extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Psychology

text:
Quantitative study of the capillaries within the white matter of the Tg2576 mouse model of Alzheimer's disease

Abstract Introduction To quantitatively investigate the capillaries within the white matter of Tg2576 Alzheimer's disease (AD) transgenic mice during the early stage. Methods In the current study, 10‐month‐old male Tg2576 AD mice were used as the early‐stage AD group and age‐matched nontransgenic littermate mice were used as the wild‐type group. Then, the Morris water maze was used to examine the spatial learning and memory abilities of the mice in both groups, and unbiased stereological methods were used to accurately quantify the volume of white matter and the parameters of the capillaries within the white matter, such as the total length, total volume, and total surface area of capillaries. Results The Morris water maze performance of the Tg2576 group was worse than that of the wild‐type group, while the white matter volume did not significantly differ between the wild‐type group and the Tg2576 group. The total length, total volume, and total surface area of the capillaries within the white matter of the Tg2576 group were significantly decreased compared to those of the wild‐type group. Conclusions The current study provide structural basis for understanding the pathological changes of the early stage of AD and cognitive decline in AD might be associated with changes in the white matter capillaries. Capillaries within the white matter might, thus, serve as a valid target for the prevention and treatment of early‐stage AD.

very critical and urgently needed (Goedert & Spillantini, 2006). Over the past 20 years, several hypotheses have been proposed regarding the pathogenesis of AD. Among them, the amyloid hypothesis is the most accepted and investigated. However, the exact role of Aβ in the pathogenesis and pathological process of AD is still not entirely clear, and it remains controversial with regard to whether the amyloid hypothesis itself is valid or not. Moreover, the amyloid hypothesis has been challenged by the vascular hypothesis (Clark et al., 2017;Farkas & Luiten, 2001;Østergaard et al., 2013;Zlokovic, 2005). Currently, the findings of an increasing amount of researches show that vascular pathological changes might play a key role in the pathogenesis and pathological process of AD (van Norden et al., 2012;de la Torre, 2002a). Capillaries, the thinnest and most widely distributed blood vessel, play a vital role role in maintaining cerebral blood flow (CBF) and regulating brain microcirculation. Studies have shown that pathological changes in capillaries in the brain play an extremely important role in the pathogenesis of this devastating disease (Østergaard et al., 2013;de la Torre, 2002b). Previous studies have reported a wide variety of vascular pathological changes in the AD brain. Cerebral capillary degeneration, included looping, twining, and braiding of vessels, has been shown to be present in pathologically confirmed cases of AD (Farkas & Luiten, 2001;Østergaard et al., 2013). These pathological changes have been shown to cause brain capillary microcirculation deficits and brain hypoperfusion, which could lead to both cognitive and memory decline in AD (Attems & Jellinger, 2004;Oshima et al., 2006).
The pathological changes in brain microvessels play a crucial role in the pathogenesis of AD. There are still questions regarding the brain regions where these microvessel changes might be most relevant to the pathogenesis of AD. People take it for granted that the pathological changes in the brain microvessels of the hippocampus decisive in AD because the hippocampus mainly responsible for storage conversion of long-term memory. However, Buѐe et al's study showed that the microvascular density of the cerebral cortex was significantly decreased in Alzheimer's disease patients compared to age-matched controls (Buѐe et al., 1994). Moreover, Moody et al. re-ported that vascular changes were more often associated with white matter lesions than other regions of the brain because the capillary distribution within the white matter is relatively smaller; thus, the white matter is more susceptible to hypoxic-ischemic injury (Moody, Brown, Challa, Ghazi-birry, & Reboussin, 1997). Recently, the findings show that, in the early stages of AD, both cognitive and memory decline are closely associated with damage to the structural integrity of white matter (Bendlin et al., 2010;Huang, Friedland, & Auchus, 2007;Rémy, Vayssière, Saint-Aubert, Barbeau, & Pariente, 2015;Zhang et al., 2013). Using clinical diffusion tensor imaging (DTI) or magnetic resonance imaging (MRI), neuroimaging studies found that ischemic changes in white matter existed in mild cognitive impairment (MCI) and AD patients (Jones et al., 1999;Medina et al., 2006). Moreover, the white matter is more susceptible to hypoxic-ischemic injury (Van Gijn, 1998;Moody et al., 2004;Nonaka et al., 2003). Van Gijin thought that vascular factors might be closely related to the damage in white matter (Van Gijn, 1998). Capillaries can be easily identified by the diameter size and complex network structure by using brain transparency technology, immunohistochemistry staining, and scanning electron microscopy to observe vascular corrosion models. In a study of 109 human brains, Nonaka et al. observed the phenomena in which the blood vessels in the white matter coiled, looped, and spiraled, while the blood vessels ran straight through the cortex (Nonaka et al., 2003). Previous studies have reported that spiral-like blood vessels and periventricular venous collagenosis, which result in vascular contortion and occlusion, are usually related to white matter lesions in AD (Brown, Moody, Thore, & Challa, 2000).
Thus, capillary microcirculation changes may be a critical structural basis for white matter lesions in AD. Therefore, the capillary pathological changes have been thought to be a crucial source of cognitive impairment in AD (Farkas & Luiten, 2001;van Norden et al., 2012;Salat et al., 2009;de la Torre, 2002ade la Torre, , 2002b. However, no study has quantified the capillary pathological changes in the white matter of early stage in AD patients or AD models.
In this study, we used the Morris water maze behavior test to investigate spatial learning and memory abilities, and then used accurate three-dimensional quantitative stereological methods to investigate the white matter and the parameters of the capillaries within the white matter of 10-month-old Tg2576 AD mice. The present study could not only provide an accurate quantitative methodological design for future studies on the capillaries changes in the AD white matter, but also provide a structural basis for capillary pathological changes in the white matter during early-stage AD.

| Animals
Eleven Tg2576 AD male mice (Overexpress the Swedish mutation of human amyloid precursor protein, APP695swe) and 11 nontransgenic littermate male mice (wild-type) were selected randomly at 10 months of age.
All mice were raised by experimental animal center of Chongqing Medical University, P. R. China. All mice were reared in a standard environment, and the animal experiment followed the National Institute of Health's guide for the care and use of laboratory animals.
Tg(APPswe) sequence：transgene = ~ 350 bp oIMR: 5′ ctg acc act cga cca ggt tct ggg t 3′ oIMR: 5′ gtg gat aac ccc tcc ccc agc cta gac ca 3′ DNA extraction: The mouse tail tip was cut and incubated with proteinase K overnight and the DNA was extracted with a genomic extraction reagent.
PCR protocol: First, a 25 μl PCR system was prepared with 9 μl ultrapure water +0.5 μl upstream primer +0.5 μl downstream prime +2.5 μl DNA template (extracted) +12.5 μl 2 × premixed Taq PCR mix. Then, the target DNA samples were added and PCR amplification was performed by using the PCR cycle program. The PCR cycle program included predenaturation at 94°C for 3 min, denaturation at 94°C for 45 s, annealing at 56°C for 60 s, extension at 72°C for 60 s, for a total of 30 cycles. Finally, the DNA samples were stored at 4°C. Electrophoresis: 1.5% agarose gel was prepared, and 3 μl target DNA and DNA maker in each panel were added. Electrophoresis at 90V for 30 min. After the end of electrophoresis, the gel was observed in the gel imaging system.

