Functional and Structural Correlates of Impaired Enrichment-Mediated Adult Hippocampal Neurogenesis in a Mouse Model of Prenatal Alcohol Exposure

Mice

C57Bl6/J or Nestin-CreER^T2: tdTomato bitransgenic mice were used for these studies. C57Bl6/J mice were purchased from Jackson Laboratories. Nestin-CreER^T2: tdTomato bitransgenic mice were obtained from breeding colonies maintained in the University of New Mexico Health Sciences Center Animal Facility. For transgenic mice, breeding colonies were maintained for homozygosity at both the Nestin-CreER^T2 [26] and Ai9 (RCL-tdT) transgene loci [27], on a C57BL/6J genetic background as previously described [21, 28]. Genotyping was routinely performed by PCR analysis of tail genomic DNA as previously described in detail [28]. All mice were housed under reverse 12-hr dark/12-hr light cycle (lights off at 08:00 h) in a humidity and temperature controlled room with food and water available ad libitum. Animal procedures were approved by the University of New Mexico Institutional Animal Care and Use Committee in accordance with NIH policies on Humane Care and Use of Laboratory Animals.

Prenatal alcohol exposure (PAE)

PAE offspring were generated within the New Mexico Alcohol Research Center Scientific Core, using a well-characterized limited-access “drinking-in-the-dark” gestational ethanol exposure paradigm as previously described in detail [29]. Food and water consumption, maternal weight gain, litter size, pup weight, pup retrieval times and time on nest does not differ between the alcohol-exposed and control animals in this drinking paradigm [29]. Briefly, 60 day old C57Bl6/J or Nestin-CreER^T2:tdTomato female mice were offered a 0.066% saccharin solution containing EtOH, instead of water, for 4 hrs per day from 10:00–14:00. (note: C57Bl/6J mice were used for c-Fos experiments, whereas Nestin-CreER^T2:tdTomato transgenic mice were used for all other experiments). Following a 5 day gradual ramp-up period (from 0–10% EtOH), the mice were subsequently maintained on a 10% EtOH drinking regimen for 2 weeks prior to pregnancy and throughout gestation. Female mice offered 0.066% saccharin (SAC) without EtOH during the same periods served as controls. Consumption volume during the 4 hr access period, as determined for dams beginning after one week of drinking 10% alcohol, was 5.74 ± 0.41 gm/kg, n = 22 (range 3.46 – 10.90 gm/kg; median 4.82 gm/kg). In this paradigm, blood alcohol concentrations directly correlate to the average amount of ethanol consumed over the 4 hr drinking period throughout gestation [29]. Based on this correlation, average daily maternal blood alcohol concentrations (BACs) were estimated to be 80–90 mg/dL throughout gestation.

Tamoxifen administration

Tamoxifen (TAM; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 10% EtOH/90% sunflower oil (Sigma-Aldrich) and administered by intraperitoneal injections (180 mg/kg) to Nestin-CreER^T2:tdTomato bitransgenic mice (SAC and PAE offspring) for five consecutive days for quantification of aDGCs as previously described [21, 22, 28] or as a single injection (180 mg/kg) for dendritic morphological analysis at the ages indicated in text.

Standard housing (SH) and Enriched environment (EE)

PAE and SAC offspring were gender segregated at weaning and housed thereafter in standard housing (SH) or enriched environment (EE) conditions until sacrifice. SH comprised a standard mouse cage (28 cm×18 cm×13 cm) without running wheels or toys (three mice/cage). EE included a larger cage (48 cm×27 cm×20 cm) containing two running wheels and multiple objects (ladder, tunnel and hanging toys that were changed out weekly; six mice/cage) as previously described [20, 22].

