity. Hence, alterations in inhibitory neurotransmission, as seen in pathological brain states, may not simply reflect a homeostatic response to disconnection.