| Morris water maze
The Morris water maze test was used to measure the spatial learning and memory abilities of the mice (Chen et al., 2000;Morris, 1984). In this study, the test lasted seven consecutive days and included two parts. Part one was the hidden platform test and was conducted within 1-6 days. A platform with a fixed position was placed ~1 to 1.5 cm underwater, with every test time up to 60 s, and the escape latency (the real-time mouse find the platform) was recorded as an evaluation of spatial learning and memory capabilities. Part two was the probe trial test, which was conducted on the seventh day. The probe trial provided an index of the mice tendency to persist around the platform's previous location and is generally considered to be a measure of retention memory. After completion of the hidden platform test, the original platform was removed, each mouse was given 60 s to freely swim, and the frequency of target zone crossings and the percentage of time spent in the target quadrant zone were recorded to evaluate spatial exploration (Chao et al., 2018).

| Brain preparation
The mice were weighed and anaesthetized with 0.4 ml/100 g pentobarbital sodium and then perfused with 0.9% sodium chloride plus heparin followed by 4% paraformaldehyde. After the perfusion, the cerebral hemispheres were removed. Then, the hemispheres were embedded with 6% agar in the Mouse Brain Matrices (Rayward life technology co. LTD; Shenzhen, China). Using knives to cut each hemisphere into continuous 1-mm-thick coronal slices (Figure 1a).

| Estimation of the white matter volume
The caudal surface of each 1-mm-thick coronal slice was photographed using anatomical microscope. Then, an equidistant counting grid probe was superimposed at random onto each captured photograph, and the points hitting the white matter were counted ( Figure 1b). The total volume of the white matter was calculated according to Cavalieri's estimator (Tang, Nyengaard, Pakkenberg, & Gundersen, 1997;Zhang et al., 2017).
where Vwm means the total volume of the white matter, a (p) means the area represented by each measuring point of the equidistant counting grid probe (0.59 mm 2 ), t means the thickness of coronal

| White matte tissue sampling and frozen sections
A transparent equidistant counting grid was superimposed at random onto each 1-mm-thick coronal slice. Based on the points that hit the white matter, three or four tissue blocks were randomly sampled ( Figure 1c). After being postfixed, dehydrated, and embedded, the tissue blocks were sectioned at 4 μm in four different planes, randomly. Using the isector technique, the sampled tissue was cut into isotropic sections, which assumed that the capillaries had the same probability of being sampled in every direction of 3D space (Nyenggard & Gundersen, 1992).

| Immunohistochemical procedures
Using the streptavidin-peroxidase (SP) method, the immunohistochemistry was performed by several steps. Briefly, the sections were fixed with acetone at 4°C for 10 min, and pretreated with 0.01 M citrate buffer solution at 99°C for 20 min for antigen retrieval, and then the sections were pretreated with 3% hydrogen peroxide at 25°C for 15 min and incubated with the working solution of normal goat serum for 30 min at 37°C. After washing thrice with PBS, a 1:500 dilution of anti-mouse CD31 primary antibody (Abcam, Cambridge, UK) was prepared and used on labeled vascular endothelial cells (Balcells et al., 2010;Cheung et al., 2015;Nojiri et al., 2015;Vecchi et al., 1994). The slices were overnight incubated with CD31 work solutions at 4°C.
The next day, after rewarming and washing, the goat anti-mouse immunoglobulin G secondary antibody working solution was added to the slices at 37°C for 30 min. Followed by washing, the sections were incubated at 37°C for 30 min with a streptavidin-horseradish peroxidase working solution. After this procedure, staining was developed with diaminobenzidine (DAB) for 60 s. In the end, the sections were dehydrated with graded ethanol, transparentized in graded dimethylbenzene alcohol solution, and mounted with neutral resins.

| Immunofluorescence procedures
The sections of both group mice were rinsed for 4 × 15 min in PBST.
After repairing in boric acid solution for 20 min, nonspecific binding sites were blocked by incubating the sections with normal goat serum for 2 hr at 37°C. The primary antibodies anti-beta amyloid (mouse, ab11132, Abcam) were then added at a dilution of 1:500 in PBS and incubated at 4°C for 72 hr and then rewarmed at 37°C for 1 hr. The secondary antibodies (1:200) DyLight 488 (Abbkine) were then added for 2 hr, followed with DAPI chemical staining. Finally, the sections were mounted on slides. The slides were then viewed and photographed under fluorescence microscope.

| Stereological sampling
The immunohistochemical sections were viewed and photographed using a modified microscope. The boundaries of the slice were drawn by using a low magnification objective lens (4×), Then, using an oil objective lens (100×), the images were captured by stereological analysis software (Glostrup, Denmark). Blood vessels with diameter less than 10 μm were defined as capillaries and were used for the stereological analysis (Alba, Vidal, Díaz, Villena, & Vargas, 2004).

| Estimation of the total length of the capillaries in the white matter
An unbiased counting frame was randomly superimposed onto each captured photograph    (Qiu et al., 2011;Shao et al., 2010;Zhang et al., 2017).
where Lv (cap/wm) is the capillary length density in the white matter, ΣQ (cap) is the total number of the capillary profiles counted per white matter sections, and ΣA is the total area of the unbiased counting frames used in the white matter. The capillary total length in the white matter then equaled the value of capillary length density in the white matter multiplied by the total volume of white matter per mouse.

| Estimation of the total volume of the capillaries in the white matter
A equidistant counting grid probe was randomly superimposed onto each captured photograph. The numbers of test points hitting the white matter capillaries and the white matter were respectively counted (Figure 2b). The capillary volume density in the white matter was calculated according to the following formula (Zhang et al., 2017).
where Vv(cap/wm) is the capillary volume density in the white matter, ΣP (cap) is the total number of counting points hitting the capillaries in the white matter, and ΣP (wm) is the total number of counting points hitting the white matter.
The capillary total volume in the white matter equaled the value of capillary volume density in the white matter multiplied by the total volume of white matter per mouse.

| Estimation of the total surface area of the capillaries in the white matter
A test line probe was randomly superimposed onto each captured photograph. The intersection numbers of the test lines and the luminal surface of the capillaries were counted, and the total test line length in the white matter was recorded (Figure 2c). The capillary surface area density in the white matter was calculated according to the following formula (Zhang et al., 2017).
where Sv(cap/wm) is the capillary surface area density in the white mattter, ΣI (cap) is the total number of intersections between capillary lumens and test lines, and ΣL is the total test lines length in the white matter.
The capillary total surface area in the white matter equaled the value of capillary surface area density in the white matter multiplied by the total volume of white matter per mouse.

| Estimation of the correlations between behavioral results and stereological measurements of the capillaries results
We analyzed the Pearson correlation coefficient to investigate the relationship between spatial learning abilities and stereological data respectively. The escape latency values of the behavioral results (swimming time) of the mice sampled for stereological analyses was adjusted by the stereological measurements of the capillaries (the total length, total volume, and total surface area of the capillaries).
And the Pearson correlation coefficient was calculated to investigate the relationship between spatial learning and the stereological results.

| Statistics
The behavioral results are presented as the mean ± standard error (SEM

| Spatial learning ability
In the hidden platform test, the escape latency of the Tg2576 group mice was significantly longer than that of the wild-type group mice (p < 0.05) (Figure 3a).

