Genetic inactivation of synaptosomal-associated protein 25 (SNAP-25) in adult hippocampal neural progenitors impairs pattern discrimination learning but not survival or structural maturation of newborn dentate granule cells

Abstract

Adult neurogenesis is necessary for proper cognition and behavior, however, the mechanisms that underlie the integration and maturation of newborn neurons into the pre-existing hippocampal circuit are not entirely known. In this study, we sought to determine the role of action potential (AP)-dependent synaptic transmission by adult-generated dentate granule cells (DGCs) in their survival and function within the existing circuitry. We used a triple transgenic mouse (NestinCreER^T2:Snap25^fl/fl: tdTomato) to inducibly inactivate AP-dependent synaptic transmission within adult hippocampal progenitors and their progeny. Behavioral testing in a hippocampal-dependent A/B contextual fear-discrimination task revealed impaired discrimination learning in mice harboring SNAP-25-deficient adult-generated dentate granule cells (DGCs). Despite poor performance on this neurogenesis-dependent task, the production and survival of newborn DGCs was quantitatively unaltered in tamoxifen-treated NestinCreER^T2:Snap25^fl/fl: tdTomato SNAP compared to tamoxifen-treated NestinCreER^T2:Snap25^wt/wt: tdTomato control mice. Although SNAP-25-deficient adult DGCs displayed a small but statistically significant enhancement in proximal dendritic branching, their overall dendritic length and distal branching complexity was unchanged. SNAP-25-deficient newborn DGCs also displayed robust efferent mossy fiber output to CA3, with normal linear density of large mossy fiber terminals (LMTs). These studies suggest that AP-dependent neurotransmitter release by newborn DGCs is not essential for their survival or rudimentary structural maturation within the adult hippocampus.

1 ∣. INTRODUCTION

Adult hippocampal neurogenesis occurs in all mammals studied to date, including humans (Spalding et al., 2013), and contributes to episodic memory, mood, and appropriate response to stress (reviewed by [Christian, Song, & Ming, 2014]). The rate of adult neurogenesis is dynamically regulated by genetic factors, hormones, drugs, and behavior (Christian et al., 2014). Hippocampal neurogenesis declines dramatically with age, and has been implicated in a growing number of brain pathologies, including epilepsy, stroke, neurodegenerative, and psychiatric diseases, implicating neurogenesis as a potential therapeutic target for mitigating cognitive decline and behavioral deficits associated with these conditions (Braun & Jessberger, 2014). Although many advances have been made in understanding the cellular and molecular mechanisms that govern the production, maturation, and integration of adult-generated dentate granule cells (DGCs), many questions still remain. For example, while it is known that the survival and circuit integration of newborn neurons is regulated by hippocampal synaptic activity, it is unknown whether action potential (AP)-dependent efferent synaptic communication between newly generated DGCs and their downstream targets is an essential component required for their survival, maturation, and integration within the existing circuitry.

In contrast to the widely held belief that the survival and maintenance of neurons in the developing central nervous system (CNS) is dependent on action potential-dependent synaptic communication (Goulding, 2004; Hanson et al., 2008; Katz and Shatz, 1996), it has been shown more recently that considerable brain development occurs in the absence of neurotransmitter release evoked by calcium-triggered neuroexocytosis. This has been particularly striking in mouse models bearing null mutations ablating the expression of various pre-synaptic proteins required for neurotransmitter release, such as the neural SNARE proteins, synaptobrevin (Schoch et al., 2001), or SNAP-25 (Blakey, Wilson, & Molnar, 2012; Molnar et al., 2002; Washbourne et al., 2002) where fetal brain development and its initial circuitry up until birth appears intact. Whether and how advanced synapse formation and neuronal circuitry would proceed in these models in situ has been difficult to establish due to early postnatal mortality. However, by exploiting heterochronic organotypic cultures to follow cortical development that occurs during the first weeks after birth, Wilson and colleagues have previously demonstrated appropriate early postnatal neural pathfinding of thalamocortical projections in Snap25^−/− constitutive null mutants (Blakey et al., 2012). These findings suggest that several rudimentary decision-making processes, including axonal guidance and target cell recognition, thought to require synaptic communication during brain development, can occur in the absence of evoked synaptic transmission.

