Adult neurogenesis modulates the hippocampus-dependent period of associative fear memory.

Acquired memory initially depends on the hippocampus (HPC) for the process of cortical permanent memory formation. The mechanisms through which memory becomes progressively independent from the HPC remain unknown. In the HPC, adult neurogenesis has been described in many mammalian species, even at old ages. Using two mouse models in which hippocampal neurogenesis is physically or genetically suppressed, we show that decreased neurogenesis is accompanied by a prolonged HPC-dependent period of associative fear memory. Inversely, enhanced neurogenesis by voluntary exercise sped up the decay rate of HPC dependency of memory, without loss of memory. Consistently, decreased neurogenesis facilitated the long-lasting maintenance of rat hippocampal long-term potentiation in vivo. These independent lines of evidence strongly suggest that the level of hippocampal neurogenesis play a role in determination of the HPC-dependent period of memory in adult rodents. These observations provide a framework for understanding the mechanisms of the hippocampal-cortical complementary learning systems.

Interregulation between fragile X mental retardation protein and methyl CpG binding protein 2 in the mouse posterior cerebral cortex.

Several X-linked neurodevelopmental disorders including Rett syndrome, induced by mutations in the MECP2 gene, and fragile X syndrome (FXS), caused by mutations in the FMR1 gene, share autism-related features. The mRNA coding for methyl CpG binding protein 2 (MeCP2) has previously been identified as a substrate for the mRNA-binding protein, fragile X mental retardation protein (FMRP), which is silenced in FXS. Here, we report a homeostatic relationship between these two key regulators of gene expression in mouse models of FXS (Fmr1 Knockout (KO)) and Rett syndrome (MeCP2 KO). We found that the level of MeCP2 protein in the cerebral cortex was elevated in Fmr1 KO mice, whereas MeCP2 KO mice displayed reduced levels of FMRP, implicating interplay between the activities of MeCP2 and FMRP. Indeed, knockdown of MeCP2 with short hairpin RNAs led to a reduction of FMRP in mouse Neuro2A and in human HEK-293 cells, suggesting a reciprocal coupling in the expression level of these two regulatory proteins. Intra-cerebroventricular injection of an adeno-associated viral vector coding for FMRP led to a concomitant reduction in MeCP2 expression in vivo and partially corrected locomotor hyperactivity. Additionally, the level of MeCP2 in the posterior cortex correlated with the severity of the hyperactive phenotype in Fmr1 KO mice. These results demonstrate that MeCP2 and FMRP operate within a previously undefined homeostatic relationship. Our findings also suggest that MeCP2 overexpression in Fmr1 KO mouse posterior cerebral cortex may contribute to the fragile X locomotor hyperactivity phenotype.

Sexually dimorphic patterns in electroencephalography power spectrum and autism-related behaviors in a rat model of fragile X syndrome.

Fragile X syndrome (FXS), a neurodevelopmental disorder with autistic features, is caused by the loss of the fragile X mental retardation protein. Sex-specific differences in the clinical profile have been observed in FXS patients, but few studies have directly compared males and females in rodent models of FXS. To address this, we performed electroencephalography (EEG) recordings and a battery of autism-related behavioral tasks on juvenile and young adult Fmr1 knockout (KO) rats. EEG analysis demonstrated that compared to wild-type, male Fmr1 KO rats showed an increase in gamma frequency band power in the frontal cortex during the sleep-like immobile state, and both male and female KO rats failed to show an increase in delta frequency power in the sleep-like state, as observed in wild-type rats. Previous studies of EEG profiles in FXS subjects also reported abnormally increased gamma frequency band power, highlighting this parameter as a potential translatable biomarker. Both male and female Fmr1 KO rats displayed reduced exploratory behaviors in the center zone of the open field test, and increased distance travelled in an analysis of 24-h home cage activity, an effect that was more prominent during the nocturnal phase. Reduced wins against wild-type opponents in the tube test of social dominance was seen in both sexes. In contrast, increased repetitive behaviors in the wood chew test was observed in male but not female KO rats, while increased freezing in a fear conditioning test was observed only in the female KO rats. Our findings highlight sex differences between male and female Fmr1 KO rats, and indicate that the rat model of FXS could be a useful tool for the development of new therapeutics for treating this debilitating neurodevelopmental disorder.

