Scientific Papers

Chronic neuroinflammation during aging leads to cholinergic neurodegeneration in the mouse medial septum | Journal of Neuroinflammation


Aging and chronic neuroinflammation are associated with increased reactive microglia in the medial septum and reduced cholinergic cell number

To investigate the effect of normal versus pathological aging, we performed immunohistochemical analysis on the Iba-1+ microglia and ChAT+ cholinergic cell population in the MS, at three different age groups corresponding to young, adult and old, in both genotypes. The qualitative differences observed from fluorescence microscopy imaging (Fig. 1A–F) were confirmed using appropriate statistical analysis.

Fig. 1
figure 1

The number of Iba-1+ microglia and ChAT+ cholinergic cells in the medial septum (MS) changes with age and chronic neuroinflammation. AF Representative fluorescence microscopy images comparing changes of the Iba1+ microglia and ChAT+ cholinergic cell population in the medial septum (MS) during aging and neuroinflammation. The images displaying the changes in ChAT+ (green) and Iba1+ (red) cells in the MS for, young, adult, and old mice from control cohorts (AC) and GFAP-IL6 cohorts (DF). Images taken under the 10 × objective using green and red channels to detect ChAT+ cholinergic cells and Iba1+ microglia cells, respectively (scale bar 100 µm). GI Grouped bar graphs representing the stereological estimation changes and septal volume, with aging and neuroinflammation; G septal Iba-1+ microglia number, H septal ChAT+ cell number, and I septal volume, of young (n = 5), adult (n = 5), and old (n = 5) mice from control and GFAP-IL6 cohorts. Data presented as mean ± SEM; two-way ANOVA, with Tukey’s post hoc test. Post hoc effect of ‘genotype’ indicated by asterisks, (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); post hoc effect of ‘age’ indicated by hash symbols (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001)

A significant main effect of ‘age’ and ‘genotype’, as well as ‘age’ x ‘genotype’ interaction was observed on the level of microglia activation in the medial septum [‘age’ F (2, 24) = 107.8, P < 0.0001; ‘genotype’ F (1, 24) = 30.54, P < 0.0001; ‘age’ x ‘genotype’ F (2, 24) = 8.378, P = 0.0017]. Post hoc test confirmed a significant increase in estimated septal Iba-1+ microglia number in the control cohort for adult (57.20%; MD = 3073 ± 616.8, ###p = 0.0006) and old (74.19%; MD = 4524 ± 616.8, ####p < 0.0001) compared to young control cohort (Fig. 1G; Additional file 1: Table S3). A much-exaggerated significant increase was observed in adult (81.20%; MD = 5307 ± 616.8, ####p < 0.0001) and old (101.82%; MD = 8053 ± 616.8, ####p < 0.0001) GFAP-IL6 cohorts compared to young GFAP-IL6 cohort. We also observed a significant increase in Iba-1+ microglia in old GFAP-IL6 cohort compared to adult GFAP-IL6 cohort (26%; MD = 2746 ± 616.8, ##p = 0.0021). Importantly significant differences were observed between adult (28.34%; MD = 2281 ± 616.8, *p = 0.0127) and old (35.24%; MD = − 3576 ± 616.8, ****p < 0.0001) GFAP-IL6 cohorts and their age-matched controls, but no significant difference for young cohorts between the two genotypes.

We observed a significant main effect of both ‘age’ and ‘genotype’ on the septal ChAT+ cholinergic cell number, with no significant ‘age’ x ‘genotype’ interaction [‘age’ F (2, 24) = 5.187, P = 0.0134; ‘genotype’ F (1, 24) = 18.88, P = 0.0002]. Post hoc test confirmed a significant decrease in ChAT+ cholinergic cell number in adult (37.20%; MD = − 1399 ± 285.7, ***p = 0.0007) and old (34.96%; MD = − 1148 ± 285.7, **p = 0.0059) GFAP-IL6 cohorts compared to their age-matched control cohorts (Fig. 1H; Additional file 1: Table S3).

A significant main effect of ‘age’ and ‘genotype’, as well as ‘age’ × ‘genotype’ interaction was observed for the medial septal volume across the cohorts [‘age’ F (2, 24) = 4.670, P = 0.0194; ‘genotype’ F (1, 24) = 20.83, P = 0.0001; ‘age’ × ‘genotype’ F (2, 24) = 16.63, P < 0.0001]. Post hoc test confirmed a significant decrease in septal volume in adult (18.60%; MD = − 0.07400 ± 0.01897, ##p = 0.0079) and old (29.27%; MD = − 0.1160 ± 0.01897, ####p < 0.0001) GFAP-IL6 cohorts compared to young GFAP-IL6 cohort (Fig. 1I; Additional file 1: Table S3). We did not observe a significant septal volume loss across normal aging cohorts. However, a significant decrease in septal volume was seen in adult (18.60%; MD = − 0.07200 ± 0.01897, *p = 0.0101) and old (29.27%; MD = − 0.1140 ± 0.01897, ****p < 0.0001) GFAP-IL6 cohorts compared to their age-matched control cohorts.

Aging and chronic neuroinflammation are associated with morphological changes in microglia in the medial septum

Following up on the significant stereological differences observed for septal Iba1+ microglia and ChAT+ cholinergic cell populations, morphological analysis was conducted. To further understand the impact of aging and neuroinflammation on the morphological features of the septal Iba-1+ microglia, single cell branched structural analysis of young, adult, and old mice from control and GFAP-IL6 cohorts was performed (Fig. 2A–F).

