It is now widely accepted that hippocampal neurogenesis underpins critical cognitive functions, such as learning and memory. To assess the behavioral importance of adult-born neurons, we developed a novel knock-in mouse model that allowed us to specifically and reversibly ablate hippocampal neurons at an immature stage. In these mice, the diphtheria toxin receptor (DTR) is expressed under control of the doublecortin (DCX) promoter, which allows for specific ablation of immature DCX-expressing neurons after administration of diphtheria toxin while leaving the neural precursor pool intact. Using a spatially challenging behavioral test (a modified version of the active place avoidance test), we present direct evidence that immature DCX-expressing neurons are required for successful acquisition of spatial learning, as well as reversal learning, but are not necessary for the retrieval of stored long-term memories. Importantly, the observed learning deficits were rescued as newly generated immature neurons repopulated the granule cell layer upon termination of the toxin treatment. Repeat (or cyclic) depletion of immature neurons reinstated behavioral deficits if the mice were challenged with a novel task. Together, these findings highlight the potential of stimulating neurogenesis as a means to enhance learning.

In Alzheimer disease and related disorders, the microtubule-associated protein tau aggregates and forms cytoplasmic lesions that impair neuronal physiology at many levels. In addition to affecting the host neuron, tau aggregates also spread to neighboring, recipient cells where the misfolded tau aggregates, in a manner similar to prions, actively corrupt the proper folding of soluble tau, and thereby impair cellular functions. One vehicle for the transmission of tau aggregates are secretory nanovesicles known as exosomes. Here, we established a simple model of a neuronal circuit using a microfluidics culture system in which hippocampal neurons A and B were seeded into chambers 1 and 2, respectively, extending axons via microgrooves in both directions and thereby interconnecting. This system served to establish two models to track exosome spreading. In the first model, we labeled the exosomal membrane by coupling tetraspanin CD9 with either a green or red fluorescent tag. This allowed us to reveal that interconnected neurons exchange exosomes only when their axons extend in close proximity. In the second model, we added exosomes isolated from the brains of tau transgenic rTg4510 mice (i.e. exogenous, neuron A-derived) to neurons in chamber 1 (neuron B) interconnected with neuron C in chamber 2. This allowed us to demonstrate that a substantial fraction of the exogenous exosomes were internalized by neuron B and passed then on to neuron C. This transportation from neuron B to C was achieved by a mechanism that is consistent with the hijacking of secretory endosomes by the exogenous exosomes, as revealed by confocal, super-resolution and electron microscopy. Together, these findings suggest that fusion events involving the endogenous endosomal secretory machinery increase the pathogenic potential and the radius of action of pathogenic cargoes carried by exogenous exosomes.Electronic supplementary materialThe online version of this article (10.1186/s40478-018-0514-4) contains supplementary material, which is available to authorized users.

The medial nucleus of the amygdala (MeA) plays a key role in innate emotional behaviors by relaying olfactory information to hypothalamic nuclei involved in reproduction and defense. However, little is known about the neuronal components of this region or their role in the olfactory-processing circuitry of the amygdala. Here, we have characterized neurons in the posteroventral division of the medial amygdala (MePV) using the GAD67-GFP mouse. Based on their electrophysiological properties and GABA expression, unsupervised cluster analysis divided MePV neurons into three types of GABAergic (Types 1-3) and two non-GABAergic cells (Types I and II). All cell types received olfactory synaptic input from the accessory olfactory bulb and, with the exception of Type 2 GABAergic neurons, sent projections to both reproductive and defensive hypothalamic nuclei. Type 2 GABAergic cells formed a chemically and electrically interconnected network of local circuit inhibitory interneurons that resembled neurogliaform cells of the piriform cortex and provided feedforward inhibition of the olfactory-processing circuitry of the MeA. These findings provide a description of the cellular organization and connectivity of the MePV and further our understanding of amygdala circuits involved in olfactory processing and innate emotions.

