Induction of Oxidative Stress and Alteration of Synaptic Gene Expression in Newborn Hippocampal Granule Cells after Developmental Exposure to Aroclor 1254.

INTRODUCTION: Hippocampal newborn neurons integrate into functional circuits where they play an important role in learning and memory. We previously showed that perinatal exposure to Aroclor 1254, a commercial mixture of polychlorinated biphenyls (PCBs) associated with alterations of cognitive function in children, disrupted the normal maturation of excitatory synapses in the dentate gyrus. We hypothesized that hippocampal immature neurons underlie some of the cognitive effects of PCBs. METHODS: We used newly generated neurons to examine the effects of PCBs in mice following maternal exposure. Newborn dentate granule cells were tagged with enhanced green fluorescent protein using a transgenic mouse line. The transcriptome of the newly generated granule cells was assessed using RNA sequencing. RESULTS: Gestational and lactational exposure to 6 mg/kg/day of Aroclor 1254 disrupted the mRNA expression of 1,308 genes in newborn granule cells. Genes involved in mitochondrial functions were highly enriched with 154 genes significantly increased in exposed compared to control mice. The upregulation of genes involved in oxidative phosphorylation was accompanied by signs of endoplasmic reticulum stress and an increase in lipid peroxidation, a marker of oxidative stress, in the subgranular zone of the dentate gyrus but not in mature granule cells in the granular zone. Aroclor 1254 exposure also disrupted the expression of synaptic genes. Using laser-captured subgranular and granular zones, this effect was restricted to the subgranular zone, where newborn neurons are located. CONCLUSION: Our data suggest that gene expression in newborn granule cells is disrupted by Aroclor 1254 and provide clues to the effects of endocrine-disrupting chemicals on the brain.

Adaptive Mossy Cell Circuit Plasticity after Status Epilepticus.

Hilar mossy cells regulate network function in the hippocampus through both direct excitation and di-synaptic inhibition of dentate granule cells (DGCs). Substantial mossy cell loss accompanies hippocampal circuit changes in epilepsy. We examined the contribution of surviving mossy cells to network activity in the reorganized dentate gyrus after pilocarpine-induced status epilepticus (SE). To examine functional circuit changes, we optogenetically stimulated mossy cells in acute hippocampal slices from male mice. In control mice, activation of mossy cells produced monosynaptic excitatory and di-synaptic GABAergic currents in DGCs. In pilocarpine-treated mice, mossy cell density and excitation of DGCs were reduced in parallel, with only a minimal reduction in feedforward inhibition, enhancing the inhibition/excitation ratio. Surprisingly, mossy cell-driven excitation of parvalbumin-positive (PV+) basket cells, primary mediators of feed-forward inhibition, was maintained. Our results suggest that mossy cell outputs reorganize following seizures, increasing their net inhibitory effect in the hippocampus.SIGNIFICANCE STATEMENT Hilar mossy cell loss in epilepsy is associated with hippocampal hyperexcitability, potentially as a result of disrupted dentate microcircuit function. We used transgenic mice, translational mouse modeling, viral vectors, and optogenetics to selectively examine functional changes to mossy cell outputs following status epilepticus (SE). Interestingly, the outputs of surviving mossy cells exhibited adaptive plasticity onto target parvalbumin-positive (PV+) interneurons, resulting in a relative increase in their inhibitory control of dentate granule cells (DGCs). Our findings suggest that residual mossy cell outputs can reorganize in a homeostatic manner, which may provide clues for therapeutic targeting of this microcircuit.

α2δ-2 is required for depolarization-induced suppression of excitation in Purkinje cells.