Development of Adult-Generated Cell Connectivity with Excitatory and Inhibitory Cell Populations in the Hippocampus.

New neurons are generated continuously in the subgranular zone of the hippocampus and integrate into existing hippocampal circuits throughout adulthood. Although the addition of these new neurons may facilitate the formation of new memories, as they integrate, they provide additional excitatory drive to CA3 pyramidal neurons. During development, to maintain homeostasis, new neurons form preferential contacts with local inhibitory circuits. Using retroviral and transgenic approaches to label adult-generated granule cells, we first asked whether a comparable process occurs in the adult hippocampus in mice. Similar to development, we found that, during adulthood, new neurons form connections with inhibitory cells in the dentate gyrus, hilus, and CA3 regions as they integrate into hippocampal circuits. In particular, en passant bouton and filopodia connections with CA3 interneurons peak when adult-generated dentate granule cells (DGCs) are ∼4 weeks of age, a time point when these cells are most excitable. Consistent with this, optical stimulation of 4-week-old (but not 6- or 8-week-old) adult-generated DGCs strongly activated CA3 interneurons. Finally, we found that CA3 interneurons were activated robustly during learning and that their activity was strongly coupled with activity of 4-week-old (but not older) adult-generated DGCs. These data indicate that, as adult-generated neurons integrate into hippocampal circuits, they transiently form strong anatomical, effective, and functional connections with local inhibitory circuits in CA3. Significance statement: New neurons are generated continuously in the subgranular zone of the hippocampus and integrate into existing hippocampal circuits throughout adulthood. Understanding how these cells integrate within well formed circuits will increase our knowledge about the basic principles governing circuit assembly in the adult hippocampus. This study uses a combined connectivity analysis (anatomical, functional, and effective) of the output connections of adult-born hippocampal cells to show that, as these cells integrate into hippocampal circuits, they transiently form strong connections with local inhibitory circuits. This transient increase of connectivity may represent an homeostatic process necessary to accommodate changes in the excitation/inhibition balance induced by the addition of these new excitatory cells to the preexisting excitatory hippocampal circuits.

Manipulating a "cocaine engram" in mice.

Experience with drugs of abuse (such as cocaine) produces powerful, long-lasting memories that may be important in the development and persistence of drug addiction. The neural mechanisms that mediate how and where these cocaine memories are encoded, consolidated and stored are unknown. Here we used conditioned place preference in mice to examine the precise neural circuits that support the memory of a cocaine-cue association (the "cocaine memory trace" or "cocaine engram"). We found that a small population of neurons (∼10%) in the lateral nucleus of amygdala (LA) were recruited at the time of cocaine-conditioning to become part of this cocaine engram. Neurons with increased levels of the transcription factor CREB were preferentially recruited or allocated to the cocaine engram. Ablating or silencing neurons overexpressing CREB (but not a similar number of random LA neurons) before testing disrupted the expression of a previously acquired cocaine memory, suggesting that neurons overexpressing CREB become a critical hub in what is likely a larger cocaine memory engram. Consistent with theories that coordinated postencoding reactivation of neurons within an engram or cell assembly is crucial for memory consolidation (Marr, 1971; Buzsáki, 1989; Wilson and McNaughton, 1994; McClelland et al., 1995; Girardeau et al., 2009; Dupret et al., 2010; Carr et al., 2011), we also found that post-training suppression, or nondiscriminate activation, of CREB overexpressing neurons impaired consolidation of the cocaine memory. These findings reveal mechanisms underlying how and where drug memories are encoded and stored in the brain and may also inform the development of treatments for drug addiction.

Rotarod training in mice is associated with changes in brain structure observable with multimodal MRI.