D-Serine is a co-agonist at the NMDA receptor glycine-binding site. Early studies have emphasized a glial localization for D-serine. However the nature of the glial cells has not been fully resolved, because previous D-serine antibodies needed glutaraldehyde-fixation, precluding co-localization with fixation-sensitive antigens. We have raised a new D-serine antibody optimized for formaldehyde-fixation. Light and electron microscopic observations indicated that D-serine was concentrated into vesicle-like compartments in astrocytes and radial glial cells, rather than being distributed uniformly in the cytoplasm. In aged animals, patches of cortex and hippocampus were devoid of immunolabeling for D-serine, suggesting that impaired glial modulation of forebrain glutamatergic signaling might occur. Dual immunofluorescence labeling for glutamate and D-serine revealed D-serine in a subset of glutamatergic neurons, particularly in brainstem regions and in the olfactory bulbs. Microglia also contain D-serine. We suggest that some D-serine may be derived from the periphery. Collectively, our data suggest that the cellular compartmentation and distribution of D-serine may be more complex and extensive than previously thought and may have significant implications for our understanding of the role of D-serine in disease states including hypoxia and schizophrenia.

Glial fibrillary acidic protein (GFAP) is an enigmatic protein; it currently has no unambiguously defined role. It is expressed in the cytoskeleton of astrocytes in the mammalian brain. We have used co-immunoprecipitation to identify in vivo binding partners for GFAP in the rat and pig brain. We demonstrate interactions between GFAP, the glutamate transporter GLAST, the PDZ-binding protein NHERF1, and ezrin. These interactions are physiologically relevant; we demonstrate in vitro that transport of D-aspartate (a glutamate analogue) is significantly increased in the presence of GFAP and NHERF1. Moreover, we demonstrate in vivo that expression of GFAP is essential in retaining GLAST in the plasma membranes of astrocytes after an hypoxic insult. These data indicate that the cytoskeleton of the astrocyte plays an important role in protecting the brain against glutamate-mediated excitotoxicity.

It is generally assumed that rodent brains can be used as representative models of neurochemical function in other species, such as humans. We have compared the distributions of the predominant glial glutamate transporters in rodents, rabbits, cats, pigs, monkeys, and humans. We identify similarities but also significant differences between species. GLT-1v, which is abundantly expressed by rodent astrocytes, is expressed only in a rare subset of astrocytes of cats and humans, and appears to be absent from brains of rabbits and monkeys. Conversely, in the pig brain GLT-1v is expressed only by oligodendrocytes. GLAST and GLT-1alpha expression differed significantly between species; while rodents and rabbits exhibited uniform expression patterns in cortex, higher species, including cats, pigs, monkeys, and humans, exhibited heterogeneities in cortical and hippocampal expression. Patches devoid of labeling intermingling with patches of strong labeling were evident in areas such as temporal cortex and frontal cortex. In addition, we noted that in human motor cortex, there were inconsistencies in labeling for the C-terminal of GLT-1alpha and common domains of GLT-1, suggesting that the C-terminal region may be missing or that an unidentified splicing is present in many human astrocytes. Collectively our data suggest that assumptions as to the roles of glutamate transporters in any species may need to be tested empirically.

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At least two splice variants of GLT-1 are expressed by rat brain astrocytes, albeit in different membrane domains. There is at present only limited data available as to the spatial relationship of such variants relative to the location of synapses and their functional properties. We have characterized the transport properties of GLT-1v in a heterologous expression system and conclude that its transport properties are similar to those of the originally described form of GLT-1, namely GLT-1alpha. We demonstrate that GLT-1alpha is localized to glial processes, some of which are interposed between multiple synapse types, including GABAergic synapses, whereas GLT-1v is expressed by astrocytic processes, at sites not interposed between synapses. Both splice variants can be expressed by a single astrocyte, but such expression is not uniform over the surface of the astrocytes. Neither splice variant of GLT-1 is evident in brain neurons, but both are abundantly expressed in some retinal neurons. We conclude that GLT-1v may not be involved in shaping the kinetics of synaptic signaling in the brain, but may be critical in preventing spillover of glutamate between adjacent synapses, thereby regulating intersynaptic glutamatergic and GABAergic transmission. Furthermore, GLT-1v may be crucial in ensuring that low levels of glutamate are maintained at extrasynaptic locations, especially in pathological conditions such as ischemia, motor neurone disease, and epilepsy.