α2δ proteins (CACNA2D1-4) are required for normal neurological function and contribute to membrane trafficking of voltage-gated calcium channels, through which calcium entry initiates numerous physiological processes. However, it remains unclear how α2δ proteins influence calcium-mediated signalling to control neuronal output. Using whole-cell recordings of mouse Purkinje cells, we show that α2δ-2 is required for functional coupling of postsynaptic voltage-dependent calcium entry with calcium-dependent effector mechanisms controlling two different outputs, depolarization-induced suppression of excitation and spike afterhyperpolarization. Our findings indicate an important role for α2δ-2 proteins in regulating functional postsynaptic calcium channel coupling in neurons, providing new context for understanding the effects of α2δ mutations on neuronal circuit function and presenting additional potential avenues to manipulate α2δ-mediated signalling for therapeutic gain. KEY POINTS: Calcium influx, via voltage-dependent calcium channels, drives numerous neuronal signalling processes with precision achieved in part by tight coupling between calcium entry and calcium-dependent effectors. α2δ proteins are important for neurological function and contribute to calcium channel membrane trafficking, although how α2δ proteins influence postsynaptic calcium-dependent signalling is largely unexplored. Here it is shown that loss of α2δ-2 proteins disrupts functional calcium coupling to two different postsynaptic calcium-dependent signals in mouse Purkinje cell neurons, retrograde endocannabinoid signalling and the action potential afterhyperpolarization. The findings provide new insights into the control of calcium coupling as well as new roles for α2δ-2 proteins in neurons.

Revisiting I-BAR Proteins at Central Synapses.

Dendritic spines, the distinctive postsynaptic feature of central nervous system (CNS) excitatory synapses, have been studied extensively as electrical and chemical compartments, as well as scaffolds for receptor cycling and positioning of signaling molecules. The dynamics of the shape, number, and molecular composition of spines, and how they are regulated by neural activity, are critically important in synaptic efficacy, synaptic plasticity, and ultimately learning and memory. Dendritic spines originate as outward protrusions of the cell membrane, but this aspect of spine formation and stabilization has not been a major focus of investigation compared to studies of membrane protrusions in non-neuronal cells. We review here one family of proteins involved in membrane curvature at synapses, the BAR (Bin-Amphiphysin-Rvs) domain proteins. The subfamily of inverse BAR (I-BAR) proteins sense and introduce outward membrane curvature, and serve as bridges between the cell membrane and the cytoskeleton. We focus on three I-BAR domain proteins that are expressed in the central nervous system: Mtss2, MIM, and IRSp53 that promote negative, concave curvature based on their ability to self-associate. Recent studies suggest that each has distinct functions in synapse formation and synaptic plasticity. The action of I-BARs is also shaped by crosstalk with other signaling components, forming signaling platforms that can function in a circuit-dependent manner. We discuss another potentially important feature-the ability of some BAR domain proteins to impact the function of other family members by heterooligomerization. Understanding the spatiotemporal resolution of synaptic I-BAR protein expression and their interactions should provide insights into the interplay between activity-dependent neural plasticity and network rewiring in the CNS.

α2δ-2 Protein Controls Structure and Function at the Cerebellar Climbing Fiber Synapse.

