The bed nucleus of the stria terminalis (BNST) plays an important role in fear, stress, and anxiety. It contains a collection of subnuclei delineated by gross cytoarchitecture features; however, there has yet to be a systematic examination of specific BNST neuronal types and their associated neurochemical makeup. The present study focuses on improved characterization of the anterior BNST based on differing molecular and chemical expression aided by mouse genetics. Specific Cre driver lines crossed with a fluorescent reporter line were used for genetic cell targeting and immunochemical staining. Using this new approach, we were able to robustly identify specific excitatory and inhibitory cell types in the BNST. The presence and distribution of excitatory neurons were firmly established; glutamatergic neurons in the anterior BNST accounted for about 14% and 31% of dorsal and ventral BNST cells, respectively. GABAergic neurons expressing different isoforms of glutamic acid decarboxylase were found to have differential subregional distributions. Almost no parvalbumin-expressing cells were found in the BNST, while somatostatin-expressing cells and calretinin-expressing cells account for modest proportions of BNST cells. In addition, vasoactive intestinal peptide-expressing axonal plexuses were prominent in the oval and juxtacapsular subregions. In addition, we discovered that corticotropin-releasing hormone-expressing cells contain GABAergic and glutamatergic subpopulations. Together, this study reveals new information on excitatory and inhibitory neurons in the BNST, which will facilitate genetic dissection and functional studies of BNST subregions. J. Comp. Neurol. 524:2379-2399, 2016. © 2016 Wiley Periodicals, Inc.

The bed nucleus of the stria terminalis (BNST) plays an important role in fear, stress, and anxiety. It contains a collection of subnuclei delineated by gross cytoarchitecture features; however, there has yet to be a systematic examination of specific BNST neuronal types and their associated neurochemical makeup. The present study focuses on improved characterization of the anterior BNST based on differing molecular and chemical expression aided by mouse genetics. Specific Cre driver lines crossed with a fluorescent reporter line were used for genetic cell targeting and immunochemical staining. Using this new approach, we were able to robustly identify specific excitatory and inhibitory cell types in the BNST. The presence and distribution of excitatory neurons were firmly established; glutamatergic neurons in the anterior BNST accounted for about 14% and 31% of dorsal and ventral BNST cells, respectively. GABAergic neurons expressing different isoforms of glutamic acid decarboxylase were found to have differential subregional distributions. Almost no parvalbumin-expressing cells were found in the BNST, while somatostatin-expressing cells and calretinin-expressing cells account for modest proportions of BNST cells. In addition, vasoactive intestinal peptide-expressing axonal plexuses were prominent in the oval and juxtacapsular subregions. In addition, we discovered that corticotropin-releasing hormone-expressing cells contain GABAergic and glutamatergic subpopulations. Together, this study reveals new information on excitatory and inhibitory neurons in the BNST, which will facilitate genetic dissection and functional studies of BNST subregions. J. Comp. Neurol. 524:2379-2399, 2016. © 2016 Wiley Periodicals, Inc.

Astrocytes play a fundamental role in synapse formation, pruning, and plasticity, which are associated with learning and memory. However, the role of astrocytes in learning and memory is still largely unknown. Our previous study showed that astrocyte-specific ephrin-B1 knock-out (KO) enhanced but ephrin-B1 overexpression (OE) in hippocampal astrocytes impaired contextual memory recall following fear conditioning. The goal of this study was to understand the mechanism by which astrocytic ephrin-B1 influences learning; specifically, learning-induced remodeling of synapses and dendritic spines in CA1 hippocampus using fear-conditioning paradigm. While we found a higher dendritic spine density and clustering on c-Fos-positive (+) neurons activated during contextual memory recall in both wild-type (WT) and KO mice, overall spine density and mEPSC amplitude were increased in CA1 neurons of KO compared to WT. In contrast, ephrin-B1 OE in hippocampal astrocytes impaired dendritic spine formation and clustering, specifically on c-Fos(+) neurons, coinciding with an overall decrease in vGlut1/PSD95 co-localization. Although astrocytic ephrin-B1 influenced learning-induced spine formation, the changes in astrocytic ephrin-B1 levels did not affect spine enlargement as no genotype differences in spine volume were observed between trained WT, KO, and OE groups. Our results suggest that a reduced formation of new spines rather than spine maturation in activated CA1 hippocampal neurons is most likely responsible for impaired contextual learning in OE mice due to abundantly high ephrin-B1 levels in astrocytes. The ability of astrocytic ephrin-B1 to negatively influence new spine formation during learning can potentially regulate new synapse formation at specific dendritic domains and underlie memory encoding.