Fig. 2
figure 2

Morphology of septal Iba-1-positive microglia during aging and the impact of chronic neuroinflammation. AE Representative confocal images of reconstructed medial septum (MS) microglia from control and GFAP-IL6 cohorts. The extended depth of focus images (A-E) obtained from collapsing 3D confocal microscopy images of Iba-1 + microglia obtained under 63 × oil immersion objective, along with the corresponding manually reconstructed images (a–e) in Neurolucida 360, of young, adult and old mice from control and GFAP-IL6 cohorts (scale bar 10 μm). GL Grouped bar graphs representing the effects of aging and neuroinflammation on Iba-1+ microglial cell morphology in the medial septum (MS), measured by branched structure analysis. Quantitative analysis of the morphological changes in Iba-1+ microglia, including G soma area (µm2), H soma perimeter (µm), I soma circularity, J number of processes from soma (n), K total length of processes (µm) and L number of nodes (n), of young (n = 30), adult (n = 30) and old (n = 30) mice from control (black) and GFAP-IL6 (red) cohorts. Data presented as mean ± SEM and analysed with two-way ANOVA followed by Tukey’s post hoc tests. Post hoc effects of ‘genotype’ are represented in asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); post hoc effects of ‘age’ are represented by hash symbols (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001). M–O Grouped bar graphs representing the effects of aging and neuroinflammation on Iba-1+ microglial cell spatial distribution in the medial septum (MS), measured by convex-hull analysis. Quantitative analysis of spatial distribution changes in Iba-1+ microglia, including M convex 3D surface area (µm2), N convex 3D volume (µm3) and O convex perimeter (µm) of young (n = 30), adult (n = 30) and old (n = 30) mice from control (black) and GFAP-IL6 (red) cohorts. Data presented as mean ± SEM and analysed with two-way ANOVA followed by Tukey’s post hoc tests. Post hoc effects of ‘genotype’ are represented in asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); post hoc effects of ‘age’ are represented by hash symbols (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001)

A significant main effect of ‘age’ and ‘genotype’ was observed in the measurements of soma area (µm2) [‘age’ F (2, 58) = 51.70, P < 0.0001; ‘genotype’ F (1, 29) = 60.37, P < 0.0001], soma perimeter (µm) [‘age’ F (2, 58) = 59.09, P < 0.0001; ‘genotype’ F (1, 29) = 88.09, P < 0.0001], and soma circularity [‘age’ F (2, 58) = 13.35, P < 0.0001; ‘genotype’ F (1, 29) = 54.69, P < 0.0001]. However, we did not observe ‘age’ × ‘genotype’ interaction for these measurements. Post hoc test confirmed significant increase in soma area and perimeter, respectively (Fig. 2G, H; Additional file 1: Supplemetary Table S4) in young (MD 5.248 ± 1.368, **p = 0.0040; MD 2.397 ± 0.6270, **p = 0.0042), 12- (MD 7.757 ± 1.368, ****p < 0.0001; MD 3.664 ± 0.6270, ****p < 0.0001), and old (MD 6.457 ± 1.368, ***p = 0.0002; MD 3.500 ± 0.6270, ****p < 0.0001) GFAP-IL6 cohorts compared to age-matched control cohorts. There was also a significant increase in soma area and perimeter, respectively, for old control compared to young control cohort (MD 9.677 ± 1.368, ####p < 0.0001; MD -3.763 ± 0.6270, ####p < 0.0001), as well as between old control compared to adult control cohort (MD 9.979 ± 1.368, ####p < 0.0001; MD -3.983 ± 0.6270, ####p < 0.0001). This significant increase in soma area and perimeter was higher between young and old GFAP-IL6 cohorts (MD 10.89 ± 1.368, ####p < 0.0001; MD 4.866 ± 0.6270, ####p < 0.0001), as well as for adult and old GFAP-IL6 cohorts (MD -8.679 ± 1.368, ####p < 0.0001; MD; 3.819 ± 0.6270, ####p < 0.0001). Furthermore, we observed a significant reduction in soma circularity (Fig. 2I) in adult (MD − 0.05100 ± 0.01376, **p = 0.0060) and old (MD -0.05967 ± 0.01376, ***p = 0.0008) GFAP-IL6 cohorts compared to their age-matched control cohorts, as well as in old GFAP-IL cohort compared to young GFAP-IL6 cohort (MD -0.05533 ± 0.01376, ##p = 0.0022). There was no significant effect of ‘age’ or ‘genotype’ on the number of processes form Iba-1+ septal microglia (Fig. 2J). However, we found a significant main effect of ‘age’, ‘genotype’, and ‘age’ x ‘genotype’ interaction, respectively, for total length of processes from soma (µm) [F (2, 58) = 73.91, P < 0.0001; F (1, 29) = 99.10, P < 0.0001; F (2, 58) = 6.581, P = 0.0027] and nodes (n) [F (2, 58) = 67.69, P < 0.0001; F (1, 29) = 68.89, P < 0.0001; F (2, 58) = 4.535, P = 0.0148]. Post hoc test confirmed significant decrease in total length of processes and nodes, respectively (Fig. 4K, L; Additional file 1: Supplementary Table S4) in young (MD − 263.9 ± 27.17, ****p < 0.0001; MD − 22.73 ± 2.842, ****p < 0.0001), adult (MD− 247.0 ± 27.17, ****p < 0.0001; MD − 18.87 ± 2.842, ****p < 0.0001), and old (MD − 135.6 ± 27.17, ****p < 0.0002; MD − 10.87 ± 2.842, **p = 0.0042) GFAP-IL6 cohorts compared to age-matched control cohorts. Moreover, there was a significant decrease in in total length of processes and nodes, respectively, within the aging cohorts for both control and GFAP-IL6 cohorts between: young and old control cohorts (MD − 250.9 ± 27.17, ####p < 0.0001; MD − 20.53 ± 2.842, ####p < 0.0001); adult and old control cohorts (MD -254.4 ± 27.17, ####p < 0.0001; MD − 23.03 ± 2.842, ####p < 0.0001); young and old GFAP-IL6 cohorts (MD − 122.6 ± 27.17, ###p = 0.0004; MD − 8.667 ± 2.842, #p = 0.0385); adult and old GFAP-IL6 cohorts (MD − 143.0 ± 27.17, ####p < 0.0001; MD − 15.03 ± 2.842, ####p < 0.0001).

