Effects of Probiotic Supplementation on Short Chain Fatty Acids in the App NL - G - F Mouse Model of Alzheimer’s Disease 1

Animals

App^NL-G-F mice were obtained from Dr. Takaomi C. Saido, Laboratory for Proteolytic Neuroscience, RIKEN Center for Brain Science, Japan. These mice were selected for use as these mice do not over express APP and expression is under endogenous promoter control. However, Aβ is elevated due to the combined effects of three mutations associated with familial AD. An APP construct containing a humanized Aβ region, includes the Swedish “NL”, the Iberian “F”, and the Arctic “G” mutations was used [22]. Fifteen female App^NL-G-F and 15 C57BL/6 (wild type) control mice were used at 6–8 months of age. Both genotypes were bred and housed under the same conditions (room, diet, bedding, lighting, etc.) to avoid potential artifacts due to the environment that are relevant in microbiome studies. Mice were weaned and used at 6–8 months age to ensure acclimation to the environment at the start of the treatment regimen. Animal use was approved by the University of North Dakota Institutional Animal Care and Use Committee (IACUC) protocol. The animals were provided food and water ad libitum and housed in a specific pathogen-free room with a 12 h light/dark cycle.

Animal groups and probiotic (VSL#3) dosing

Animals were randomly assigned into four groups: 1) wild type supplemented with vehicle (WT Vehicle), 2) wild type supplemented with VSL#3 (WT VSL), 3) App^NL-G-F supplemented with vehicle (App^NL-G-F Vehicle), and 4) App^NL-G-F supplemented with VSL#3 (App^NL-G-F VSL#3). The vehicle used was MediGel® (Clear H₂O, Portland, ME, USA) and VSL#3 was dissolved in the vehicle and administered to animals daily at estimated mean water consumption of 5 mL/25 g mouse/day. The treatment was given for eight weeks and the dose of probiotic VSL#3 (0.32×10⁹ CFU bacteria/25 g mice) was calculated based on the body surface area normalization method from the recommended human dose of VSL#3 [23]. VSL#3 is a commercially available probiotic cocktail (Leadiant Biosciences, Inc.., Gaithersburg, MD, USA) of eight strains of lactic acid-producing bacteria: Lactobacillus plantarum, Lactobacillus delbrueckii subsp. Bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, and Streptococcus salivarius subspecies, thermophilus. VSL#3 is a well-known medical food that has been shown to improve disease symptoms in the treatment of ulcerative colitis and pouchitis, an inflammation of the ileal pouch in colectomy patients, liver cirrhosis and hepatic encephalopathy [24, 25].

Antibodies and reagents

Antibodies against GFAP and Aβ were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Anti-c-Fos antibody was purchased from Abcam (Cambridge, MA, USA). Anti-Iba-1 antibody was purchased from Wako Chemicals USA, Inc. (Richmond, VA, USA). Elite Vectastain ABC reagents, Vector VIP, biotinylated anti-rabbit, and anti-mouse antibodies were purchased from Vector Laboratories Inc (Burlingame, CA, USA). The Aβ_1–40 and Aβ_1–42 enzyme-linked immunosorbent assay (ELISA) kits were obtained from EMD Millipore (Burlington, MA). Standards used in mass spectroscopy analysis were purchased from Millipore-Sigma Burlington, MA, USA. Auto sampler vials were obtained from ThermoFisher Scientific, (Waltham, MA, USA), Silanized micro-vial inserts from Agilent (Santa Clara CA; part #5181-8872) and inserts were from VWR (Radnor, PA, USA).

