Lifelong absence of microglia alters hippocampal glutamatergic networks but not synapse and spine density.

Microglia sculpt developing neural circuits by eliminating excess synapses in a process called synaptic pruning, by removing apoptotic neurons, and by promoting neuronal survival. To elucidate the role of microglia during embryonic and postnatal brain development, we used a mouse model deficient in microglia throughout life by deletion of the fms-intronic regulatory element (FIRE) in the Csf1r locus. Surprisingly, young adult Csf1rΔFIRE/ΔFIRE mice display no changes in excitatory and inhibitory synapse number and spine density of CA1 hippocampal neurons compared with Csf1r+/+ littermates. However, CA1 neurons are less excitable, receive less CA3 excitatory input and show altered synaptic properties, but this does not affect novel object recognition. Cytokine profiling indicates an anti-inflammatory state along with increases in ApoE levels and reactive astrocytes containing synaptic markers in Csf1rΔFIRE/ΔFIRE mice. Notably, these changes in Csf1rΔFIRE/ΔFIRE mice closely resemble the effects of acute microglial depletion in adult mice after normal development. Our findings suggest that microglia are not mandatory for synaptic pruning, and that in their absence pruning can be achieved by other mechanisms.

Differential contribution of THIK-1 K+ channels and P2X7 receptors to ATP-mediated neuroinflammation by human microglia.

Neuroinflammation is highly influenced by microglia, particularly through activation of the NLRP3 inflammasome and subsequent release of IL-1ß. Extracellular ATP is a strong activator of NLRP3 by inducing K+ efflux as a key signaling event, suggesting that K+-permeable ion channels could have high therapeutic potential. In microglia, these include ATP-gated THIK-1 K+ channels and P2X7 receptors, but their interactions and potential therapeutic role in the human brain are unknown. Using a novel specific inhibitor of THIK-1 in combination with patch-clamp electrophysiology in slices of human neocortex, we found that THIK-1 generated the main tonic K+ conductance in microglia that sets the resting membrane potential. Extracellular ATP stimulated K+ efflux in a concentration-dependent manner only via P2X7 and metabotropic potentiation of THIK-1. We further demonstrated that activation of P2X7 was mandatory for ATP-evoked IL-1ß release, which was strongly suppressed by blocking THIK-1. Surprisingly, THIK-1 contributed only marginally to the total K+ conductance in the presence of ATP, which was dominated by P2X7. This suggests a previously unknown, K+-independent mechanism of THIK-1 for NLRP3 activation. Nuclear sequencing revealed almost selective expression of THIK-1 in human brain microglia, while P2X7 had a much broader expression. Thus, inhibition of THIK-1 could be an effective and, in contrast to P2X7, microglia-specific therapeutic strategy to contain neuroinflammation.

Characterisation of C101248: A novel selective THIK-1 channel inhibitor for the modulation of microglial NLRP3-inflammasome.

Neuroinflammation, specifically the NLRP3 inflammasome cascade, is a common underlying pathological feature of many neurodegenerative diseases. Evidence suggests that NLRP3 activation involves changes in intracellular K+. Nuclear Enriched Transcript Sort Sequencing (NETSseq), which allows for deep sequencing of purified cell types from human post-mortem brain tissue, demonstrated a highly specific expression of the tandem pore domain halothane-inhibited K+ channel 1 (THIK-1) in microglia compared to other glial and neuronal cell types in the human brain. NETSseq also showed a significant increase of THIK-1 in microglia isolated from cortical regions of brains with Alzheimer's disease (AD) relative to control donors. Herein, we report the discovery and pharmacological characterisation of C101248, the first selective small-molecule inhibitor of THIK-1. C101248 showed a concentration-dependent inhibition of both mouse and human THIK-1 (IC50: ∼50 nM) and was inactive against K2P family members TREK-1 and TWIK-2, and Kv2.1. Whole-cell patch-clamp recordings of microglia from mouse hippocampal slices showed that C101248 potently blocked both tonic and ATP-evoked THIK-1 K+ currents. Notably, C101248 had no effect on other constitutively active resting conductance in slices from THIK-1-depleted mice. In isolated microglia, C101248 prevented NLRP3-dependent release of IL-1ß, an effect not seen in THIK-1-depleted microglia. In conclusion, we demonstrated that inhibiting THIK-1 (a microglia specific gene that is upregulated in brains from donors with AD) using a novel selective modulator attenuates the NLRP3-dependent release of IL-1ß from microglia, which suggests that this channel may be a potential therapeutic target for the modulation of neuroinflammation in AD.

