Inhibition of colony-stimulating factor 1 receptor improves synaptic plasticity and cognitive performance in aged mice

doi:10.21203/rs.3.rs-4859575/v1

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Inhibition of colony-stimulating factor 1 receptor improves synaptic plasticity and cognitive performance in aged mice

https://doi.org/10.21203/rs.3.rs-4859575/v1

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Depletion of microglia in the aged mouse brain is known to improve cognitive functions. However, even temporal ablation of microglia puts the brain at a high risk of infection. Hence, in the present work, we studied if the partial reduction of microglia with PLX3397 (pexidartinib), an inhibitor of the colony-stimulating factor 1 receptor (CSF1R), could bring similar benefits as reported for microglia ablation. Aged (two-year-old) mice were treated with PLX3397 for 28 days, which reduced microglia numbers in the hippocampus to the levels seen in young mice and resulted in layer-specific ablation in the expression of microglial complement protein C1q mediating synaptic remodeling. This treatment boosted long-term potentiation in the CA1 region and improved performance in the hippocampus-dependent novel object location recognition task. Although PLX3397 treatment did not alter the number or total intensity of Wisteria floribundaagglutinin-positive perineuronal nets (PNN) in the CA1 region of the hippocampus, it changed the fine structure of PNNs and elevated the expression of perisynaptic proteoglycan brevican, presynaptic vGluT1 and postsynaptic PSD95 proteins at the excitatory synapses in the CA1 stratum radiatum. Thus, targeting the CSF1R may provide a safe and efficient strategy to boost synaptic and cognitive functions in the aged brain.

Aging

Glia

Extracellular matrix

Synapses

Parvalbumin

Brevican

Perineuronal nets

perisynaptic ECM

Brain aging and its underlying molecular and cellular processes have been gaining continuous attention over the last few decades. In various brain regions, aging is accompanied by a decline in neural resources resulting in impairment of learning and memory. In the hippocampus, aging is associated with a loss of neurons [1], impaired neurogenesis in the dentate gyrus [2, 3] as well as alterations in dendrites and synaptic transmission [4–6][7]. Functionally, it is connected to cognitive decline [8, 9], as exemplified by a recent study of 20-month-old mice showing difficulties in different behavioral paradigms, including object recognition and spatial navigation tasks linked to the hippocampal function [10].

An important characteristic of brain aging is the increasing low-grade neuroinflammation, the so-called “inflammaging” [11]. Activation of the cGAS-STING signaling by cytosolic DNA released from perturbed mitochondria in old microglia is a critical driver of microglia activation during aging [12]. It is manifested in a shift in the cytokine balance towards higher levels of pro- and reduction in anti-inflammatory cytokines and in impaired cognitive flexibility and stress responses in humans and animals [13]. Aging also changes the morphology of microglia, the innate immune cells of the brain, towards a more amoeboid phenotype associated with activation upon injury or inflammation. The cell body increases in size while the normally long and versatile processes shorten and thicken [14]. Secondly, the motility and with it the ability to react to injury and phagocytize cellular debris are decreased. Finally, they lose their ability to proliferate. This cellular senescence is typical to a variety of aging brain cells [15] and leads to increased iron storage and ferritin overexpression as well as to the increased release of neurotoxic molecules [16].

Pharmacological depletion of microglia CSF1R inhibitors has gained attention, as this approach allows one to rather specifically study the effects of microglia reduction at any time point within the lifespan of mice [17]. It has been shown that microglia repopulation after withdrawal of the CSF1R inhibitor resulted in improved spatial memory function in the Morris water maze in aged animals and a rescue of age-related deficits in the long-term potentiation following theta-burst stimulation to Schaffer collateral-commissural projections [18].

Microglia secrete the extracellular matrix (ECM) molecules as well as extracellular proteases and these mechanisms are altered with age and in age-associated pathologies. Functionally, attenuation of ECM can either promote or inhibit synaptic plasticity [19]. For example, mice deficient in the ECM glycoprotein tenascin-R, chondroitin sulfate proteoglycans (CSPGs) brevican or versican, or treated with chondroitinase ABC to digest chondroitin sulfates, all display impaired long-term potentiation in the CA1 [20, 21]. On the other hand, a study by Vegh and colleagues has shown that ECM proteins, including brevican, are progressively upregulated with aging, both on genetic and proteomic levels, coinciding with an impairment of hippocampus-dependent spatial memory [22]. Furthermore, attenuation of ECM with chondroitinase ABC reversed the age-dependent cognitive deficits in motor learning [23]. This indicates that the optimal levels of ECM support the synaptic plasticity, but excessive loss or accumulation of ECM results in impaired LTP and learning.

Several research studies undertaken over the past years have shown an interaction between microglia and the brain ECM in young and aged mice [24, 25]. For example, a gene expression analysis showed that long-term depletion of microglia enhances the mRNA levels of brevican in the hippocampus of wild-type mice [26] and we recently confirmed this increase at a protein level using immunohistochemistry in young adult mice [27]. Microglia remodeling of ECM enhances synaptic plasticity by engulfing and remodeling ECM, especially at synapses, and this is essential for long-term memory consolidation [25]. However, this concept of microglia and ECM interaction has not been fully explored in terms of age-dependency, although both microglia senescence and hippocampal perisynaptic ECM were reported to increase with age[22, 28].