Fecal sample collection and microbiome analysis

After 8 weeks of probiotic feeding, fecal samples were obtained by placing each mouse separately in a clean cage for 10–30 min and fecal pellets were collected in a sterile 1.5 mL Eppendorf tube. More than 90% of the mice excreted a fecal pellet within one min of being placed into a clean cage. In those instances, in which a mouse did not pass stool within 30 min, the mouse was gently picked up vertically by the tail for 20–30 s until a stool pellet was excreted and collected. Samples were stored at 4°C until all samples were collected and later stored at –80°C until microbiome analysis. The fecal DNA samples were sent to RTL Genomics (Research and Testing Laboratory, Lubbock, TX, USA) for 16S rRNA sequencing. Hypervariable regions V1 to V3 of 16S rRNA gene were amplified for sequencing at RTL Genomics in a reaction using HotStarTaq Master Mix Kit (Qiagen, Inc., Germantown, MD, USA) with the universal primer set 27F/519R.

Short chain fatty acid analysis

Carboxylic acids moiety of SCFAs were analyzed as nitrophenyl hydrazide derivatives (CANPD). Proteins from serum (10μL) and hippocampus (10 mg) were precipitated after sonication with 280μL of methanol containing butyrate-¹³C₄, propionate-¹³C₃, and lactate-¹³C₃ (20 ng each) as internal standards. After centrifugation at 10,000×g, the supernatant was subjected to derivatization to obtain carboxylic acids nitrophenyl hydrazides as described in [26] with modifications. Because ethanol showed high levels of short chain carboxylic acid contamination, it was substituted with methanol for the derivatization reaction. All reagents for derivatization were from Millipore-Sigma Burlington, MA, USA. Briefly, the supernatant volume was brought up to 300μL with methanol, mixed with 300μL of pyridine, 300μL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (250 mM in methanol), 300μL of 2-Nitrophenyl hydrazine hydrochloride (20 mM in methanol), and incubated at 60°C for 20  min. After quenching with 200μL of potassium hydroxide (15% in 20% methanol) at 60°C for 20 min, 3 mL of Phosphoric acid (0.5 mM in water) were added to each sample. CANPD were extracted with 4 mL of ether and the extract was washed with 4 mL of water. Centrifugation at 1,500×g was used to separate phases during extraction and washing steps.

An LC-MS method was used for CANPD quantification. CANPD extracts were evaporated under nitrogen stream, transferred to silanized microvial inserts (Agilent, Santa Clara CA; part #5181-8872) with 100μL of ether, evaporated, re-dissolved in 20μL of 50% methanol in water, and 10μL was analyzed on UPLC-MS/MS in the selected ion monitoring mode.

UPLC separation was performed on a Waters ACUITY UPLC HSS T3 column (1.8μM, 100 Å pore diameter, 2.1×150 mm, Waters, Milford, MA) with an ACUITY UPLC HSS T3 precolumn (1.8μM, 100 Å pore diameter, 2.1×5 mm, Waters) at a temperature of 55°C using a previously described method [2, 3]. The LC system consisted of a Waters ACUITY UPLC pump with a well plate autosampler at 8°C. Solvent A was water containing 0.1% formic acid and solvent B was acetonitrile with 0.1% formic acid. The flow rate was maintained at 0.45 mL/min. CANPD were eluted with increasing gradient of solvent B from 39% to 98% as we previously described [27]. A quadrupole time-of-flight mass spectrometer (Q-TOF, Synapt G2-S, Waters) was used for detection as described previously [28]. The electrospray ionization was set in negative ion mode with the cone voltage at 20 V, and the capillary voltage at 1.51 kV. The source and desolvation temperatures were 110°C and 350°C, respectively. MSE mode was used to collect data. Leucine enkephalin (400 pg/μL in ACN: water, 50:50 by volume) was used for mass correction. MassLynx V4.1 software (Waters) was used for instrument control, acquisition, and sample analysis. Nitrophenyl hydrazide derivatives of acetate (m/z = 194.0566), and propionate (m/z = 208.0722) were quantified against propionate-13C3(m/z = 211.0823), lactate (m/z = 224.0671) and pyruvate (m/z = 222.0515) were quantified against lactate-13C3,(m/z = 227.0772), and butyrate and isobutyrate (m/z = 222.0879) - against butyrate-13C4 (m/z = 226.1013).