| Spatial memory abilities
In the probe trial test, there was no significant difference in the cross platform frequency between the Tg2576 group mice and the wildtype group mice (p > 0.05) (Figure 3b). There was no significant difference between the Tg2576 group mice and the wild-type group mice in the quadrant percentage time (p > 0.05) (Figure 3c).

| Total volume of the white matter
The total white matter volume of the wild-type group mice was 12.87 ± 0.44 mm 3 , and the total white matter volume of the Tg2576 ∑ L group mice was 12.33 ± 1.01 mm 3 . The statistical results indicated that the white matter volume between the Tg2576 group mice and the wild-type group mice was not significant difference (p > 0.05) ( Figure 4).

| Immunofluorescent results of Aβ
The results of the immunofluorescent-stained Aβ in the white matter of both groups of mice are shown in Figure 5. High-intensity Aβ fluorescence was observed in the white matter of the Tg2576 group mice.

| Immunohistochemical results of white matter capillaries
The results of the immunohistochemical-stained capillary profiles in the white matter of both groups of mice are shown in Figure 6.

| Total length of the capillaries in the white matter
The total length of the capillaries in the white matter was 12.44 ± 2.64 m in the wild-type group mice and 6.90 ± 0.82 m in the Tg2576 group mice. The total length of the capillaries in the white matter of the Tg2576 group mice was significantly shorter than that of the age-matched wild-type group mice (p < 0.01) (Figure 7a).

| Total volume of the capillaries in the white matter
The total volume of the capillaries in the white matter was 0.0891 ± 0.0145 mm 3 in the wild-type group mice and 0.0648 ± 0.0167 mm 3 in the Tg2576 group mice. The total volume of the capillaries in the white matter of the Tg2576 group mice was significantly smaller than that of the age-matched wild-type group mice (p < 0.05) (Figure 7b).

| Total surface area of the capillaries in the white matter
The total surface area of the capillaries in the white matter was 106.81 ± 20.17 mm 2 in the wild-type group mice and 61.77 ± 13.80 mm 2 in the Tg2576 group mice. The total surface area of the capillaries in the white matter of the Tg2576 group mice was F I G U R E 3 The Morris water maze behavior results. (a) The escape latency of Morris water maze positioning navigation test in 10-month-old wild-type AD mice, Tg2576 AD mice is shown. Each point represents the average of the four escape latencies (x ± sem). * indicates p < 0.05. ** indicates p < 0.01. (b) The target zone frequency in target quadrant zone of in wild-type AD mice and Tg2576 AD mice(x ± sem.). (c) The percentage of time in target quadrant zone of wild-type AD mice and Tg2576 AD mice (x ± sem) F I G U R E 4 The total volume of the white matter of wild-type AD mice and Tg2576 AD mice (x ± SD) significantly smaller than that of the age-matched wild-type group mice (p < 0.01) (Figure 7c).

| Correlations between behavioral results and capillary results
As shown in Table 1, our data indicated that the all the stereological measurements of the capillaries within the white matter, the total length, total volume, and total surface area of the capillaries, were significantly correlated with the last day escape latency data of the water maze behavioral results both in the wild-type group and the Tg2576 group (p < 0.01) ( Table 1).