To further confirm the morphological changes displayed with branched structure analysis, the convex-hull analysis was performed. There was a significant main effect of ‘age’ and ‘genotype’ as well as ‘age’ x ‘genotype’ interaction, respectively, for the measurements of convex 3D surface area (µm2) [F (2, 58) = 56.25, P < 0.0001; F (1, 29) = 87.82, P < 0.0001; F (2, 58) = 11.72, P < 0.0001], volume (µm3) [F (2, 58) = 32.78, P < 0.0001; F (1, 29) = 83.08, P < 0.0001; F (2, 58) = 14.36, P < 0.0001], and perimeter (µm) [F (2, 58) = 67.15, P < 0.0001; F (1, 29) = 69.29. P < 0.0001; F (2, 58) = 5.492, P = 0.0065] (Fig. 2; Additional file 1: Supplementary Table S4). Post hoc test confirmed significant reduction in microglia arborization field in terms of surface area (Fig. 2M) for young (MD -2269 ± 255.0, ****p < 0.0001), adult (MD -2399 ± 255.0, ****p < 0.0001), and old (MD − 826.4 ± 255.0, *p = 0.0231) GFAP-IL6 cohorts compared to their age-matched control cohorts. There was also a significant reduction in surface area within the aging cohorts between; young and old control cohorts (MD − 2809 ± 255.0, ####p < 0.0001); adult and old control mice (MD − 2146 ± 255.0, ####p < 0.0001); young and adult GFAP-IL6 cohorts (MD − 791.9 ± 255.0, #p = 0.0332); young and old GFAP-IL6 cohorts (MD − 1366 ± 255.0, ####p < 0.0001). In terms of volume (Fig. 2N), we found a significant reduction in young (MD − 16,530 ± 1781, ****p < 0.0001), and adult (MD − 16,219 ± 1781, ****p < 0.0001) GFAP-IL6 cohorts compared to age-matched control cohorts. A significant reduction in volume within the aging cohorts were visible between; young and old control cohorts (MD − 15,592 ± 1781, ####p < 0.0001); adult and old control cohorts (MD − 14,984 ± 1781, ####p < 0.0001). Furthermore, in terms of perimeter (Fig. 2O), a significant reduction was found in young (MD -31.41 ± 6.003, ****p < 0.0001), and adult (MD − 45.44 ± 6.003, ****p < 0.0001) GFAP-IL6 cohorts compared to age-matched control cohort. There was also a significant reduction in perimeter (µm) within the aging cohorts between; young and adult control cohorts (MD − 19.57 ± 6.003, #p = 0.0219); young and old control cohorts (MD − 55.55 ± 6.003, ####p < 0.0001); adult and old control cohorts (MD -35.98 ± 6.003, ####p < 0.0001); young and adult GFAP-IL6 cohorts (MD -33.60 ± 6.003, ####p < 0.0001); young and old GFAP-IL6 cohorts (MD − 41.45 ± 6.003, ####p < 0.0001).

Finally, to further investigate the effects of aging and neuroinflammation on the complexity of microglia arborization, a segmental (Sholl) analysis was performed to examine the changes as a function of radial distance from the microglia cell body. We observed less complex microglial arborization in GFAP-IL6 mice with aging, mostly confined to 0–30 µm radius, compared to age-matched control mice which showed interactions beyond the 30 µm radius (Fig. 3). After analysing the area under the curve (AUC) for each cumulative line graph (Fig. 3A–F; Additional file 1: Supplementary Table S5), overall significant mean peak differences were observed between GFAP-IL6 mice and their age-matched controls for the measurements of intersections (n) [F (5, 2424) = 7.781, P < 0.0001], length (µm) [F (5, 2424) = 10.80, P < 0.0001], surface area (µm2) [F (5, 2424) = 7.311, P < 0.0001], volume (µm3) [F (5, 2424) = 3.599, P = 0.0030], and nodes (n) [F (5, 2424) = 5.955, P < 0.0001]. No significant differences were observed among the cohorts for the average diameter (µm). Post hoc comparisons displayed a significant reduction in AUC for intersections (Fig. 3A) in young (MD -27.30 ± 8.888, *p = 0.0262) and adult (MD − 28.24 ± 8.888, *p = 0.0188) GFAP-IL6 cohorts when compared to their age-matched control cohorts. A significant reduction was also observed among the aging cohorts between young and old control cohorts (MD − 26.63 ± 8.888, #p = 0.0329), and adult and old control cohorts (MD − 28.67 ± 8.888, #p = 0.0161). Measurements of AUC for length (Fig. 3B) displayed a significant reduction in young (MD − 264.0 ± 7.81, **p = 0.0014) and adult (MD − 247.0 ± 67.81 ± 8.888, **p = 0.0037) GFAP-IL6 cohorts when compared to their age-matched control cohorts. A significant length reduction was also observed among the aging cohorts between young and old control cohorts (MD − 250.8 ± 67.81, ##p = 0.0030), and adult and old control cohorts (MD -254.4 ± 67.81, ##p = 0.0025). We also observed a significant reduction in AUC for surface area (Fig. 3C) in young GFAP-IL6 cohort compared to their age-matched control cohort (MD − 524.0 ± 145.8, **p = 0.0045). Moreover, a significant reduction in surface area was observed among the aging cohorts between young and old control cohorts (MD − 434.0 ± 145.8, #p = 0.0348), and adult and old control cohorts (MD -448.0 ± 145.8, #p = 0.0261). Furthermore, there was a significant reduction in the number of nodes (Fig. 3F) in the young GFAP-IL6 cohort compared to young control cohort (MD − 22.73 ± 7.741, *p = 0.0393), as well as significant reduction between adult and old control cohorts (MD − 23.03 ± 7.741, #p = 0.0350).