Ki-67 stereology and counting in hippocampus

Immunopositive Ki-67 nuclei were counted in the subventricular zone (SVZ) using an Olympus BX51WI microscope with a motorized XYZ stage. Unbiased quantification of nuclei was conducted using the optional fractionator workflow in StereoInvestigator11.0 (Micro Brightfield, Inc., Wiliston, VT). For cell counting, a tight contour was outlined around the subvertical zone at the same region for all the treatment conditions. Positive nuclei were counted in every 24th section in systematically selected frames based on an optical dissector frame (50×50μm) and a grid size (125×125μm) that were empirically determined by initial oversampling. The section thickness was measured at each sampling site. The sampling and counting paradigm resulted in a Coefficient of Error (CE) less than 0.1 (10%) for all samples counted. The total numbers were estimated in Stereo Investigator with the optical fractionator formula (N = 1/ssf.1/asf.1/hsf.ΣQ–) where ssf = section sampling fraction (10), asf = area sampling fraction (area sampled/total area), hsf = height sampling fraction (counting frame height/30μm), and ΣQ–(total particle count). Four sections (40μm) per brain were used at a counting interval of every 24th section and 7 brains were quantified per treatment condition. Cells in the dentate gyrus were counted manually due to the few Ki-67 positive cells being present in hippocampus.

Immunohistochemistry

Following the eight-week treatment paradigm, the left brain hemisphere of each animal was fixed in 4% paraformaldehyde and prepared for histologic analysis as described previously [29]. Briefly, paraformaldehyde-fixed tissue was embedded in a 15% gelatin (in 0.1 M phosphate buffer) matrix and immersed in a 4% paraformaldehyde for 2 days to harden the gelatin matrix followed by two changes of 30% sucrose for cryoprotection for two days each. The blocks were then flash frozen using dry ice/isomethylpentane, and 40μm serial sections were cut using a freezing microtome. Serial sections of 40μm were stored at 4°C in PBS with 0.1% sodium azide until immunostaining was performed. Anti-Aβ (1:1000), anti-GFAP (1:700), anti-Iba-1 (1:750), anti-cFos (1:2000), and anti-Ki-67 (1:500, used sodium citrate, heat mediated antigen retrieval) antibodies were used for immuno detection in sections followed by incubation with biotinylated secondary antibodies (1:2000 dilution; Vector Laboratories) and an avidin/biotin complex (Vector ABC kit). Immunoreactivity was visualized using Vector VIP as the chromogen. The stained sections were mounted onto gelatin-coated slides and cover slipped using Permount (Vector Laboratories) following a standard dehydrating procedure through a series of ethanol concentrations and Histo-Clear (National Diagnostics, Atlanta, GA, USA). A Leica DM1000 microscope and Leica DF320 digital camera system (Leica Microsystems Inc., Buffalo Grove, IL, USA) were used to take images and quantification of immuno staining was performed as described previously [30]. Briefly, optical densities from serial hippocampus sections from each condition were measured using Adobe Photoshop software (Adobe Systems). The average values for each condition were analyzed and plotted. This entailed 2 hippocampus sections per brain, 7 brains per condition, or 14 sections from each condition that were used for immunohistochemistry analysis.