Here, we aimed to study whether inhibition of CSF1R could reverse age-related deficits in the mouse brain and what mechanisms might be underlying the rescue. Using the CSF1R-inhibitor PLX3397 we reduced microglia numbers to the level seen in young mice and showed increased long-term potentiation in the CA1 region and cognitive improvement in the novel object location recognition task. These changes were associated with brain-subregion-specific changes in the phagocytic cues produced and secreted by microglia.

2.1. Animals

Twenty male C57BL6J mice aged between 24 and 26 months were used in this study. During the time of observation, four animals (3 controls and 1 PLX-treated) died and were not part of further experiments. Animals were housed individually under standard conditions, including ad libitum access to food and water and a 12-hour light/12-hour dark cycle. All animal experiments were conducted in accordance with the ethical animal research standards defined by German law and the recommendations of the Ethical Committee on Animal Health and Care of the State of Saxony-Anhalt, Germany (license number: 42502-2-1346).

2.2. PLX3397 treatment

The animals were treated with the CSF1R inhibitor PLX3397 (pexidartinib; MedChemExpress). The drug was mixed into the animal chow at a nominal concentration of 290 mg/kg (ssniff Spezialdiäten GmbH; [17]. The mice received fresh control (product number 1534-70) or PLX-chow daily and the food intake was recorded. On average mice consumed 5g of chow per day with no significant difference between groups. The treatment started 28 days before the first experiment and mice were fed with PLX3397 until they were sacrificed 8 weeks later (see timeline, Fig. 1).

2.3. Behavior

2.3.1. Behavioral analysis

To test different forms of memory, we performed behavioral tests including the novel object location task (NOLT), the novel object recognition task (NORT), and the labyrinth task (dry maze). All behavioral tests were videorecorded and the animal’s performance was analyzed using Anymaze 4.99 (Stoelting Co., Wood Dale, IL, USA).

2.3.2. Open field

To study locomotor activity and anxiety-associated behavior the mice were put into an open field arena (50 x 50 x 30 cm) [29, 30] and studied using an overhead camera for 10 minutes. The arena was virtually divided into a central area (30 x 30 cm) and a peripheral area (the 10 cm area adjacent to the wall of the recording chamber). The overall physical and emotional state of mice was determined using the total distance moved and time spent in both the central and peripheral areas.

2.3.3. Novel object location test

The novel object location test was carried out as previously described[31] including the encoding phase and retrieval phases [32]. The encoding phase lasted 10 minutes, during which mice were allowed to explore two indistinguishable objects. During the 10-minute retrieval phase, which was carried out 24 hours later, one of the objects was repositioned to a novel position. To analyze the animal’s behavior, the exploration time for an object located at familiar (F) and novel (N) position, as well as thediscrimination ratio [(N-F)/(N + F)] x 100 % were used.

2.3.4. Novel object discrimination test

The novel object recognition test was also conducted in the open field arena and included the encoding and retrieval phases as described previously [31, 33]. Briefly, the animals were allowed to explore the arena with two identical objects for 10 minutes. After 24 hours one of the objects was replaced by a novel one and the exploration time was measured again. Exploration time for familiar (F) and novel (N) objects, as well as the discrimination ratio [(N-F)/(N + F)] x 100 %, wereused to evaluate animals’ recognition memory.

2.3.5. Labyrinth task (dry maze)

Quadrant-based labyrinth task was designed to measure spatial learning and memory as well as spatial navigational strategies in aged mice. This task was designed and implemented as an alternative to the Morris water maze (MWM) task that uses imaginary quadrants (software) compared to actual quadrants. Comparatively, this task is less stressful and capable of distinguishing between navigational strategies used by mice, namely egocentric (route) and allocentric (place) [34, 35].

Using the labyrinth set-up to study hippocampus-dependent spatial learning and memory in PLX-treated mice and aged-matched controls, the labyrinth was designed with 4 entry points and divided into quadrants of equal size but with different intra-quadrant configurations. In each quadrant configuration, zones were defined to quantify errors (with pink highlights). As outlined above, the asymmetrical-quadrant-based labyrinth was carried out with distal cues placed on curtains to enclose the maze and reward (water) placed in the 4th quadrant. Two entry paths (start1 and 2) were used for all daily training sessions from day 1(D1) to day 3 (D3) and a different path for probe tests (Probe). On D1, mice were allowed to explore the maze for 10min with reward. Subsequent training sessions, that is 2 trails per day for 2 days (D2 & D3), were ended upon entry into the reward zone. An inter-trial delay of 1 hour was implemented for each daily training session. With the probe test, mice entered the maze through the probe entry path, and the session was manually ended upon entry into the reward zone. Using an overhead camera with animal tracking software (Anymaze 4.99: Stoelting Co., Wood Dale, IL, USA), parameters such as the distance traveled, errors made and latency to the reward were recorded and quantified.

