Early life stress (ES) increases vulnerability to psychopathology and impairs cognition in adulthood. These ES-induced deficits are associated with lasting changes in hippocampal plasticity. Detailed information on the neurobiological basis, the onset, and progression of such changes and their sex-specificity is currently lacking but is required to tailor specific intervention strategies. Here, we use a chronic ES mouse model based on limited nesting and bedding material from postnatal day (P) 2-9 to investigate; (1) if ES leads to impairments in hippocampus-dependent cognitive function in adulthood and (2) if these alterations are paralleled by changes in developmental and/or adult hippocampal neurogenesis. ES increased developmental neurogenesis (proliferation and differentiation) in the dentate gyrus (DG) at P9, and the number of immature (NeurD1(+)) cells migrating postnatally from the secondary dentate matrix, indicating prompt changes in DG structure in both sexes. ES lastingly reduced DG volume and the long-term survival of developmentally born neurons in both sexes at P150. In adult male mice only, ES reduced survival of adult-born neurons (BrdU/NeuN(+) cells), while proliferation (Ki67(+)) and differentiation (DCX(+)) were unaffected. These changes correlated with impaired performance in all learning and memory tasks used here. In contrast, in female mice, despite early alterations in developmental neurogenesis, no lasting changes were present in adult neurogenesis after ES and the cognitive impairments were less prominent and only apparent in some cognitive tasks. We further show that, although neurogenesis and cognition correlate positively, only the hippocampus-dependent functions depend on changes in neurogenesis, whereas cognitive functions that are not exclusively hippocampus-dependent do not. This study indicates that chronic ES has lasting consequences on hippocampal structure and function in mice and suggests that male mice are more susceptible to ES than females. Unraveling the mechanisms that underlie the persistent ES-induced effects may have clinical implications for treatments to counteract ES-induced deficits.

Development of tools to manipulate activity of specific neurons is important for dissecting the function of neural circuits. Viral vectors and conditional transgenic animal lines that target recombinases to specific cells facilitate the successful manipulation and recording of specific subsets of neurons. So far, it has been possible to target neuronal subtypes within a certain brain region based on transcriptional control regions from a gene selectively expressed in those cells or based upon its projections. Nevertheless, there are only a few tools available that combine this and target a neuronal subtype within a projection. We tested a viral vector system, consisting of a canine adenovirus type 2 expressing a Cre-dependent Flp recombinase (CavFlexFlp) and an adeno-associated viral (AAV) vector expressing a Flp-dependent cDNA, which targets neurons in a subtype- and projection-specific manner. As proof of principle we targeted expression of a Designer Receptor Exclusively Activated by Designer Drugs (DREADD) to the dopamine neurons of the mesolimbic projection, which allows the transient activation of neurons by the ligand Clozapine-N-Oxide (CNO). We validated that the system specifically targets dopamine neurons and that chemogenetic activation of these neurons induces an increase in locomotor activity. We thus validated a valuable tool that allows in vivo neuronal activation in a projection- and subtype-specific manner.

Targeting specific neuronal cell types is a major challenge for unraveling their function and utilizing specific cells for gene therapy strategies. Viral vector tools are widely used to target specific cells or circuits for these purposes. Here, we use viral vectors with short promoters of neuropeptide genes to target distinct neuronal populations in the hypothalamus of rats and mice. We show that lowering the amount of genomic copies is effective in increasing specificity of a melanin-concentrating hormone promoter. However, since too low titers reduce transduction efficacy, there is an optimal titer for achieving high specificity and sufficient efficacy. Other previously identified neuropeptide promoters as those for oxytocin and orexin require further sequence optimization to increase target specificity. We conclude that promoter-driven viral vectors should be used with caution in order to target cells specifically.

Hypothalamic nuclei which regulate homeostatic functions express leptin receptor (LepR), the primary target of the satiety hormone leptin. Single-cell RNA sequencing (scRNA-seq) has facilitated the discovery of a variety of hypothalamic cell types. However, low abundance of LepR transcripts prevented further characterization of LepR cells. Therefore, we perform scRNA-seq on isolated LepR cells and identify eight neuronal clusters, including three uncharacterized Trh-expressing populations as well as 17 non-neuronal populations including tanycytes, oligodendrocytes and endothelial cells. Food restriction had a major impact on Agrp neurons and changed the expression of obesity-associated genes. Multiple cell clusters were enriched for GWAS signals of obesity. We further explored changes in the gene regulatory landscape of LepR cell types. We thus reveal the molecular signature of distinct populations with diverse neurochemical profiles, which will aid efforts to illuminate the multi-functional nature of leptin’s action in the hypothalamus.