| D ISCUSS I ON
Alzheimer's disease (AD) is a central nervous system neurodegenerative disease; however, the pathological changes during the early stage of AD remain inconclusive (Querfurth & LaFerla, 2010). Past studies indicated that brain capillaries play a key role in the onset  Torre , 2002b,2004). However, the quantitative changes in the capillaries in AD brain are unknown. Moreover, it is nearly impossible to investigate the quantitative changes in the capillaries in the human early-stage AD brain. Therefore, we used a mouse model of earlystage AD to quantitatively investigate the capillary changes in the early-stage AD brain.
To study early-stage AD in mice, the first key is the choice of the right type and the right age of AD mice. Furthermore, appropriate and accurate behavioral tests should be used to detect the typical learning and memory ability decline which are related to the AD. The Tg2576 transgenic mice (APP695swe) are Swedish mutants which could overexpress human amyloid precursor protein. This type of AD mouse is considered one of the ideal models for studying the cellular and molecular pathology of AD in early-stage Alzheimer's disease (Bilkei-Gorzo, 2014). Moreover, both the memory and cognitive functions of Tg2576 transgenic AD mice have been shown to decline in association with aging procession (Corcoran, Lu, & Turner, 2002).
The Morris water maze is used to study brain function of Alzheimer's disease. It has been widely recognized in the world and is the preferred classical experiment for behavioral research, especially learning and memory research about age-related memory impairment in transgenic mouse models of AD (Morris, 1984). In this study, the spatial learning and memory abilities of Tg2576 AD mice were detected using the Morris water maze. Previous studies have showed that most of the animal models of AD need at least 6-10 months to begin with deposition of senile plaques or to show learning and memory deficits behavior (Hsiao et al., 1996;Jacobsen et al., 2006;Savonenko et al., 2005;Toledo & Inestrosa., 2010 and memory abilities by 9 or 10 months of age (Hsiao et al., 1996). In this study, we used Tg2576 transgenic mice to simulate early-stage AD, and the Morris water maze was used to test behavior defects in our AD mice. The escape latency of the hidden platform test showed that the spatial learning ability of the 10-month-old Tg2576 group mice declined, and the cross platform frequency and quadrant percentage time of the probe trial test showed that the memory ability of the 10-month-old Tg2576 group mice declined. All the behavioral tests showed that the behavioral changes occurred in 10-month-old Tg2576 AD mice, which was consistent with the results of our previous study, which found that the Tg2576 group mice did not show neurobehavioral changes until 10 months of age (Zhang et al., 2013).
The immunofluorescence results of Aβ and our previous ELISA results of soluble Aβ40 and Aβ42 protein from Zhang et al. both indicated the presence of characteristic Aβ deposition of AD in Tg2576 mice (Zhang et al., 2013). Our present findings, together with the findings from previous studies, further suggested that 10-month-old Tg2576 AD mice could be meaningfully used to investigate the morphological changes during early-stage AD.
What might cause the spatial learning and memory ability decline during early-stage AD? Traditionally, white matter lesions were considered as a secondary pathological event following gray matter damage (Pierpaoli et al., 2001). However, the role of the white matter play in learning and memory ability get more and more attention by researchers. Evidence showed that the volume of white matter were increased after adults learning to read, indicated that altered The stereological results of white matter capillaries. (a) The total length of the capillaries in the white matter of wildtype AD mice and Tg2576 AD mice (x ± SD). ** indicates p < 0.01. (b) The total volume of the capillaries in the white matter of wild-type AD mice and Tg2576 AD mice (x ± SD). * indicates p < 0.05. (c) The total surface area of the capillaries in the white matter of wild-type AD mice, and Tg2576 AD mice (x ± SD). ** indicates p < 0.01 white matter structure was associated with the learning and cognitive function (Fields, 2010). Alzheimer's disease-related researches showed that both cognitive and memory decline were associated with white matter lesions (Bendlin et al., 2010;Medina et al., 2006;Salat et al., 2009). In particular, rather than hippocampal atrophy, white matter changes predict incident AD (Brickman et al., 2012).
The clinical evidences indicated that there was a lesion in the white matter of the mild cognitive impairment (MCI) and early AD (Rémy et al., 2015;Zhuang et al., 2013). As early as 2000, Brown et al. re-ported that the spiral-like blood vessels and periventricular venous collagenosis existed, which caused vascular contortion and occlusion in the white matter in AD (Brown et al., 2000). Later, Jellinger reported that arteriolosclerosis, fibrohyalinosis, lipohyalinosis, and adventitial calcifications were present in the white matter of the AD brain (Jellinger, 2002;Jellinger & Attems, 2003). What might be the relationship between the white matter changes in AD and the capillary changes in the white matter in AD? The vascular supply in the brain was not uniform and the capillary distribution within the white matter is relatively small, and the white matter is more vulnerable than other brain regions to chronic hypoperfusion and hypoxic-ischemic injury (Van Gijn, 1998;Nonaka et al., 2003), we speculated that capillary microcirculation changes might play a vital role in the white matter lesions in the early-stage AD. In this study, we used stereological methods to quantitate capillaries within the white matter of 10-month-old Tg2576 AD mice. The results showed that the geometric parameters of the capillaries within the white matter of 10-month-old Tg2576 AD mice, such as total length, total volume, and total surface area of capillaries in the white matter, were significantly lower than those in the wild-type mice of the same age. These results indicated that the marked decrease in the capillaries existed in the white matter of Tg2576 AD mice at 10 months of age.
There have been some studies that have investigated the vascular changes in the AD brain (Bourasset et al., 2009;Buѐe, Hof, & Delacourte, 1997;Desai, Schneider, Li, Carvey, & Hendey, 2009;Lee et al., 2005;Paris et al., 2004). In 2004, Paris et al. examine the vascular density and found that the vascular density in the hippocampus and cortex of the APP mutation mice was decreased by approximately 30% compared to control littermates (Paris et al., 2004).
Using the vascular space markers, [ 3 H]-inulin and [ 14 C]-sucrose, for in situ brain perfusion, Bourasset reported that the cerebrovascular volume was significantly lower (−26% and −27%) in the brains of 3xTg-AD mice than in those of nontransgenic littermates (Bourasset et al., 2009). In contrast to the above studies, Desai et al. found that ανβ3, a marker of angiogenesis, exhibited elevated immunoreactivity, and vascular densities were increased in the hippocampus, midfrontal cortex, and other brain regions of AD cases compared to controls (Desai et al., 2009 (Lee et al., 2005). However, from the methodological point of view, capillary number could not have been obtained with their method.
In fact, they obtained the capillary profiles in the corpus callosum.
Our results quantify the total absolute value of parameters of the capillaries other than density value, which can avoid the problems related to the "reference space trap" induced by the density values.
Second, capillary distributions in the white matter were not of random orientation; this type of distribution may cause a potential estimation bias. In the current study, we used the isector technique to guarantee isotropic and uniform, random sections so capillaries had the same probability of being sampled in every direction of 3D space (Nyenggard & Gundersen, 1992). Therefore, this study provided accurate stereological data of the white matter capillaries in the earlystage AD mice. Moreover, the current methods can be used to study postmortem human AD brain. When the methods are used in human brain, the researcher needs to adjust the sampling interval according to human brain. There were several differences between our study and the study of Lee et  TA B L E 1 Correlations between behavioral tests and stereological measurements systematically quantify the white matter vessels of AD. Moreover, in animal research, it is possible to conduct research in the early stage of AD, which is very important for the prevention of AD. Using dynamic susceptibility contrast magnetic resonance imaging, Eskildsen et al. studied microvascular blood flow in white matter without damaging the brain (Eskildsen et al., 2017). Our stereological vascular study is a quantitative study of the internal structure of blood vessels, which is more reflective of the details of AD-damaged blood vessels than just local blood flow. In the future, we need to figure out how the stereological methods can be used together with the image techniques so that the microvascular changes in vivo can be studied.
The marked changes in the capillary microcirculation in the white matter might induce an insufficient blood supply to the white matter in AD. The findings of a previous study indicated that the vascular changes were related to white matter lesions (Brown et al., 2000). Using the imaging techniques, researchers found that the white matter volume in AD patients was decreased (Guo et al., 2010;Medina et al., 2006;Salat et al., 2009). In contrast, our results showed that there was no significant difference in the white matter volume between the Tg2576 AD mice and the wild-type group mice at 10 months of age. Even though we could not directly compare our results in the AD mice with the previous findings in human AD, we suspected that the following reasons might partly explain the difference. First, the results from AD patients were from aged subjects, while our results were from the early-stage AD mice. Second, the current study used three-dimensional quantitative methods, namely, the stereological methods, while, all of the above imaging studies (Guo et al., 2010;Medina et al., 2006;Salat et al., 2009), from a methodological point of view, were qualitative or semiquantitative. Combining the current results with our previous results, we could see that the capillary microcirculation changes were present before the white matter atrophy and the myelinated fiber changes in the white matter of Tg2576 AD mice, and we suggested that the capillary microcirculation changes might play a vital role in the white matter lesions in early-stage AD. The results of a previous study indicated that white matter changes predicted incident AD in early stages (Brickman et al., 2012). What is more, studies have shown that the white matter pathological changes before the deposition of amyloid plaque and neurofibrillary tangles formed in gray matter in AD animal models (Redwine et al., 2003). The previous results mentioned above, together with our current results and our previous results, suggest that capillary microcirculation changes in the white matter may be one of the earliest changes in the AD brain, and they might play a vital role in the progression of AD.

| CON CLUS ION
In conclusion, the pathological progression of cognitive decline in AD mice was associated with the capillary changes within the white matter. The capillary microcirculation changes were present before white matter atrophy and the myelinated fiber changes in the white matter of AD mice. The current results might provide structural basis for understanding the pathological changes of the early stage of AD. Capillaries within the white matter might, thus, serve as a valid target for the prevention and treatment of earlystage AD. University.

This study was approved by the Animal Care and Research
Committee of Chongqing Medical University, China.

CO N FLI C T S O F I NTE R E S T
The authors declare that there is no conflict of interest regarding publication of this article.

AUTH O R CO NTR I B UTI O N S
Conception and design of the study: Yi Zhang, Yong Tang


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IL-17a promotes sociability in mouse models for neurodevelopmental disorders

A subset of children with autism spectrum disorder appear to show an improvement in their behavioural symptoms during the course of a fever, a sign of systemic inflammation1,2. Here we elucidate the molecular and neural mechanisms that underlie the beneficial effects of inflammation on social behaviour deficits in mice. We compared an environmental model of neurodevelopmental disorders in which mice were exposed to maternal immune activation (MIA) during embryogenesis3,4 with mouse models that are genetically deficient for contactin-associated protein-like 2 (Cntnap2)5, fragile X mental retardation-1 (Fmr1)6 or Sh3 and multiple ankyrin repeat domains 3 (Shank3)7. We establish that the social behaviour deficits in offspring exposed to MIA can be temporarily rescued by the inflammatory response elicited by the administration of lipopolysaccharide (LPS). This behavioural rescue was accompanied by a reduction in neuronal activity in the primary somatosensory cortex dysgranular zone (S1DZ), the hyperactivity of which was previously implicated in the manifestation of behavioural phenotypes associated with offspring exposed to MIA8. By contrast, we did not observe an LPS-induced rescue of social deficits in the monogenic models. We demonstrate that the differences in responsiveness to the LPS treatment between the MIA and the monogenic models emerge from differences in the levels of cytokine production. LPS treatment in monogenic mutant mice did not induce amounts of interleukin-17a (IL-17a) comparable to those induced in MIA offspring; bypassing this difference by directly delivering IL-17a into S1DZ was sufficient to promote sociability in monogenic mutant mice as well as in MIA offspring. Conversely, abrogating the expression of IL-17 receptor subunit a (IL-17Ra) in the neurons of the S1DZ eliminated the ability of LPS to reverse the sociability phenotypes in MIA offspring. Our data support a neuroimmune mechanism that underlies neurodevelopmental disorders in which the production of IL-17a during inflammation can ameliorate the expression of social behaviour deficits by directly affecting neuronal activity in the central nervous system.