A-B Contextual-fear discrimination

Behavioral testing was performed in the University of New Mexico Center for Brain Recovery and Repair Preclinical Core Facility using a protocol blinded to treatment. Mice were trained on the A-B contextual-fear discrimination learning task as previously described [22, 30, 31], and originally modified from Sahay et al., [32] (Fig. 1A and 1B). This behavioral assay tests the cognitive ability of mice to learn to discriminate between two similar contexts, using a protocol that is dependent upon the activity of aDGCs [24, 31–33]. All experiments were performed using the automated NIR Video Fear Conditioning System for Mouse (Med Associates, Fairfax, VT). Briefly, Context A (shock condition) was comprised of a standard chamber with a stainless steel floor with steel grid rods arranged in a straight horizontal plane, clear Plexiglas front wall, and aluminum side and back walls. A similar chamber comprised Context B (non-shock), except for a non-shock floor comprised of stainless steel grid rods arranged in 2 offset horizontal planes, and side and back walls covered in striped black and white contact paper. All mice were habituated in a red-light holding room for one hour before the first training session and were returned to their home cages in the animal facility following the final training session each day. Each mouse was individually placed into either Context A (foot shock) or Context B (no foot shock) for 90 s, followed immediately by foot shock (0.5 mA) for 2 s in Context A only. Following a 90 s interval, mice in Context A received a second foot shock and were removed from the chamber 30 s later. Mice in Context B received no shock or other aversive stimulus during the trial and remained within the chamber for the same period of time. Three hours following the first session, mice were exposed to a second identical training session, except that the mouse initially subjected to Context A (shock) was subjected to Context B (no shock), and vice versa. This sequence was repeated once per day for 7 consecutive days, with the order of testing in Context A vs. Context B reversed every day for each individual mouse. Freezing time was measured with an automated software system (Video Fear Conditioning “Video Freeze^®” Software, Med Associate, Fairfax, VT) for analysis of digitally-recorded motion (30 frames per second) under red-light conditions. Freezing was defined using a motion threshold of 10 and minimum freeze duration of 30 frames (i.e., subject must remain immobile for one second to be registered as a freeze). A discrimination score was automatically calculated for each mouse for each day of training [(freezing score Context A - freezing score Context B)/(freezing score Context A + freezing score Context B)], during the first 90 s within either chamber. Thus, higher discrimination scores indicate better contextual discrimination. All mice were returned to home cages (EE or SH) and sacrificed 1 week later (∼11 weeks post-tamoxifen and ∼4.0 months of age) and brains from 4 mice per experimental group were randomly selected for histological quantification of tdTomato⁺ aDGCs as described below.

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Fig. 1

PAE impairs EE-mediated improvement A-B context fear discrimination learning. A. Experimental timeline. SAC and PAE Nestin-CreERT2:tdTomato mice received tamoxifen dosing to induce reporter expression in aDGCs and were subjected to SH or EE housing conditions for 10 weeks prior to behavioral testing. B. Experimental design for the A-B context fear discrimination task. Mice received two 2-second 0.5 mA foot shocks separated by a 90 second interval in Context A only. The first shock occurred 90 seconds following placement into the chamber and mice were removed from the chamber 30 seconds following the 2^nd foot shock. In the non-shock context B, mice received no foot shock or other aversive event. Each mouse experienced one daily session in Context A and one daily session in Context B, separated by 3 hours. The order of context exposure was alternated each day for 7 days. Daily discrimination scores were calculated as: (freezing time context A - freezing time context B) / (freezing time context A + freezing time context B) during the first 90 seconds of each testing session. C. Discrimination learning over time. Data are plotted as the mean daily discrimination scores per group±SEM. Group n’s were as follows: SAC-SH (n = 8 mice) and SAC-EE (n = 8 mice) sampled across 5 separate litters; PAE-SH (n = 15 mice) and PAE-EE (n = 15 mice) sampled across 7-8 separate litters. Three way ANOVA statistics: testing day [F(6,250) = 325, p < 0.0001], alcohol treatment [F(1,250) = 175, p < 0.0001], housing [F(1,250) = 12.49, p = 0.0005)], alcohol treatment x housing [F(1,250) = 98.73, p < 0.0001], alcohol treatment x housing x day [F(6,250) = 18.38, p < 0.0001]. *p < 0.01 SAC-EE vs. all other groups (Tukey’s multiple comparison). D. Mean discrimination scores at testing day 7. *p < 0.01 SAC-EE vs. all other groups (Tukey’s post-hoc comparison). E. Freeze time in Context A (shock) and Context B (non-shock) across all experimental groups on testing day 7. Data expressed as mean±SEM.