Enzyme linked immunosorbent assay (ELISA) for amyloid-β

Following the eight-weeks treatment paradigm, the animals were euthanized via CO₂ asphyxiation followed by cervical dislocation and cardiac exsanguination. The hippocampi of each animal were collected for biochemical procedures. The right hippocampus of each animal was collected, flash frozen in liquid nitrogen, and stored at –80°C for subsequent use. A small part of the hippocampus was weighed and lysed in ice cold radio immuno precipitation assay buffer (RIPA) buffer (20 mM Tris, pH 7.4, 150 mM sodium chloride (NaCl), 1 mM sodium orthovanadate (Na₃VO₄), 10 mM sodium fluoride, 1 mM ethylene diaminetetraacetic acid (EDTA), 1 mM (ethylene glycol-bis (β-aminoethyl ether)-N, N, N^′, N^′-tetraacetic acid) EGTA, 0.2 mM phenylmethylsulfonyl fluoride, 1% Triton, 0.1% sodium dodecyl sulfate (SDS), and 0.5% deoxycholate) with protease inhibitors (4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride 1 mM, aprotinin 0.8μM, leupeptin 21μM, bestatin 36μM, pepstatin A 15μM, E-64 14μM). The samples were homogenized in a Bullet Blender Storm homogenizer 24 (Next Advance, Inc. Troy, NY, USA) and later centrifuged to remove the insoluble content. The resulting clear tissue lysate (supernatant) was used for soluble Aβ_1–40 and Aβ_1–42 ELISAs. The pellet or insoluble content was re-suspended in 5 M guanidine HCl/50 mM Tris HCl, pH 8.0 and samples were again bullet blended and centrifuged (21,000 g, 4°C, 10 min) and the supernatant was removed and used to determine insoluble Aβ concentrations (Aβ_1–40 and Aβ_1–42 ELISA). The levels of soluble and insoluble Aβ_1–40 and Aβ_1–42 were quantified by using commercially available ELISA kits from Millipore Sigma. The levels of Aβ_1–40 and Aβ_1–42 (soluble and insoluble) were reported as pg/mL per mg protein derived from a standard curve established for each protein. Protein concentrations of cell lysates were determined using the bicinchoninic acid (BCA) protein determination assay (ThermoFisher Scientific).

Behavioral analysis

Light-dark box (Stoelting) testing was performed to assess anxiety-like behavior in the mice [31, 32]. The light/dark box consisted of two compartments, light and dark, connected by a door in the middle of the wall that separated the two compartments. Each animal was placed individually in the light compartment and allowed to move freely in the apparatus for 10 min. The movement was recorded and analyzed by Anymaze software (Stoelting Co., Wood Dale, IL). The total number of entries into the dark and light chamber, the total time spent in the light chamber (percentage of time spent in light) and latency to first enter the dark compartment were examined. After each trial, the chambers were cleaned, and the next mouse was placed for testing.

Statistical analyses

Statistical analyses were performed using Graphpad Prism (Prism version 7.00 for Windows, GraphPad Software, La Jolla California USA). The tests for outliers (Grubb’s test) and normality (Shapiro-Wilk test) were performed on each data set. If the data followed normal distribution a t-test (two tailed) was performed. If the data was not normally distributed, a nonparametric Mann Whitney test was performed to compare the two conditions. All plots in the manuscript were generated using GraphPad software. Results are presented as mean values±the standard error of the mean (SEM). Differences were considered significant when p < 0.05 and indicated in the figure legend as appropriate.

Probiotic supplementation altered the fecal microbiome of both C57BL/6 (WT) and App^NL-G-F mice

To investigate the existence of gut dysbiosis and the effect of eight weeks of VSL#3 probiotic supplementation in AD mice, fecal samples of WT and App^NL-G-F (AD) mice were subjected to 16S rRNA sequencing. The relative abundance results showed a dramatic difference across treatments in control and AD mice (Fig. 1A). App^NL-G-Fmice had a significant increase in Clostridia, Lachnospiracea, and Akkermansia genera and both an increase and a decrease in order Bacteroidales following probiotic treatment (Fig. 1B). While a decrease in Lachnospiracea, Alistepes, and Oscillibacter was observed in WT mice treated with probiotics (Fig. 1B).

OPEN IN VIEWER

Fig.1

Effects of VSL#3 probiotic supplementation on fecal bacterial genus profile in App^NL-G-Fand wild type mice. Fecal samples were collected from female C57BL/6 wild type and App^NL-G-Fmice with and without ad libitum VSL#3 in the drinking water from 6–8 months of age. Bacterial genera were determined from 16S rRNA sequencing. A) Relative abundances of the 30 most abundant genera in stool samples of wild type (WT) and App^NL-G-Fmice are graphed. B) The relative abundances of the top 15 most abundant genera across the treatment groups were statistically compared. Operational taxonomic units (OTUs) of some samples lacked a taxonomy label at the genus level and are represented as order or family only. 6 animals per group were examined. Data are presented as mean values±standard error of mean (SEM). Significant differences between vehicle and VSL#3 treated animals in each genotype were determined by Mann–Whitney test, *p < 0.05.