2.4. Electrophysiology

Four randomly chosen animals from each group were used for electrophysiological analysis. Animals were deeply anesthetized with isoflurane and perfused transcardially with ice-cold sucrose-based cutting solution containing (in mM): 230 sucrose, 2 KCl, 1 MgCl₂, 2 MgSO_4, 1.25 NaH₂PO₄, 26 NaHCO₃, 1 CaCl₂ and 10 D-Glucose, then decapitated. Hippocampi were isolated and placed on an agar block to cut transverse slices (350 µm thick) using a vibrating microtome (VT1200S, Leica). Slices were transferred and incubated in a submerged chamber with a solution containing (in mM): 113 NaCl, 2.38 KCl, 1.24 MgSO₄, 0.95 NaH₂PO₄, 24.9 NaHCO₃, 1 CaCl₂, 1.6 MgCl₂, 27.8 D-glucose for at least 2 hours at room temperature. Next, the slices were transferred to the recording chamber and were continuously perfused with the solution containing (in mM) 120 NaCl, 2.5 KCl, 1.5 MgCl₂, 1.25 NaH₂PO₄, 24 NaHCO₃, 2 CaCl₂ and 25 D-Glucose. All solutions were saturated with 95% O₂ and 5% CO₂ and the osmolality was adjusted to 300 ± 5 mOsm. Thin glass electrodes filled with ACSF were used for stimulation and recording of fEPSPs. The CA3-CA1 pathway was stimulated by two times of the theta-burst stimulation (TBS) trains to induce LTP. The stimulation intensity was determined based on the input-output curve and was set to give fEPSPs with a slope of ~ 30% and ~ 50% of the supramaximal fEPSP for the paired-pulse facilitation (PPF) and LTP induction, respectively. Single stimuli were repeated every 20 s for at least 10 min for baseline recording before and for 60 min after LTP induction. The paired-pulse ratio (PPR) was evaluated at different time intervals under the same conditions. All recordings were obtained at room temperature using an EPC-10 amplifier (HEKA Elektronik). The recordings were filtered at 1–3 kHz and digitized at 10–20 kHz.

2.5. Tissue preparation

For tissue preparation, animals were anesthetized with isoflurane and then transcardially perfused with PBS, followed by 4% paraformaldehyde (PFA). Brains were dissected and fixed in 4% PFA in phosphate-buffered saline (PBS) overnight at 4°C. Afterwards, the tissue was transferred to 30% sucrose in PBS for two nights before being frozen in methylbutane at -80°C. Using a cryostat at -20°C coronal brain sections of 40-µm thickness were prepared and stored in a cryoprotectant solution (ethylene glycol based; 30% ethylene glycol, 30% glycerol, 10% 0.2 M sodium phosphate buffer pH 7.4, in dH2O) at 4°C.

2.6. Immunohistochemistry

Immunohistochemistry (IHC) was performed based on previous protocols [27]. In short, all sections were washed 3 times with 120 mM phosphate buffer (PB), pH = 7.2 for 10 min at room temperature (RT), followed by permeabilization with 0.5% Triton X-100 (Sigma T9284) in phosphate buffer for 10 min and blocking in PB supplemented with 0.1% Triton X-100 and 5% normal goat serum (Gibco 16210‐064) for 1 hr at RT. For primary antibody delivery, the sections were incubated for 20h at 37°C. After washing in PB, sections were incubated with conjugated secondary antibodies for 90 min at RT. Labeled sections were washed again and then mounted and cover-slipped on glass slides using Fluoromount medium (Sigma; Cat no. F4680).

Table 1

Primary and secondary antibodies used for IHC
Primary reagents	Supplier, product number	Dilution
Chicken anti-PV	Synaptic Systems, 195006	1:500
Rabbit anti-Brevican	Seidenbecher et al., 1995; John et al., 2006	1:1000
Rabbit anti-vGAT	Synaptic Systems, 131002	1:500
Guinea pig anti-Iba1	Synaptic Systems, 234004	1:500
Rabbit anti-cFos	Synaptic Systems, 226003	1:500
Guinea pig anti-vGluT1	Synaptic Systems, 235304	1:1500
Chicken anti-GFAP	Sigma Aldrich, AB5541	1:400
Rabbit anti-Olig2	Sigma Aldrich, AB9610	1:200
Mouse anti-APC	Merck Millipore, OP80-100UG	1:500
Rabbit anti-C1q	Abcam, AB182451	1:500
Rat anti-CD68	AbD Serotec, MCA1957	1:500
Biotinylated WFA	Vector Laboratories, B-1355	1:1000
Secondary reagents	Supplier	Dilution
Alexa Fluor 405 goat Anti-Chicken IgG	Abcam, ab175675	1:500
Alexa Fluor 647 goat Anti-Chicken IgG	Invitrogen, A-21449	1:1000
Alexa Fluor 647 goat Anti-Rabbit IgG	Invitrogen, S32351	1:1000
Alexa Fluor 405 donkey Anti-Guinea pig IgG	Sigma Aldrich, SAB4600230	1:500
Alexa Fluor 647 goat Anti-Mouse IgG	Invitrogen, A21236	1:1000
Alexa Fluor 488 goat Anti-Guinea pig IgG	Invitrogen, A21044	1:1000
Alexa Fluor 546 goat Anti-Rabbit IgG	Invitrogen, A11035	1:500
Streptavidin Alexa Fluor 405 conjugated	Invitrogen, S32351	1:1000

2.7. Confocal microscopy and image processing

Images were obtained using a Zeiss confocal microscope (LSM 700). Brain regions were determined in accordance with the Allen mouse brain atlas. All images were then further processed and analyzed using the open software ImageJ (Fiji) version 1.54f.