Exposure to novel environment

Mice were transferred in their home cage to a nearby behavioral testing room and acclimated for one hour before exposure to novel environment. Each mouse was individually removed from its home cage and placed into an open field novel environment for 30 minutes, followed by return to home cage for 80 minutes prior to sacrifice for c-Fos expression analysis. All experiments were performed under infrared lighting conditions during the reversed light/dark cycle. Control mice not exposed to novel environment remained within their home cages in their home room undisturbed until sacrifice.

Immunohistochemistry

All mice were overdosed with sodium pentobarbital (150 mg/kg, Fort Dodge Animal Health, Fort Dodge, IA) administered by intraperitoneal injection, and transcardially perfused with phosphate-buffered saline (PBS) containing 0.1% procaine and 2 U/ml heparin, followed by 4% paraformaldehyde (w/v) in 0.1 M PBS. The brains were post-fixed overnight, cryoprotected with 30% sucrose (w/v) in 0.1 M PBS for 48 hours at 4°C and sectioned in the coronal plane at 40μm (quantification of cell numbers) or 100μm (dendritic morphology analysis) as indicated in text, using a freezing sliding knife mictrotome. Free-floating tissue sections were immunostained using primary antibodies directed against the neuronal nuclear antigen, NeuN (1:1000, EMD Millipore, MAB 377) or against the immediate early gene, c-Fos (1:1000, Calbiochem, PC-38T) and visualized using FITC-conjugated or Cy3-conjugated secondary antibodies (1:250; Jackson Immunoresearch Laboratories, Westgrove, PA), respectively. Where indicated, sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, Carlsbad, CA). All sections were mounted onto glass slides and cover slipped using Fluoromount G mounting media (Electron Microscopy Sciences, Hatfield, PA). Z-stack maximum projection images were obtained using LASX acquisition software linked to a Leica DMi8 TCS SP8 confocal microscope (Wetzlar, Germany) using a 20X oil immersion objective. Images were subsequently tiled and stitched using LASX or Adobe Photoshop Software (Adobe Systems Inc., San Jose, CA) to generate representative photomicrographs.

Quantification of tdTomato⁺/NeuN⁺ aDGCs and c-Fos⁺ nuclei

The number of tdTomato⁺/NeuN⁺ co-labelled aDGCs or c-Fos⁺ nuclei were quantified using the Optical Fractionator probe in StereoInvestigator software (MicroBrightfield Biosciences, Williston, VT) linked to an Olympus IX-51 DSU spinning disk confocal microscope using a 40X objective as previously described [28]. For comparing tdTomato⁺/NeuN⁺ aDGCs across groups, the number of co-labeled aDGCs within the dentate gyrus of the dorsal hippocampus were estimated across two coronal sections per mouse. Regions of interest (ROI) were manually outlined using a 10X objective and followed the outer limits of the granule cell layer and 1 cell thickness into the hilus. Two sections spaced 480μm apart between –1.28 and –2.12 mm relative to bregma were quantified per mouse using an exhaustive counting scheme with 150×150μm grid size. The numbers of c-Fos positive nuclei within the suprapyramidal blade of the dorsal dentate gyrus were similarly estimated utilizing three coronal sections (between –1.28 and –2.12 to bregma) spaced 240μm apart and an identical exhaustive counting scheme. The optical disector height was set at 15μm with 2μm top and bottom guard zones.