Fig. 3
figure 3

Cumulative line graphs and corresponding area under the curve (AUC) bar graphs, representing the effects of aging and neuroinflammation on Iba-1+ microglial cell arborization complexity in the medial septum (MS), measured by Sholl analysis. Quantitative analysis of Iba-1+ microglia arborization complexity, including A intersections (n), B length (µm), C surface area (µm2), D volume (µm3), E average diameter (µm), and F nodes (n) of young (n = 30), adult (n = 30) and old (n = 30) mice from control and GFAP-IL6 cohorts. Data presented as mean ± SEM for AUC graphs and analysed with one-way ANOVA followed by Tukey’s post hoc tests. Post hoc effects of ‘genotype’ are represented in asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); post hoc effects of ‘age’ are represented by hash symbols (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001)

Aging and chronic neuroinflammation are associated with morphological changes in cholinergic cells in the medial septum

To further interrogate the impact of aging and chronic neuroinflammation on the morphological differences of the septal cholinergic neurons (Fig. 4A–F), we performed quantitative morphological measurements, including branch structure, convex-hull, and Sholl analysis.

Fig. 4
figure 4

Morphology of septal ChAT-positive neurons during aging and the impact of chronic neuroinflammation. AF Representative confocal images of reconstructed medial septum (MS) cholinergic cells from control and GFAP-IL6 cohorts. The extended depth of focus images (AE) obtained from collapsing 3D tile scanned confocal microscopy images of ChAT + cholinergic cells obtained under 63 × oil immersion objective, along with the corresponding manually reconstructed images (a-e) in Neurolucida 360, of young, adult and old mice from control and GFAP-IL6 cohorts (scale bar 20 μm). GL Grouped bar graphs representing the effects of aging and neuroinflammation on ChAT+ cholinergic cell morphology in the medial septum (MS), measured by branched structure analysis. Quantitative analysis of the morphological changes in ChAT+ cholinergic cells, including G soma area (µm2), H soma perimeter (µm), I soma circularity, J number of processes from soma (n), K total length of processes (µm) and L number of nodes (n), of young (n = 25), adult (n = 25) and old (n = 25) mice from control (black) and GFAP-IL6 (green) cohorts. Data presented as mean ± SEM and analysed with two-way ANOVA followed by Tukey’s post hoc tests. Post hoc effects of ‘genotype’ are represented in asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); post hoc effects of ‘age’ are represented by hash symbols (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001). M–O Grouped bar graphs representing the effects of aging and neuroinflammation on ChAT+ cholinergic cell spatial distribution in the medial septum (MS), measured by convex-hull analysis. Quantitative analysis of spatial distribution changes in ChAT+ cholinergic cells, including M convex 3D surface area (µm2), N convex 3D volume (µm3) and O convex perimeter (µm) of young (n = 25), adult (n = 25) and old (n = 25) mice from control (black) and GFAP-IL6 (green) cohorts. Data presented as mean ± SEM and analysed with two-way ANOVA followed by Tukey’s post hoc tests. Post hoc effects of ‘genotype’ are represented in asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); post hoc effects of ‘age’ are represented by hash symbols (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001)

We observed a significant main effect of ‘age’, ‘genotype’, and ‘age’ x ‘genotype’ interaction, respectively, in the ChAT+ cell measurements of processes from soma (n) [‘age’ F (2, 48) = 7.129, P = 0.0019; ‘genotype’ F (1, 24) = 10.75, P = 0.0032; ‘age’ x ‘genotype’ F (2, 48) = 5.152, P = 0.0094], and total length of dendrites (µm) [‘age’ F (2, 48) = 72.87, P < 0.0001; ‘genotype’ F (1, 24) = 70.24, P < 0.0001; ‘age’ x ‘genotype’ F (2, 48) = 4.981, P = 0.0108]. A significant main effect of only ‘genotype’ [F (1, 24) = 8.076, P = 0.0090] and ‘age’ x ‘genotype’ interaction [F (2, 48) = 5.570, P = 0.0067] was observed for soma area (µm2). Cholinergic cell soma perimeter was not significantly affected by ‘age’ or ‘genotype’. However, we observed a significant main effect of ‘age’ and ‘genotype’ on soma circularity [‘age’ F (2, 48) = 11.15, P = 0.0001; ‘genotype’ F (1, 24) = 4.825, P = 0.0379], and nodes (n) [‘age’ F (2, 48) = 34.83, P < 0.0001; ‘genotype’ F (1, 24) = 8.101, P = 0.0089], with no significant ‘age’ x ‘genotype’ interaction. Post hoc test confirmed significant decrease in ChAT+ cholinergic cell soma area (Fig. 4G) only in old control compared to adult control cohort (MD − 29.96 ± 9.295, #p = 0.0261). Soma circularity (Fig. 4I) displayed a significant increase in adult (MD 0.05559 ± 0.01677, #p = 0.0204) and old (MD 0.05305 ± 0.01677, #p = 0.0304) compared to young GFAP-IL6 cohort. A significant decrease was observed for the number of processes from soma (Fig. 4J), between adult control and adult GFAP-IL6 cohorts (MD − 1.080 ± 0.2645, **p = 0.0022); young and adult GFAP-Il6 cohorts (MD − 1.000 ± 0.2645, ##p = 0.0055); young and old GFAP-IL6 cohorts (MD − 1.000 ± 0.2645, ##p = 0.0055). Importantly, we observed a significant decrease in total length of dendrites (Fig. 4K) in young (MD -167.7 ± 24.80, ****p < 0.0001) and adult (MD − 144.4 ± 24.80, ****p < 0.0001) GFAP-IL6 cohorts, respectively, compared to their age-matched control cohorts. There was also a significant decrease in total length of dendrites within the aging cohorts for both control and GFAP-IL6 cohorts between; young and adult control cohorts (MD − 195.8 ± 24.80, ####p < 0.0001); young and old control cohorts (MD − 292.3 ± 24.80, ####p < 0.0001); adult and old control cohorts (MD − 96.51 ± 24.80, ##p = 0.0039); young and adult GFAP-IL6 cohorts (MD − 172.4 ± 24.80, ####p < 0.0001); young and old GFAP-IL6 cohorts (MD -186.9 ± 24.80, ####p < 0.0001). Complementary, there was a significant decrease in nodes (Fig. 4L) in young GFAP-IL6 cohort compared to their age-matched control cohort (MD − 1.160 ± 0.3711, *p = 0.0336). Moreover, we observed a significant decrease in nodes within the aging cohorts for both control and GFAP-IL6 mice between young and adult control cohorts (MD − 2.680 ± 0.3711, ####p < 0.0001); young and old control cohorts (MD − 2.680 ± 0.3711, ####p < 0.0001); young and adult GFAP-IL6 cohorts (MD − 2.040 ± 0.3711, ####p < 0.0001); young and old GFAP-IL6 cohorts (MD − 2.120 ± 0.3711, ####p < 0.0001) (see Additional file 1: Supplementary Table S6).