2.8. Glial cells counting and microglia analysis

For the counting of microglia, astrocytes, and oligodendrocytes images were required at 20x objective for hippocampal CA1, CA2, CA3 and the dentate gyrus (DG) as well as the retrosplenial cortex (RSC). Glial cells were then counted manually using the Cell Counter Plugin in ImageJ. For analysis of the oligodendrocytes (OLGs) two markers were used, Olig2, which labels all OLGs, and APC, which specifically labels myelinating mature OLGs. Then we calculated the percentage of APC+/Olig2 + cells in one ROI.

2.9. Analysis of PNNs around PV + cells and perisynaptic ECM

Parvalbumin-positive cells (PV + cells) and their surrounding PNNs were counted using the Cell Counter in ImageJ. Biotinylated Wisteria floribunda agglutinin (WFA) was used to label the ECM of perineuronal nets (PNN). Furthermore, we used antibodies against various PNN components such as aggrecan, brevican, neurocan, versican, and phosphacan and measured the ECM mean intensity around PV + cells.

The single PNN units were analyzed as described previously [27, 36]. In short, this semi-automatic algorithm creates an automatic outline of a PNN hole and its surrounding ECM after manual selection. The results table then contains various key characteristics of PNN units such as size, density, and circularity in 2D as well as 3D. To ensure a seamless analysis of the fine structure, images were collected using 63x objective and a z-stack of 40 images with an interval of 0.1 um.

For analysis of perisynaptic ECM molecules, we first measured global ECM intensity inside randomly selected ROIs. Only the intensity of brevican was significantly changed, therefore we extended our analysis using a macro that specifically measured perisynaptic brevican around single presynaptic puncta.

2.10. Analysis of inflammatory markers

To study the effect of microglia reduction on neuroinflammation and phagocytosis, we used IHC to stain for IL1ß, C1q, CD68, and TREM2. For C1q and TREM2 global mean intensity was measured. Furthermore, we measured C1q inside microglia cell bodies. For CD68 and IL1ß we only analyzed particles inside microglia cell bodies by outlining the cell soma and measuring inside the ROIs.

2.11. Statistical analysis

All bar graphs are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software Inc., La Jolla, CA, USA) and XLSTAT software (Addinsoft Inc, N.Y., USA). Gaussian distributions of the data points were verified using the Kolmogorov-Smirnov, Shapiro-Wilk, or D’Agostino tests. Pairwise comparisons were performed using a two-tailed Student’s t-test for Gaussian distributions unless otherwise stated; in case the data did not follow the Gaussian law, the Mann-Whitney test was employed. Comparison of vGlut1 expression between groups was done using the one-tailed t-test for the alternative hypothesis that the intensity is higher in the PLX-treated than in control mice because our previous study revealed an increase in the intensity of vGluT1 puncta [27].

For PNN unit analysis a principal factor analysis was performed to detect the most informative parameters. Spearman correlation analysis to detect the correlation between measured parameters, Kolmogorov-Smirnov test for cumulative frequency distribution functions, and two-way analysis of variance (ANOVA) nested with animal ID to investigate the interdependencies and differences between variables. Statistical significance was set at p < 0.05.

3.1. PLX treatment reduces microglia density

Oral administration of the CSF1R inhibitor PLX3397 (pexidartineb) in two-year-old mice for 8 weeks led to a partial reduction in microglia in the hippocampus and retrosplenial cortex (RSC). Using two-way RM ANOVA, we observed that PLX treatment accounted for about 30% of the total variance observed (p < 0.0001), while the brain region identity (hippocampal CA1, DG, or RSC) accounted for 44% (p < 0.0001). We then performed post hoc tests to evaluate the PLX effects for each region and revealed a microglia reduction by 14% in the CA1 (Fig. 1B, C; 368.1 ± 11.2 vs. 317.2 ± 11.3 cells/mm², t-test with the Bonferroni correction for multiple comparisons here and below, p = 0.021), by 27% in the dentate gyrus (Fig. 1D, E; 361.1 ± 13.1 vs. 263.0 ± 11.7 cells/mm², t-test, p < 0.0003) and by 25% in the RSC (Fig. 1F,G; 272.6 ± 15.08 vs. 206.1 ± 4.72 cells/mm², Welch’s t-test, p = 0.0114). As aging leads to an increase in microglia numbers (Vaughan und Peters 1974), we included a group of 3-month-old mice to compare microglia in young and aged mice. Interestingly, after the PLX treatment the level of microglia in the aged animals was equal to that of the young controls (Supp. Figure 1), suggesting that our treatment rejuvenates microglia, at least in terms of cell density.