α2δ proteins (Cacna2d1-4) are auxiliary subunits of voltage-dependent calcium channels that also drive synapse formation and maturation. Because cerebellar Purkinje cells (PCs) predominantly, if not exclusively, express one isoform of this family, α2δ-2 (Cacna2d2), we used PCs as a model system to examine roles of α2δ in excitatory synaptic function in male and female Cacna2d2 knock-out (KO) mice. Whole-cell recordings of PCs from acute cerebellar slices revealed altered climbing fiber (CF)-evoked complex spike generation, as well as increased amplitude and faster decay of CF-evoked EPSCs. CF terminals in the KO were localized more proximally on PC dendrites, as indicated by VGLUT2+ immunoreactive puncta, and computational modeling demonstrated that the increased EPSC amplitude can be partly attributed to the more proximal location of CF terminals. In addition, CFs in KO mice exhibited increased multivesicular transmission, corresponding to greater sustained responses during repetitive stimulation, despite a reduction in the measured probability of release. Electron microscopy demonstrated that mutant CF terminals had twice as many vesicle release sites, providing a morphologic explanation for the enhanced glutamate release. Though KO CFs evoked larger amplitude EPSCs, the charge transfer was the same as wild-type as a result of increased glutamate reuptake, producing faster decay kinetics. Together, the larger, faster EPSCs in the KO explain the altered complex spike responses, which degrade information transfer from PCs and likely contribute to ataxia in Cacna2d2 KO mice. Our results also illustrate the multidimensional synaptic roles of α2δ proteins.SIGNIFICANCE STATEMENT α2δ proteins (Cacna2d1-4) regulate synaptic transmission and synaptogenesis, but coexpression of multiple α2δ isoforms has obscured a clear understanding of how various α2δ proteins control synaptic function. We focused on roles of the α2δ-2 protein (Cacna2d2), the deletion of which causes cerebellar ataxia and epilepsy in mice and humans. Because cerebellar Purkinje cells (PCs) only express this single isoform, we studied excitatory climbing fiber synaptic function onto PCs in Cacna2d2 KO mice. Using optical and electrophysiological analysis, we provide a detailed description of the changes in PCs lacking α2δ-2, and provide a comprehensive mechanistic explanation for how functional synaptic phenotypes contribute to the altered cerebellar output.

Autoimmune receptor encephalitis in mice induced by active immunization with conformationally stabilized holoreceptors.

Autoimmunity to membrane proteins in the central nervous system has been increasingly recognized as a cause of neuropsychiatric disease. A key recent development was the discovery of autoantibodies to N-methyl-d-aspartate (NMDA) receptors in some cases of encephalitis, characterized by cognitive changes, memory loss, and seizures that could lead to long-term morbidity or mortality. Treatment approaches and experimental studies have largely focused on the pathogenic role of these autoantibodies. Passive antibody transfer to mice has provided useful insights but does not produce the full spectrum of the human disease. Here, we describe a de novo autoimmune mouse model of anti-NMDA receptor encephalitis. Active immunization of immunocompetent mice with conformationally stabilized, native-like NMDA receptors induced a fulminant encephalitis, consistent with the behavioral and pathologic characteristics of human cases. Our results provide evidence for neuroinflammation and immune cell infiltration as components of the autoimmune response in mice. Use of transgenic mice indicated that mature T cells and antibody-producing cells were required for disease induction. This active immunization model may provide insights into disease induction and a platform for testing therapeutic approaches.

Exercise-induced enhancement of synaptic function triggered by the inverse BAR protein, Mtss1L.

Exercise is a potent enhancer of learning and memory, yet we know little of the underlying mechanisms that likely include alterations in synaptic efficacy in the hippocampus. To address this issue, we exposed mice to a single episode of voluntary exercise, and permanently marked activated mature hippocampal dentate granule cells using conditional Fos-TRAP mice. Exercise-activated neurons (Fos-TRAPed) showed an input-selective increase in dendritic spines and excitatory postsynaptic currents at 3 days post-exercise, indicative of exercise-induced structural plasticity. Laser-capture microdissection and RNASeq of activated neurons revealed that the most highly induced transcript was Mtss1L, a little-studied I-BAR domain-containing gene, which we hypothesized could be involved in membrane curvature and dendritic spine formation. shRNA-mediated Mtss1L knockdown in vivo prevented the exercise-induced increases in spines and excitatory postsynaptic currents. Our results link short-term effects of exercise to activity-dependent expression of Mtss1L, which we propose as a novel effector of activity-dependent rearrangement of synapses.

Early detonation by sprouted mossy fibers enables aberrant dentate network activity.