It has been described that a subset of children with ASD show an improvement in their behavioral symptoms during the course of a fever, a sign of systemic inflammation 1,2 . Here we elucidate the molecular and neural mechanisms that underlie the beneficial effects of inflammation on social behavior deficits. We compared an environmental model of neurodevelopmental disorders in which mice were exposed to maternal immune activation (MIA) during embryogenesis 3,4 with mice genetically deficient for Contactin-associated protein-like 2 (Cntnap2) 5 , Fragile X mental retardation-1 (Fmr1) 6 , and Sh3 and multiple ankyrin repeat domains 3 (Shank3) 7 . We establish that social behavior deficits in MIAexposed offspring can be temporarily rescued by the inflammatory response elicited by lipopolysaccharide (LPS) administration. This behavioral rescue was accompanied by a reduction in neural activity in the primary somatosensory cortex dysgranular zone (S1DZ), whose hyperactivity was previously implicated in the manifestation of MIA behavioral phenotypes 8 . In contrast, we did not observe an LPS-induced rescue of social deficits in monogenic models. We demonstrate that the differences in responsivity to the LPS treatment between the MIA and the monogenic models emerge from differences in the levels of cytokine production. LPS treatment in monogenic mutant mice did not induce comparable amounts of interleukin-17a (IL-17a) as in MIA offspring, and bypassing this difference by directly delivering IL-17a into S1DZ was sufficient to promote sociability in monogenic mutant mice as well as in MIA offspring. Conversely, abrogating IL-17 receptor subunit a (IL-17Ra) expression in the S1DZ neurons eliminated the ability of LPS to reverse the sociability phenotypes in MIA offspring. Our data support a novel neuroimmune mechanism underlying neurodevelopmental disorders, whereby production of IL-17a during inflammation can ameliorate the expression of social behavior deficits by directly affecting neural activity in the central nervous system.
The beneficial effects of infection and ensuing inflammation on neurological disorders have been noted 9 . For example, a subset of children with autism spectrum disorder (ASD) exhibit temporary but significant improvement of their behavioral symptoms during episodes of fever, a sign of systemic inflammation 1,2 . However, a mechanistic understanding of how fever-associated immune responses translate into behavioral relief, both at the molecular and neural level, is still lacking. ASD etiology includes both environmental and genetic risk factors 10 . Animal models of ASD in which mice harbor mutations in ASD risk genes including Cntnap2 5 , Fmr1 6 , and Shank3 7 show behavioral abnormalities including social interaction deficits 11,12 . Similar behavioral abnormalities are observed in mouse models for environmental ASD risk factors like exposure to maternal inflammation 13 . We sought to explore the mechanisms that allow for the fever-associated rescue of sociability deficits using both genetic and environmental mouse models for neurodevelopmental disorders.

LPS rescues sociability in MIA offspring
A febrile response can be exogenously induced by injecting animals with a low dose of LPS 14 . Indeed, intraperitoneal (i.p.) LPS administration (50μg/kg) in control animalsoffspring born to mothers injected with PBS -led to a significant increase in body temperature (~0.5-1.0 °C) ~4-5 hours after the treatment (Fig. 1a). To investigate whether LPS injection can ameliorate sociability deficits, we compared sociability in adult males one day before LPS injection (pretest, Pre) and 4 hours after injection (Test), when the increase in body temperature initiates (Fig. 1b). As previously reported, MIA offspring born to mothers injected with Poly(I:C) (polyinosinic:polycytidylic acid) at embryonic day 12.5 (E12.5) exhibited impaired social approach behavior during the pretest [12][13][14][15] . The Cntnap2, Fmr1 and Shank3 monogenic models also displayed sociability deficits albeit with a marked variability during the pretest (Extended Data Fig. 1). We therefore compared MIA offspring to monogenic mutant animals that exhibited a sociability index lower than ~62%, the mean value for the three monogenic lines (Extended Data Fig. 1a). and Extended Data Fig. 2). LPS injection did not affect baseline sociability in control offspring. LPS-induced sociability rescue was absent 72 hours following the treatment, paralleling the transient nature of fever-associated improvement observed in ASD children 1 (Extended Data Fig. 3a-e). Furthermore, LPS-induced rescue was observed in both young (2-5 months) and aged (9-12 month) mice ( Fig. 1c and Extended Data Fig. 3f-j), suggesting that this is a generalizable phenomenon, not confined to early adulthood. LPS-induced rescue of MIA sociability deficits was also evident when assayed using a reciprocal social interaction test (Extended Data Fig. 3k). Lastly, LPS treatment in MIA offspring also rescued an enhanced marble burying behavior (Extended Data Fig. 3l), showing that inflammation can relieve several MIA-associated behavioral phenotypes. Intriguingly, unlike in MIA offspring, LPS treatment failed to restore sociability in mutant mice deficient for Cntnap2, Fmr1, and Shank3 ( Fig. 1c and Extended Data Fig. 2), indicating that rescue by LPS-driven inflammation may be applicable only to a subset of animal models for neurodevelopmental disorders.

Acute fever does not restore sociability
We next probed whether fever has a role in the observed behavioral rescue. Unlike in control offspring, LPS treatment did not induce changes in body temperature in MIA animals (Extended Data Fig. 4a), suggesting that the febrile response might not be the main factor contributing to the rescue. To directly test whether fever is dispensable, we sought to increase the animals' body temperature without inducing systemic inflammation by targeting inhibitory DREADDs (designer receptors exclusively activated by designer drugs) 17 to GABAergic neurons in the ventral part of the lateral preoptic nucleus (vLPO) of Vgat-Cre mice 18 (Fig. 1d). As previously reported 19 , inhibition of these neurons led to an increase in body temperature of ~1°C (Fig. 1e). Induction of febrile response alone, however, failed to promote social preference in MIA offspring (Fig. 1f, g and Extended Data Fig. 4b-e), confirming that fever per se is not the main driver of the LPS-induced rescue. Of note, MIA Vgat-Cre mice exhibited comparable sociability deficits, were unaffected by CNO treatment and showed an increase in sociability upon LPS treatment (Extended Data Fig. 4f-j).