3.2. Effects of PLX treatment on other glial cells

It has been shown that in the brain of young animals, PLX administration is specific to microglia and does not change the number of other glial cells [17]. To explore this in aged animals, we used GFAP as an astrocytic marker. We could not detect any differences in GFAP density in the hippocampal CA1 nor DG, but in the RSC the microglia reduction resulted in the increase in GFAP numbers (Fig. 1F, G; 173.0 ± 16.5 vs. 271.8 ± 19.3 cells/mm², U-test, p = 0.0051). Using antibodies against the transcription factor Olig2, which is expressed in oligodendrocytes (OLGs) during early development and labels OLGs at all developmental stages[37] and APC (adenomatous polyposis coli) that is crucial for oligodendrocyte differentiation and myelination [38], we explored changes in OLG density. We did not detect differences in the overall Olig2 + cell number or the proportion of APC+/Olig2 + oligodendrocytes, suggestingnormal myelination after the PLX treatment.

3.3. Morphological changes in microglia and characteristics of cell senescence

To further characterize the morphological and activation state of microglia, we focused on microglia analysis in the CA1 region. We observed that PLX treatment leads to a slight increase in the Iba1 intensity in the remaining microglia (Fig. 2B; 1.0 ± 0.01 vs. 1.14 ± 0.06, U-test, p = 0.0076). We could not detect a difference in the area occupied by a microglial soma (240.1 ± 3.6 µm² vs. 239.7 ± 4.4 µm², KS-test, p = 0.62) or branches (45.3 ± 2.2 µm² vs. 52.9 ± 1.9 µm², KS-test, p = 0.13).

It has been reported that the microglia that repopulate the brain after PLX5622 treatment show similar characteristics to young cells, i.e. there is a reversal of the senescent state of microglia [18]. Hence, we performed an immunohistochemical analysis for ferritin storage and lipofuscin aggregates, both biomarkers for cellular senescence. The mean intensity of ferritin inside Iba1 + cells per animal was not significantly changed in PLX-treated animals (1 ± 0.09 vs. 1.2 ± 0.1; t-test, p = 0.18), but looking at the cumulative frequency distribution of single Iba1 + cells we observed a higher accumulation of Ferritin signal after PLX treatment (KS-test, p = 0.0003). Likewise, the intensity of the autofluorescence inside microglia cell bodies was higher in the remaining microglia in the PLX-treated group (1 ± 0.01 vs. 1.18 ± 0.06; Welch’s t-test, p = 0.027; KS-test, p = 0.025). This finding suggests that PLX treatment in our conditions does not reduce the number of senescent microglia or the senescent state.

3.4. PLX treatment improves performance in novel object location task in aged animals

It is known that aging leads to hippocampus-dependent learning deficits in humans and mice (Végh et al. 2014). To test for hippocampal-dependent learning and memory we performed behavioral tests 28 days after starting the PLX3397 treatment. In the open fieldtest, we could not detect any difference between groups in the distance traveled (Fig. 3B; 29.2m ± 2.3m vs. 26.8 ± 1.2m; t-test, p = 0.38), whereas mice from the control group spent slightly more time in the periphery (505s ± 10s vs. 480s ± 11s; U-test, p = 0.043). In the novel object location task (NOLT), PLX-treated animals spent significantly more time exploring the displaced objects compared to age-matched controls (Fig. 3C; discrimination ratio: 9.46 ± 6.5 % vs. 28.8 ± 5.9 %; t-test,p = 0.043). In the novel object recognition task (NORT), on the other hand, mice from both groups spent a significant time exploring the new objects and we could not detect any differences in the discrimination ratio between both groups (Fig. 3D; 27.79 ± 10.9 % vs. 25.36 9.6 %; t-test, p = 0.86).

In the more cognitively challenging Labyrinth task (dry maze), an adaption of the established Morris water maze, mice use egocentric and allocentric spatial navigation strategies to reach a reward (Fig. 3E). Here, in comparison to the Morris water maze, one can quantify the numbers of errors made which is an ideal metric to estimate memory performance in mice [39]. Mice in both groups showed good learning and we found no differences in spatial memory acquisition and in probe tests between groups in the latency to reward (Fig. 3F) or number of errors made (Fig. 3G).

These data suggests that the mild microglia reduction after PLX treatment rescued impaired hippocampus-dependent memory in NOLT in aged mice but did not affect performance in NORT and dry maze, in which both control and PLX-treated aged mice performed quite well.

3.5. PLX treatment enhances synaptic plasticity in aged mice

Next, to test whether the microglial changes in PLX-treated mice impact basal synaptic transmission and synaptic plasticity in the aging brain, we performed extracellular recordings in CA3-CA1 synapses in transverse hippocampal slices. Input-output characteristics of fEPSPs in PLX-treated and control groups were plotted for different stimulus intensities and no treatment effect was detected (Fig. 4A; two-way RM ANOVA, p = 0.99). The comparison of the paired-pulse facilitation (PPF) ratio in different stimulus intervals, likewise, did not yield any differences (Fig. 4B; two-way RM-ANOVA, p = 0.35), suggesting no effects of PLX on this form of short-term plasticity in the efficacy of transmitter release. Lastly, LTP was induced using theta-burst stimulation (TBS) and was higher in PLX-treated mice in comparison to the control group (Fig. 4C; % above the baseline level during the last 10 minutes of 60 minutes’ recordings: 22.81 ± 4.71 % vs. 39.16 ± 5.67 %; t-test, p = 0.039). hat indicates thattargeting microglia in the aging brain may be instrumental to improve synaptic plasticity.