In temporal lobe epilepsy, sprouting of hippocampal mossy fiber axons onto dentate granule cell dendrites creates a recurrent excitatory network. However, unlike mossy fibers projecting to CA3, sprouted mossy fiber synapses depress upon repetitive activation. Thus, despite their proximal location, relatively large presynaptic terminals, and ability to excite target neurons, the impact of sprouted mossy fiber synapses on hippocampal hyperexcitability is unclear. We find that despite their short-term depression, single episodes of sprouted mossy fiber activation in hippocampal slices initiated bursts of recurrent polysynaptic excitation. Consistent with a contribution to network hyperexcitability, optogenetic activation of sprouted mossy fibers reliably triggered action potential firing in postsynaptic dentate granule cells after single light pulses. This pattern resulted in a shift in network recruitment dynamics to an "early detonation" mode and an increased probability of release compared with mossy fiber synapses in CA3. A lack of tonic adenosine-mediated inhibition contributed to the higher probability of glutamate release, thus facilitating reverberant circuit activity.

Preferential Targeting of Lateral Entorhinal Inputs onto Newly Integrated Granule Cells.

Mature dentate granule cells in the hippocampus receive input from the entorhinal cortex via the perforant path in precisely arranged lamina, with medial entorhinal axons innervating the middle molecular layer and lateral entorhinal cortex axons innervating the outer molecular layer. Although vastly outnumbered by mature granule cells, adult-generated newborn granule cells play a unique role in hippocampal function, which has largely been attributed to their enhanced excitability and plasticity (Schmidt-Hieber et al., 2004; Ge et al., 2007). Inputs from the medial and lateral entorhinal cortex carry different informational content. Thus, the distribution of inputs onto newly integrated granule cells will affect their function in the circuit. Using retroviral labeling in combination with selective optogenetic activation of medial or lateral entorhinal inputs, we examined the functional innervation and synaptic maturation of newly generated dentate granule cells in the mouse hippocampus. Our results indicate that lateral entorhinal inputs provide the majority of functional innervation of newly integrated granule cells at 21 d postmitosis. Despite preferential functional targeting, the dendritic spine density of immature granule cells was similar in the outer and middle molecular layers, which we speculate could reflect an unequal distribution of shaft synapses. However, chronic blockade of neurotransmitter release of medial entorhinal axons with tetanus toxin disrupted normal synapse development of both medial and lateral entorhinal inputs. Our results support a role for preferential lateral perforant path input onto newly generated neurons in mediating pattern separation, but also indicate that medial perforant path input is necessary for normal synaptic development.SIGNIFICANCE STATEMENT The formation of episodic memories involves the integration of contextual and spatial information. Newly integrated neurons in the dentate gyrus of the hippocampus play a critical role in this process, despite constituting only a minor fraction of the total number of granule cells. Here we demonstrate that these neurons preferentially receive information thought to convey the context of an experience. Each newly integrated granule cell plays this unique role for ∼1 month before reaching maturity.

Trajectory Analysis Unveils Reelin's Role in the Directed Migration of Granule Cells in the Dentate Gyrus.

Reelin controls neuronal migration and layer formation. Previous studies in reeler mice deficient in Reelin focused on the result of the developmental process in fixed tissue sections. It has remained unclear whether Reelin affects the migratory process, migration directionality, or migrating neurons guided by the radial glial scaffold. Moreover, Reelin has been regarded as an attractive signal because newly generated neurons migrate toward the Reelin-containing marginal zone. Conversely, Reelin might be a stop signal because migrating neurons in reeler, but not in wild-type mice, invade the marginal zone. Here, we monitored the migration of newly generated proopiomelanocortin-EGFP-expressing dentate granule cells in slice cultures from reeler, reeler-like mutants and wild-type mice of either sex using real-time microscopy. We discovered that not the actual migratory process and migratory speed, but migration directionality of the granule cells is controlled by Reelin. While wild-type granule cells migrated toward the marginal zone of the dentate gyrus, neurons in cultures from reeler and reeler-like mutants migrated randomly in all directions as revealed by vector analyses of migratory trajectories. Moreover, live imaging of granule cells in reeler slices cocultured to wild-type dentate gyrus showed that the reeler neurons changed their directions and migrated toward the Reelin-containing marginal zone of the wild-type culture, thus forming a compact granule cell layer. In contrast, directed migration was not observed when Reelin was ubiquitously present in the medium of reeler slices. These results indicate that topographically administered Reelin controls the formation of a granule cell layer.SIGNIFICANCE STATEMENT Neuronal migration and the various factors controlling its onset, speed, directionality, and arrest are poorly understood. Slice cultures offer a unique model to study the migration of individual neurons in an almost natural environment. In the present study, we took advantage of the expression of proopiomelanocortin-EGFP by newly generated, migrating granule cells to analyze their migratory trajectories in hippocampal slice cultures from wild-type mice and mutants deficient in Reelin signaling. We show that the compartmentalized presence of Reelin is essential for the directionality, but not the actual migratory process or speed, of migrating granule cells leading to their characteristic lamination in the dentate gyrus.