Probiotic supplementation increased serum SCFAs inWT and App^NL-G-Fmice

In order to investigate the effect of altered gut bacterial composition on bacterial fermentation end-products, we elected to quantify levels of SCFAs, acetate, butyrate, isobutyrate, lactate, and propionate in serum from WT and App^NL-G-F mice. We also examined the impact of probiotic supplementation on the concentration of SCFAs in serum. Supplementation with probiotic significantly increased acetate, butyrate, lactate, isobutyrate, and propionate in the serum of VSL#3 fed App^NL-G-Fmice compared to their vehicle controls (Fig. 2). The dietary response was highly variable between genotypes, as probiotic supplementation increased only acetate and lactate in WT mice (Fig. 2). To understand the association between alterations in serum SCFAs and changes in gut bacteria, a Pearson’s correlation analysis was performed, and the results graphed as a correlation map (Fig. 3A) and as a p value map (Fig. 3B) for comparison. The results showed that an increase in serum propionate in vehicle fed App^NL-G-F mice correlated with a high abundance of Clostridia class bacteria and a low abundance of the Parasutterella genus. However, there was no significant correlation between increased other serum SCFAs and any of the bacteria genera. Following probiotic supplementation, the order Bacteroidales positively correlated with an increase in serum butyrate and isobutyrate in App^NL-G-F mice (Fig. 3). A decrease in the genus Parasutterella negatively correlated with an increase in lactate in brains of App^NL-G-F mice treated with probiotics (Fig. 3).

OPEN IN VIEWER

Fig.2

Effects of VSL#3 supplementation on serum concentrations of short chain fatty acids (SCFAs) acetate (A), butyrate (B), isobutyrate (C), lactate (D), and propionate (E). Female C57BL/6 wild type and App^NL-G-F mice were provided normal drinking water or water supplemented with probiotic, VSL#3, ad libitum from 6–8 months of age. Blood was collected upon termination and centrifuged to collect serum for SCFA analysis performed using UPLC-MS/MS. Seven animals per group were examined. Data are presented as mean±SEM. Significant differences between vehicle and VSL#3 treated animals in each genotype were determined by t-test; *p < 0.05.

OPEN IN VIEWER

Fig.3

Comparison of serum SCFAs and fecal microbiome bacteria in C57BL/6 (WT) and App^NL-G-F mice. Heat maps are presented of the Pearson correlation (A) and the p value (B) of serum SCFAs and fecal bacterial composition. Asterisks in (A) indicate statistically significant positive or negative correlation.

Probiotic supplementation increased hippocampal SCFAs in App^NL-G-F mice

The impact of probiotic supplementation on levels of acetate, butyrate, isobutyrate, lactate, and propionate were assessed in hippocampi from WT and App^NL-G-F mice. Surprisingly, probiotic supplementation decreased acetate and propionate SCFAs in wild type mice (Fig. 4). Similar to the serum findings, elevated levels of acetate and lactate were observed in the hippocampi ofVSL#3 fed App^NL-G-F mice (Fig. 4). Correlation analysis was performed comparing fecal bacterial composition and brain SCFAs using Pearson’s correlation and graphed as a correlation maps (Fig. 5A) or a p value map (Fig. 5B). The increase in hippocampal acetate positively correlated with the genus Lactobacillus and negatively correlated with the genus Parasutterella in App^NL-G-F mice treated with probiotics (Fig. 5). The VSL#3-induced increase in hippocampal lactate levels negatively correlated with class Clostridia (Fig. 5).

