Characterization of human P2X7 receptor function on microglia by longitudinal in vivo two-photon microscopy and behavioural phenotyping in a conditional, humanized mouse line

Mücke-Heim, Iven-Alex von (2026): Characterization of human P2X7 receptor function on microglia by longitudinal in vivo two-photon microscopy and behavioural phenotyping in a conditional, humanized mouse line. Dissertation, LMU München: Faculty of Medicine

Abstract

Depression resembles burden to individuals and healthcare systems alike. As an interface linking environmental psychosocial stress to immune activation, the transmembrane P2X7 receptor (P2X7R) been repeatably discussed to play a role in depression genesis. P2X7R is an ionotropic adenosine triphosphate (ATP) receptor located primarily on immune cells. In microglia and cerebral macrophages (M/Ms), the resident immune cells of the brain, P2X7R expression is high, particularly compared to expression levels of other P2X and P2Y receptors. Upon ATP binding, the P2X7R acts as selective cation channel allowing rapid influx of Na+ and Ca2+ into the respective cell, along with K+ efflux. These changes in ion concentrations activate the NLRP3 inflammasome among others intracellular pathways, which primarily and jointly cause proinflammatory cytokine release (foremost: IL-1-β and IL-18), followed by the production of inflammatory molecules like cyclooxygenase 2, TNFα, IL-6, prostaglandin E2, phospholipase A2, and reactive nitrogen and oxygen species. Thus, P2X7R is considered one of the most powerful activators of the NLRP3 cascade and IL-1β production in the brain. Still, little in vivo evidence exists on the select role of the human P2X7R (hP2X7R) on M/M in the context of chronic stress and its etiopathological transition to depression. The aim of this thesis was thus to study the impact of psychosocial stress and sterile immune trauma on behaviour and in vivo cell morphology using a humanized (hP2X7R), conditional (Tamoxifen-dependent knockout (KO)) and cell-specific (M/Ms) mouse model. Here, the focus was to clarify our current understanding of the link between chronic psychosocial stress and the M/Ms morphological response as an indirect function of hP2X7R activity and expression, respectively, using comprehensive behavioural profiling tailored to depressive- and anxiety-like features and in vivo TPM for single-cells morphometry. Prior to data collection, the genetic, surgical, and imaging aspects of the mouse model of choice were extensively validated using PCR of different tissues and in vivo magnetic resonance imaging (MRI) incl. time of flight angiography (TOF MRA). In addition, technical suitability of different optical implants tailored to access frontolimbic or limbic regions, namely (i) cranial windows, (ii) subcortical cannulas, and (iii) optical micro-prisms was experimentally evaluated. The behavioural assessments were performed without (baseline) and following stress (= 21 days chronic social defeat stress (CSDS); 5 days i.p. lipopolysaccharides (LPS)). Longitudinal in vivo two photon microscopy (TPM) via micro-prisms implanted in the murine prefrontal cortex was obtained over 6 weeks before, during, and after CSDS. In the validation experiments, the genetic construct of the hP2X7R mouse line proved ideal for the planned experiments because red fluorescence protein (RFP = tdTomato) expression was constitutive in M/Ms, while hP2X7R KO remained TAM-dependent. Regarding the model’s surgical aspects, micro-prisms implanted into the ventromedial prefrontal cortex and adjacent frontolimbic areas (VMPFC/FLA) proved most suitable for longitudinal in vivo TPM. In vivo Gadolinium-enhanced MRI demonstrated correct localisation of the micro-prisms in the VMPFC/FLA accompanied by minimal and only local foreign-body-reaction and scarring. TOF MRA confirmed no relevant vessel obstruction, particularly of the superior sagittal sinus. In the baseline behavioural experiments, hP2X7R wildtype animals (WT) demonstrated less anxiety-like features, increased locomotion, and superior short-term memory compared to KO controls. In the LPS experiments, though a clear genotype-dependent effect on body weight over time was observed in both sexes and sex along with stress regularly impacted behaviours, no clear behavioural profile difference was seen between genotypes. In the CSDS experiments, which evoked clear post mortem adrenal weight increase in both sexes, primarily sex- and stress-related changes were found thought most of the behavioural tests. In select assessments, however, genotype-related effects were seen. In the open field test, the elevated plus maze, and dark-light box discrete genotype and stress-genotype interaction effects were detected regarding locomotion and anxiety-like features. In the social aversion test, weak genotype, sex-genotype and sex-stress-genotype interaction effects were revealed. In the in vivo TPM experiments at baseline (42 days imaging, no stress), clear morphological differences were observed: female WT M/Ms occupied less area, were smaller, had less end- and branchpoints, and possessed shorter average, maximum, and minimum branch length than M/Ms in male WTs. Even though the latter were the most consistent pattern across the baseline cell morphology parameters (= 7 / 8 morphology criteria differed significantly), KO males and females were also distinguishable yet in a much smaller subset of 3 / 8 morphology criteria. For the in vivo TPM before, during, and after CSDS over a total of 42 days, female WT M/Ms displayed a significant increase in territory, volume, and ramification peaking around day 21. For KOs, a somewhat comparable pattern was observed, with endpoint and branch points, as well as average and minimum branch lengths, increasing over time and reaching their maximum at day 21. Robust Z scores calculated for KO females referenced to same-day WTs found statistically significant differences for cell territory, volume, ramification, average and maximum branch length over time. These differences were most prominent at d21 and d42. For WT and KO males, aside from variations in ramification lacking conclusive directionality, no definable pattern emerged before, during, and after CSDS over a total of 42 days. Taken together, the presented findings confirm a baseline hP2X7R-dependent behavioural phenotype, which conceptually aligns with the baseline in vivo TPM results concerning M/Ms morphology. Stress experiments (LPS, CSDS) produced limited evidence that might suggest an impact of microglial hP2X7R on behaviour. Still, longitudinal in vivo TPM revealed notable sex- and genotype-dependent changes in microglial morphology. Future studies are needed to further clarify the functional and molecular pathways linking microglial hP2X7R expression to genotype-dependent behaviours at baseline as well as to genotype- and sex-dependent M/Ms morphology changes at baseline as well as downstream of psychosocial stress.

• Item Type: Theses (Dissertation, LMU Munich)
• Subjects: 600 Technology, Medicine; 600 Technology, Medicine > 610 Medical sciences and medicine
• Faculties: Faculty of Medicine
• Language: English
• Date of oral examination: 19. March 2026
• 1. Referee: Binder, Elisabeth
• Signature of the printed copy: 0700/UMD 22744