The convex-hull analysis further confirmed changes to the septal ChAT+ cholinergic cell dendritic arbor field size (Fig. 4M–O). We observed a significant main effect of ‘age’ and ‘genotype’ for the measurements of convex 3D surface area (µm2) [‘age’ F (2, 48) = 17.94, P < 0.0001; ‘genotype’ F (1, 24) = 61.34, P < 0.0001], volume (µm3) [‘age’ F (2, 48) = 8.320, P = 0.0008; ‘genotype’ F (1, 24) = 33.78, P < 0.0001], and perimeter (µm) [ ‘age’ F (2, 48) = 41.14, P < 0.0001; ‘genotype’ F (1, 24) = 97.21, P < 0.0001). However, the ‘age’ x ‘genotype’ interaction was only significant for convex 3D surface area (µm2) [F (2, 48) = 3.490, P = 0.0385] and convex perimeter (µm) [F (2, 48) = 5.034, P = 0.0104]. Post hoc test confirmed significant reduction in convex 3D surface area (Fig. 4M), in young (MD − 5694 ± 1431, **p = 0.0030), and adult (MD − 8607 ± 1431, ****p < 0.0001) GFAP-IL6 mice compared to their age-matched control cohorts. There was also a significant reduction in surface area (µm2) within the aging cohorts between young and old control cohorts (MD -7916 ± 1431, ####p < 0.0001); adult and old control cohorts (MD − 6140 ± 1431, ##p = 0.0011); young and adult GFAP-IL6 cohorts (MD -4688 ± 1431, #p = 0.0227); young and old GFAP-IL6 cohorts (MD − 5489 ± 1431, ##p = 0.0046). In terms of volume (Fig. 4N), we found a significant reduction in young (MD − 38,918 ± 11,251, *p = 0.0138), and adult (MD − 56,168 ± 11,251, ***p = 0.0001) GFAP-IL6 mice compared to their age-matched controls. A significant reduction in volume within the aging cohorts were visible between young and old control cohorts (MD − 42,042 ± 11,251, ##p = 0.0062), and adult and old control cohorts (MD − 38,769 ± 11,251, #p = 0.0143). Furthermore, in terms of perimeter (Fig. 4O), a significant reduction was found in young (MD − 81.11 ± 18.26, ***p = 0.0007), adult (MD − 140.1 ± 18.26, ****p < 0.0001), and old (MD − 61.32 ± 18.26, *p = 0.0182) GFAP-IL6 cohorts compared to their age-matched control cohorts. There was also a significant reduction in perimeter (µm) within the aging cohorts between young and old control cohorts (MD − 144.5 ± 18.26, ####p < 0.0001); adult and old control cohorts (MD − 108.7 ± 18.26, ####p < 0.0001); young and adult GFAP-IL6 cohorts (MD − 94.75 ± 18.26, ####p < 0.0001); young and old GFAP-IL6 cohorts (MD − 124.7 ± 18.26, ####p < 0.0001).

Finally, we carried out the Sholl analysis on septal cholinergic neurons to study the changes in radial distribution of dendritic arborization from the cell soma, with aging and neuroinflammation. We observed less complex cholinergic dendritic arborization in GFAP-IL6 mice with aging, mostly confined to 0–80 µm radius, compared to age-matched control mice which showed interactions beyond the 90 µm radius (Fig. 5). After analysing AUC for each cumulative line graph for ChAT+ cells (Fig. 5A–F; Additional file 1: Supplementary Table S7), overall significant mean peak differences were observed between GFAP-IL6 mice and their age-matched controls for the measurements of intersections (n) [F (5, 2580) = 10.93, P < 0.0001], length (µm) [F (5, 2580) = 16.15, P < 0.0001], surface area (µm2) [F (5, 2580) = 8.290, P < 0.0001], and volume (µm3) [F (5, 2580) = 2.370, P = 0.0372]. No significant differences were observed among the cohorts for the average diameter (µm), and nodes (n). Post hoc comparisons displayed a significant reduction in AUC for intersections (Fig. 5A) in adult GFAP-IL6 cohort when compared to their age-matched control cohort (MD − 12.32 ± 3.633, **p = 0.0092). A significant reduction was also observed among the aging cohorts between young and old control cohorts (MD − 16.88 ± 3.633, ####p < 0.0001); young and adult GFAP-IL6 cohorts (MD − 11.12 ± 3.633, #p = 0.0271); young and old GFAP-IL6 cohorts (MD − 11.92 ± 3.633, #p = 0.0134). Measurements of AUC length (Fig. 5B) displayed a significant reduction in young (MD − 167.7 ± 47.04, **p = 0.0050), and adult (MD − 144.1 ± 47.04, *p = 0.0268) GFAP-IL6 cohorts when compared to their age-matched control cohorts. A significant reduction was also observed among the aging cohorts between young and adult control cohorts (MD − 96.0 ± 47.04, ###p = 0.0005); young and old control cohorts (MD − 292.3 ± 47.04, ####p < 0.0001); young and adult GFAP-IL6 cohorts (MD − 172.4 ± 47.04, ##p = 0.0034); young and old GFAP-IL6 cohorts (MD − 186.9 ± 47.04, ##p = 0.0010). Finally, AUC measurements for surface area (Fig. 5C) displayed a significant reduction in adult GFAP-IL6 cohorts compared to age-matched control cohorts (MD − 406.1 ± 125.9, #p = 0.0161). There was also a significant reduction seen between young and old control aging cohorts (MD − 511.9 ± 125.9, ###p = 0.0007) (see Additional file 1: Supplementary Table S8).

