The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

To conclude, the brain antigen-directed AB tested here are comparably detectable in healthy subjects and the disease groups studied here, thus questioning an upfront pathological role of these serum AB.

In 2007, a multifaceted syndrome, associated with anti-NMDA receptor autoantibodies (NMDAR-AB) of immunoglobulin-G isotype, has been described, which variably consists of psychosis, epilepsy, cognitive decline and extrapyramidal symptoms. Prevalence and significance of NMDAR-AB in complex neuropsychiatric disease versus health, however, have remained unclear. We tested sera of 2817 subjects (1325 healthy, 1081 schizophrenic, 263 Parkinson and 148 affective-disorder subjects) for presence of NMDAR-AB, conducted a genome-wide genetic association study, comparing AB carriers versus non-carriers, and assessed their influenza AB status. For mechanistic insight and documentation of AB functionality, in vivo experiments involving mice with deficient blood-brain barrier (ApoE(-/-)) and in vitro endocytosis assays in primary cortical neurons were performed. In 10.5% of subjects, NMDAR-AB (NR1 subunit) of any immunoglobulin isotype were detected, with no difference in seroprevalence, titer or in vitro functionality between patients and healthy controls. Administration of extracted human serum to mice influenced basal and MK-801-induced activity in the open field only in ApoE(-/-) mice injected with NMDAR-AB-positive serum but not in respective controls. Seropositive schizophrenic patients with a history of neurotrauma or birth complications, indicating an at least temporarily compromised blood-brain barrier, had more neurological abnormalities than seronegative patients with comparable history. A common genetic variant (rs524991, P=6.15E-08) as well as past influenza A (P=0.024) or B (P=0.006) infection were identified as predisposing factors for NMDAR-AB seropositivity. The >10% overall seroprevalence of NMDAR-AB of both healthy individuals and patients is unexpectedly high. Clinical significance, however, apparently depends on association with past or present perturbations of blood-brain barrier function.

Erythropoietin (EPO), named after its role in hematopoiesis, is also expressed in mammalian brain. In clinical settings, recombinant EPO treatment has revealed a remarkable improvement of cognition, but underlying mechanisms have remained obscure. Here, we show with a novel line of reporter mice that cognitive challenge induces local/endogenous hypoxia in hippocampal pyramidal neurons, hence enhancing expression of EPO and EPO receptor (EPOR). High-dose EPO administration, amplifying auto/paracrine EPO/EPOR signaling, prompts the emergence of new CA1 neurons and enhanced dendritic spine densities. Singlecell sequencing reveals rapid increase in newly differentiating neurons. Importantly, improved performance on complex running wheels after EPO is imitated by exposure to mild exogenous/inspiratory hypoxia. All these effects depend on neuronal expression of the Epor gene. This suggests a model of neuroplasticity in form of a fundamental regulatory circle, in which neuronal networks-challenged by cognitive tasks-drift into transient hypoxia, thereby triggering neuronal EPO/EPOR expression. 1 1234567890():,;E rythropoietin (EPO) is a hypoxia-inducible growth factor in mammalian kidney, named after its role in hematopoiesis 1,2 . Unexpectedly, both EPO and its receptor (EPOR) were later detected in the brain, where they are upregulated by injury conditions. High-dose recombinant human (rh) EPO, a drug in clinical use for anemic patients, exerts neuroprotective and neuroregenerative effects that are independent of the hematocrit, which is mechanistically unexplained 3-8 . Moreover, rhEPO improves cognitive function and reduces gray matter loss in a range of neuropsychiatric conditions 9-13 . Even in healthy mice, rhEPO treatment improves cognition, which is associated with enhanced hippocampal long-term potentiation [14][15][16] . Surprisingly, rhEPO increases the number of mature hippocampal pyramidal neurons without underlying effect on cell proliferation or cell death 17 . This effect is mediated in neurons mainly by JAK-STAT, PI3K/AKT/PKB, Ras-MEK, and ERK1/2, as well as NF-κB; pathways widely comparable to the hematopoietic system [18][19][20] . This raises the question whether the expression of EPO and its receptor serves a physiological function in the nervous system, and what could be the triggering factors of EPO expression under physiological conditions. ResultsGeneration of pyramidal neurons in adult mice and amplification by rhEPO. First, we developed a method to directly label and quantify newly generated neurons in the hippocampal cornu ammonis (CA) field of adult mice. This was possible by permanently labeling all mature pyramidal neurons present at P27 using a tamoxifen-inducible reporter gene in NexCreERT2::R26R-tdT mice (Fig. 1a, b) 21 . Thus, all neurons differentiating and maturing after termination of the tamoxifen-induced Cre recombination lack tdTomato, but can be positively identified by Ctip2, a specific marker of pyramidal neurons, thereby revealing adult 'neurogenesis' independent of DNA synt...

3 3 a r t I C l e SA main rate-limiting step in synaptic transmission is the retrieval of synaptic vesicles from the presynaptic membrane for further rounds of use. Several modes of synaptic vesicle retrieval have been proposed, but the main pathway is considered to be clathrin-mediated endocytosis 1,2 . This process is relatively slow because after fusion the recycling machinery has to resort different vesicle membrane proteins in the right stoichiometry to generate fusion-competent synaptic vesicles 3 . As a result, this process occurs with a time constant of tens of seconds to minutes 4 . However, to sustain transmission during continuous activity, it was suggested that synaptic vesicles might 'kiss and run' with a time constant of 1-2 s, whereby the vesicles transiently fuse with the membrane without full collapse and hence retain their molecular identity [5][6][7][8][9] .The fast 'kiss and run' recycling mechanism not only would provide a kinetic advantage but would spatially and temporally couple exoand endocytosis. Using a green fluorescent protein (GFP) fused with the coat-forming clathrin light chain, we previously found evidence that during the first 10 s of prolonged stimulation, clathrin is not being recruited from the cytosol to form coated pits, although the rate of endocytosis measured with styryl (FM) dyes is high 3 , suggesting that vesicles during this first phase are either retrieved by a clathrin-independent mechanism (kiss and run) or by preassembled coat structures at the periphery of the active zone.Support for such a 'readily retrievable pool' (RRetP) of preassembled structures came from experiments using fusion constructs of the synaptic vesicle proteins synaptobrevin 2 (Syb2) and synaptotagmin 1 (Syt1) with a pH-sensitive GFP, pHluorin 10 . These studies showed that synaptic vesicles lose their protein complement after fusion, and the molecular identity of synaptic vesicles exocytosed and subsequently endocytosed is not conserved [11][12][13] . On the basis of these observations, we suggested that exocytosis and subsequent endocytosis are uncoupled and that there is a pool of preassembled vesicle proteins on the presynaptic surface that is preferentially retrieved on exocytosis 3,13 . Previous studies using activity-dependent markers in snake neuromuscular terminals have proposed that the accumulation of such probes at the bouton margins upon stimulation might represent endocytic active zones 14,15 . This is in agreement with other ultrastructural and high-resolution microscopy analyses that describe the presence of several synaptic vesicle proteins on the presynaptic membrane of resting synapses 16,17 . Likewise, the first reconstruction of the endocytic time course from electron micrographs of frog neuromuscular junctions quick-frozen at different times after stimulation revealed a first wave of clathrin-mediated endocytosis lasting ~10 s (ref. 18), in line with the notion of a preclustered pool being immediately available for this first wave of endocytosis upon stimulation 13 . In hi...