Distinct temporal filters in mitral cells and external tufted cells of the olfactory bulb.

KEY POINTS: The release probability of the odorant receptor neuron (ORN) is reportedly one of the highest in the brain and is predicted to impose a transient temporal filter on postsynaptic cells. Mitral cells responded to high frequency ORN stimulation with sustained transmission, whereas external tufted cells responded transiently. The release probability of ORNs (0.7) was equivalent across mitral and external tufted cells and could be explained by a single pool of slowly recycling vesicles. The sustained response in mitral cells resulted from dendrodendritic amplification in mitral cells, which was blocked by NMDA and mGluR1 receptor antagonists, converting mitral cell responses to transient response profiles. Our results suggest that although the afferent ORN synapse shows strong synaptic depression, dendrodendritic circuitry in mitral cells produces robust amplification of brief afferent input, and thus the relative strength of axodendritic and dendrodendritic input determines the postsynaptic response profile. ABSTRACT: Short-term synaptic plasticity is a critical regulator of neural circuits, and largely determines how information is temporally processed. In the olfactory bulb, afferent olfactory receptor neurons respond to increasing concentrations of odorants with barrages of action potentials, and their terminals have an extraordinarily high release probability. These features suggest that during naturalistic stimuli, afferent input to the olfactory bulb is subject to strong synaptic depression, presumably truncating the postsynaptic response to afferent stimuli. To examine this issue, we used single glomerular stimulation in mouse olfactory bulb slices to measure the synaptic dynamics of afferent-evoked input at physiological stimulus frequencies. In cell-attached recordings, mitral cells responded to high frequency stimulation with sustained responses, whereas external tufted cells responded transiently. Consistent with previous reports, olfactory nerve terminals onto both cell types had a high release probability (0.7), from a single pool of slowly recycling vesicles, indicating that the distinct responses of mitral and external tufted cells to high frequency stimulation did not originate presyaptically. Rather, distinct temporal response profiles in mitral cells and external tufted cells could be attributed to slow dendrodendritic responses in mitral cells, as blocking this slow current in mitral cells converted mitral cell responses to a transient response profile, typical of external tufted cells. Our results suggest that despite strong axodendritic synaptic depression, the balance of axodendritic and dendrodendritic circuitry in external tufted cells and mitral cells, respectively, tunes the postsynaptic responses to high frequency, naturalistic stimulation.

Short-Term Depression of Sprouted Mossy Fiber Synapses from Adult-Born Granule Cells.