Fig. 5
figure 5

AF Cumulative line graphs and corresponding area under the curve (AUC) bar graphs, representing the effects of aging and neuroinflammation on ChAT+ cholinergic cell dendritic arborization complexity in the medial septum (MS), measured by Sholl analysis. Quantitative analysis of individual ChAT+ cell dendritic arborization complexity including: A intersections (n), B length (µm), C surface area (µm2), D volume (µm3), E average diameter (µm), and F nodes (n) of young (n = 25), adult (n = 25) and old (n = 25) mice from control and GFAP-IL6 cohorts. Data presented as mean ± SEM and analysed with two-way ANOVA followed by Tukey’s post hoc tests. Post hoc effects of ‘genotype’ are represented in asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); post hoc effects of ‘age’ are represented by hash symbols (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001)

Aging and chronic neuroinflammation are associated with degenerating fibres in the medial septum

To further investigate the medial septal cholinergic degenerating axons due to aging and chronic neuroinflammation, we performed a silver staining for degenerating cells to assess the medial septum. Background silver-staining of nuclei was evident in all sections. However, silver-stained fibres were not prominently observed in the sections from young control cohort compared to adult and old control cohorts (Fig. 6A–C). Moreover, the GFAP-IL6 cohorts exhibited darkly stained fibres at all three time points, with highest intensity staining in the old cohort (Fig. 6D–F).

Fig. 6
figure 6

A–F Representative bright-field microscopy images revealing degenerating fibres in the medial septum (MS) during aging and neuroinflammation. While background silver-staining of nuclei was visible in all sections, silver-stained fibres were not prominently observed in the sections from A young control mice compared to much aged control mice B adult and C old. The GFAP-IL6 mice D–F presented with darkly stained fibres at all three time points, with highest intensity staining in old mice. Images taken under the 20 × objective (scale bar 40 µm)

Aging and chronic neuroinflammation are associated with pyramidal dendritic spine loss in the hippocampus

To investigate if the changes observed within the septal ChAT+ cholinergic cell population have reciprocal effects on the hippocampal pyramidal neuronal spine density, we performed a spine density analysis (Fig. 7A–F). A main effect of ‘age’ and ‘genotype’ was observed in the measurements of spine density (1/µm), with no ‘age’ x ‘genotype’ interaction [‘age’ F (2, 138) = 21.90, P < 0.0001; ‘genotype’ F (1, 138) = 76.04, P < 0.0001]. Post hoc test confirmed a significant reduction in spine density (Fig. 7G) in young (MD − 0.2477 ± 0.06388, **p = 0.0022), adult (MD − 1.020 ± 0.2787, ***p = 0.0004), and old (MD − 0.4384 ± 0.06388, ****p < 0.0001) GFAP-IL6 mice when compared to their age-matched control cohorts. Furthermore, spine density significantly reduced within the aging cohorts between adult and old control cohorts (MD − 0.2014 ± 0.06388, #p = 0.0238); young and old GFAP-IL6 cohorts (MD − 0.3235 ± 0.06388, ####p < 0.0001); adult and old GFAP-IL6 cohorts (MD − 0.3611 ± 0.06388, ####p < 0.0001).

Fig. 7
figure 7

A-G Representative images of reconstructed hippocampal pyramidal spines revealing changes during aging and neuroinflammation. The bright-field Golgi-stained images of reconstructed pyramidal spines (AF) represent changes in young, adult, and old mice from control and GFAP-IL6 cohorts. The grouped bar graph (G) represents the estimated spine density per µm of reconstructed pyramidal dendrite in the hippocampus of young (n = 20), adult (n = 20) and old (n = 20) mice from control and GFAP-IL6 cohorts. Data presented as mean ± SEM and analysed with two-way ANOVA followed by Tukey’s post hoc tests. Post hoc effects of ‘genotype’ are represented in asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); post hoc effects of ‘age’ are represented by hash symbols (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001). Images taken under the 60× oil-objective (scale bar 100 µm)

The impact of aging and neuroinflammation on the intrinsic passive properties of cholinergic neurons in the MS