3.6. PNN numbers and composition

Mild microglia reduction in PLX-treated mice does not change the overall number of either PV + interneurons (Fig. 5A, B; 64.09 ± 4.3 per mm² vs. 63.02 ± 4.7 per mm²; U-test, p = 0.83) or WFA + perineuronal nets (58.89 ± 3.7 per mm² vs. 52.43 ± 2.9 per mm²; U-test, p = 0.16) in the CA1 of aged mice. That finding is in accordance with our and another groups previous studies [27, 40]. Additionally, the PV intensity (1 ± 0.03 vs. 1.07 ± 0.09; Welch’s t-test, p = 0.5) and WFA intensity (1 ± 0.03 vs. 1.01 ± 0.07; Welch’s t-test, p = 0.88) were not changed.

The results of the PNN unit analysis of the fine structural organization of PNNs were similar to what we previously observed in young animals (Strackeljan et al. 2021). After microglia reduction, the hole of a single PNN unit was slightly smaller (2.35 ± 0.03 vs. 2.21 ± 0.03; KS-test, p < 0.0001), and the intensity of ECM borders was higher (Fig. 5C, D; 95.75 ± 1.2 vs. 102.6 ± 1.3; KS-test, p = 0.001), which means more ECM is associated with synapses located in PNN holes.

3.7. Microglia reduction increases brevican in perisynaptic ECM but not as part of PNNs

In our previous study in young adult animals, we showed that complete microglia depletion leads to an increase in brevican around excitatory presynapses in the str. radiatum but not in the str. oriens of the CA1 region [27]. Similarly, in this current study, we observed that PLX treatment in aged mice changes the intensity of brevican around excitatory presynaptic terminals in the str. radiatum (Fig. 6A, B, C; 1 ± 0.05 vs. 1.25 ± 0.04; t-test, p = 0.002), but not in the str. oriens (1 ± 0.05 vs. 1.15 ± 0.06; t-test, p = 0.1). The mean intensity of brevican integrated in PNNs in the hippocampal CA1 remains unchanged (1 ± 0.03 vs. 1.03 ± 0.06; t-test, p = 0.66).

3.8. Changes in pre- and postsynaptic markers

Next, we analyzed the effects of PLX treatment on pre- (vGlut1) and postsynaptic (PSD-95) markers in both the CA1 str. radiatum and str. oriens to identify synaptic correlates of the NOLT memory and LTP enhancement observed in aged mice treated with PLX. We could not detect any changes in the mean vGluT1 intensity per ROI (str. oriens: 1 ± 0.06 vs. 0.9 ± 0.04; t-test, p = 0.89; str. radiatum: 1 ± 0.06 vs. 1.04 ± 0.03; t-test; p = 0.54), density of vGluT1 puncta (str. oriens: 40.6 ± 1.7 /100 µm² vs. 41.6 ± 1.3 /100 µm²; t-test, p = 0.6; str. radiatum: 40.7 ± 1.8 /100 µm² vs. 41.1 ± 1.3 /100 µm²; t-test, p = 0.88) or size of puncta (str. oriens: 0.317 ± 0.008 µm² vs. 0.315 ± 0.007 µm²; t-test, p = 0.82; str. radiatum: 0.32 ± 0.01 vs. 0.322 ± 0.01µm² µm²; t-test, p = 0.88. However, we detected a higher mean vGluT1 puncta intensity after PLX treatment using the analysis of cumulative frequency distributions (Fig. 6C; 66.3 ± 0.42 vs. 72.6 ± 0.4; KS-test, p < 0.0001).

At the postsynaptic terminals, we also observed in the CA1 str. radiatum a slight increase in the PSD-95 intensity (Fig. 6D, E; 102.5 ± 0.2 vs. 104.4 ± 0.2; KS-test, p < 0.0001) after PLX treatment.

3.9. Microglia reduction leads to downregulation of C1q in str. radiatum

Microglia are the main source of the complement factor C1q, a protein involved in synaptic pruning (Fonseca et al. 2017; Hong et al. 2016). Here, we revealed that PLX treatment led to a prominent reduction in C1q immunofluorescence in the CA1 str. radiatum (Fig. 7; 1.0 ± 0.15 vs. 0.29 ± 0.1; t-test, p = 0.0014) but not in str. oriens (1.0 ± 0.18 vs. 1.0 ± 0.06; U-test, p = 0.21). However, the expression levels of CD68, a lysosomal protein enriched in active phagocytic microglia, and the expression of TREM2, which is known to have anti-inflammatory properties, were not altered by PLX treatment (Fig. 7), pointing to rather specific changes in the microglial activity towards synapses.