Dendritic morphological analysis

For dendritic morphological analysis, PAE and SAC NestinCreER^T2:tdTomato mice received a single dose of tamoxifen (180 mg/kg, intraperitoneal) at 47–51 days of age and were maintained in EE housing conditions until sacrifice at 6 weeks post-tamoxifen (∼3.5 months of age at sacrifice). Mice were sacrificed by sodium pentobarbital followed by transcardial perfusion with 4% paraformaldehyde, with post-fixation and tissue cryoprotection as described above. Brains were sectioned in the coronal plane at 60–100μm thickness, mounted on glass slides and coverslipped with Fluoromount G as described above. Z-stack images were obtained using the Leica DMi8 TCS SP8 confocal microscope and LASX acquisition software. A 20× oil immersion objective (1μm optical sections) was used to capture the dendritic tree and a 63× oil immersion objective (zoom 5, and z-step size of 0.15μm) was used to capture spine images. Z stacks of dendrites were subsequently processed into maximum projection 2D images for further analysis. Spine images were processed using Huygens Deconvolution (Scientific Volume Imaging, Hilversum, Netherlands) prior to 3D reconstruction using Imaris^TM image analysis software (Bitplane, Concord, MA).

Dendrites from individual tdTomato⁺ aDGCs were traced using 2D Neurolucida software (MBF Bioscience, Williston, VT) and analyzed for branching complexity (Sholl analysis), total dendritic length and cell body area using Neurolucida Explorer. Dendritic protrusions (spines) were imaged from segments 20–75μm in length (40μm average) located within the middle 1/3 of the dendritic tree of tdTomato⁺ aDGCs located within the suprapyramidal blade of the dentate gyrus. Dendritic segments were digitally reconstructed in 3D utilizing Imaris Filament Tracer and Spine Detection Software (Bitplane, Concord, MA), and subjected to automated spine quantification and classification using Imaris Classify Spines Xtension. Dendritic protrusions were binned into the following categories using default measurement rules that are based on morphological maturity (Toni et al., 2007; Berry and Navadi, 2017): Stubby spines (<1μm in length), long thin spines (protrusion > 3μm in length with head width > neck width), mushroom spines (protrusion length < 3μm and a head width > 2× neck width) and filopodia (remaining dendritic protrusions). Two to five tdTomato⁺ aDGCs were sampled and averaged for each mouse for Sholl analysis (n/group = mouse number/group), and 3 dendritic sections were sampled per mouse for spine analysis (n/group = mouse number/group), with 4–6 mice per group across 2–6 distinct litters as indicated in figure legends.

Statistical Analysis

This study was restricted to male offspring for consistency due to limited availability of mice for some experiments. Future work will focus on characterization of female mice, given that prior research has demonstrated sex differences in vulnerability to alcohol toxicity [34, 35].

All data were analyzed using ANOVA with appropriate post-hoc analyses as indicated in text using GraphPad Prism 8.3.0 (La Jolla, CA). Data are expressed as means±SEM, with p-values < 0.05 considered significant.

Impaired EE-mediated improvement in discriminative learning in PAE mice is directly correlated with impaired neurogenesis

The A-B contextual-fear discrimination behavioral assay tests the cognitive ability of mice to learn to discriminate between two similar contexts [24, 36] and requires the activity of adult-generated DGCs (aDGCs) for optimal performance [30–32, 37–39]. Here, we tested whether impairment of EE-mediated neurogenesis in PAE mice is directly correlated with impaired performance on this behavioral task. Figures 1A and 1B depict the experimental timeline and 7 day behavioral testing protocol, respectively. Briefly, SAC and PAE NestinCreER^T2:tdTomato male offspring received daily tamoxifen injections for five consecutive days at one week post-weaning to induce tdTomato reporter gene expression in hippocampal nestin⁺ stem/progenitor cells and their downstream progeny [21, 22, 28, 30, 31], and were subsequently placed into standard housing (SH) or enriched environment (EE) for 10 weeks. We then tested all mice on a neurogenesis-dependent version of the A-B context-fear discrimination learning task, where mice learn to discriminate the shock context (context A) from a similar but distinct non-shock context (context B). Discrimination was quantified using an automated scoring method based on freezing behavior as previously described [22, 30, 31]. We began testing all mice at 10 weeks post-tamoxifen, as prior work demonstrated that PAE-EE mice display ∼50% fewer aDGCs compared to SAC-EE mice at this time point [20–22].