An important caveat for these studies is that they have been limited to the very small window of late fetal and early neonatal brain development and have not been tested with regard to neurogenesis that occurs within the context of the adult brain. It is well known that during adult hippocampal neurogenesis, newly generated DGCs form functional efferent connections, but whether the survival, maturation, and integration of these developing neurons within the existing circuitry of the adult hippocampus requires efferent AP-dependent synaptic communication between newborn DGCs and their downstream targets has not been tested. Here, we focus on the role of action potential (AP)-dependent neurotransmission on the survival and structural maturation of adult-generated DGCs using Nestin-CreER^T2: Snap^fl/fl:tdTomato triple transgenic mice. This approach allows for conditional, tamoxifen-inducible inactivation of SNAP-25, a protein necessary for AP-dependent neurotransmission (Washbourne et al., 2002), in adult hippocampal progenitors and their progeny.

2 ∣. MATERIALS AND METHODS

2.1 ∣. Animals

Animal procedures were approved by the University of New Mexico Animal Care and Use Committee according to the NIH Animal Welfare Regulations and Public Health Service Policy on Humane Care and Use of Laboratory Animals. Nestin-CreER^T2:Snap25^fl/fl:tdTomato triple transgenic mice were maintained with homozygosity for all three alleles on a C57Bl/6 J genetic background. These mice harbor the tamoxifen-inducible Cre-ER^T2 fusion protein under control of the nestin promoter (Lagace et al., 2007), a mutated floxed Snap25 gene sequence in which loxP sites flank the alternatively spliced 5a/b exon (Marques-Smith et al., 2016), and a Rosa26-STOP-tdTomato reporter allel (Madisen et al., 2010). Snap25^fl/fl:tdTomato and Snap25^wt/wt: tdTomato homozygous bitransgenic mice were used for all experiments that invlolved stereotaxic delivery of retrovirus to confer bacterial Cre Recombinase to hippocampal progenitors in vivo. Snap25^fl/wt mice and Snap25^fl/fl embryos were used for culture of neural stem cells and embryonic hippocampal neurons, respectively. All mice were maintained in a temperature and humidity controlled facility with reverse 12-h dark/light cycle (lights off at 0800 hours), with food and water available ad libitum. For in vivo experiments of adult hippocampal neurogenesis, mice were tail clipped and genotyped, gender segregated and placed into enriched environment living conditions with access to running wheels to maximally promote hippocampal neurogenesis, using a previously described protocol (Choi, Allan, & Cunningham, 2005; Kajimoto et al., 2016; Kajimoto, Allan, & Cunningham, 2013). Enriched conditions included a large cage (48 cm × 27 cm × 20 cm) with 4–6 mice/cage, access to 2 running wheels per cage and various toys exchanged weekly. Only female mice were used to avoid potential gender-specific effects on learning performance and neurogenesis (Chow, Epp, Lieblich, Barha, & Galea, 2013).

2.2 ∣. Cell culture and electrophysiology

For PCR analysis of Cre-mediated recombination in culture, neural stem/progenitor cultures were established from microdissected sub-ventricular zone of postnatal day 28 Snap^fl/wt heterozygote mice, and the cultures were incubated for 2 days with adenovirus to confer expression of Cre-recombinase (ad-CMV-Cre; Vector Biolabs, Malvern, PA) as previously described (Harms, Li, & Cunningham, 2010). Genomic DNA was isolated from Ad-CMV-Cre-transduced (+CRE) and non-transduced control cultures and assayed by qualitative PCR using forward primer 5’-TGA CCA GGT TAG TGA GCA GGT 3’ and reverse primer 5’-TCC TTG TAG CAA TGG AGC AAT GCC-3’. Successful Cre-mediated DNA recombination in heterozygote Snap25^fl/wt neural stem/progenitor cells resulted in amplification products for the wildtype Snap25 allele (2,250 bp) and the recombined Snap25 allele (800 bp, Figure 1).

FIGURE 1.