The brain has been shown to remain structurally plastic even throughout adulthood. However, little is known how motor-skill training affects different MRI modalities in the adult mouse brain. The aim of this study is to investigate whether rotarod training, a simple motor training task taken from the standard test battery, is associated with structural plasticity observable with different MRI modalities in adult C57BL/6 mice. The rotarod is a standard test that taxes motor coordination and balance. We use T2-weighted MRI followed by deformation-based morphometry to assess local volume and fractional anisotropy (FA) derived from diffusion MRI to assess microstructure ex-vivo. Using deformation-based morphometry we found that the hippocampus, frontal cortex and amygdala are larger in rotarod-trained mice compared to untrained controls. Surprisingly, the cerebellum and white matter in the corpus callosum underlying the primary motor cortex are smaller after training. We also found that the volume of the motor cortex is positively correlated with better rotarod performance. Diffusion imaging indicates group differences and behavioral correlations with FA, a measure of microstructure. Trained mice have higher FA in the hippocampus. Better rotarod performance is associated with higher FA in the hippocampus and lower FA in the primary visual cortex. This is the first study to reveal the substantial structural reorganization of the adult mouse brain following only a relatively brief period of motor-skill training by using complementary measures of microstructure and volume.

In silico prediction and in vivo testing of promoters targeting GABAergic inhibitory neurons.

Impairment of GABAergic inhibitory neuronal function is linked to epilepsy and other neurological and psychiatric disorders. Recombinant adeno-associated virus (rAAV)-based gene therapy targeting GABAergic neurons is a promising treatment for GABA-associated disorders. However, there is a need to develop rAAV-compatible gene-regulatory elements capable of selectively driving expression in GABAergic neurons throughout the brain. Here, we designed several novel GABAergic gene promoters. In silico analyses, including evolutionarily conserved DNA sequence alignments and transcription factor binding site searches among GABAergic neuronal genes, were carried out to reveal novel sequences for use as rAAV-compatible promoters. rAAVs (serotype 9) were injected into the CSF of neonatal mice and into the brain parenchyma of adult mice to assess promoter specificity. In mice injected neonatally, transgene expression was detected in multiple brain regions with very high neuronal specificity and moderate-to-high GABAergic neuronal selectivity. The GABA promoters differed greatly in their levels of expression and, in some brain regions, showed strikingly different patterns of GABAergic neuron transduction. This study is the first report of rAAV vectors that are functional in multiple brain regions using promoters designed by in silico analyses from multiple GABAergic genes. These novel GABA-targeting vectors may be useful tools to advance gene therapy for GABA-associated disorders.

GABAergic Gene Regulatory Elements Used in Adeno-Associated Viral Vectors.

Several neurological and psychiatric disorders have been associated with impairments in GABAergic inhibitory neurons in the brain. Thus, in the current era of accelerated development of molecular medicine and biologically-based drugs, there is a need to identify gene regulatory sequences that can be utilized for selectively manipulating the expression of nucleic acids and proteins in GABAergic neurons. This is particularly important for the use of viral vectors in gene therapy. In this Mini Review, we discuss the use of various gene regulatory elements for targeting GABAergic neurons, with an emphasis on adeno-associated viral vectors, the most widely used class of viral vectors for treating brain diseases.

Gene therapy using an ortholog of human fragile X mental retardation protein partially rescues behavioral abnormalities and EEG activity.

Fragile X syndrome (FXS), a neurodevelopmental disorder with no known cure, is caused by a lack of expression of the fragile X mental retardation protein (FMRP). As a single-gene disorder, FXS is an excellent candidate for viral-vector-based gene therapy, although that is complicated by the existence of multiple isoforms of FMRP, whose individual cellular functions are unknown. We studied the effects of rat and mouse orthologs of human isoform 17, a major expressed isoform of FMRP. Injection of neonatal Fmr1 knockout rats and mice with adeno-associated viral vectors (AAV9 serotype) under the control of an MeCP2 mini-promoter resulted in widespread distribution of the FMRP transgenes throughout the telencephalon and diencephalon. Transgene expression occurred mainly in non-GABAergic neurons, with little expression in glia. Early postnatal treatment resulted in partial rescue of the Fmr1 KO rat phenotype, including improved social dominance in treated Fmr1 KO females and partial rescue of locomotor activity in males. Electro-encephalogram (EEG) recordings showed correction of abnormal slow-wave activity during the sleep-like state in male Fmr1 KO rats. These findings support the use of AAV-based gene therapy as a treatment for FXS and specifically demonstrate the potential therapeutic benefit of human FMRP isoform 17 orthologs.

Sexually Divergent Mortality and Partial Phenotypic Rescue After Gene Therapy in a Mouse Model of Dravet Syndrome.