Although all mice learned the discrimination task over time, there were significant differences across groups. Analysis of the discrimination ratios by three-way repeated measures ANOVA revealed significant discrimination learning across all groups over time (testing day F(6,250) = 325, p < 0.0001, Fig. 1C). Significant main effects of alcohol treatment [F(1,250) = 175, p < 0.0001] and housing [F(1,250) = 12.49, p = 0.0005)] were detected, with significant interactions of treatment x housing [F(1,250) = 98.73, p < 0.0001] and treatment x housing x day [F(6,250) = 18.38, p < 0.0001]. As shown in Fig. 1D, by testing day 7 SAC-EE mice displayed an approximate 2-fold increase in discrimination ability compared to their SAC-SH controls (p < 0.0001; Tukey’s post-hoc multiple comparison). In contrast, discrimination scores of PAE-EE mice were no different from PAE-SH mice at day 7. Figure 1E depicts average freezing times in context A and context B across all groups on testing day 7, demonstrating that the improved discrimination scores were due to reduced freezing time in the non-shock context, with SAC-EE mice showing greatest improvement compared to all other groups. These data demonstrate that EE-mediated improvement in a neurogenesis-dependent version of the A-B contextual fear discrimination task is impaired in PAE mice.

To assess the relationship between behavioral performance and adult hippocampal neurogenesis, we sacrificed all mice within one week of the final behavioral testing day (mice age ∼4 months) and quantified the number of tdTomato⁺ aDGCs within the dorsal dentate gyrus from 4 randomly selected mice per group. As shown in Fig. 2A, tdTomato⁺/NeuN⁺ aDGCs were robustly labeled across all groups, with an obvious increase in the number of aDGCs in SAC-EE mice. As shown in Fig. 2B, SAC-EE mice displayed a > 2-fold increase in the number of tdTomato⁺ aDGCs compared to SAC-SH controls (p = 0.02), whereas PAE-EE mice displayed no change in the number of tdTomato⁺ aDGCs compared to PAE-SH mice, consistent with previous reports [20–22]. Two-way ANOVA of tdTomato⁺ aDGC cell counts revealed main effects of alcohol treatment [F (1,11) = 5.83, p = 0.03)], housing [F(1,11) = 6.74, p = 0.02] and a significant treatment x housing interaction [F(1,11) = 8.07, p = 0.01]. Importantly, the number of aDGCs across individual mice was directly correlated with the level of performance on the neurogenesis-dependent A-B contextual fear-discrimination task at training day 7 (R² = 0.9422), such that mice with the highest number of aDGCs displayed the highest discrimination scores and vice versa (Fig. 2C).

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Fig. 2

Impaired EE-mediated discrimination learning in PAE mice is directly correlated with impaired neurogenesis. A. Representative confocal microscopy images of coronal sections through the dorsal dentate gyrus demonstrating tdTomato⁺ aDGCs (red), NeuN⁺ mature postmitotic neurons (green), DAPI⁺ nuclear counterstain (blue). B. Number of tdTomato+/NeuN+aDGCs across groups (means±SEM). Two-way ANOVA statistics: alcohol treatment [F (1,11) = 5.83, p = 0.03)], housing [F(1,11) = 6.74, p = 0.02], alcohol treatment x housing interaction [F(1,11) = 8.07, p = 0.01]. *p = 0.02 SAC-SH vs. SAC-EE; p < 0.01 SAC-EE vs. PAE-EE (Tukey’s post-hoc analysis). N = 4 mice/group. C. Behavioral performance as a function of neurogenesis. Colored circles correspond to data from individual mice from SAC-SH (blue), SAC-EE (red), PAE-SH (black) and PAE-EE (green); i.e., color convention as in B. Pearson correlation, R² = 0.9422.