CRE-mediated excision of Snap25^fl results in loss of SNAP-25 protein expression and reduced excitatory neurotransmission. (a) NSPCs were generated from Snap25^wt/wt (WT) or Snap25^fl/wt heterozygote mice. The latter were infected with an ad-CRE vector (+CRE) or were uninfected (-CRE). Two days post-infection genomic DNA was probed with a primer set positioned up- and downstream of the LoxP sites (orange triangles) flanking tandem arranged exons 5a/b (red/green). CRE-excision leads to the deletion of ~1,550 bp of endogenous sequence, distinguished as a PCR product of ~800 bp (+CRE, CRE-excised) compared to the >2,000 bp wild type allele (full length) amplified from WT and noninfected (-CRE) Snap25^fl/wt NSPCs. (b) Epifluorescence images of Snap25^fl/fl hippocampal cultures transduced with GFP-expressing control vector or with CRE-GFP-expressing lentiviral vector. Arrows indicate SNAP-25 immunofluorescent puncta in control cultures (left panel), which were undetectable in GFP⁺ SNAP-25 deficient neurons (right panel). (c) Representative traces of whole cell patch recordings from control (upper trace) and SNAP-25-deficient (lower trace) hippocampal cultures. (d) Pooled data demonstrate a significant reduction in the frequency of sEPSCs from SNAP-25-deficient cultures (SNAP25 KO; n = 19 cells across three separate cultures) compared to controls (GFP; n =16 cells across three separate cultures) with no significant change in amplitude of recorded events. All recordings were performed in the presence of 50 μM picrotoxin to block GABA-mediated inhibitory events. ***p < .01, unpaired Student’s t-test [Color figure can be viewed at wileyonlinelibrary.com]

For electrophysiological assessment of synaptic neurotransmission, dissociated hippocampal neuronal cultures were established from 1–2 day old Snap25^fl/fl mouse pups as previously described (Weick, Groth, Isaksen, & Mermelstein, 2003). At 4 days in vitro (DIV), neuronal cultures were transduced with lentiviral vectors to confer expression of Cre-recombinase and green fluorescent protein (GFP) or expression of GFP alone. One week after lentiviral transduction, cultures were used for immunostaining or for electrophysiological recordings. Immunostaining was performed using anti-SNAP25 monoclonal primary antibody SMI 81 (1:1,000; Abcam, Cambridge, UK,) and Alexa Fluor-conjugated goat anti-mouse secondary IgG (1:200; Molecular Probes, Eugene, OR) as previously described (Shimada et al., 2007). Whole cell patch-clamp recordings of spontaneous excitatory post-synaptic currents (sEPSCs) were obtained in the presence of 50 μM picrotoxin from GFP+ neurons after ~10 DIV as previously described (Weick et al., 2003).

2.3 ∣. Tamoxifen administration

Tamoxifen was administered to young adult female Nestin-CreER^T2:Snap25^fl/fl:tdTomato (n = 6) or Nestin-CreER^T2:Snap25^wt/wt:tdTomato (n = 8) mice at 6 weeks of age as previously described (Kajimoto et al., 2013). Tamoxifen was dissolved in 10% EtOH/90% sunflower seed oil and administered intraperitoneal (i.p.; 180 mg/kg) once per day for 5 consecutive days. Tamoxifen (T5648), 200 proof EtOH (459836), and sunflower oil (S5007) were obtained from Sigma-Aldrich (St. Louis, MO). At 4 weeks following the final tamoxifen injection, all mice were tested on an A-B contextual fear discrimination learning paradigm and subsequently sacrificed for histological assessment at 8 weeks post-recombination, as described below.

2.4 ∣. Contextual fear discrimination learning

Four weeks following the final tamoxifen injection, Nestin-CreER^T2:Snap25^fl/fl:tdTomato and Nestin-CreER^T2:Snap25^wt/wt:tdTomato mice were tested for context discrimination learning using an A-B contextual fear-discrimination learning task, as modified from Sahay et al. (2011). Mice received two training sessions/day for 7 consecutive days, during their active dark cycle. At least 1 h prior to each daily training session, the mice were transported to a low-light holding room located adjacent to the context behavioral testing room. At the end of the second daily training session, mice were returned to their home enrichment cages and animal rooms. For testing, each mouse was placed into either Context A (foot shock) or Context B (no foot shock) for 90 s, followed by applied foot shock (0.8 mA) to only Context A for 2 s. After a second 90 s interval, another foot shock was applied only in Context A, followed by a 30-s exploration period to the end of the session. Three hours after the first session, mice were exposed to a second identical training session, except that the mouse initially subjected to Context A with foot shock was now subjected to Context B without foot shock, and vice versa. The order of testing in Context A versus Context B was altered every day for each mouse (Figure 2b). Context A was a standard chamber with a stainless steel floor, clear Plexiglas front wall, and aluminum side and back walls. Context B was a similar chamber, except the floor was a wire mesh nonshock floor and the side and back walls were covered in striped black and white contact paper. Trials were recorded using a digital camera and mice were scored for freezing behavior at 5 s intervals throughout the duration of each training session by an observer blinded to genotype. A discrimination score was 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]. Thus, higher discrimination scores indicate better contextual discrimination. Mice were returned to EE housing and sacrificed 3 weeks later (8 weeks post-tamoxifen) for histological analysis (see below).