Epileptic seizures potently modulate hippocampal adult neurogenesis, and adult-born dentate granule cells contribute to the pathologic retrograde sprouting of mossy fiber axons, both hallmarks of temporal lobe epilepsy. The characteristics of these sprouted synapses, however, have been largely unexplored, and the specific contribution of adult-born granule cells to functional mossy fiber sprouting is unknown, primarily due to technical barriers in isolating sprouted mossy fiber synapses for analysis. Here, we used DcxCreERT2 transgenic mice to permanently pulse-label age-defined cohorts of granule cells born either before or after pilocarpine-induced status epilepticus (SE). Using optogenetics, we demonstrate that adult-born granule cells born before SE form functional recurrent monosynaptic excitatory connections with other granule cells. Surprisingly, however, although healthy mossy fiber synapses in CA3 are well characterized "detonator" synapses that potently drive postsynaptic cell firing through their profound frequency-dependent facilitation, sprouted mossy fiber synapses from adult-born cells exhibited profound frequency-dependent depression, despite possessing some of the morphological hallmarks of mossy fiber terminals. Mature granule cells also contributed to functional mossy fiber sprouting, but exhibited less synaptic depression. Interestingly, granule cells born shortly after SE did not form functional excitatory synapses, despite robust sprouting. Our results suggest that, although sprouted mossy fibers form recurrent excitatory circuits with some of the morphological characteristics of typical mossy fiber terminals, the functional characteristics of sprouted synapses would limit the contribution of adult-born granule cells to hippocampal hyperexcitability in the epileptic hippocampus.SIGNIFICANCE STATEMENT In the hippocampal dentate gyrus, seizures drive retrograde sprouting of granule cell mossy fiber axons. We directly activated sprouted mossy fiber synapses from adult-born granule cells to study their synaptic properties. We reveal that sprouted synapses from adult-born granule cells have a diminished ability to sustain recurrent excitation in the epileptic hippocampus, which raises questions about the role of sprouting and adult neurogenesis in sustaining seizure-like activity.

Presynaptic gain control by endogenous cotransmission of dopamine and GABA in the olfactory bulb.

In the olfactory bulb, lateral inhibition mediated by local juxtaglomerular interneurons has been proposed as a gain control mechanism, important for decorrelating odorant responses. Among juxtaglomerular interneurons, short axon cells are unique as dual-transmitter neurons that release dopamine and GABA. To examine their intraglomerular function, we expressed channelrhodopsin under control of the DAT-cre promoter and activated olfactory afferents within individual glomeruli. Optical stimulation of labeled cells triggered endogenous dopamine release as measured by cyclic voltammetry and GABA release as measured by whole cell GABAA receptor currents. Activation of short axon cells reduced the afferent presynaptic release probability via D2 and GABAB receptor activation, resulting in reduced spiking in both mitral and external tufted cells. Our results suggest that short axon cells influence glomerular activity not only by direct inhibition of external tufted cells but also by inhibition of afferent inputs to external tufted and mitral cells.NEW & NOTEWORTHY Sensory systems, including the olfactory system, encode information across a large dynamic range, making synaptic mechanisms of gain control critical to proper function. Here we demonstrate that a dual-transmitter interneuron in the olfactory bulb controls the gain of intraglomerular afferent input via two distinct mechanisms, presynaptic inhibition as well as inhibition of a principal neuron subtype, and thereby potently controls the synaptic gain of afferent inputs.

Modulating synaptic NMDA receptors.

Recent structural information on ligand-gated glutamate receptors and newly-discovered clinical uses for NMDA receptor antagonists has renewed interest in understanding the mechanisms of drug action at these receptors. Although the voltage-dependence and calcium permeability of NMDA receptors are well-studied, the mechanisms affecting the time course of synaptic NMDA receptor activation may be of more therapeutic value by serving as a rheostat for the total synaptic response. The NMDA receptor-mediated EPSC time course has been thought of as a fixed parameter based simply on receptor subunit composition as variably constrained by anatomical and developmental expression patterns, albeit subject to modification by kinetic behaviors such as modal gating. However, the EPSC time course also can be manipulated by endogenous and exogenous ligands. In this commentary we discuss insights into the in situ composition and kinetic behavior of synaptic NMDA receptors and propose new opportunities to target modulatory sites on NMDA receptors and to develop useful therapeutics. The emerging data on the atomic structure of NMDA receptors and knowledge of the kinetics of native receptors in neurons provide a roadmap in this regard. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

Parallel processing of afferent olfactory sensory information.