Increased c-Fos expression within the dentate gyrus granule cell layer of PAE-EE mice following exposure to novel environment

Recent work suggests that young aDGC contribute to pattern discrimination by facilitating sparse activation within the dentate granule cell layer via local feedback inhibition and/or via direct monosynaptic input onto mature DGCs [40, 41]. To determine whether impaired EE-mediated neurogenesis in PAE mice is associated with alterations in behavioral activation of pre-existing, mature DGCs, we compared expression of the immediate early gene, c-Fos, within the dentate granule cell layer of SAC-EE and PAE-EE mice following exposure to novel environment. For these experiments, we exposed SAC-EE and PAE-EE mice (both groups reared under EE for 8 weeks) to a novel open field environment for 30 minutes and sacrificed them 80 minutes after return to their home cage (Fig. 3A). Control mice were not exposed to the novel environment and remained undisturbed within their home cage until sacrifice. We then quantified the number of c-Fos⁺ nuclei within the superior blade of the dentate granule cell layer of all mice. As shown in Fig. 3C, c-Fos⁺ cells were sparsely distributed throughout the dentate granule cell layer in both groups exposed to novelty; however, PAE-EE mice displayed a > 2-fold increase in the mean number of c-Fos⁺ DGCs compared to SAC-EE mice (Fig. 3B). Two-way ANOVA revealed a significant alcohol treatment x novelty interaction [[F (1,21) = 10.03, p = 0.005] with a significant impact of novelty on c-Fos⁺ expression only in PAE-EE mice (p = 0.01, PAE-EE home cage vs. PAE-EE novelty, Tukey’s post-hoc analysis).

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Fig. 3

PAE-EE mice display broadened novelty-induced c-Fos expression within the dentate gyrus compared to SAC-EE mice. A. Experimental timeline. SAC and PAE C57Bl6/J mice were exposed to EE housing conditions for 8 weeks (SAC-EE and PAE-EE). Mice were then exposed to a novel environment for 30 minutes and sacrificed 80 minutes following return to home cage. SAC-EE and PAE-EE mice not exposed to novelty remained in home cage until sacrifice and served as controls. B. c-Fos immunofluorescence. Representative confocal images of c-Fos immunoreactivity (pink) within dorsal dentate gyrus in SAC-EE and PAE-EE mice following exposure to novel environment. Histological sections were counterstained with DAPI nuclear dye (blue). C. Quantification of c-Fos⁺ nuclei within the suprapyramidal blade of the dorsal dentate gyrus across groups (mean±SEM). Two-way ANOVA statistics: alcohol treatment x novelty interaction [[F (1,21) = 10.03, p = 0.005]. Tukey’s post-hoc analysis revealed a significant impact of novelty on c-Fos+expression only in PAE-EE mice (*p = 0.01). Group n’s are as follows: SAC-EE home cage (n = 5 mice across 5 litters) and SAC-EE novelty (n = 7 mice across 7 litters); PAE-EE home cage (n = 5 mice across 5 litters) and PAE-EE novelty (n = 8 mice across 8 litters).

PAE impairs EE-mediated structural maturation of aDGCs

EE and spatial learning enhance dendritic complexity of aDGCs in mice [42–48]. Here, we examined the impact of PAE on EE-mediated dendritic branching complexity, using Sholl analysis and computer-assisted reconstruction of dendritic spines. For these studies, Nestin-CreER^T2:tdTomato reporter mice received a single injection of tamoxifen at approximately 3 months of age to sparsely label aDGCs. Such sparse labeling facilitates dendritic tracing by minimizing dendritic tree overlap among labeled aDGCs. All mice were sacrificed at 6 weeks post-labeling (mice ∼4.5 months of age at sacrifice), dendritic trees were traced and their morphology assessed by computer assisted Sholl analysis. As shown in Fig. 4A, dendritic branching was highest in SAC-EE mice compared to all other groups. Three-way ANOVA of dendritic branching complexity revealed a significant effect of distance from cell soma [F(26, 216) = 53.90, p < 0.0001), alcohol treatment [F(1,216) = 12.27, p < 0.0006] and housing [F(1,162) = 9.40, p < 0.0025] on dendritic branching, with a significant alcohol treatment x housing interaction [(F(1,162) = 24.70), p < 0.0001], but no change in total dendritic length (Fig. 4D). Two-way ANOVA comparison of total intersections across groups revealed a significant alcohol treatment x housing interaction [F(1,14) = 6.05, p = 0.027] with SAC-EE>PAE-EE (p = 0.016, Sidak’s multiple comparison). Representative traces of aDGC dendrites in 2D are shown in Fig. 1B. The increased dendritic complexity in SAC-EE mice was also accompanied by a significant increase in cell body size compared to SAC-SH controls, which was not observed in PAE-EE mice (Fig. 4C).