LPS reduces c-Fos induction in the S1DZ
We previously established that adult MIA offspring display cortical abnormalities preferentially localized to S1DZ, a subregion of the S1 cytoarchitecturally defined by the absence of a discernable fourth layer (Extended Data Fig. 5). The cortical phenotype is characterized by an overall increase in neural activity, that, when reduced, can acutely rescue MIA-induced deficits in social behaviors 8 . We therefore investigated whether LPS-induced behavioral rescue in MIA offspring is accompanied by changes in S1DZ neural activity. MIA offspring exhibited an increase in the number of S1DZ cells expressing c-Fos, a marker for neuronal activation, relative to control offspring. However, in LPS-treated MIA offspring, the number of c-Fos + S1DZ neurons was reduced to the level of control offspring (Fig. 2a, b and Extended Data Fig. 6a-c). LPS injections did not elicit a generalized, brainwide effect on c-Fos expression in MIA offspring; the number of c-Fos + neurons either remained unchanged, as in several cortical regions examined, or increased, such as in the central amygdala (CeA), a region known to be activated by LPS 20 (Fig. 2a,c and Extended Data Fig. 6d,e). Therefore, LPS-induced behavioral rescue in MIA offspring was accompanied by a reduction in S1DZ neural activity.
Dysregulation of neural activity and deficits in interneuron function in S1 have been previously associated with various genetic mouse models for neurodevelopmental disorders [21][22][23] . We, therefore, sought to determine whether increased neural activity can also be observed in the S1DZ of monogenic mutant mice. The number of c-Fos + S1DZ neurons was increased compared to that of WT animals, and the magnitude of this increase correlated with the severity of the sociability deficits, notably in Cntnap2 and Fmr1 mutant animals (Extended Data Fig. 7a,b). These data suggest that increased neural activity in S1DZ may contribute to the expression of sociability deficits also in monogenic mutant mice, similarly to what we have described for MIA offspring 8 . Consistent with this idea, optogenetically reducing neural activity in the S1DZ could rescue sociability deficits in Cntnap2 and Fmr1 mutant animals. Shank3 mutant mice also showed an increase in sociability upon photoinhibition, but it was not significantly different from that of control animals expressing EYFP ( Fig. 2d-f and Extended Data Fig. 7c-f). Therefore, reducing neural activity in the S1DZ was sufficient to restore sociability in Cntnap2 and Fmr1 mutant mice as well as in MIA offspring 8 . Unlike in MIA offspring, however, LPS treatment failed to reduce the number of c-Fos + S1DZ neurons in monogenic mutant mice ( Fig. 2g-i).

IL-17a restores sociability deficits
LPS injection is known to increase production of inflammatory cytokines 24 . We observed that administration of LPS results in a robust increase in plasma levels of IFN-γ, IL-6, and TNF-α (Fig. 3a). Intriguingly, IL-17a, whose orthologue in C. elegans 25 has previously been implicated in promoting social behaviors, was prominently upregulated in MIA offspring, but not in monogenic mutant mice or in control animals (Fig. 3a). Furthermore, we noted that the receptor subunit A for IL-17a (IL-17Ra) is expressed in cortical neurons, including in the S1DZ (Fig. 3b-d and Extended Data Fig.8a-c). These data suggested that the increased levels of IL-17a upon LPS treatment in MIA, but not in monogenic mutant mice, may directly impact the S1DZ to restore sociability. Consistent with this idea, direct administration of recombinant IL-17a into the S1DZ was sufficient to increase sociability in not only MIA offspring, but also in Cntnap2 and Fmr1 mutant mice (

LPS-induced rescue requires IL-17a
To further determine whether IL-17a mediates LPS-driven behavioral rescue, we inhibited IL-17a activity in the brain via intracerebroventricular (i.c.v.) injection of blocking antibodies. Antibodies against IL-17a prevented both the LPS-induced rescue of sociability ( Fig. 4a and Extended Data Fig. 9a-e) and the reduction of c-Fos expression in the S1DZ of MIA offspring (Extended Data Fig. 9f). To directly assay the effects of LPS on neural activity we used multi-electrode arrays to measure the firing rate of S1DZ neurons in awake animals (Extended Data Fig. 10a, b). Upon LPS treatment, we observed a decrease in overall firing rate that was prevented by blocking IL-17a in MIA offspring. Neither LPS nor IL-17a blocking antibody treatment changed the firing rate in the S1DZ of control offspring ( Fig.  4b-d and Extended Data Fig. 10c). Furthermore, LPS injection failed to restore sociability to MIA offspring deficient for IL-17Ra in the S1DZ neurons (Fig. 4e, f and Extended Data Fig.  10d-j). Our data collectively demonstrate that IL-17a mediates the restoring effects of inflammation on social behaviors by directly acting on IL-17Ra + S1DZ neurons.

Discussion
Our and other group's previous data suggested that increased IL-17a production in the pregnant mothers may present as a risk factor for neurodevelopmental disorders in offspring 26,27 . Based on our current findings, we propose that in adult MIA offspring the same type of cytokine is beneficial and ameliorates sociability phenotypes during episodes of inflammation. LPS treatment led to an increase in IL-17a levels in the blood selectively in MIA offspring, but not in other monogenic mutant mice, suggesting that inflammatory responses may result in beneficial effects only for those individuals who have their immune systems primed by prenatal exposure to immune activation or by other environmental factors. A better understanding of the role of a primed immune system among patients with neurodevelopmental disorders may help identify those whose behavioral symptoms are likely to improve upon exposure to fever-associated inflammation. Furthermore, elucidating the mechanisms by which IL-17a can induce two opposing behavioral outcomes depending on when its upregulation occurs, during embryonic fetal brain development or in the adult brain, may provide opportunities to devise novel therapeutic as well as preventive treatments for ASD-associated behavioral symptoms.

Animals
All experiments were performed according to Guide for the Care and Use of Laboratory Animals and were approved by the National Institutes of Health and the Committee on Animal Care at Massachusetts Institute of Technology. C57BL/6 were purchased from Taconic Biosciences for generating PBS and MIA offspring. Vgat-Cre (028862) were purchased from Jackson Laboratories and inbred. For monogenic experiments, C57BL/6, Cntnap2 (017482), Fmr1 (003025), and Shank3 (017688) mice were purchased from Jackson Laboratories and inbred after colonizing with SFB from donor mice. IL-17Ra fl/fl and IL-17Ra KO were previously described 28,29 . All mice were males aged 2-5 months, unless otherwise specified.

Maternal immune activation
Mice were mated overnight with females carrying SFB in their guts 26 . On E12.5, pregnant female mice were weighed and injected with a single dose (20 mg/kg i.p.) of poly(I:C) (P9582, Sigma Aldrich) or PBS vehicle. Each dam was returned to its cage and left undisturbed until the birth of its litter. All pups remained with the mother until weaning on postnatal day 21-28 (P21-P28), at which time mice were group housed at a maximum of 5 per cage with same-sex littermates. Mating between Vgat-Cre(c/c) males and WT females were used to make MIA Vgat-Cre mice.

Tracking of body temperature
Body temperature was measured using the Anipill remote temperature monitoring system (007894-001, DSI). Adult male mice were implanted with an Anipill capsule in the abdominal cavity. Experiments were carried out >3 weeks following surgery. Mice were singly housed the day before the experiment. Ambient temperature was maintained at 23.5°C, consistent with the ambient temperature of the vivarium. Body temperature was sampled in 5-minute increments. LPS or saline (Veh) injections occurred between 11:00-13:00 for experiments assaying the effects of LPS on body temperature in PBS and MIA offspring.

