Activation of serotonin receptors promotes microglial injury-induced motility but attenuates phagocytic activity

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Abstract

Microglia, the brain immune cell, express several neurotransmitter receptors which modulate microglial functions. In this project we studied the impact of serotonin receptor activation on distinct microglial properties as serotonin deficiency not only has been linked to a number of psychiatric disease like depression and anxiety but may also permeate from the periphery through blood–brain barrier openings seen in neurodegenerative disease. First, we tested the impact of serotonin on the microglial response to an insult caused by a laser lesion in the cortex of acute slices from Cx3Cr1-GFP-/+ mice. In the presence of serotonin the microglial processes moved more rapidly towards the laser lesion which is considered to be a chemotactic response to ATP. Similarly, the chemotactic response of cultured microglia to ATP was also enhanced by serotonin. Quantification of phagocytic activity by determining the uptake of microspheres showed that the amoeboid microglia in slices from early postnatal animals or microglia in culture respond to serotonin application with a decreased phagocytic activity whereas we could not detect any significant change in ramified microglia in situ. The presence of microglial serotonin receptors was confirmed by patch-clamp experiments in culture and amoeboid microglia and by qPCR analysis of RNA isolated from primary cultured and acutely isolated adult microglia. These data suggest that microglia express functional serotonin receptors linked to distinct microglial properties.

Highlight

► This work shows that microglial cells express functional serotonin receptors linked to modulation of migration and phagocytic activity in vitro and in situ.

Introduction

Microglia are considered as the primary immune competent cell of the central nervous system. They respond to any type of brain injury by a process termed microglial activation. Generally, two types of signals which control microglial activation have been designated, the ‘on’ signals, which newly appear or increase in response to injury and the ‘off’ signals, which keep microglia in their resting state and antagonize activation (Biber et al., 2007). Neurotransmitters can act as such ‘off’ signals. Acetylcholine, norepinephrine and dopamine act as anti-inflammatory agents for microglia affecting LPS-induced cytokine and nitric oxide release (Pocock and Kettenmann, 2007). Indeed, a number of recent studies have substantiated that microglial cells can express diverse neurotransmitter and hormone receptors which control microglial functions including phagocytosis and migration (Kettenmann et al., 2011). Among the prominent transmitter systems expressed by microglia are purinergic receptors which sense ATP. Microglia express a variety of ionotropic P2X and metabotropic P2Y receptors which control migration, process movement, phagocytosis and cytokine release (Inoue, 2008). ATP is released under physiological conditions as a neurotransmitter or cotransmitter by neurons and also by astrocytes (Burnstock, 2009, Butt, 2011). Under pathological conditions ATP is released in high amounts by damaged cells. Microglial cells also express functional receptors for epinephrine, dopamine, glutamate and GABA whose activation can modulate cytokine release like TNF-α, IL-6 and IL-12p40 (Pocock and Kettenmann, 2007).

In the present study, we investigated the impact of serotonin receptor activation on microglial function. Serotonin is an important neurotransmitter of the monoamine family which is involved in a variety of physiological and behavioral functions. Dysregulation of the serotonergic system can lead to many psychiatric and neurological disorders (Green, 2006, Risch and Nemeroff, 1992, Temel et al., 2007). The principal source of serotonin in the brain is the neurons of the Raphe nuclei projecting to nearly all brain regions including cortex and brain stem. Serotonin receptors are, however, not restricted to the central nervous system, but are also expressed by immune cells such as T-cells, macrophages and dendritic cells. Here, serotonin acts as an anti-inflammatory component on peripheral immune cells by influencing cytokine release (Muller et al., 2009), maturation to dendritic cells and apoptosis induction (Katoh et al., 2006).

Receptors for serotonin have been classified in distinct types, 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7 which have various subtypes (for review, see (Filip and Bader, 2009). So far, serotonin receptors have not been identified on microglia. Only in a cell line with microglial properties, 5-HT7 was found to be expressed (Mahe et al., 2005).

In the healthy brain microglial cells are distributed evenly and recent evidence indicates that they continuously survey their environment by rapid movement of their processes (Nimmerjahn et al., 2005). In case of local injury, the processes rapidly (within less than one hour) move to the lesion site. Experimentally, acute injury can be induced by focal laser light projected on cortical tissue. Microglial process extension was suggested to reduce local damage. This response is diminished by volume sensitive chloride channel blockers like NPPB, tamoxifen and DIDS (Hines et al., 2009). Another rapid response is the uptake of damaged material by phagocytosis. Both types of events are controlled by ATP signaling through distinct purinergic receptors (Davalos et al., 2005, Koizumi et al., 2007).

In the present study we addressed the question whether serotonin affects the microglial process extension towards a lesion site or phagocytosis. Therefore we performed two-photon microscopy on acute brain slices taken from Cx3Cr1-GFP-/+ mice and compared microglial response towards cortical laser lesion in the presence or absence of serotonin. Additionally we characterized the phagocytic activity in different microglia preparations. The expression of functional serotonin receptors was verified by electrophysiological recordings and quantitative PCR.

Section snippets

Microglial cell culture

Microglial cultures were prepared from cerebral cortex of newborn C57BL/6 mice as described previously (Prinz et al., 1999). In brief, cortical tissue of newborn mice was trypsinized (Biochrom, Berlin, Germany) and dissociated with a fire-polished pipette. After cultivation of mixed glial cells for 9–12 days, microglial cells were separated from the underlying astrocytic layer by gentle shaking of the flasks for one hour at 37 °C in a shaker-incubator (100 rpm). Cultures usually contained >95%

Serotonin increases process motility in response to laser lesion

Microglial cells are constantly moving their processes in the intact brain. In response to an injury, such as a laser induced lesion, microglia respond with a targeted movement towards the lesion site (Nimmerjahn et al., 2005). To test whether serotonin can modulate this property, we studied microglial process motility in response to a laser lesion by two-photon imaging in acute brain slices. We used heterozygous fractalkine receptor-deficient mice which express EGFP under the Cx3Cr1 promoter

Discussion

Our findings indicate that neonatal, amoeboid as well as adult microglia express functional serotonin receptors consistent in the expression of the 5-HT2 subgroup. Activation of these receptors enhanced process motility in adult microglia in acute brain slices but decreased phagocytic activity as well as the inward current in neonatal and amoeboid microglia.

It was shown that microglia directly contact neuronal synapses for minutes before process retraction (Wake et al., 2009). In ischemic

Conflict of interest statement

All authors declare that there are no conflicts of interest.

Acknowledgment

This work was supported by Deutsche Forschungsgemeinschaft (SFB-TRR43, research school GRK 1258). We thank Prof. Michael Bader and Daniel Beis from the Max Delbrueck Center for Molecular Medicine in Berlin for discussion and material supply. Further we thank the microscopy core facility at the Max Delbrueck Center for Molecular Medicine for technical assistance.

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