KEY POINTS: The functional synaptic connectivity between olfactory receptor neurons and principal cells within the olfactory bulb is not well understood. One view suggests that mitral cells, the primary output neuron of the olfactory bulb, are solely activated by feedforward excitation. Using focal, single glomerular stimulation, we demonstrate that mitral cells receive direct, monosynaptic input from olfactory receptor neurons. Compared to external tufted cells, mitral cells have a prolonged afferent-evoked EPSC, which serves to amplify the synaptic input. The properties of presynaptic glutamate release from olfactory receptor neurons are similar between mitral and external tufted cells. Our data suggest that afferent input enters the olfactory bulb in a parallel fashion. ABSTRACT: Primary olfactory receptor neurons terminate in anatomically and functionally discrete cortical modules known as olfactory bulb glomeruli. The synaptic connectivity and postsynaptic responses of mitral and external tufted cells within the glomerulus may involve both direct and indirect components. For example, it has been suggested that sensory input to mitral cells is indirect through feedforward excitation from external tufted cells. We also observed feedforward excitation of mitral cells with weak stimulation of the olfactory nerve layer; however, focal stimulation of an axon bundle entering an individual glomerulus revealed that mitral cells receive monosynaptic afferent inputs. Although external tufted cells had a 4.1-fold larger peak EPSC amplitude, integration of the evoked currents showed that the synaptic charge was 5-fold larger in mitral cells, reflecting the prolonged response in mitral cells. Presynaptic afferents onto mitral and external tufted cells had similar quantal amplitude and release probability, suggesting that the larger peak EPSC in external tufted cells was the result of more synaptic contacts. The results of the present study indicate that the monosynaptic afferent input to mitral cells depends on the strength of odorant stimulation. The enhanced spiking that we observed in response to brief afferent input provides a mechanism for amplifying sensory information and contrasts with the transient response in external tufted cells. These parallel input paths may have discrete functions in processing olfactory sensory input.

Transcriptional Profiling of Newly Generated Dentate Granule Cells Using TU Tagging Reveals Pattern Shifts in Gene Expression during Circuit Integration.

Despite representing only a small fraction of hippocampal granule cells, adult-generated newborn granule cells have been implicated in learning and memory (Aimone et al., 2011). Newborn granule cells undergo functional maturation and circuit integration over a period of weeks. However, it is difficult to assess the accompanying gene expression profiles in vivo with high spatial and temporal resolution using traditional methods. Here we used a novel method ["thiouracil (TU) tagging"] to map the profiles of nascent mRNAs in mouse immature newborn granule cells compared with mature granule cells. We targeted a nonmammalian uracil salvage enzyme, uracil phosphoribosyltransferase, to newborn neurons and mature granule cells using retroviral and lentiviral constructs, respectively. Subsequent injection of 4-TU tagged nascent RNAs for analysis by RNA sequencing. Several hundred genes were significantly enhanced in the retroviral dataset compared with the lentiviral dataset. We compared a selection of the enriched genes with steady-state levels of mRNAs using quantitative PCR. Ontology analysis revealed distinct patterns of nascent mRNA expression, with newly generated immature neurons showing enhanced expression for genes involved in synaptic function, and neural differentiation and development, as well as genes not previously associated with granule cell maturation. Surprisingly, the nascent mRNAs enriched in mature cells were related to energy homeostasis and metabolism, presumably indicative of the increased energy demands of synaptic transmission and their complex dendritic architecture. The high spatial and temporal resolution of our modified TU-tagging method provides a foundation for comparison with steady-state RNA analyses by traditional transcriptomic approaches in defining the functional roles of newborn neurons.

Functional Integration of Adult-Born Hippocampal Neurons after Traumatic Brain Injury(1,2,3).