In this study, we investigated the effects of pharmacological inhibition of CSF1R in aged mice on alleviating various age-associated hippocampal abnormalities. Strikingly, we found the enhancement of hippocampus-dependent cognitive function and synaptic plasticity in the aged mice treated with PLX. Moreover, this treatment increased the expression of presynaptic vGluT1, postsynaptic PSD-95, and perisynaptic brevican, previously reported to influence excitatory synaptic transmission. These changes were specifically detected in the CA1 str. radiatum, but not in str. oriens, correlating with a layer-specific reduction in the complement protein C1q that tags synapses for synaptic modifications by microglia.

Several previous works have studied the effects of altered density of microglia in young and aged mice. The inhibition of CSF1R could deplete up to 99% of CNS microglia, although they rapidly repopulate after the termination of treatment [17, 41, 42],with a recovery rate of 20% and 80% for 7 and 14 days’ post-PLX treatment, respectively[41, 43]. Therefore, two microglia populations can exist after PLX treatment, the resident and the newly formed. Several studies using different CSF1R inhibitors showed varying degrees of microglia depletion depending on the length of treatment and concentration. Interestingly, there seem to be age-related differences in the response of microglia to PLX3397 treatment. In a study by Yegla and colleagues the aged rats showed a more robust reduction in microglia after 21 days of PLX3397 treatment compared to young animals [44]. Noteworthy, the number of Iba1 + cells were significantly higher in the aged compared to young controls, where PLX treatment led to a reduction rate of approximately 50%. This study also reported that microglia depletion leads to impaired synaptic and cognitive function in aged rats. A new study explored how PLX5622 affects microglia in young versus old mice. For a week, mice received either a low (300 mg/kg) or high (1200 mg/kg) dose. Notably, both doses significantly reduced microglial cells in the motor cortex. Young mice showed reductions of 44% and 84.4% for low and high doses, respectively. Similarly, aged mice experienced reductions of 32% and 80%. Interestingly, the low dose appeared to reduce inflammation by preventing astrocyte activation. However, the effects on synaptic plasticity and cognition were not elucidated [45]. Another recent study investigating the effects of a 7-day treatment with PLX5622, which resulted in 89% microglial depletion in aged mice, suggests that microglial depletion followed by repopulation leads to the rescue effects in the context of spatial learning and memory, and LTP [18]. Similarly, our study highlights the beneficial effects of PLX treatment in aged mice.

One critical question is whether 90% microglia depletion without repopulation is clinically friendly to CNS functions in aged humans considering the risks of infection. Moreover, it has been shown that complete inhibition of the CSF1R results in the death of mice in adulthood [46, 47], indicating the survival of mice beyond adulthood critically depends on the presence of microglia. Therefore, we attempted to study the effects of PLX3397 in the aged mice, at concentration which did not deplete microglia but rather resulted ina modest reduction of microglia by 14%, 27%, and 25% in the CA1, DG, and RSC, respectively, after 28days of treatment, bringing microglia numbers back to the level of young controls.

After depleting 14% of the aged microglia in the CA1 of aged mice, the resident microglial population had slightly modified structural features. One such feature is the increase in the size of resident-aged microglia without any alteration in the degree of activation, as we observed no differences in soma and branching area by using Iba1 + signals [48, 49]. We hypothesized that the CSF1R inhibitor might preferentially target senescent microglia, but to our surprise, the treatment increased the amount of ferritin and AF, subcellular structures in the soma of aged microglia [50, 51], despite no changes in CD68 and TREM2 were detected. A study by Burns and colleagues showed that the level of AF increases with aging and strongly correlates with microglial size [52]. Also, the ferritin protein is critical in aged microglia, regulating the iron content in the brain by binding to iron [53, 54]. With 14% depletion of microglia without room for repopulation coupled with the enhanced production of iron in the aged brain [54], it is possible that the increased sequestering of iron by the resident-aged microglia together with the increased AF content might explain the observed increase in the overall microglial size in aged mice treated with PLX.

The aged brain is highly populated by microglia that are in a perpetually activated state that coincides with age-related cognitive and synaptic decline [55]. Additionally, activated aged microglia are in a senescent state and continuously produce inflammatory cytokines and express phagocytic phenotype [54, 56]. Aging is strongly associated with the decline in hippocampus-dependent cognitive functions, in aged mice and humans. A study by Vegh and colleagues showed reduced object recognition as well as spatial learning and memory in the hippocampus of aged mice [10, 22]. Studies have also shown that the depletion of microglia with repopulation, other than reducing neuroinflammation [57], also enhances cognition and synaptic transmission in aged mice [18]. Likewise, in the present study, although aged mice failed to discriminate between the stably located and displaced objects, the PLX-treated aged mice displayed an enhanced long-term NOLT memory. Noteworthy, microglial depletion enhanced neither NORT nor spatial learning and memory in aged mice. One explanation might be that according to the study by Elmore et. al in 2018, the repopulation of microglia after depletion is essential for enhancing spatial learning and memory performance in aged mice. Also, the newly formed microglial population in the aged mice brain can attain the morphology and functions similar to the resident microglia [41] or young mice, including mRNA profiles [43]. In the context of synaptic plasticity and transmission, which are the mechanisms underlying memory acquisition and storage [58], reports indicate that LTP enhancement in aged mice brains after PLX treatment relies on microglial repopulation [18]. That indicates that microglia functions in the aging brain impair synaptic plasticity. Contrarily, here we report for the first time that mild microglial depletion without repopulation can enhance LTP-dependent synaptic transmission in the hippocampus of aged mice. To better explain these findings, we investigated the effect of PLX treatment on both synaptic and synaptic pruning markers in the hippocampal CA1, aiming to identify correlates for the enhancement of NOLT performance and LTP. Consistent with these findings, the reduction of microglia led to the elevated intensity of vGluT1 and PSD-95. Whilst vGluT1 is normally reduced in the hippocampus of aged rodents and contributes to the impairment of memory formation and synaptic transmission[59–61] contradictory data has been reported about the expression of PSD-95 depending on the observed age point and brain region [62–64]. One interesting observation was that these effects were exclusive to the CA1 str. radiatum, the region that contains synapses potentiated during LTP experiments and facilitates memory formation[65, 66] .