FIGURE 2.

Mice with SNAP-25-deficient adult-generated DGCs display impaired contextual fear-discrimination learning. (a) Experimental design of a/B contextual fear discrimination learning paradigm. (b) Mice with SNAP-25-deficient adult generated DGCs are slower in learning to discriminate between two similar but distinct contexts. (means ± SEM; control n = 8 mice, SNAP-25^−/− n = 6; F[91,12] = 11.79, p = .0049; two-way repeated measures ANOVA) [Color figure can be viewed at wileyonlinelibrary.com]

2.5 ∣. Retroviral delivery of Cre recombinase

Replication incompetent VSVG-G pseudotyped MMLV-based retroviral vectors were prepared according to the manufacturer's protocol using the GP2–293 retroviral packaging cell line transfected with pCMV-VSV-G (Cat. No. 631457; Clonetech, Mountain View, CA) and a retroviral plasmid construct harboring the expression sequence for bacterial Cre recombinase (kind gift from Dr. Shaoyu Ge, State University of New York, Stoneybrook, NY). Female Snap25^fl/fl:tdTomato (n = 6) or Snap25^wt/wt:tdTomato (n = 6) mice aged 6–8 weeks received stereotaxic injection of retrovirus delivered to two sites in the subgranular zone/hilus of the dorsal hippocampus (0.5 μL/site using a 1 μL Hamilton syringe), at the following coordinates relative to bregma (−2.0 mm AP, ±2.0 mm L, and – 2.0 mm DV). Mice were single housed for 2 days post-surgery and placed back into enrichment cages for 8 weeks prior to sacrifice for histological assessment of dendritic and axonal morphology as described below.

2.6 ∣. Histology and stereology analysis

Mice were overdosed with sodium pentobarbital (150 mg/kg, i.p.; Fort Dodge Animal Health, Fort Dodge, IA), and transcardially perfused with 0.1 M phosphate-buffered saline (PBS) containing 0.1% procaine and 2 U/mL heparin followed by 4% paraformaldehyde (PFA) in 0.1 M PBS. Brains were post-fixed in 4% paraformaldehyde overnight and cryoprotected by immersion in 30% sucrose in PBS for approximately 2 days at 4 °C. Using a freezing sliding knife microtome, brains were sectioned coronally at either 30 μm for immunostaining and stereological cell counting or at 60 μm for dendritic and axonal morphological analysis. Sections were stored in cryoprotectant solution (25% glycerol, 25% ethylene glycol, and 50% 0.1 M PBS) at −20 °C. Immunostaining was performed using mouse anti-NeuN primary antibody (1:1,000; EMD Millipore, Billerica, MA) and Cy5- or FITC-conjugated donkey anti-mouse IgG secondary antibody (1:250 Jackson Immunoresearch, West Grove, PA), using a previously published protocol (Newville, Valenzuela, Li, Jantzie, & Cunningham, 2017). Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) nuclear dye, mounted onto glass slides and coverslipped with Fluoromount G (ThermoFisher Scientific, Waltham, MA).

The number of newborn DGCs (NeuN⁺/tdTomato⁺) were estimated within the right dentate gyrus of the dorsal hippocampus using the Optical Fractionator probe in Stereoinvestigator™ software (Microbrightfield, Williston, VT) linked to an Olympus DSU spinning disk confocal microscope and a 40× objective as previously described (Kajimoto et al., 2013; Newville et al., 2017). The region of interest was traced in each histological section at 10×, which included the dentate granule cell layer and subgranular zone. At least 3 histological sections spaced approximately 120 μm apart were quantified from each mouse between stereotaxic coordinates −1.3 to −2.3 mm relative to bregma using an exhaustive counting method. Morphological analysis of dendritic branching was performed using Sholl analysis and Neurolucida™ software (Microbrightfield, Williston, VT). Confocal images of tdTomato⁺ DGCs were acquired using a Leica TCS SP8 confocal microscope. Z-stacks were acquired using a 20× objective with 2 μm optical intervals, and then collapsed into a single image plane using a maximum image intensity projection function. Dendrites were traced using Neurolucida™ software. Dendritic intersections crossing each concentric ring, spaced 5.75 μm apart and beginning 10 μm from the center of cell soma, were quantified by Neurolucida Explorer program (Kajimoto et al., 2016). Analysis was performed on 4–6 cells/ mouse across 3–6 histological sections/mouse.