OPEN IN VIEWER

Fig.4

Effect of VSL#3 supplementation on hippocampal concentrations of short chain fatty acids (SCFA) acetate (A), butyrate (B), isobutyrate (C), lactate (D), and propionate (E). Female C57BL/6 wild type and App^NL-G-Fmice were provided normal drinking water or water supplemented with probiotic, VSL#3, ad libitum for four weeks from 6–8 months of age. Hippocampi were isolated, and concentrations of SCFA, were analyzed using UPLC-MS/MS. Seven animals per group were examined. Data are presented as mean±SEM. Significant differences between vehicle and VSL#3 treated animals in each genotype were determined by t-test; *p < 0.05.

OPEN IN VIEWER

Fig.5

Comparison of hippocampal SCFAs and fecal microbiome bacteria in C57BL/6 (WT) and App^NL-G-F mice. Heat maps are presented of the Pearson correlation of (A) and the p value for hippocampal SCFAs and fecal bacterial composition. Asterisks in (A) indicate statistically significant positive or negative correlation.

Probiotic supplementation had no effect on Ki-67 immunoreactivity in App^NL-G-F mice

Recently, gut microbiota have been reported to influence neurogenesis in the brain[33]. Using Ki-67 c-Fos immunoreactivity as a maker of proliferation and a surrogate for possible neurogenesis changes, we investigated whether altered gut microbiota in App^NL-G-F mice had any effect on proliferation in the dentate gyrus and subventricular zone. Although there appeared to be less overall Ki-67 immunoreactivity in App^NL-G-Fdentate gyrus compared to WT, the probiotic supplementation for eight weeks showed no effect on Ki-67 immunoreactivity in either the dentate gyrus or the subventricular zone ofApp^NL-G-F or WT mice (Fig. 6).

OPEN IN VIEWER

Fig.6

Effect of VSL#3 supplementation on neurogenesis in the subventricular zone (SVZ) and dentate gyrus of the hippocampus. Brain sections from female C57BL/6 wild type and App^NL-G-Fmice with and without VSL#3 supplementation from 6–8 months were stained with Ki-67 antibody. Seven animals per group were examined and Ki-67 positive cells were counted in 4 sections from each animal. Stereological quantification was done in the (A) SVZ region to count Ki-67 positive cells. B) However, manual counting was done in the dentate gyrus due to the limited numbers of detectable Ki-67 positive cells. Representative images are shown, and data are presented as mean±SEM. Significant differences between vehicle and VSL#3 treated animals in each genotype were determined by t-test; * p < 0.05.

Probiotic supplementation increased c-Fos immunoreactivity in App^NL-G-F mice

As a measure of assessing probiotic supplementation effects on neuronal activity, c-Fos immunostaining was performed on brain sections. c-Fos is commonly used as a functional marker of activity in neurons [34]. Eight weeks of probiotic supplementation resulted in a significant increase in c-Fosimmunoreactivity in App^NL-G-F mice treated with VSL#3 with no change in WT mice (Fig. 7).

OPEN IN VIEWER

Fig.7

Effect of VSL#3 dietary supplementation on hippocampal c-Fos immunoreactivity. Female C57BL/6 wild type and App^NL-G-F mice were provided normal drinking water or water supplemented with probiotic, VSL#3, ad libitum from 6–8 months of age. Brain sections from each treatment group were stained with anti-c-Fos antibody (purple labeling). Representative images of dentate gyrus (DG) of hippocampi are shown at 10X, 20X, and 100X magnification with the positive cells clearly visible at the higher magnifications and the anatomical references points indicated for dentate gyrus. The positive cells were counted and values were averaged from 3 sections per brain. Six animals per group were examined and the data are presented as the mean±SEM. Significant differences between vehicle and VSL#3 treated animals in each genotype were determined by t-test; *p < 0.05.