In the CA1 str. radiatum, we also identified a reduced expression of the complement protein C1q after PLX treatment that may explains the increase in numbers of synaptic puncta. Microglia cells are the primary source of C1q in the brain [67]. Previous research has shown that high doses of PLX5622 (1200 mg/kg) reduce C1q levels in the hippocampus of adult mice [68]. Our study suggests that even lower doses may have a similar effect. During development, the interaction between microglia and components of the complement cascade, including C1q and C3, are known to be involved in the pruning of synapses in an activity-dependent manner and therefore in the maturation of synaptic circuits [69–71]. A more recent study shows that these mechanisms also play a part in Alzheimer’s disease where the inhibition of either C1q, C3, or microglia prevents the early synapse loss characteristic of the disease pathology [72]. Our time-lapse analysis directly demonstrated the elevated elimination of spines after inoculation of Tau proteins derived from AD patients, with the rate of spine elimination correlating with the expression of complement proteins [73]. In the hippocampus of aged mice, C1q is strongly upregulated in proximity to synapses and this is associated with cognitive decline. However, aged C1q-deficient mice have similar spine numbers as wild-type controls, suggesting that C1q may induce synaptic modifications rather than changes in the balance in spine formation/elimination in these conditions.

CSF1R colony-stimulating factor 1 receptor

CSPG chondroitin

DG dentate gyrus

ECM extracellular matrix

fEPSP excitatory postsynaptic potential

NOLT novel object location task

NORT novel object recognition task

PB phosphate Buffer

PBS phosphate-buffered saline

PFA paraformaldehyde

PNN perineuronal net

PPF paired-pulse facilitation

PPR paired-pulse ratio

PV parvalbumin

OLGs oligodendrocytes

RSC retrosplenial cortex

RT room temperature

SEM standard error of mean

TBS theta-burst stimulation

WFA Wisteria floribunda agglutinin

Acknowledgments

We are thankful to Katrin Boehm for technical assistance.

Funding

This research was supported by the DFG (362321501/RTG 2413 SynAGE, TP5, TP6, A1 and B1) to A.D., DZNE Stiftung (T0531/43703/2023/hhe) to C.C..

Availability of data and materials

The data supporting the findings of this study are available from the corresponding authors upon request.

Authors’ contributions

A.D. conceptualized the study and secured funding. L.S. and A.D. wrote the first draft of the manuscript. L.S., D.B.-A., H.M., R.K. performed or contributed to different aspects of the experimental analysis. A.D., R.K. and C.C. supervised the research, L.S.., C.C. and A.D. critically revised the manuscript, and all authors approved the final version of the manuscript for submission.

Ethics approval and consent to participate

All animal experiments were conducted in accordance with
ethical animal research standards defined by the Directive 2010/63/EU, the German law and the recommendations of the Ethical Committee on Animal Health and Care of the State of Saxony-Anhalt, Germany (license number: 42502-2-1346).

Competing interests

The authors declare that they have no competing interests.

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Inhibition of colony-stimulating factor 1 receptor improves synaptic plasticity and cognitive performance in aged mice

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Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Animals

2.2. PLX3397 treatment

2.3. Behavior

2.3.1. Behavioral analysis

2.3.2. Open field

2.3.3. Novel object location test

2.3.4. Novel object discrimination test

2.3.5. Labyrinth task (dry maze)

2.4. Electrophysiology

2.5. Tissue preparation

2.6. Immunohistochemistry

2.7. Confocal microscopy and image processing

2.8. Glial cells counting and microglia analysis

2.9. Analysis of PNNs around PV + cells and perisynaptic ECM

2.10. Analysis of inflammatory markers

2.11. Statistical analysis

3. Results

3.1. PLX treatment reduces microglia density

3.2. Effects of PLX treatment on other glial cells

3.3. Morphological changes in microglia and characteristics of cell senescence

3.4. PLX treatment improves performance in novel object location task in aged animals

3.5. PLX treatment enhances synaptic plasticity in aged mice

3.6. PNN numbers and composition

3.7. Microglia reduction increases brevican in perisynaptic ECM but not as part of PNNs

3.8. Changes in pre- and postsynaptic markers

3.9. Microglia reduction leads to downregulation of C1q in str. radiatum

Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1