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Fig. 4

PAE impairs EE-mediated dendritic branching in aDGCs. A. Sholl analysis of dendritic branching complexity in tdTomato⁺ aDGCs across experimental groups. Three way ANOVA statistics: alcohol treatment [F(1,216) = 12.27, p < 0.0006], housing [F(1,162) = 9.40, p < 0.0025], alcohol treatment x housing interaction [(F(1,162) = 24.70), p < 0.0001]. n’s were as follows: SAC-SH (n = 4 mice; 14 neurons sampled) and SAC-EE (n = 6 mice; 21 neurons sampled) across 6 separate litters; PAE-SH (n = 4 mice; 16 neurons sampled) and PAE-EE (n = 4 mice; 14 neurons sampled) across 2 separate litters. 2-5 tdTomato⁺ cells were sampled/mouse and averaged such that n = mouse constitutes the unit of statistical determination. B. Representative Neurolucida^tm traces from compressed Z-stack confocal images for each group. C. Quantification of cell soma size of tdTomato+aDGCs across groups. Mean±SEM *p < 0.02 Tukey’s post-hoc. D. Quantification of total dendritic length in tdTomato⁺ aDGCs across groups. Mean±SEM.

To determine whether impairment of EE-mediated dendritic branching in aDGCs from PAE mice was associated with alterations in dendritic spine density or morphology, we sampled 20–75μm dendritic segments (average 40μm) located within the middle third of the dendritic tree from randomly selected SAC-EE and PAE-EE tdTomato⁺ aDGCs. We digitally reconstructed dendritic segments and subjected the reconstructions to automated quantification and classification of dendritic protrusions based on pre-determined morphological criteria as outlined in Methods and depicted in Fig. 5D. Total spine density of 6 week old aDGCs did not differ significantly between SAC-EE and PAE-EE mice (Fig. 5A). However, classification of spine type by morphology revealed 50% fewer filopodia-like spines in 6 week old aDGCs from PAE-EE compared to SAC-EE mice (Fig. 5B). When calculated as percentage of total spines (Fig. 5C), the reduced number of filopodial protrusions was accompanied by an increase in percentage of mushroom spines in PAE-EE (4.14% filopodial, 10.17% mushroom) compared to SAC-EE mice (8.44% filopodial, 8.77% mushroom); however, this increased percentage of mushroom spines did not reach statistical significance (Chi-square statistical analysis), and there were also no significant differences in the distribution of other spine types.

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Fig. 5

Comparison of spine density and morphology in tdTomato⁺ aDGCs from SAC-EE vs. PAE-EE mice. A. Total dendritic spine densities in SAC-EE vs. PAE-EE mice. SAC-EE (n = 6 mice from 5 litters), PAE-EE (n = 4 mice from 2 litters). Mean #spines/μm±SEM. B. Densities of stubby, long thin, filopodial and mushroom protrusions in SAC-EE vs. PAE-EE mice. Classification are based on criteria as outlined in Methods. Mean #spines/μm±SEM. *p < 0.01 unpaired t-test with Welch’s correction. C. Mean percent distribution of spine classes in tdTomato+aDGCs from SAC-EE and PAE-EE mice. D. Representative 3D reconstruction of dendritic segment with filopodial, long thin, stubby and mushroom spines. Image reconstructed with Imaris^tm Filament Tracer software.