Probiotic supplementation had no effect on Aβ, GFAP, and Iba-1 immunoreactivityin App^NL-G-F mice

Since we observed a remarkable difference in serum and brain SCFA levels following probiotic supplementation, we examined whether changes occurred in plaque load and gliosis in App^NL-G-F mice. Immunohistochemistry for Aβ, GFAP, and Iba-1 was performed using brain sections from WT and App^NL-G-F mice. Surprisingly, probiotic supplementation did not alter microgliosis, astrogliosis, or Aβ plaque load in App^NL-G-Fmice (Fig. 8).

OPEN IN VIEWER

Fig.8

Effect of VSL#3 supplementation on Aβ accumulation and gliosis in brains of C57BL/6 (WT) and App^NL-G-F mice. Female C57BL/6 wild type and App^NL-G-F mice were provided normal drinking water or water supplemented with probiotic, VSL#3, ad libitum from 6–8 months of age. Brain sections were immunolabeled for Aβ (A), GFAP (B), and Iba-1 (C). Representative images are shown from the hippocampus region and were taken at 20X. Quantitation of immunolabeling was performed using optical density values and the data are presented as mean±SEM. Significant differences between vehicle and VSL#3 treated animals in each genotype were determined by t-test; * p < 0.05 (n = 7). Scale bar is 50μm.

Probiotic feeding had no effect on Aβ levels in the hippocampus App^NL-G-F mice

In order to provide a more quantifiable assessment of brain Aβ levels, ELISAs were next performed. The levels of Aβ_1–40 and Aβ_1–42, both soluble and insoluble, were assessed from the hippocampi of WT and App^NL-G-Fmice. As expected, App^NL-G-F mice demonstrated high levels of soluble and insoluble Aβ_1–40 and Aβ_1–42 (Fig. 9). However, probiotic supplementation of App^NL-G-Fanimals had no effect on Aβ levels, consistent with our immunolabeling findings (Fig. 9).

OPEN IN VIEWER

Fig.9

Effect of VSL#3 dietary supplementation on hippocampal Aβ levels. Female C57BL/6 wild type and App^NL-G-Fmice were provided normal drinking water or water supplemented with probiotic, VSL#3, ad libitumfrom 6–8 months of age. Hippocampi were isolated, lysed, and levels of Aβ_1–40 and Aβ_1–42, both soluble and insoluble, were quantified by ELISA. Seven animals per group were examined and the data are presented as mean±SEM. Significant differences between vehicle and VSL#3 treated animals in each genotype were determined by t-test; *p < 0.05.

Probiotic supplementation altered anxiety-like behavior in App^NL-G-F mice

Even though we did not observe a change in plaque load or gliosis in VSL#3 fed App^NL-G-Fmice, the increased c-Fos immunoreactivity and changes in brain SCFA levels demonstrated clear brain effects. Since we previously did not observe probiotic feeding effects on memory in App^NL-G-Fmice [35], we elected to quantify whether VSL#3 feeding might influence a distinct behavioral dysfunction associated with AD, anxiety. Anxiety-like behavior was quantified using a light-dark apparatus. The total number of entries into the dark chamber, total time spent in the light chamber (percentage of time spent in light) and latency to first enter the dark compartment were examined. VSL#3 supplementation of the App^NL-G-Fmice decreased the number of entries into the dark and normalized the latency to first enter the dark to WT levels demonstrating an anxiolytic effect of the probiotic feeding (Fig. 10).

OPEN IN VIEWER

Fig.10

Effect of VSL#3 supplementation on anxiety in C57BL/6 (WT) and App^NL-G-Fmice. Female C57BL/6 wild type and App^NL-G-F mice were provided normal drinking water or water supplemented with probiotic, VSL#3, ad libitum from 6–8 months of age. All mice were subjected to light-dark test after 8 weeks of treatment and (A) total number of entries into the dark compartment, (B) latency of first entry into the dark compartment, (C) time spent in the light compartment, and (D) the percentage of time spent in the light were determined. Data are represented as the mean±SEM (n = 14). Significant differences between vehicle and VSL#3 treated animals in each genotype were determined by t-test; * p < 0.05.