In situ hybridization
Animals were transcardially perfused with cold PBS. Brains were extracted and embedded in optimal cutting temperature (OCT) compound on dry ice. Sections were cut at 20μm thickness on a cryostat. In situ hybridizations were performed using RNAscope 2.5 HD Assay-Red kit (322350, Advanced Cell Diagnostics) using a probe targeting the Il17ra transcript (Mm-Il17ra-O1, 566131, Advanced Cell Diagnostics). The probe was designed to target region 444-882 of the Il17ra transcript (NM_008359.2). Modifications to the kit protocol to improve adherence of tissue to the slide include an extension of fixation time to 30min and the addition of a humidified bake step at 40°C immediately prior to probe hybridization. Sections were counterstained with DAPI. Images were acquired using a confocal microscope (LSM710, Carl Zeiss) with a 10x or 20x objective lens. Il17ra and DAPI expression was quantified using QuPath 30 . Cells were divided into the following categories based on level of Il17ra expression: low = 1-3 puncta, medium = >3-9 puncta, high = >9-15 puncta, highest = >15 puncta.

In situ hybridization followed by immunohistochemistry
For experiments assaying the overlap of Il17ra and NeuN expression, immunohistochemistry for NeuN was performed following a modified in situ hybridization protocol. The RNAscope 2.5 HD Assay-Red kit in situ protocol was modified in the following ways: Sections were baked at 60°C, followed by a 10min fixation step with 4% PFA at room temperature. Sections were then stored in 70% ethanol at 4°C overnight. Sections were permeabilized in 8% SDS for 10min. Sections were washed twice with PBS between each step. Following SDS treatment, the RNAscope 2.5 HD Assay-Red kit protocol was followed from the probe hybridization step. Following the completion of the in situ protocol, sections were incubated in blocking buffer containing NeuN antibody overnight at 4°C. Sections were then incubated in blocking buffer containing DAPI and secondary antibody for 2hrs at room temperature.

Behavioral analysis
Male mice were tested during the light cycle in a room with lighting maintained at 230 Lux. Animals were transferred to the testing area at least one hour prior to the initiation of experiments. Tracking of mouse behavior was done using EthoVision XT (Noldus) tracking system.
Three-chamber social approach assay-Adult male mice were assayed for sociability using a three-chamber social approach assay. The arena was constructed of white acrylic (50cm × 35cm × 30cm). Wire cups (Spectrum Diversified) were placed in the back left and right corner of the arena beneath water-filled 1L bottles (Nalgene). On day 0, mice were habituated to the arena for 10min. Immediately following habituation, mice were singly housed. On day 1 (Pre), mice were placed in the center of the arena and allowed to freely explore. Following 10min, mice were confined to the center of the arena. An inanimate object (rubber stopper) or a male conspecific were placed beneath the wire cups. Placement of the inanimate object and social target were alternated. Mice were then allowed to freely explore the arena for 10min. Interaction time was defined as time spent in the areas circumscribing the wire cups (<2cm). Sociability was defined as interaction time with the social target divided by total interaction time and expressed as a percentage. For experiments involving LPS injections, mice were injected with either saline (Veh) or LPS (50μg/kg, i.p., L2630, Sigma) on day 2 (Test), four hours prior to testing. For the experiment assaying sociability 72hrs following LPS injection, mice used for 4hr LPS sociability experiments were tested for sociability again at 72hrs.
Three-chamber social approach assay with DREADD manipulation-Adult male Vgat-Cre MIA offspring were bilaterally injected with virus encoding the inhibitory DREADD receptor fused to mCherry into the vLPO. Following >3 weeks of recovery, mice were assayed on the three-chamber social approach assay outlined above. Baseline sociability was assayed on day 1 (Pre). On day 2 and day 3, mice were injected with either Saline (Veh) or CNO (1.5mg/kg i.p., BML-NS105, Enzo Life Sciences), two hours prior to initiation of behavior. Injection order was counterbalanced. Following behavioral experiments, post-mortem histology was used to confirm mCherry expression within the vLPO. For experiments assaying the effect of CNO and LPS injection in Vgat-Cre PBS and MIA offspring that have not undergone surgery, baseline sociability was assessed on day 1. On day 2 and 3 mice received counterbalanced injections of CNO or vehicle. On day 4, mice were injected with LPS.
Three-chamber social approach assay with S1DZ IL-17a administration-Adult male mice were implanted with a cannula into S1DZ bilaterally and allowed to recover for >2 weeks prior to the behavioral experiments. On day 1 (Pre) mice were assayed for sociability. On day 2 (Test), mice were anesthetized briefly using isoflurane and either saline (Veh) or IL-17a (50ng/side in 1μl at a rate of 180nl/min, 7956-ML/CF, R&D) was administered bilaterally into S1DZ through 250μm-projecting injector tips (PlasticsOne). Four hours after vehicle or IL-17a administration, mice were assayed for sociability. Cannula placements were verified using histology.
Three-chamber social approach assay with S1DZ optogenetic inhibition-Enhanced yellow fluorescent protein or halorhodopsin were virally targeted to the S1DZ. After two weeks of recovery mice were allowed to freely explore the arena for 10 min. The following day, the mice were given 3 min of no stimulation ('off' session) and 3 min of laser stimulation ('on' session) (594 nm, 6mW).

Three-chamber social approach assay with IL-17a blocking antibody-For
central cytokine blockade experiments, adult male mice were implanted with a cannula into the lateral ventricle and allowed to recover for >2 weeks prior to the behavioral experiments. On day 1 (Pre), mice were tested for baseline sociability. On day 2 (Test) mice were injected with IL-17a blocking antibody (clone 50104; R&D) or isotype control antibody (IgG2a, clone 54447; R&D). Antibodies were dissolved in saline and administered intracerebroventricularly at 1mg/kg in 500nl at a rate of 180nl/min through 750μmprojecting injector tips (PlasticsOne). Blocking antibody was administered 30min prior to LPS administration. Four hours following LPS administration, mice were assayed for sociability.
Three-chamber social approach assay in mice deficient for IL17R in the S1DZ -IL-17Ra knockout was mediated by viral delivery of Cre-recombinase, expressed under the control of the human synapsin (hSyn) promoter, into the S1DZ of IL-17Ra fl/fl MIA offspring. Following at least three weeks of recovery, mice were assayed for sociability. On the next day sociability was assessed four hours following LPS administration.
Marble burying assay-On day 1, mice were tested for their baseline marble burying phenotype. On day 2, four hours prior to beginning the marble burying assay, mice were treated with either LPS or vehicle. Marble burying assay was carried out as described previously 8 . Mice were placed into testing arenas (arena size: 40cm×20cm×30cm, bedding depth: 3cm) each containing 20 glass marbles (laid out in four rows of five marbles equidistant from one another). At the end of the 15min exploration period mice were carefully removed from the testing cages and the number of marbles buried was recorded. The marble burying index was arbitrarily defined as the following: 1 for marbles covered >50% with bedding, 0.5 for marbles covered <50% with bedding, or 0 for anything less.
Reciprocal social interaction assay-Four hours prior to testing, mice were injected with vehicle or LPS. Two unfamiliar mice of the same treatment and background were placed in a fresh mouse cage and allowed to freely interact for 10min. Videos were acquired using IC Capture (The Imaging Source) at 640×480 aspect ratio and 25fps. Social interaction (close following, push-crawl, nose-nose sniffing, and nose-anus sniffing) was scored by an observer blind to treatment and background 31 .

