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...

The exposure to aversive experiences during early life influences brain development and leads to altered behavior. Moreover, the combination of these experiences with subtle alterations in neurodevelopment may contribute to the emergence of psychiatric disorders, such as schizophrenia. Recent hypotheses suggest that imbalances between excitatory and inhibitory (E/I) neurotransmission, especially in the prefrontal cortex and the amygdala, may underlie their etiopathology. In order to understand better the neurobiological bases of these alterations, we studied the impact of altered neurodevelopment and chronic early-life stress on these two brain regions. Transgenic mice displaying fluorescent excitatory and inhibitory neurons, received a single injection of MK801 (NMDAR antagonist) or vehicle solution at postnatal day 7 and/or were socially isolated from the age of weaning until adulthood (3 months old). We found that anxiety-related behavior, brain volume, neuronal structure, and the expression of molecules related to plasticity and E/I neurotransmission in adult mice were importantly affected by early-life stress. Interestingly, many of these effects were potentiated when the stress paradigm was applied to mice perinatally injected with MK801 ("double-hit" model). These results clearly show the impact of early-life stress on the adult brain, especially on the structure and plasticity of inhibitory networks, and highlight the double-hit model as a valuable tool to study the contribution of early-life stress in the emergence of neurodevelopmental psychiatric disorders, such as schizophrenia.

Chronic stress has an important impact on the adult brain. However, most of the knowledge on its effects is focused on principal neurons and less on inhibitory neurons. Consequently, recent reports have begun to describe stress-induced alterations in the structure, connectivity and neurochemistry of interneurons. Some of these changes appear to be mediated by certain molecules particularly associated to interneurons, such as the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) and components of the perineuronal nets (PNN), specialized regions of the extracellular matrix. These plasticity-related molecules modulate interneuronal structure and connectivity, particularly of parvalbumin expressing basket interneurons, both during development and adult life. These inhibitory neurons are specially affected after chronic stress and in some stress-related disorders, in which the expression of PSA-NCAM and certain components of PNN are also altered. For these reasons we have decided to study PSA-NCAM, PNN and parvalbumin expressing interneurons after 10 days of chronic restraint stress, a time point in which its behavioral consequences are starting to appear. We have focused initially on the medial prefrontal cortex (mPFC), basolateral amygdala (BLA) and hippocampus, regions affected by stress and stress-related psychiatric diseases, but we have also explored the habenula and the thalamic reticular nucleus (TRN) due to the important presence of PNN and their relationship with certain disorders. PSA-NCAM expression was increased by stress in the stratum lacunosum-moleculare of CA1. Increases in parvalbumin immunoreactive cells were detected in the mPFC and the BLA, but were not accompanied by increases in the number of parvalbumin expressing perisomatic puncta on the somata of principal neurons. The number of PNN was also increased in the mPFC and the habenula, although habenular PNN were not associated to parvalbumin cells. Increased expression of parvalbumin and components of PNN were also detected in the TRN after chronic restraint stress, revealing for the first time substantial effects on this region. Our study shows that, even a short chronic stress protocol, can induce consistent changes in interneuronal plasticity-related molecules in cortical and extracortical regions, which may represent initial responses of inhibitory circuits to counteract the effects of this aversive experience.

The prefrontal cortex (PFC) continues its development during adolescence and alterations in its structure and function, particularly of inhibitory networks, have been detected in schizophrenic patients. Since cannabis use during adolescence is a risk factor for this disease, our main objective was to investigate whether THC administration during this period might exacerbate alterations in prefrontocortical inhibitory networks in mice subjected to a perinatal injection of MK801 and postweaning social isolation. This double-hit model (DHM) combines a neurodevelopmental manipulation and the exposure to an aversive experience during early life; previous work has shown that DHM mice have important alterations in the structure and connectivity of PFC interneurons. In the present study we found that DHM had reductions in prepulse inhibition of the startle reflex (PPI), GAD67 expression and cingulate 1 cortex volume. Interestingly, THC by itself induced increases in PPI and decreases in the dendritic complexity of somatostatin expressing interneurons. Both THC and DHM reduced the density of parvalbumin expressing cells surrounded by perineuronal nets and, when combined, they disrupted the ratio between the density of puncta expressing excitatory and inhibitory markers. Our results support previous work showing alterations in parameters involving interneurons in similar animal models and schizophrenic patients. THC treatment does not modify further these parameters, but changes some others related also to interneurons and their plasticity, in some cases in the opposite direction to those induced by the DHM, suggesting a protective effect.