Dravet syndrome (DS) is a neurodevelopmental genetic disorder caused by mutations in the SCN1A gene encoding the α subunit of the NaV1.1 voltage-gated sodium channel that controls neuronal action potential firing. The high density of this mutated channel in GABAergic interneurons results in impaired inhibitory neurotransmission and subsequent excessive activation of excitatory neurons. The syndrome is associated with severe childhood epilepsy, autistic behaviors, and sudden unexpected death in epilepsy. Here, we compared the rescue effects of an adeno-associated viral (AAV) vector coding for the multifunctional ß1 sodium channel auxiliary subunit (AAV-NaVß1) with a control vector lacking a transgene. We hypothesized that overexpression of NaVß1 would facilitate the function of residual voltage-gated channels and improve the DS phenotype in the Scn1a+/- mouse model of DS. AAV-NaVß1 was injected into the cerebral spinal fluid of neonatal Scn1a+/- mice. In untreated control Scn1a+/- mice, females showed a higher degree of mortality than males. Compared with Scn1a+/- control mice, AAV-NaVß1-treated Scn1a+/- mice displayed increased survival, an outcome that was more pronounced in females than males. In contrast, behavioral analysis revealed that male, but not female, Scn1a+/- mice displayed motor hyperactivity, and abnormal performance on tests of fear and anxiety and learning and memory. Male Scn1a+/- mice treated with AAV-NaVß1 showed reduced spontaneous seizures and normalization of motor activity and performance on the elevated plus maze test. These findings demonstrate sex differences in mortality in untreated Scn1a+/- mice, an effect that may be related to a lower level of intrinsic inhibitory tone in female mice, and a normalization of aberrant behaviors in males after central nervous system administration of AAV-NaVß1. The therapeutic efficacy of AAV-NaVß1 in a mouse model of DS suggests a potential new long-lasting biological therapeutic avenue for the treatment of this catastrophic epilepsy.

The Application of Adeno-Associated Viral Vector Gene Therapy to the Treatment of Fragile X Syndrome.

Viral vector-mediated gene therapy has grown by leaps and bounds over the past several years. Although the reasons for this progress are varied, a deeper understanding of the basic biology of the viruses, the identification of new and improved versions of viral vectors, and simply the vast experience gained by extensive testing in both animal models of disease and in clinical trials, have been key factors. Several studies have investigated the efficacy of adeno-associated viral (AAV) vectors in the mouse model of fragile X syndrome where AAVs have been used to express fragile X mental retardation protein (FMRP), which is missing or highly reduced in the disorder. These studies have demonstrated a range of efficacies in different tests from full correction, to partial rescue, to no effect. Here we provide a backdrop of recent advances in AAV gene therapy as applied to central nervous system disorders, outline the salient features of the fragile X studies, and discuss several key issues for moving forward. Collectively, the findings to date from the mouse studies on fragile X syndrome, and data from clinical trials testing AAVs in other neurological conditions, indicate that AAV-mediated gene therapy could be a viable strategy for treating fragile X syndrome.

Mammalian Susceptibility to a Neonicotinoid Insecticide after Fetal and Early Postnatal Exposure.

Neonicotinoids have become the most widely used class of insecticides world-wide. Although numerous studies have documented neonicotinoid toxicity in bees and other insects, the effects of exposure during early development in mammals remain largely unexplored. We assessed the effects of the neonicotinoid imidacloprid (IMI) in adult male and female mice after in utero and early postnatal exposure. Pregnant mice were infused with IMI (0.5 mg/kg/day) from gestational day 4 to the end of nursing at postnatal day 21. The young adult offspring were studied in a series of biochemical and behavioral tests. To assess reproducibility, the behavioral analyses were conducted in three separate studies using multiple exposed litters. Exposure to IMI reduced fecundity, and in adult offspring, decreased body weight in male but not female pups. Offspring from IMI-treated mothers displayed lower triglycerides, elevated motor activity, enhanced social dominance, reduced depressive-like behavior, and a diminution in social aggression compared to vehicle treated controls. Low levels of IMI were detected in the brains and livers of the treated mothers, while trace levels were detected in some offspring. Our results demonstrate that transient exposure to a neonicotinoid over the early developmental period induces long-lasting changes in behavior and brain function in mice.

