Serotonin inputs to the dorsal BNST modulate anxiety in a 5-HT1A receptor-dependent manner.

Serotonin (5-HT) neurons project from the raphe nuclei throughout the brain where they act to maintain homeostasis. Here, we study 5-HT inputs into the bed nucleus of the stria terminalis (BNST), a major subdivision of the extended amygdala that has been proposed to regulate responses to anxiogenic environments in humans and rodents. While the dorsal part of the BNST (dBNST) receives dense 5-HT innervation, whether and how 5-HT in the dBNST normally modulates anxiety remains unclear. Using optogenetics, we demonstrate that activation of 5-HT terminals in the dBNST reduces anxiety in a highly anxiogenic environment. Further analysis revealed that optogenetic inhibition of 5-HT inputs into the dBNST increases anxiety in a less anxiogenic environment. We found that 5-HT predominantly hyperpolarizes dBNST neurons, reducing their activity in a manner that can be blocked by a 5-HT1A antagonist. Finally, we demonstrate that activation of 5-HT1A receptors in the dBNST is necessary for the anxiolytic effect observed following optogenetic stimulation of 5-HT inputs into the dBNST. These data reveal that 5-HT release in the dBNST modulates anxiety-like behavior via 5-HT1A receptors under naturalistic conditions.

Disruption of 5-HT1A function in adolescence but not early adulthood leads to sustained increases of anxiety.

Current evidence suggests that anxiety disorders have developmental origins. Early insults to the circuits that sub-serve emotional regulation are thought to cause disease later in life. Evidence from studies in mice demonstrate that the serotonergic system in general, and serotonin 1A (5-HT1A) receptors in particular, are critical during the early postnatal period for the normal development of circuits that subserve anxious behavior. However, little is known about the role of serotonin signaling through 5-HT1A receptors between the emergence of normal anxiety behavior after weaning, and the mature adult phenotype. Here, we use both transgenic and pharmacological approaches in male mice, to identify a sensitive period for 5-HT1A function in the stabilization of circuits mediating anxious behavior during adolescence. Using a transgenic approach we show that suppression of 5-HT1A receptor expression beginning in early adolescence results in an anxiety-like phenotype in the open field test. We further demonstrate that treatment with the 5-HT1A antagonist WAY 100,635 between postnatal day (P)35 and P50, but not at later timepoints, results in altered anxiety in ethologically based conflict tests like the open field test and elevated plus maze. This change in anxiety behavior occurs without impacting behavior in the more depression-related sucrose preference test or forced swim test. The treatment with WAY 100,635 does not affect adult 5-HT1A expression levels, but leads to increased expression of the serotonin transporter in the raphe, along with enhanced serotonin levels in both the prefrontal cortex and raphe that correlate with the behavioral changes observed in adult mice. This work demonstrates that signaling through 5-HT1A receptors during adolescence (a time when pathological anxiety emerges), but not early adulthood, is critical in regulating anxiety setpoints. These data suggest the possibility that brief interventions in the serotonergic system during adolescence could lead to profound and enduring changes in physiology and behavior.

Antidepressants recruit new neurons to improve stress response regulation.

Recent research suggests an involvement of hippocampal neurogenesis in behavioral effects of antidepressants. However, the precise mechanisms through which newborn granule neurons might influence the antidepressant response remain elusive. Here, we demonstrate that unpredictable chronic mild stress in mice not only reduces hippocampal neurogenesis, but also dampens the relationship between hippocampus and the main stress hormone system, the hypothalamo-pituitary-adrenal (HPA) axis. Moreover, this relationship is restored by treatment with the antidepressant fluoxetine, in a neurogenesis-dependent manner. Specifically, chronic stress severely impairs HPA axis activity, the ability of hippocampus to modulate downstream brain areas involved in the stress response, the sensitivity of the hippocampal granule cell network to novelty/glucocorticoid effects and the hippocampus-dependent negative feedback of the HPA axis. Remarkably, we revealed that, although ablation of hippocampal neurogenesis alone does not impair HPA axis activity, the ability of fluoxetine to restore hippocampal regulation of the HPA axis under chronic stress conditions, occurs only in the presence of an intact neurogenic niche. These findings provide a mechanistic framework for understanding how adult-generated new neurons influence the response to antidepressants. We suggest that newly generated neurons may facilitate stress integration and that, during chronic stress or depression, enhancing neurogenesis enables a dysfunctional hippocampus to restore the central control on stress response systems, then allowing recovery.