2.7 ∣. Statistics

Data were analyzed using unpaired parametric t-test and two-way ANOVA using GraphPad Prism 7 or SPSS 2.0 software. Power analysis was done using GPower 3.1. Data are expressed as means ±S.E.M. with p < .05 considered statistically significant.

3 ∣. RESULTS

3.1 ∣. Cre-mediated excision of floxed Snap25 exon 5 a/b results in impaired synaptic neurotransmission

Cre-mediated excision of the floxed Snap25 allele and its impact on protein expression and synaptic transmission were assessed in neural cells in culture. To confirm Cre-mediated excision, neural progenitor cells cultured from subventricular zone of postnatal day 28 Snap25^fl/wt heterozygote mice were transduced with Ad-CMV-Cre. Qualitative PCR on isolated genomic DNA revealed successful Cre-mediated DNA recombination in heterozygote Snap25^fl/wt cultures, as indicated by the appearance an 800 bp PCR amplification product representing the shortened, Cre-recombined floxed Snap25 allele (Figure 1a). Excision of the floxed exon 5a/b has previously been shown to result in a frame-shift mutation with premature termination of SNAP-25 mRNA, rendering the gene inactive (Washbourne et al., 2001). To verify loss of SNAP-25 protein expression and its impact on excitatory synaptic neurotransmission, dissociated hippocampal mixed neuronal cultures established from 1- to 2-day old Snap25^fl/fl mouse pups were incubated with lentiviral-Cre-GFP or control lentiviral-GFP vector. As shown in Figure 1b, SNAP-25 immunofluorescent puncta were readily detectable in control cultures, but not in those transduced with Cre-GFP lentiviral vector. Whole cell patch clamp recordings from GFP⁺ neurons also demonstrated a marked reduction in the average frequency of recorded sEPSCs in SNAP-25-deficient cultures (n = 19 cells) compared to control cultures (n = 16 cells), although the average amplitude of recorded events remained similar indicating intact postsynaptic signaling (Figure 1c and d). The very small number of residual events in Cre-infected SNAP-25^fl/fl cultures may be due to slightly less than 100% efficiency in viral transduction and recombination. Overall, these data validate Cre-mediated recombination of the floxed Snap25 allele, and nearly complete loss of synaptic neurotransmission following Snap25 gene inactivation.

3.2 ∣. Conditional SNAP-25 deficient mice display impaired contextual fear-discrimination learning

Contextual fear-discrimination learning has previously been demonstrated to be dependent upon the function of adult-generated DGCs (Kheirbek, Tannenholz, & Hen, 2012; McHugh et al., 2007; Niibori et al., 2012; Sahay, Scobie, et al., 2011; Tronel et al., 2012). Here we used Nestin-CreER^T2:Snap25^fl/fl:tdTomato mice to determine whether tamoxifen-induced Snap25-deficiency in adult hippocampal progenitors and their downstream progeny impairs learning in an A/B contextual fear-discrimination paradigm. We treated adult Nestin-CreER^T2:Snap25^fl/fl:tdTomato mice with tamoxifen for 5 consecutive days and behaviorally tested them 4 weeks following the final tamoxifen injection. We chose this time frame based on previous studies indicating that immature adult-generated DGCs between 4 and 6 weeks of cellular age play a predominant role in pattern discrimination learning [reviewed in Anacker & Hen (2017)]. Nestin-CreER^T2:Snap25^wt/wt:tdTomato mice treated identically with tamoxifen served as controls. All mice were subjected to two learning trials each day for 7 consecutive days, which alternated between Context A (foot shock) and Context B (no foot shock; Figure 2a). As shown in Figure 2b, tamoxifen-treated Nestin-CreER^T2:Snap25^fl/fl:tdTomato mice displayed marked impairment in their ability to discriminate between these similar contexts, as indicated by significantly lower daily discrimination scores over time compared to control mice (genotype x time, F[1,12] = 11.79, p = .0049), suggesting impairment in the survival and/or function of SNAP-25 deficient adultgenerated DGCs.