Promoting Family Integrated Early Childhood Development During the First 1000 Days in Urban Slums of Delhi: Feasibility and Early Implementation Experience.

The first 1000 days are critical for human development. Children residing in urban slums are at higher risk of suboptimal development. This article reports the feasibility and early implementation experiences of child stimulation directed counselling integrated with nutrition and health services by community functionaries in Delhi slums. This implementation research with cohort study design has two groups. The intervention group includes: pregnant women; infants and children in second year. The comparison group (without intervention) includes: recently delivered women, children aged 13-25 months. The feedback from the community functionaries and parents of children were also obtained. 44 videos with Hindi voiceover, demonstration of tools/toys/props, and 9 calendars depicting age-appropriate child stimulation activities were prepared and packaged with health and nutrition services for delivery by the frontline functionaries. These functionaries are coaching the families on child stimulation using these audio-visual messages using Tablets and demonstration tools/toys/props twice every three months. Monitoring and feedback data on service delivery are being collected. The data from the intervention cohort shall be compared with that from the comparison group to document the impact. From 63 anganwadi centres, 130 pregnant women, 142 infants and 136 children have been recruited for the intervention. 82.9% of the families received the intervention within 2 weeks and average time taken was 15 minutes. The initial feedback from the community functionaries and parents are very encouraging. The integrated child development package is acceptable and feasible for implementation through community functionaries and possible integration into the existing programs at scale.

P2Y13 receptors regulate microglial morphology, surveillance, and resting levels of interleukin 1ß release.

Microglia sense their environment using an array of membrane receptors. While P2Y12 receptors are known to play a key role in targeting directed motility of microglial processes to sites of damage where ATP/ADP is released, little is known about the role of P2Y13 , which transcriptome data suggest is the second most expressed neurotransmitter receptor in microglia. We show that, in patch-clamp recordings in acute brain slices from mice lacking P2Y13 receptors, the THIK-1 K+ current density evoked by ADP activating P2Y12 receptors was increased by ~50%. This increase suggested that the P2Y12 -dependent chemotaxis response should be potentiated; however, the time needed for P2Y12 -mediated convergence of microglial processes onto an ADP-filled pipette or to a laser ablation was longer in the P2Y13 KO. Anatomical analysis showed that the density of microglia was unchanged, but that they were less ramified with a shorter process length in the P2Y13 KO. Thus, chemotactic processes had to grow further and so arrived later at the target, and brain surveillance was reduced by ~30% in the knock-out. Blocking P2Y12 receptors in brain slices from P2Y13 KO mice did not affect surveillance, demonstrating that tonic activation of these high-affinity receptors is not needed for surveillance. Strikingly, baseline interleukin-1ß release was increased fivefold while release evoked by LPS and ATP was not affected in the P2Y13 KO, and microglia in intact P2Y13 KO brains were not detectably activated. Thus, P2Y13 receptors play a role different from that of their close relative P2Y12 in regulating microglial morphology and function.

Introducing a complex health innovation--primary health care reforms in Estonia (multimethods evaluation).

All post-Soviet countries are trying to reform their primary health care (PHC) systems. The success to date has been uneven. We evaluated PHC reforms in Estonia, using multimethods evaluation: comprising retrospective analysis of routine health service data from Estonian Health Insurance Fund and health-related surveys; documentary analysis of policy reports, laws and regulations; key informant interviews. We analysed changes in organisational structure, regulations, financing and service provision in Estonian PHC system as well as key informant perceptions on factors influencing introduction of reforms. Estonia has successfully implemented and scaled-up multifaceted PHC reforms, including new organisational structures, user choice of family physicians (FPs), new payment methods, specialist training for family medicine, service contracts for FPs, broadened scope of services and evidence-based guidelines. These changes have been institutionalised. PHC effectiveness has been enhanced, as evidenced by improved management of key chronic conditions by FPs in PHC setting and reduced hospital admissions for these conditions. Introduction of PHC reforms - a complex innovation - was enhanced by strong leadership, good co-ordination between policy and operational level, practical approach to implementation emphasizing simplicity of interventions to be easily understood by potential adopters, an encircling strategy to roll-out which avoided direct confrontations with narrow specialists and opposing stakeholders in capital Tallinn, careful change-management strategy to avoid health reforms being politicized too early in the process, and early investment in training to establish a critical mass of health professionals to enable rapid operationalisation of policies. Most importantly, a multifaceted and coordinated approach to reform - with changes in laws; organisational restructuring; modifications to financing and provider payment systems; creation of incentives to enhance service innovations; investment in human resource development - was critical to the reform success.