Genetics of affective and anxiety disorders.

The study of the genetics of complex behaviors has evolved dramatically from the days of the nature versus nurture debates that dominated much of the past century. Here we discuss advances in our understanding of the genetics of affective and anxiety disorders. In particular, we highlight our growing understanding of specific gene-environment interactions that occur during critical periods in development, setting the stage for later behavioral phenotypes. We review the recent literature in the field, focusing on recent advances in our understanding of the role of the serotonin system in establishing normal anxiety levels during development. We emphasize the importance of understanding the effect of genetic variation at the level of functional circuits and provide examples from the literature of how such an approach has been exploited to study novel genetic endpoints, including genetically based variation in response to medication, a potentially valuable phenotype that has not received much attention to date.

Molecular heterogeneity along the dorsal-ventral axis of the murine hippocampal CA1 field: a microarray analysis of gene expression.

There has been increasing interest in functional heterogeneity along the septotemporal, dorsal-ventral (D-V) axis of the hippocampus. Although anatomical connectivity and lesion studies point to discrete roles for these sub-regions, the contribution of differential gene expression across this axis has not been systematically studied. Here we present findings from an Affymetrix microarray screen aimed at identifying genes in the CA1 region of the adult murine hippocampus that show significant differential expression along the D-V axis. Our results indicate that the vast majority of monitored genes (>90%) had tissue expression levels that differed by less than 20% between regions, while less than 0.1% of genes had expression levels that varied more than three-fold by sub-region. Only 23 probes showed a CA1 dorsoventral signal intensity ratio greater than three: 18 enriched dorsally and five enriched ventrally. Probes with the greatest difference in expression levels represent a range of genes with known functions in patterning and signaling, as well as genes without known function. Selective screening with digoxigenin-labeled in situ hybridization confirms the existence of CA1 sub-regionalized expression, with some genes exhibiting a graded expression pattern across the D-V axis, and others restricted to a discrete region. Our findings demonstrate that there are gene expression differences across the D-V axis of the adult murine hippocampus within traditionally recognized cytoarchitecturally defined boundaries. Combined with the previously recognized differences in connectivity and results from lesion studies, our data further confirm the existence of functional heterogeneity along the D-V axis.

Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors.

In the developing nervous system, migrating cells and axons are guided to their targets by cues in the extracellular environment. The netrins are a family of phylogenetically conserved guidance cues that can function as diffusible attractants and repellents for different classes of cells and axons. In vertebrates, insects and nematodes, members of the DCC subfamily of the immunoglobulin superfamily have been implicated as receptors that are involved in migration towards netrin sources. The mechanisms that direct migration away from netrin sources (presumed repulsions) are less well understood. In Caenorhabditis elegans, the transmembrane protein UNC-5 (ref. 14) has been implicated in these responses, as loss of unc-5 function causes migration defects and ectopic expression of unc-5 in some neurons can redirect their axons away from a netrin source. Whether UNC-5 is a netrin receptor or simply an accessory to such a receptor has not, however, been defined. We now report the identification of two vertebrate homologues of UNC-5 which, with UNC-5 and the product of the mouse rostral cerebellar malformation gene (rcm), define a new subfamily of the immunoglobulin superfamily, and whose messenger RNAs show prominent expression in various classes of differentiating neurons. We provide evidence that these two UNC-5 homologues, as well as the rcm gene product, are netrin-binding proteins, supporting the hypothesis that UNC-5 and its relatives are netrin receptors.

Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system.