3.3 ∣. SNAP-25 deficiency does not impair survival of adult-generated DGCs

To determine whether impaired context discrimination learning in tamoxifen-treated Nestin-CreER^T2:Snap25^fl/fl:tdTomato mice was due to impaired survival of adult-generated DGCs, we sacrificed mice 3 weeks following behavioral testing and performed stereological cell counts of NeuN⁺/tdTomato⁺ co-labeled cells (8 weeks postrecombination). As shown in Figure 3a, tdTomato was robustly expressed in tamoxifen-treated mice from both genotypes. Stereological analysis of cell numbers revealed no significant effect of genotype on the number of NeuN⁺/tdTomato⁺ newborn DGCs. These findings indicate that SNAP-25 expression is not required for the survival of adult generated DGCs, at least out to 8 weeks postrecombination.

FIGURE 3.

Induced SNAP-25-deficiency in adult hippocampal progenitors does not impair adult neurogenesis. (a) Confocal images depicting robust tdTomato⁺/NeuN⁺ adult-generated DGCs in both genotypes. (b) Quantification of adult neurogenesis reveals no difference between tamoxifen-treated genotypes at 8 weeks post-recombination. (control n = 8; SNAP-25^−/− n = 6; statistical comparison using unpaired Student's t-test) [Color figure can be viewed at wileyonlinelibrary.com]

3.4 ∣. Snap25 gene inactivation does not impair structural maturation of adult-generated DGCs

To determine whether SNAP-25 is critical for the morphological maturation of adult-generated DGCs, we stereotaxically delivered a retrovirus conferring Cre-recombinase to adult hippocampal progenitors in Snap25^fl/fl:tdTomato bitransgenic mice. Retroviral Cre was used to achieve sparse labeling of adult-generated DGCs for detailed dendritic and axonal morphological analysis. Snap25^wt/wt:tdTomato mice with stereotaxic delivery of Cre retrovirus served as controls. All mice were sacrificed at 8 weeks post retroviral injection. As shown in Figure 4, adult-generated tdTomato⁺ DGCs with SNAP-25 deficiency displayed a small but significant increase in the branching of proximal dendrites (Figure 4b and d), but no change in total dendritic length (Figure 4e) compared to control DGCs. Axons from SNAP-25-deficient DGCs also extended through the hilus and CA3 to reach the CA2/3 border (Figure 5a). Quantification of axon terminals at higher magnification revealed no difference in the linear density of large mossy fiber terminals (LMTs) in SNAP-25^−/− DGCs compared to controls (Figure 5b and c). These results indicate a surprisingly negligible impact of cell-intrinsic Snap25 gene inactivation on the general morphological differentiation of adult-generated DGCs.

FIGURE 4.

SNAP-25-deficient adult-generated DGCs show increased proximal dendritic branching. (a) Cre retrovirus was delivered stereotaxically to the subgranular/hilar region to initiate recombination and sparsely label newborn DGCs cells for dendritic and axonal morphological analysis. (b) Sample images from each genotype demonstrating newborn DGC dendritic morphology. (c) Sholl analysis of dendritic complexity demonstrates increased proximal branching at 47–110 μm from the soma in SNAP-25-deficient DGCs (control n = 6 mice; Snap25^−/− n = 5 mice; F[60,540] = 1.624, p = .0031). (d) Significant increases in number of dendritic intersections in SNAP-25-deficient DGCs, binned at 47–110 μm from cell body. (e) Total dendritic length was not different between genotypes (control n = 6 mice, SNAP25^−/− n = 5 mice). Statistical comparisons were made using a two-way repeated measures ANOVA with Fisher's LSD post-hoc analysis on fixed distance data and unpaired Student's t-test for dendritic length [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 5.