PCR for assaying IL-17Ra knockout
IL17Ra fl/fl male mice were bilaterally injected with virus encoding nuclear Cre fused to EGFP or control virus encoding only EYFP into the S1DZ. Following >3 weeks, injection sites were dissected from the primary somatosensory cortex. Single cells were dissociated from brain tissue using a modified version 34 of the Papain Dissociation Kit protocol (LK003153, Worthington) and sorted on a BD FACS Aria (BD biosciences) based on EGFP/ EYFP expression. RNA was extracted from sorted cells using a Quick-RNA micro-prep kit (Zymo). 20ng of RNA was converted into cDNA using oligodT (Protoscript First Strand CDNA Synthesis Kit, NEB). Il17ra mRNA expression was analyzed using PCR. 1μl of cDNA was diluted in a 20ul reaction volume. Il17ra and Gapdh mRNA expression were assessed using the following primers: Il17ra 5′-AGATGCCAGCATCCTGTACC-3′ and 5′-CACAGTCACAGCGTGTCTCA-3′; Gapdh 5′-

In vivo electrophysiology
Electrophysiological experiments were conducted in head-fixed mice trained to walk on a rotating running wheel. Prior to training, mice were implanted with custom crowns permitting head-fixing above the wheel and allowed to recover for one week before training. Mice were trained to walk on the wheel for three 10min sessions daily for at least one week. Following training, mice were implanted with a multi-electrode array targeting the S1DZ and allowed to recover before testing. To assess IL-17a dependence of LPS-induced changes in neural activity, mice were injected i.p. with control antibodies or blocking antibody against IL-17a (1mg/mouse), 30min prior to i.p. vehicle or LPS injection. Baseline neural activity was measured while mice were running on the wheel immediately prior to the first injection. Following the first injection, mice were returned to their home cage. Post-injection (Test) neural activity during wheel running was measured 4hrs after vehicle or LPS injection.
Electrophysiological recordings and spike sorting-Signals were acquired using a Neuralynx multiplexing digital recording system (Neuralynx) through a combination of 32and 64-channel digital multiplexing headstages plugged into the EIB of the implant. Signals from each electrode were amplified, filtered between 0.1 Hz and 9 kHz and digitized at 30 kHz. Initial spike sorting was performed using MountainSort, followed by manual quality control using MClust toolbox (http://redishlab.neuroscience.umn.edu/mclust/MClust.html).
Analysis of firing rate-Firing rate was calculated across one second time windows during which animals were walking stably on the rotating wheel averaged across 25-50 trials per condition. The first second after wheel rotation onset was omitted to avoid firing rate changes due to acceleration. Firing rate was sampled with a 1ms bin width passed through a box car filter (100ms). The resulting PSTHs were then smoothed with a 50ms gaussian. To assess the effect of treatment on neural activity, changes in firing rates during wheel running 4hrs after injection were normalized to the pre-injection baseline. Cells showing firing rate below 0.1Hz were excluded from analysis.

Statistics and reproducibility
Statistical analyses were performed using GraphPad Prism. Sample size was chosen based on similar previous studies and not based on statistical methods to predetermine sample size 8,15 . Within each iteration of an experiment, animals were randomly assigned to groups with approximately balanced sample size. Behavioral results from mice with inaccurate targeting of viral infection or cannula implantations were excluded. Experimenters were blind to subject treatment during data collection and analysis.  test (f,h,j,l). Graphs indicate mean ± s.e.m. Extended Data Figure 4. Acute increase in body temperature is insufficient to promote sociability. a, Body temperature profile following Veh or LPS injection in MIA offspring (Veh n=10, LPS n=10; from 4 independent experiments). Initial spike in body temperature is due to handling stress. b-e, Data expressed as time spent investigating social (S) versus inanimate (I) objects (b), total interaction time (c), time spent in social (S), center (C) or inanimate (I) chamber (d), and distance traveled (e) during three-chambered sociability experiments described in Fig. 1g (n=9 for all groups; from 2 independent experiments). f-j, Sociability performance in Vgat-Cre PBS and MIA offspring following Veh, CNO, or LPS treatment. Data expressed as percent sociability (f), time spent investigating social (S) versus inanimate (I) objects (g), total interaction time (h), time spent in social (S), center (C) or inanimate (I) chamber (i), and distance traveled (j) during three-chambered sociability experiments (PBS n=11, MIA n=7; from 2 independent experiments). *P<0.05, **P<0.01 calculated by twoway repeated measures ANOVA with Bonferroni's (a) and Dunnett's (f,h,j) post-hoc tests, and two-way ANOVA with Sidak's (b,g) and Dunnett's (d,i) post-hoc tests, and one-way repeated measures ANOVA with Tukey's post-hoc test (c,e). Graphs indicate mean ± s.e.m. Extended Data Figure 5. Histological identification of S1DZ. a, Coronal section of the cortex counterstained with DAPI to highlight the abrupt reduction in cell density in layer 4 between the S1DZ and the S1BF at AP −0.46mm. (n=5, from 1 independent experiment). b, Coronal section of the cortex imaged with differential interference contrast further highlighting the reduced layer 4 in the S1DZ at AP −0.46mm (n=3, from 2 independent experiments). White arrows indicate borders of S1DZ. Scare bar represents 500μm (a) and 300μm (b). D: dorsal, V: ventral. a, Quantification of c-Fos expressing cells in the S1DZ of monogenic mutant mice (WT n=6, Cntnap2 n=21, Fmr1 n=17, Shank3 n=15: from 5 independent experiments). b, Correlation of c-Fos expression in the S1DZ with severity of sociability deficits across monogenic mutant mice (Cntnap2 n=21, Fmr1 n=17, Shank3 n=15; from 4 independent experiments). Black solid lines represent regression line; grey lines indicate 90% confidence intervals. c, Individual data for experiments in Fig. 2f. d-f, Data expressed as time spent investigating social (S) versus inanimate (I) objects (d), total interaction time (e) and distance traveled (f) during three-chambered sociability experiments described in Fig. 2f (WT-EYFP n=7, WT-eNpHR n=8, Cntnap2-EYFP n=11, Cntnap2-eNpHR n=9, Fmr1-EYFP n=8, Fmr1-eNpHR n=12, Shank3-EYFP n=8, Shank3-eNpHR n=10; from 6 independent experiments). Statistics calculated by one-way ANOVA with Dunnett's post-hoc test (a), linear regression (b), one-way repeated measures ANOVA with Dunnet's post-hoc test (c), two-way ANOVA with Sidak's post-hoc test (d), and two-way repeated measures ANOVA with Sidak's post-hoc test (e,f). Graphs indicate mean ± s.e.m.
Extended Data Figure 8. Il17ra expression in the S1DZ of PBS and MIA offspring. Further behavioral analyses of S1DZ IL-17a rescue of sociability in MIA offspring and monogenic mutant mice. a, Representative images of Il17ra expression in the S1DZ of PBS and MIA offspring. Scare bar represents 1mm. b, Quantification of Il17ra expression within the S1DZ of MIA offspring according to cortical layer (n=6; from 2 independent experiments). c, Quantification of overall Il17ra expression in the S1DZ of PBS and MIA offspring (PBS n=8, MIA n=6; from 2 independent experiments). d-h, Further behavioral analyses of experiments described in Fig. 3f; Time spent investigating social (S) versus inanimate (I) Extended Data Figure 9. IL-17a is necessary for LPS-induced behavioral rescue and reduction of c-Fos expression in MIA offspring.