Traumatic brain injury (TBI) increases hippocampal neurogenesis, which may contribute to cognitive recovery after injury. However, it is unknown whether TBI-induced adult-born neurons mature normally and functionally integrate into the hippocampal network. We assessed the generation, morphology, and synaptic integration of new hippocampal neurons after a controlled cortical impact (CCI) injury model of TBI. To label TBI-induced newborn neurons, we used 2-month-old POMC-EGFP mice, which transiently and specifically express EGFP in immature hippocampal neurons, and doublecortin-CreER(T2) transgenic mice crossed with Rosa26-CAG-tdTomato reporter mice, to permanently pulse-label a cohort of adult-born hippocampal neurons. TBI increased the generation, outward migration, and dendritic complexity of neurons born during post-traumatic neurogenesis. Cells born after TBI had profound alterations in their dendritic structure, with increased dendritic branching proximal to the soma and widely splayed dendritic branches. These changes were apparent during early dendritic outgrowth and persisted as these cells matured. Whole-cell recordings from neurons generated during post-traumatic neurogenesis demonstrate that they are excitable and functionally integrate into the hippocampal circuit. However, despite their dramatic morphologic abnormalities, we found no differences in the rate of their electrophysiological maturation, or their overall degree of synaptic integration when compared to age-matched adult-born cells from sham mice. Our results suggest that cells born after TBI participate in information processing, and receive an apparently normal balance of excitatory and inhibitory inputs. However, TBI-induced changes in their anatomic localization and dendritic projection patterns could result in maladaptive network properties.

Localized hypoxia within the subgranular zone determines the early survival of newborn hippocampal granule cells.

The majority of adult hippocampal newborn cells die during early differentiation from intermediate progenitors (IPCs) to immature neurons. Neural stem cells in vivo are located in a relative hypoxic environment, and hypoxia enhances their survival, proliferation and stemness in vitro. Thus, we hypothesized that migration of IPCs away from hypoxic zones within the SGZ might result in oxidative damage, thus triggering cell death. Hypoxic niches were observed along the SGZ, composed of adult NSCs and early IPCs, and oxidative byproducts were present in adjacent late IPCs and neuroblasts. Stabilizing hypoxia inducible factor-1α with dimethyloxallyl glycine increased early survival, but not proliferation or differentiation, in neurospheres in vitro and in newly born SGZ cells in vivo. Rescue experiments in Bax(fl/fl) mutants supported these results. We propose that localized hypoxia within the SGZ contributes to the neurogenic microenvironment and determines the early, activity-independent survival of adult hippocampal newborn cells.

The IGF-derived tripeptide Gly-Pro-Glu is a weak NMDA receptor agonist.

Glutamate acts as the universal agonist at ionotropic glutamate receptors in part because of its high degree of conformational flexibility. Other amino acids and small peptides, however, can activate N-methyl-d-aspartate (NMDA) receptors, albeit usually with lower affinity and efficacy. Here, we examined the action of glycine-proline-glutamate (GPE), a naturally occurring tripeptide formed in the brain following cleavage of IGF-I. GPE is thought to have biological activity in the brain, but its mechanism of action remains unclear. With its flanking glutamate and glycine residues, GPE could bind to either the agonist or coagonist sites on NMDA receptors, however, this has not been directly tested. Using whole cell patch-clamp recordings in combination with rapid solution exchange, we examined both steady-state currents induced by GPE as well as the effects of GPE on synaptically evoked currents. High concentrations of GPE evoked inward currents, which were blocked either by NMDA receptor competitive antagonists or the voltage-dependent channel blocker Mg(2+). GPE also produced a slight attenuation in the NMDA- and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-mediated excitatory postsynaptic currents without altering the paired-pulse ratio. Our results suggest that GPE can activate NMDA receptors but at concentrations well above the expected concentration of GPE in the brain. Therefore, it is unlikely that endogenous GPE interacts with glutamate receptors under normal conditions.