Astrocytes are implicated in synapse formation and elimination, which are associated with developmental refinements of neuronal circuits. Astrocyte dysfunctions are also linked to synapse pathologies associated with neurodevelopmental disorders and neurodegenerative diseases. Although several astrocyte-derived secreted factors are implicated in synaptogenesis, the role of contact-mediated glial-neuronal interactions in synapse formation and elimination during development is still unknown. In this study, we examined whether the loss or overexpression of the membrane-bound ephrin-B1 in astrocytes during postnatal day (P) 14-28 period would affect synapse formation and maturation in the developing hippocampus. We found enhanced excitation of CA1 pyramidal neurons in astrocyte-specific ephrin-B1 KO male mice, which coincided with a greater vGlut1/PSD95 colocalization, higher dendritic spine density, and enhanced evoked AMPAR and NMDAR EPSCs. In contrast, EPSCs were reduced in CA1 neurons neighboring ephrin-B1-overexpressing astrocytes. Overexpression of ephrin-B1 in astrocytes during P14-28 developmental period also facilitated evoked IPSCs in CA1 neurons, while evoked IPSCs and miniature IPSC amplitude were reduced following astrocytic ephrin-B1 loss. Lower numbers of parvalbumin-expressing cells and a reduction in the inhibitory VGAT/gephyrin-positive synaptic sites on CA1 neurons in the stratum pyramidale and stratum oriens layers of KO hippocampus may contribute to reduced inhibition and higher excitation. Finally, dysregulation of excitatory/inhibitory balance in KO male mice is most likely responsible for impaired sociability observed in these mice. The ability of astrocytic ephrin-B1 to influence both excitatory and inhibitory synapses during development can potentially contribute to developmental refinement of neuronal circuits.SIGNIFICANCE STATEMENT This report establishes a link between astrocytes and the development of excitatory and inhibitory balance in the mouse hippocampus during early postnatal development. We provide new evidence that astrocytic ephrin-B1 differentially regulates development of excitatory and inhibitory circuits in the hippocampus during early postnatal development using a multidisciplinary approach. The ability of astrocytic ephrin-B1 to influence both excitatory and inhibitory synapses during development can potentially contribute to developmental refinement of neuronal circuits and associated behaviors. Given widespread and growing interest in the astrocyte-mediated mechanisms that regulate synapse development, and the role of EphB receptors in neurodevelopmental disorders, these findings establish a foundation for future studies of astrocytes in clinically relevant conditions.

Astrocyte-derived factors can control synapse formation and functions, making astrocytes an attractive target for regulating neuronal circuits and associated behaviors. Abnormal astrocyte-neuronal interactions are also implicated in neurodevelopmental disorders and neurodegenerative diseases associated with impaired learning and memory. However, little is known about astrocyte-mediated mechanisms that regulate learning and memory. Here, we propose astrocytic ephrin-B1 as a regulator of synaptogenesis in adult hippocampus and mouse learning behaviors. We found that astrocyte-specific ablation of ephrin-B1 in male mice triggers an increase in the density of immature dendritic spines and excitatory synaptic sites in the adult CA1 hippocampus. However, the prevalence of immature dendritic spines is associated with decreased evoked postsynaptic firing responses in CA1 pyramidal neurons, suggesting impaired maturation of these newly formed and potentially silent synapses or increased excitatory drive on the inhibitory neurons resulting in the overall decreased postsynaptic firing. Nevertheless, astrocyte-specific ephrin-B1 knock-out male mice exhibit normal acquisition of fear memory but enhanced contextual fear memory recall. In contrast, overexpression of astrocytic ephrin-B1 in the adult CA1 hippocampus leads to the loss of dendritic spines, reduced excitatory input, and impaired contextual memory retention. Our results suggest that astrocytic ephrin-B1 may compete with neuronal ephrin-B1 and mediate excitatory synapse elimination through its interactions with neuronal EphB receptors. Indeed, a deletion of neuronal EphB receptors impairs the ability of astrocytes expressing functional ephrin-B1 to engulf synaptosomes in vitro Our findings demonstrate that astrocytic ephrin-B1 regulates long-term contextual memory by restricting new synapse formation in the adult hippocampus.SIGNIFICANCE STATEMENT These studies address a gap in our knowledge of astrocyte-mediated regulation of learning and memory by unveiling a new role for ephrin-B1 in astrocytes and elucidating new mechanisms by which astrocytes regulate learning. Our studies explore the mechanisms underlying astrocyte regulation of hippocampal circuit remodeling during learning using new genetic tools that target ephrin-B signaling in astrocytes in vivo On a subcellular level, astrocytic ephrin-B1 may compete with neuronal ephrin-B1 and trigger astrocyte-mediated elimination of EphB receptor-containing synapses. Given the role EphB receptors play in neurodevelopmental disorders and neurodegenerative diseases, these findings establish a foundation for future studies of astrocyte-mediated synaptogenesis in clinically relevant conditions that can help to guide the development of clinical applications for a variety of neurological disorders.