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Systemic inflammation induced by lipopolysaccharide reduces the number of hippocampal GABA-ergic synapses and leads to memory impairment (animal study)

Neuroinflammation is a complex combination of acute and chronic responses of cells within the central nervous system (CNS) initiated by various stimuli, including infection, traumatic brain injury, toxic metabolites, and autoimmunity. The primary responders are microglia, the resident macrophages in the brain that play critical roles in the onset and progression of neuroinflammation. Microglia are rapidly activated and undergo cellular and molecular changes. In this study, Korean researchers used several methods to investigate the temporal sequence of brain pathology associated with neuroinflammation in the hyppocampus, from microglial activation to synaptic dysfunction and memory-related behavioral changes. 

The authors pointed out that they have shown before that two daily intraperitoneal injections of 0.5 mg/kg lipopolysaccharide (LPS) activated microglia throughout the brain, including the hippocampus. In addition, a recent study on the consequences of nasal SARS-CoV-2 inoculation on the mouse brain has shown that intraperitoneal administration of the LPS markedly reduced the expression of calbindin, a marker of γ-aminobutyric acid (GABA)-ergic neurons.  https://discovermednews.com/s1-protein-causes-brain-inflammation-and-decreases-the-acetylcholine-levels/

Synapses are fundamental information-processing units of neuronal circuits. They form the basis for all brain functions by controlling the excitation-to-inhibition balance. Various cellular and molecular mechanisms that underly synapse formations have been identified. Microglia shape the number, morphology and/or connections of synapses under neuroinflammatory conditions. It was initially reported that microglia contribute to the reconstruction of neuronal circuits by eliminating synapses, a process mediated by phagocytosis of excess neuronal synapses and newborn neurons during brain development. However, according to recent data, microglia are actively involved in synapse dynamics in healthy brains. A reduction in hippocampal GABA-ergic synapses, induced by an altered excitation-to-inhibition balance, results in cognitive and memory impairments in rodents. 

             Multiple physiological roles of microglia in the CNS. Original figure from the article of Wang H et al. Journal of Neuroinflammation, 2022; 19(1). DOI:10.1186/s12974-022-02492-0

About the study

The scientists used several methods to examine the temporal sequences of brain pathology associated with neuroinflammation in the hippocampus, like microglial activation, synaptic dysfunction, and memory-related behavioral changes. They focused on changes in hippocampal CA1 regions, known for their role in memory.

At the beginning of the study, to induce systemic inflammation, the researchers injected intraperitoneally 0.5 mg/kg LPS (from Escherichia coli) to eight-week-old C57BL/6J mice once a day for two days. Behavior in the acute phase of the disease, locomotor activity, and body weight were monitored daily until the mice were back in the normal range. The open-field test that assesses general locomotor activity, the novel object-recognition test that assesses memory deficit, and the light and dark transition test that assesses anxiety-related behavior were used to investigate behavioral changes related to memory.

The hippocampal slices were analyzed for the number of microglia positive for the microglial marker, Iba-1 (ionized calcium-binding adapter molecule-1), and the expression level of CD68 (a lysosomal protein, frequently used as a marker for actively phagocytic microglia) at 3, 4, 6, and 8 days post-injection (dpi). The excitatory and inhibitory synaptic puncta in various layers of the hippocampal CA1 region were analyzed to assess whether LPS-induced microglial activation contributes to synaptic dynamics. After a novel object-recognition test, the whole-cell electrophysiological recording of spontaneous and evoked inhibitory postsynaptic currents (sIPSCs and eIPSCs) was conducted in hippocampal slices from LPS-treated and control mice at 6 dpi.

Results

After intraperitoneal injection of LPS, mice lost about 20% of their body weight, but they reached 90% of their body weight after four days. 

The results of the overall locomotor activity measured by the open-field test and anxiety-related behavior assessed by the light and dark transition test did not differ between LPS-treated and control mice at 6 dpi. However, the results of the novel object-recognition test demonstrated a severe memory deficit in LPS-treated mice compared with control mice at 6 dpi, indicating that memory impairment lasted for 6 days.

Immunohistochemical analysis of the microglial marker Iba-1, and the expression of CD68, a marker for actively phagocytizing microglia, showed a transient activation of hippocampal microglia at 3 dpi in the LPS-injected mice, which was absent at 4–8 dpi. The morphological changes of microglia from ramified to a bushy form were also observed at 3 dpi, but they returned to normal at 8 dpi. These findings demonstrate a significant microglial activation in the hippocampal CA1 regions at 3 days after LPS injection, with changes in cellular morphology, microglial density, and expression of phagocytic markers. However, all of these parameters gradually returned to normal at 8 dpi.

A significant decrease in density and the average size of VGAT (vesicular GABA transporter; inhibitory presynaptic marker) puncta were found in hippocampal CA1 regions at 4-6 dpi by quantitative immunofluorescence. Minor changes were seen in the density and the average area of GABAARγ2 (inhibitory postsynaptic marker) puncta. Similarly, a semiquantitative immunohistochemical analysis with antibodies against VGAT (GABA inhibitory presynaptic marker) and gephyrin (scaffold responsible for organizing the inhibitory postsynaptic density) demonstrated the significant reduction in GABAergic synaptic components in all examined layers of hippocampal CA1 regions at 6 dpi in LPS-treated mice.

However, the density and average size of VGLUT1 (vesicular glutamate transporter 1; excitatory presynaptic marker) and PSD-95 (excitatory postsynaptic marker) puncta in all examined layers of hippocampal CA1 regions in LPS-treated mice were unchanged.

These results show that GABAergic synaptic deficits were detected 4 days after LPS treatment, after a transient hippocampal microglial activation. The persistence of GABAergic synaptic deficits up to 8 dpi suggests a long-lasting synaptic alteration. The persistent structural alteration of GABAergic synapses was parallel with the memory impairment seen at 6 dpi after the LPS treatment.

The whole-cell electrophysiological recording of spontaneous and evoked inhibitory postsynaptic currents (sIPSCs and eIPSCs) showed no difference between LPS-treated mice and control mice in the amplitude or frequency of sIPSCs recorded from CA1 hippocampal neurons.

Conclusion

This study has shown that microglial activation in the hippocampus after systemic LPS administration preceded a GABA-ergic synaptic deficit. Furthermore, the alterations in the structure and function of GABA-ergic synapses, induced by neuroinflammation, resulted in a memory deficit in experimental animals.

According to the authors, the loss of GABA-ergic synapse in LPS-treated mice could be caused by microglia-mediated synapse elimination rather than by pro/anti-inflammatory cytokines released from activated microglia.

This research provides mechanistic insights that may explain the persistent cognitive deficit observed in individuals with neuroinflammation. The authors focused on changes in the hippocampus, but they emphasized that future investigations should examine the temporal sequence of biological events in other brain regions. Furthermore, future research is needed to explore why LPS-induced microglial activation specifically affects GABA-ergic synapses.

The article was published in Scientific Reports (Nature)

Journal Reference

Jung H, Lee D, You H et al. LPS induces microglial activation and GABAergic synaptic deficits in the hippocampus accompanied by prolonged cognitive impairment. Scientific Reports (2023) 13:6547.  https://doi.org/10.1038/s41598-023-32798-9

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