Ectopic expression of aPKC-mediated phosphorylation in p300 modulates hippocampal neurogenesis, CREB binding and fear memory differently with age.

Epigenetic modifications have become an emerging interface that links extrinsic signals to alterations of gene expression that determine cell identity and function. However, direct signaling that regulates epigenetic modifications is unknown. Our previous work demonstrated that phosphorylation of CBP at Ser 436 by atypical protein kinase C (aPKC) regulates age-dependent hippocampal neurogenesis and memory. p300, a close family member of CBP, lacks the aPKC-mediated phosphorylation found in CBP. Here, we use a phosphorylation-competent p300 (G442S) knock-in (KI) mouse model that ectopically expresses p300 phosphorylation in a homologous site to CBP Ser436, and assess its roles in modulating hippocampal neurogenesis, CREB binding ability, and fear memory. Young adult (3 months) p300G422S-KI mice exhibit enhanced hippocampal neurogenesis due to increased cell survival of newly-generated neurons, without alterations in CREB binding and contextual fear memory. On the other hand, mature adult (6 months) p300G422S-KI mice display reduced CREB binding, associated with impaired contextual fear memory without alterations in hippocampal neurogenesis. Additionally, we show that repulsive interaction between pS133-CREB and pS422-p300G422S may contribute to the reduced CREB binding to p300G422S. Together, these data suggest that a single phosphorylation change in p300 has the capability to modulate hippocampal neurogenesis, CREB binding, and associative fear memory.

Alternative poly(A) site-selection regulates the production of alternatively spliced vesl-1/homer1 isoforms that encode postsynaptic scaffolding proteins.

The vesl-1/homer1 gene encodes a scaffold protein that interacts with several receptors to modulate synaptic functions. The gene also encodes two shorter forms that counteract the functions of the long form of Vesl. Expression of the shorter forms is driven by neural activities such as long-term potentiation. Here we analyzed the mechanism regulating vesl-1 alternative splicing. Each functional poly(A) site was in a different part of the 3'-terminal exon, with promoter-proximal and promoter-distal sites at the end of exons corresponding to the short and long form Vesl-1, respectively. 3'-End-processing at proximal poly(A) site, specifically at the vesl-1M poly(A) site, was enhanced by extracellular stimuli, thereby switching transcription termination from promoter-distal to -proximal poly(A) site. This switch was not specifically coupled to the vesl-1 promoter and was independent of de novo protein synthesis. Analysis of transcripts from mini-genes that mimic the structure of endogenous vesl-1 revealed that the vesl-1M poly(A) region plays a crucial role in switching to the alternative pre-mRNA splicing that is triggered by extracellular stimuli. Therefore, a 3'-end-processing event regulates the neural activity-dependent alternative splicing of vesl-1. This is the first report of a gene in which alternative poly(A) site-selection regulates alternative splicing in a protein synthesis-independent manner.

FMRP Expression Levels in Mouse Central Nervous System Neurons Determine Behavioral Phenotype.

Fragile X mental retardation protein (FMRP) is absent or highly reduced in Fragile X Syndrome, a genetic disorder causing cognitive impairment and autistic behaviors. Previous proof-of-principle studies have demonstrated that restoring FMRP in the brain using viral vectors can improve pathological abnormalities in mouse models of fragile X. However, unlike small molecule drugs where the dose can readily be adjusted during treatment, viral vector-based biological therapeutic drugs present challenges in terms of achieving optimal dosing and expression levels. The objective of this study was to investigate the consequences of expressing varying levels of FMRP selectively in neurons of Fmr1 knockout and wild-type (WT) mice. A wide range of neuronal FMRP transgene levels was achieved in individual mice after intra-cerebroventricular administration of adeno-associated viral vectors coding for FMRP. In all treated knockout mice, prominent FMRP transgene expression was observed in forebrain structures, whereas lower levels were present in more caudal regions of the brain. Reduced levels of the synaptic protein PSD-95, elevated levels of the transcriptional modulator MeCP2, and abnormal motor activity, anxiety, and acoustic startle responses in Fmr1 knockout mice were fully or partially rescued after expression of FMRP at about 35-115% of WT expression, depending on the brain region examined. In the WT mouse, moderate FMRP over-expression of up to about twofold had little or no effect on PSD-95 and MeCP2 levels or on behavioral endophenotypes. In contrast, excessive over-expression in the Fmr1 knockout mouse forebrain (approximately 2.5-6-fold over WT) induced pathological motor hyperactivity and suppressed the startle response relative to WT mice. These results delineate a range of FMRP expression levels in the central nervous system that confer phenotypic improvement in fragile X mice. Collectively, these findings are pertinent to the development of long-term curative gene therapy strategies for treating Fragile X Syndrome and other neurodevelopmental disorders.