To assess alterations in the intrinsic electrophysiological properties of cholinergic neurons during aging and chronic neuroinflammation, we have recorded both passive (resting membrane potential (RMP), input resistance (Rin), membrane time constant-(tau)) and active membrane properties of BFCN neurons in the MS of control and GFAP-IL6 mice (Fig. 8A–C). Our results suggest that the RMP was affected by both aging and chronic neuroinflammation (F(2,108) = 4.32, p < 0.05 for the factor aging; F(1,108) = 6.26, p < 0.01 for the factor chronic neuroinflammation, and F(2,108) = 2.88, p = 0.06 for the factor interaction, two-way ANOVA; Fig. 8E). However, while the RMP in ChAT mice was comparable between age groups (p > 0.05, two-way ANOVA, Fig. 1D), it depolarized throughout aging in IL6-ChAT mice (− 62.76 ± 0.65 mV, n = 19 in young mice vs. − 60.35 ± 0.26 mV, n = 20 in aged mice, p < 0.05, two-way ANOVA with Tukey’s post hoc test), indicating that the aging process itself in control mice is not sufficient to induce changes in the RMP. In addition, aging alone and combined with chronic inflammation had a significant impact on the input resistance (F(2,112) = 24.29, p < 0.0001 for the factor aging; F(1,112) = 1.68, p = 0.2 for the factor chronic neuroinflammation; F(2,112) = 4.84, p = 0.01 for the factor interaction, two-way ANOVA). Post hoc analysis indicated that the average input resistance significantly increased with age in the ChAT mice (604.8 ± 31.99 MΩ, n = 18 in Young mice vs 908.8 ± 65.06 MΩ, n = 17, in aged mice p < 0.001 and adult 638.5 ± 40.77 MΩ, n = 21; p < 0.01, two-way ANOVA with Tukey’s post hoc test, Fig. 8E). Similarly, the young IL6-ChAT mice had a lower input resistance (531.4 ± 41.77 MΩ, n = 20) compared to both the adult (853.7 ± 49.85 MΩ, n = 22, p < 0.0001) and aged group (921.6 ± 56.48 MΩ, n = 20, p < 0.0001, two-way ANOVA with Tukey’s post hoc test, Fig. 8E).

Fig. 8
figure 8

The impact of aging and chronic neuroinflammation on the passive membrane properties of cholinergic neurons in the MS. A Representative confocal image depicting the MS in control mouse obtained under 2.5× magnification. Inset−10 × magnification of the MS. B Representative picture (× 40) depicting a cholinergic neuron with the recording electrode during whole-cell patch-clamp. C Sample traces of electrophysiological membrane properties in response to depolarizing and hyperpolarizing current injections. DF Box plots depicting the intrinsic passive properties of the cholinergic neurons in the MS of control and GFAP-IL6 mice at young (3–4 months, light grey), adults (9–10 months, pink), and old (18–19 months, dark red) groups. The properties of resting membrane potential (D), input resistance (E), and time constant tau (F). Data are shown as box plot, the box upper and lower limits are the 25th and 75th quartiles, respectively. The whiskers depict the lowest and highest data points, the horizontal line through the box is the median and the + sign represents the mean, two-way ANOVA, with Tukey’s post hoc test. Post hoc effect of ‘genotype’ indicated by asterisks, (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); post hoc effect of ‘age’ indicated by hash symbols (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001)

Similarly, chronic neuroinflammation during aging had significant impact on the membrane time constant (F(2,116) = 2.31, p = 0.1 for the factor aging; F(1,116) = 4.1, p = 0.05 for the factor chronic neuroinflammation; F (2,116) = 5.99, p = 0.003 for the factor interaction, two-way ANOVA). Post hoc analysis indicated that the average tau in the aged L6-ChAT mice (69.95 ± 3.73 ms, n = 20) was significantly higher than both adults (54.02 ± 4.01 ms, n = 22, p < 0.01, two-way ANOVA with Tukey’s post hoc test) and young mice (56.18 ± 2.78 ms, n = 22, p < 0.05, two-way ANOVA with Tukey’s post hoc test, Fig. 8F). While the tau in ChAT mice was comparable between age groups (p > 0.05, two-way ANOVA, Fig. 8F), the aged group showed an increase between ChAT and IL6-ChAT (aged ChAT 52.69 ± 2.79 ms, n = 17; aged IL6-ChAT 69.95 ± 3.73 ms, n = 20, **p < 0.01, two-way ANOVA with Tukey’s post hoc test) indicating that this age-group is specifically susceptible for chronic neuroinflammation. These results highlight the impact of chronic neuroinflammation on the passive membrane properties of BFCNs at different age-groups, specifically the adult and aged groups that express an increased excitability profile.

The impact of aging and neuroinflammation on the active membrane properties of cholinergic neurons in the MS

To further understand the impact of aging and neuroinflammation on the excitability profile of BCFN neurons, we recorded their active membrane properties, including spike rheobase (the minimum current required to elicit an action potential), time to spike and spike amplitude (Fig. 9A). Aging had an impact on all the active membrane properties. Two-way ANOVA analysis for spike rheobase showed an impact factor for aging equal to F(2,101) = 5.23, p = 0.007; time to spike had a significant value of F(2,110) = 8.76, p = 0.0003; and the spike amplitude was significant as well, F(2,111) = 13.39, p < 0.0001 (Fig. 9B–D). Chronic neuroinflammation significantly affected the spike rheobase F(1,101) = 11.89, p = 0.0008, but not the time to spike (F(1,110) = 0.47, p = 0.49, two-way ANOVA) or the spike amplitude (F(1,111) = 0.35, p = 0.55, two-way ANOVA). Similarly, the interaction factor between aging and chronic neuroinflammation was significant for the spike rheobase (F(2,101) = 12.61, p < 0.0001, two-way ANOVA) but not for the time to spike (F(2,110) = 2.45, p = 0.09, two-way ANOVA) and spike amplitude (F(2,111) = 0.37, p = 0.069, two-way ANOVA).