During nervous system development, spinal commissural axons project toward floor plate cells and trochlear motor axons extend away from these cells. Netrin-1, a diffusible protein made by floor plate cells, can attract spinal commissural axons and repel trochlear axons in vitro, but its role in vivo is unknown. Netrin-1 deficient mice exhibit defects in spinal commissural axon projections that are consistent with netrin-1 guiding these axons. Defects in several forebrain commissures are also observed, suggesting additional guidance roles for netrin-1. Trochlear axon projections are largely normal, predicting the existence of additional cues for these axons, and evidence is provided for a distinct trochlear axon chemorepellent produced by floor plate cells. These results establish netrin-1 as a guidance cue that likely collaborates with other diffusible cues to guide axons in vivo.

Hepatocyte growth factor/scatter factor is an axonal chemoattractant and a neurotrophic factor for spinal motor neurons.

In the embryonic nervous system, developing axons can be guided to their targets by diffusible factors secreted by their intermediate and final cellular targets. To date only one family of chemoattractants for developing axons has been identified. Grafting and ablation experiments in fish, amphibians, and birds have suggested that spinal motor axons are guided to their targets in the limb in part by a succession of chemoattractants made by the sclerotome and by the limb mesenchyme, two intermediate targets that these axons encounter en route to their target muscles. Here we identify the limb mesenchyme-derived chemoattractant as hepatocyte growth factor/scatter factor (HGF/SF), a diffusible ligand for the c-Met receptor tyrosine kinase, and we also implicate HGF/SF at later stages as a muscle-derived survival factor for motoneurons. These results indicate that, in addition to functioning as a mitogen, a motogen, and a morphogen in nonneural systems, HGF/SF can function as a guidance and survival factor in the developing nervous system.

Deleted in Colorectal Cancer (DCC) encodes a netrin receptor.

The guidance of developing axons in the nervous system is mediated partly by diffusible chemoattractants secreted by axonal target cells. Netrins are chemoattractants for commissural axons in the vertebrate spinal cord, but the mechanisms through which they produce their effects are unknown. We show that Deleted in Colorectal Cancer (DCC), a transmembrane protein of the immunoglobulin superfamily, is expressed on spinal commissural axons and possesses netrin-1-binding activity. Moreover, an antibody to DCC selectively blocks the netrin-1-dependent outgrowth of commissural axons in vitro. These results indicate that DCC is a receptor or a component of a receptor that mediates the effects of netrin-1 on commissural axons, and they complement genetic evidence for interactions between DCC and netrin homologs in C. elegans and Drosophila.

Semaphorin III can function as a selective chemorepellent to pattern sensory projections in the spinal cord.

Distinct classes of primary sensory neurons in dorsal root ganglia subserve different sensory modalities, terminate in different dorsoventral locations in the spinal cord, and display different neurotrophin response profiles. Large diameter muscle afferents that terminate in the ventral spinal cord are NT-3 responsive, whereas small diameter afferents subserving pain and temperature are NGF responsive and terminate in the dorsal spinal cord. Previous in vitro studies showed that the developing ventral spinal cord secretes a diffusible factor that inhibits the growth of sensory axons. Here we show that this factor repels NGF-responsive axons but has little effect on NT-3-responsive axons. We also provide evidence implicating semaphorin III/collapsin, a diffusible guidance molecule expressed by ventral spinal cord cells, in mediating this effect. These results suggest that semaphorin III functions to pattern sensory projections by selectively repelling axons that normally terminate dorsally.

A new vector for cloning large eukaryotic DNA segments in Escherichia coli.

We investigated the relationship between plasmid size and electroporation efficiency in E. coli and found that even very large plasmids can be transfected efficiently. The efficiencies are well above the minimum required to construct representative libraries of complex eukaryotic genomes. To exploit this observation we constructed a novel mammalian-E. coli shuttle vector whose replication in E. coli is driven by the F sex factor episome origin. This new vector system should accept inserts well in excess of 100 kb, thus putting the cloning of mammalian genes by direct phenotypic complementation within reach.