The aPKC-CBP Pathway Regulates Adult Hippocampal Neurogenesis in an Age-Dependent Manner.

While epigenetic modifications have emerged as attractive substrates to integrate environmental changes into the determination of cell identity and function, specific signals that directly activate these epigenetic modifications remain unknown. Here, we examine the role of atypical protein kinase C (aPKC)-mediated Ser436 phosphorylation of CBP, a histone acetyltransferase, in adult hippocampal neurogenesis and memory. Using a knockin mouse strain (CbpS436A) in which the aPKC-CBP pathway is deficient, we observe impaired hippocampal neuronal differentiation, maturation, and memory and diminished binding of CBP to CREB in 6-month-old CbpS436A mice, but not at 3 months of age. Importantly, elevation of CREB activity rescues these deficits, and CREB activity is reduced whereas aPKC activity is increased in the murine hippocampus as they age from 3 to 6 months regardless of genotype. Thus, the aPKC-CBP pathway is a homeostatic compensatory mechanism that modulates hippocampal neurogenesis and memory in an age-dependent manner in response to reduced CREB activity.

Optimization of CLARITY for Clearing Whole-Brain and Other Intact Organs

The development, refinement, and use of techniques that allow high-throughput imaging of whole brains with cellular resolution will help us understand the complex functions of the brain. Such techniques are crucial for the analysis of complete neuronal morphology-anatomical and functional-connectivity, and repeated molecular phenotyping. CLARITY is a recently introduced technique that produces structurally intact, yet optically transparent tissue, which may be labeled and imaged without sectioning. However, the utility of this technique depends on several procedural variables during the process in which the light-scattering lipids in a tissue are replaced by a transparent hydrogel matrix. Here, we systematically varied a number of factors (including temperature, hydrogel composition, and polymerization conditions) to provide an optimized, highly replicable CLARITY procedure for clearing mouse brains. We found that for these preparations optimal tissue clearing requires electrophoresis (and cannot be achieved with passive clearing alone) for 5 d with a combination of 37 and 55°C temperature. Although this protocol is optimized for brains, we also show that it can be used to clear and analyze a variety of organs. Brain or other tissue prepared using this protocol is suitable for high-throughput imaging with confocal or single-plane illumination microscopy.

Hippocampal neurogenesis regulates forgetting during adulthood and infancy.

Throughout life, new neurons are continuously added to the dentate gyrus. As this continuous addition remodels hippocampal circuits, computational models predict that neurogenesis leads to degradation or forgetting of established memories. Consistent with this, increasing neurogenesis after the formation of a memory was sufficient to induce forgetting in adult mice. By contrast, during infancy, when hippocampal neurogenesis levels are high and freshly generated memories tend to be rapidly forgotten (infantile amnesia), decreasing neurogenesis after memory formation mitigated forgetting. In precocial species, including guinea pigs and degus, most granule cells are generated prenatally. Consistent with reduced levels of postnatal hippocampal neurogenesis, infant guinea pigs and degus did not exhibit forgetting. However, increasing neurogenesis after memory formation induced infantile amnesia in these species.

Suppression of adult neurogenesis impairs population coding of similar contexts in hippocampal CA3 region.

Different places may share common features, but are coded by distinct populations of CA3 neurons in the hippocampus. Here we show that chemical or genetic suppression of adult neurogenesis in the hippocampus impairs this population-based coding of similar (but not dissimilar) contexts. These data provide a neural basis for impaired spatial discrimination following ablation of adult neurogenesis, and support the proposal that adult neurogenesis regulates the efficiency of a pattern separation process in the hippocampus.