SNAP-25-deficient adult-generated DGCs display robust mossy fiber axonal outgrowth and normal linear density of large mossy fiber terminals (LMTs). (a) Confocal images demonstrating the presence of tdTomato+ mossy fiber axons passing through the hilus and CA3 to reach CA2/3 border in both genotypes. (b) Higher magnification of tdTomato+ LMTs in CA3 region. (c) SNAP-25-deficiency had no significant effect on the linear density of LMTs in CA3 (control n = 6 mice, SNAP25^−/− n = 6 mice, 5 axon sections per mouse; data analyzed by unpaired Student's t-test) [Color figure can be viewed at wileyonlinelibrary.com]

4 ∣. DISCUSSION

In this study, we demonstrate impaired pattern discrimination learning following selective and inducible Snap25 gene inactivation in adult hippocampal progenitors and their downstream progeny. Despite impaired learning of a neurogenesis-dependent task, we found no impairment in the survival of SNAP-25-deficient DGCs derived from these hippocampal progenitors out to 8 weeks post-recombination. Morphologically, SNAP-25-deficient DGCs appeared relatively normal in terms of dendritic complexity and axonal morphology, except for a small increase in proximal dendritic branching. Taken together, these studies suggest that AP-dependent synaptic transmission is not necessary for the survival or basic structural maturation of developing neurons within the context of the adult brain.

SNAP-25 is a component of the well-characterized SNARE core complex that mediates docking and vesicular release of neurotransmitters. Constitutive SNAP-25 null mice generated by germline deletion of the alternatively spliced exons 5a/b display lethality at birth due to respiratory failure (Washbourne et al., 2002). Neither abnormally spliced mRNA nor truncated polypeptide accumulate within fetal brains of these mice, indicating an essentially null mutation. SNAP-25 is not required for nerve growth or stimulus-independent neurotransmitter release, but is essential for evoked AP-dependent synaptic transmission (Washbourne et al., 2002). In contrast to the constitutive SNAP-25 null mice, Snap25^fl/fl mice are viable and fertile, allowing for conditional and inducible Cre-mediated Snap25 gene inactivation in distinct neuronal populations postnatally. Recent work using SST-ires-Cre:Snap25^fl/fl bitransgenic mice, in which Cre-mediated excision of exon 5a/b was driven by the somatostatin (SST) gene promoter, demonstrated conditional loss of AP-dependent GABA neurotransmission from SST⁺ inhibitory interneurons of developing neonatal cortex (Marques-Smith et al., 2016). In this study, we provide evidence that glutamatergic neurotransmission is also essentially abolished following conditional inactivation of SNAP-25, as assessed by a marked reduction of EPSCs in Snap25^fl/fl hippocampal cultures infected with lentiviral Cre.

Despite loss of glutamatergic transmission in SNAP-25 deficient neurons, we found no effect on the survival of adult-generated DGCs out to 8 weeks post-recombination in tamoxifen-treated Nestin-CreER^T2:Snap25^fl/fl:tdTomato mice. However, SNAP-25 deficient newborn DGCs were functionally impaired in these mice as indicated by diminished behavioral performance on a neurogenesis-dependent A/B contextual fear-discrimination task tested at 4–5 weeks postrecombination. Previous work has demonstrated that immature DGCs uniquely impact hippocampal network activity and are critically for pattern discrimination learning (Anacker & Hen, 2017). Indeed, many studies have demonstrated that the ability to discriminate similar episodic memories with overlapping features, such as required in the A/B contextual fear-discrimination and other pattern separation learning tasks, depends upon functional hippocampal neurogenesis (Clelland et al., 2009; Nakashiba et al., 2012; Sahay, Scobie, et al., 2011; Sahay, Wilson, & Hen, 2011; Tronel et al., 2012). Our observation of impaired pattern discrimination learning without numerical loss of adultgenerated SNAP25-deficient DGCs strongly suggests that the behavioral deficit is a consequence of impaired activity-dependent neurotransmission by SNAP-25-deficient newborn DGCs in vivo.