Fig. 9
figure 9

The impact of aging and chronic neuroinflammation on the active membrane properties of cholinergic neurons in the MS. A Example action potential trace, detailing the parameters of spike amplitude and time to spike. BD Box plots depicting the intrinsic active properties of the cholinergic neurons in the MS of control and GFAP-IL6 mice at young (light grey), adults (pink), and old (dark red) groups. The properties shown are rheobase (B), time to spike (C), spike amplitude (D). Data are shown as box plot, the box upper and lower limits are the 25th and 75th quartiles, respectively. The whiskers depict the lowest and highest data points, the horizontal line through the box is the median and the + sign represents the mean, two-way ANOVA, with Tukey’s post hoc test. Post hoc effect of ‘genotype’ indicated by asterisks, (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); post hoc effect of ‘age’ indicated by hash symbols (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001)

The analysis of the spike rheobase revealed significant differences in the GFAP-IL6 mice, as in the old (33.75 ± 2.46 pA, n = 20, ##p < 0.01, two-way ANOVA with Tukey’s post hoc test) and in the adult groups (26.90 ± 1.6 pA, n = 21, ####p < 0.0001, two-way ANOVA with Tukey’s post hoc test) the rheobase current decreased compared to the young GFAP-IL6 (47.19 ± 2.67 pA, n = 16), with the lowest current amplitude reached by the adult group (Fig. 9B). Post hoc analysis indicated that in the adult age group, the spike amplitude (Fig. 9D) increased in both genotypes compared to the young group [(control adult, 68.77 ± 3 mV, n = 21; control young, 56.71 ± 2.97 mV, n = 19, #p < 0.05, two-way ANOVA with Tukey’s post hoc test); (GFAP-IL6 adult, 72.62 ± 1.8 mV, n = 20; GFAP-IL6 young, 57.81 ± 3.43 mV, n = 21, ##p < 0.01, two-way ANOVA with Tukey’s post hoc test)]. These results indicate an increase in the excitability profile of the old and adult GFAP-IL6, which is also associated with an increase in the time to spike compared to the young group (Fig. 9C) (GFAP-IL6 young, 35.37 ± 2.87 ms, n = 21; GFAP-IL6 adult, 45.73 ± 0.94 ms, n = 19, ##p < 0.01; GFAP-IL6 old, 46.63 ± 2 ms, n = 20, ##p < 0.01, two-way ANOVA with Tukey’s post hoc test).

Neuronal firing properties are highly dependent on their excitability profile and reflect the dynamic interplay between their passive and active characteristics (Fig. 10). We therefore assessed the impact of chronic neuroinflammation and aging on the firing patterns of cholinergic neurons in the MS. To this extent, we measured the firing frequency–current relationship (F–I curves) following injections of increasing step currents, as illustrated in (Fig. 10A). Our results indicate comparable gains between all age groups of control mice, suggesting that aging itself does not affect the firing properties (Fig. 10B, C). The gain of the F–I curve showed a tendency to increase in the young (2.63 ± 0.33, n = 21) and adult (2.42 ± 0.2, n = 20) GFAP-IL6 mice compared to their age-matched control mice (young control, 2.06 ± 0.2, n = 21; adult control, 1.88 ± 0.22, n = 21 (Fig. 10D). Under physiological conditions, neuronal firing patterns are embedded within network oscillations termed brain waves. In order to assess the firing patterns of BCFN neurons under oscillatory regimes, we measured the relationship between membrane oscillation frequencies and spike threshold through injection of sinusoidal currents at increasing frequencies (0.1–100 Hz) (Fig. 10E) and different intensities (30–250 pA chirp current). This protocol allows an evaluation of the relationship between neuronal excitability and oscillatory behaviour, depicted by the frequency spiking curve (SFC), as we previously described [59]. Comparison between SFC’s recorded in control mice indicated an age-dependent biphasic pattern, in which neurons from adult mice show a decrease in the max firing frequency under high stimulation (250 pA) (Fig. 10F, G), which increased in old mice. However, a comparison between the oscillatory activity in control and IL-6 mice indicated a significant increase of the SFC’s gain only in the adult age group (Fig. 10H). Our data show that following 60, 125, and 250 pA chirp stimulation, cholinergic neurons of the GFAP-IL6 adult group are capable of firing action potentials at higher oscillation frequencies compared to the adult control mice and overall are more excitable than their age-matched controls (gain of SFC adult control, 0.096 ± 0.01, n = 22,; adult GFAP-IL6, 0.16 ± 0.01, n = 26, ***p < 0.001, two-way ANOVA with Tukey’s post hoc test (Fig. 10H). These results suggest that chronic neuroinflammation effects the excitability profile of cholinergic neurons in the MS, specifically the adult age group.

Fig. 10
figure 10

The effect of aging and chronic neuroinflammation on the excitability profile of cholinergic neurons in the MS. A Sample voltage traces recorded after step current injections from 10 pA to 300 pA representing the firing frequency–current relationship (F–I curve). B, C Graphs of the F–I curves for all the groups describing the relationship between the current injected into cholinergic neurons and the firing frequency associated with the stimulus. The upward shift of the F–I curve in the young and adult group of IL6-control is indicative of increased excitability. D Box plot depicting the average gain of the F–I curves in all groups tested. E Sample voltage traces recorded following sinusoidal chirp stimulation (0.1–100 Hz) at different intensities (from bottom to top: 30 pA, 60 pA, 125 pA, 250 pA). F, G Graphs of the SFC curves for all the groups describing the relationship between the oscillation intensity and the maximal frequency at which the cell is still excitable. Note the upward shift in the maximal oscillation frequency in adult GFAP-IL6 mice, indicative of increased responsiveness compared to the age-matched control group. H Box plot depicting the average gain of the SFC curves in all groups tested. Data are shown as box plot, the box upper and lower limits are the 25th and 75th quartiles, respectively. The whiskers depict the lowest and highest data points, the horizontal line through the box is the median and the + sign represents the mean, two-way ANOVA, with Tukey’s post hoc test. Post hoc indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)



Source link