Although the general morphological features of SNAP-25-deficient DGCs were relatively normal, we did observe a small but significant increase in the branching of proximal but not distal dendrites, suggesting a potential shift in their functional synaptic innervation. Previous studies have demonstrated that engagement of hippocampal network activity by spatial learning sculpts the dendritic arbor of adult-generated DGCs (Lemaire et al., 2012; Tronel et al., 2010). Monosynaptic afferents connecting with newborn DGCs originate from both local and distal brain regions. The first synaptic inputs reaching young maturing DGCs originate from local SGZ GABAergic interneurons and exert a depolarizing effect important for neuroblast survival (Song et al., 2012, 2013). A few days later, the first glutamatergic input occurs from local excitatory mossy cells (Chancey, Poulsen, Wadiche, & Overstreet-Wadiche, 2014). As newborn DGCs extend their apical dendrites, they begin to receive inputs from dendritic-targeting interneurons, followed by long-range excitatory inputs from distant structures such as the medial and lateral entorhinal cortex and medial septum (Song, Olsen, Sun, Ming, & Song, 2016). Importantly, immature DGCs are thought to recruit feedback inhibition onto the mature circuit, such that the overall activity of the dentate gyrus likely represents a regulated balance of firing between mature and immature neurons (Drew et al., 2016). Thus, it is possible that increased proximal branching of SNAP-25 deficient DGCs may represent a compensatory shift in monosynaptic afferent input due to a disruption in circuit mechanisms regulating overall dentate activity. It is important to note that afferent input to adultgenerated DGCs is dynamically regulated by experience (Bergami et al., 2015) and that experience can alter hippocampal circuitry in complex ways (Adlaf et al., 2017). Therefore, it is not possible to interpret the current work outside of the context of environmental enrichment, since all mice were housed under EE conditions to maximally promote neurogenesis and neurogenic function. Future studies using rabies-based retrograde tracing of monosynaptic input onto SNAP-deficient adult-generated DGCs might be useful in assessing whether the pattern of afferent innervation is altered.

As mentioned, SNAP-25 deficient DGCs appeared to undergo relatively normal structural maturation, including the extension of mossy fiber axons that reached the CA3/CA2 border. Mossy fibers are the axons of DGCs that synaptically link DGCs with downstream target neurons, including hilar and CA3 interneurons and CA3 pyramidal neurons. Newborn DGCs extend mossy fiber axons to form functional synaptic connections with CA3 by 2 weeks after their birth, and these connections become stable by 4 weeks of cellular age (Gu et al., 2012). In this study, we found that the numbers and linear densities of large mossy fiber terminals (LMTs) were unaffected by SNAP-25 deficiency in adult-generated DGCs, out to 8 weeks post-recombination. This observation is consistent with a recent report demonstrating that permanent and selective genetic silencing of glutamatergic neurotransmission in DGCs using tetanus neurotoxin (TeNT) during early postnatal development also does not impair the formation or maintenance of LMTs along mossy fiber terminals (Sando et al., 2017). However, mossy fibers also form synaptic connections with inhibitory interneurons via filopodia-like extensions from LMTs and en passant boutons (Acsady, Kamondi, Sik, Freund, & Buzsaki, 1998), which are dynamically regulated during the maturation of adult-generated DGCs (Restivo, Niibori, Mercaldo, Josselyn, & Frankland, 2015). It will be important in future studies to determine whether SNAP-25 deficiency in adult-generated DGCs alters the size of LMTs and/or the formation of other types of presynaptic specializations, as well as the long-term maintenance of these connections past 8 weeks post-recombination in the adult brain.

During early postnatal development of the dentate gyrus, genetic silencing of a subset of granule neurons results in retraction of their processes and cell death within approximately 25 days due to inability to effectively compete with axons from younger DGCs (Yasuda et al., 2011). In contrast, inducible expression of tetanus toxin in DGCs older than 4–6 weeks of cellular age in adult mice, after stable efferent synaptic connections have formed, does not result in axonal retraction. Indeed, these genetically silenced DGCs display remarkable stability in their morphological and electrophysiological properties even after prolonged synaptic silencing for 3–6 months (Lopez et al., 2012). Likewise, recent studies using transgenic mice that allow for selective induction of tetanus toxin expression in Emx1-derived neurons, (which represent >95% of principal neurons in the dorsal telencephalon), demonstrate remarkable assembly and maintenance of excitatory synapses in the absence of glutamatergic neurotransmission in most principle cells of the hippocampal trisynaptic circuit during early postnatal development (Sando et al., 2017). Pertinent to the current research, a recent study has found that conditional loss of SNAP-25 from long-range projecting cortical neurons from embryonic stages allows normal axonal projections but results in eventual cell death months after silencing, although DGCs appeared to remain intact (Hoerder-Suabedissen et al., 2018). Our findings add to this literature to demonstrate the ability of adult-generated DGCs to survive, undergo structural maturation and form anatomical efferent connections when AP-dependent neurotransmission is blocked at the early hippocampal progenitor stage in adult mice. Further studies are warranted to evaluate whether SNAP-25 gene deletion alters more detailed aspects of connectivity including synapse number or ultra-structural morphological differentiation.