Temporal patterning of apical progenitors and their daughter neurons in the developing neocortex.

During corticogenesis, distinct subtypes of neurons are sequentially born from ventricular zone progenitors. How these cells are molecularly temporally patterned is poorly understood. We used single-cell RNA sequencing at high temporal resolution to trace the lineage of the molecular identities of successive generations of apical progenitors (APs) and their daughter neurons in mouse embryos. We identified a core set of evolutionarily conserved, temporally patterned genes that drive APs from internally driven to more exteroceptive states. We found that the Polycomb repressor complex 2 (PRC2) epigenetically regulates AP temporal progression. Embryonic age-dependent AP molecular states are transmitted to their progeny as successive ground states, onto which essentially conserved early postmitotic differentiation programs are applied, and are complemented by later-occurring environment-dependent signals. Thus, epigenetically regulated temporal molecular birthmarks present in progenitors act in their postmitotic progeny to seed adult neuronal diversity.

Correction: SSRIs target prefrontal to raphe circuits during development modulating synaptic connectivity and emotional behavior.

This article was originally published under standard licence, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the paper have been modified accordingly.

SSRIs target prefrontal to raphe circuits during development modulating synaptic connectivity and emotional behavior.

Antidepressants that block the serotonin transporter, (Slc6a4/SERT), selective serotonin reuptake inhibitors (SSRIs) improve mood in adults but have paradoxical long-term effects when administered during perinatal periods, increasing the risk to develop anxiety and depression. The basis for this developmental effect is not known. Here, we show that during an early postnatal period in mice (P0-P10), Slc6a4/SERT is transiently expressed in a subset of layer 5-6 pyramidal neurons of the prefrontal cortex (PFC). PFC-SERT+ neurons establish glutamatergic synapses with subcortical targets, including the serotonin (5-HT) and GABA neurons of the dorsal raphe nucleus (DRN). PFC-to-DRN circuits develop postnatally, coinciding with the period of PFC Slc6a4/SERT expression. Complete or cortex-specific ablation of SERT increases the number of functional PFC glutamate synapses on both 5-HT and GABA neurons in the DRN. This PFC-to-DRN hyperinnervation is replicated by early-life exposure to the SSRI, fluoxetine (from P2 to P14), that also causes anxiety/depressive-like symptoms. We show that pharmacogenetic manipulation of PFC-SERT+ neuron activity bidirectionally modulates these symptoms, suggesting that PFC hypofunctionality has a causal role in these altered responses to stress. Overall, our data identify specific PFC descending circuits that are targets of antidepressant drugs during development. We demonstrate that developmental expression of SERT in this subset of PFC neurons controls synaptic maturation of PFC-to-DRN circuits, and that remodeling of these circuits in early life modulates behavioral responses to stress in adulthood.

Excess of serotonin affects neocortical pyramidal neuron migration.

The serotonin transporter (SERT) is a key molecule involved in the homeostasis of extracellular levels of serotonin and is regulated developmentally. Genetic deletion of SERT in rodents increases extracellular levels of serotonin and affects cellular processes involved in neocortical circuit assembly such as barrel cortex wiring and cortical interneuron migration. Importantly, pharmacological blockade of SERT during brain development leads to phenotypes relevant to psychiatry in rodents and to an increased risk for autism spectrum disorders in humans. Furthermore, developmental adversity interacts with genetically-driven variations of serotonin function in humans and nonhuman primates to increase the risk for a variety of stress-related phenotypes. In this study, we investigate whether an excess of serotonin affects the migration of neocortical pyramidal neurons during development. Using in utero electroporation combined with time-lapse imaging to specifically monitor pyramidal neurons during late mouse embryogenesis, we show that an excess of serotonin reversibly affects the radial migration of pyramidal neurons. We further identify that the serotonin receptor 5-HT(6) is expressed in pyramidal neuron progenitors and that 5-HT(6) receptor activation replicates the effects of serotonin stimulation. Finally, we show that the positioning of superficial layer pyramidal neurons is altered in vivo in SERT knockout mice. Taken together, these results indicate that a developmental excess of serotonin decreases the migration speed of cortical pyramidal neurons, affecting a fundamental step in the assembly of neural circuits. These findings support the hypothesis that developmental dysregulation of serotonin homeostasis has detrimental effects on neocortical circuit formation and contributes to increased vulnerability to psychiatric disorders.

Acute decrease in net glutamate uptake during energy deprivation.

The extracellular glutamate concentration ([glu](o)) rises during cerebral ischemia, reaching levels capable of inducing delayed neuronal death. The mechanisms underlying this glutamate accumulation remain controversial. We used N-methyl-D-aspartate receptors on CA3 pyramidal neurons as a real-time, on-site, glutamate sensor to identify the source of glutamate release in an in vitro model of ischemia. Using glutamate and L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) as substrates and DL-threo-beta-benzyloxyaspartate (TBOA) as an inhibitor of glutamate transporters, we demonstrate that energy deprivation decreases net glutamate uptake within 2-3 min and later promotes reverse glutamate transport. This process accounts for up to 50% of the glutamate accumulation during energy deprivation. Enhanced action potential-independent vesicular release also contributes to the increase in [glu](o), by approximately 50%, but only once glutamate uptake is inhibited. These results indicate that a significant rise in [glu](o) already occurs during the first minutes of energy deprivation and is the consequence of reduced uptake and increased vesicular and nonvesicular release of glutamate.

Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin.

Maintaining glutamate at low extracellular concentrations in the central nervous system is necessary to protect neurons from excitotoxic injury and to ensure a high signal-to-noise ratio for glutamatergic synaptic transmission. We have used DL-threo-beta-benzyloxyaspartate (TBOA), an inhibitor of glutamate uptake, to determine the role of glutamate transporters in the regulation of extracellular glutamate concentration. By using the N-methyl-D-aspartate receptors of patched CA3 hippocampal neurons as "glutamate sensors," we observed that application of TBOA onto organotypic hippocampal slices led to a rapid increase in extracellular glutamate concentration. This increase was Ca(2+)-independent and was observed in the presence of tetrodotoxin. Moreover, prevention of vesicular glutamate release with clostridial toxins did not affect the accumulation of glutamate when uptake was inhibited. Inhibition of glutamine synthase, however, increased the rate of accumulation of extracellular glutamate, indicating that glial glutamate stores can serve as a source in this process. TBOA blocked synaptically evoked transporter currents in astrocytes without inducing a current mediated by the glutamate transporter. This indicates that this inhibitor is not transportable and does not release glutamate by heteroexchange. These results show that under basal conditions, the activity of glutamate transporters compensates for the continuous, nonvesicular release of glutamate from the intracellular compartment. As a consequence, acute disruption of transporter activity immediately results in significant accumulation of extracellular glutamate.

BDNF stimulates expression, activity and release of tissue-type plasminogen activator in mouse cortical neurons.

Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor involved in neuronal development and synaptic plasticity. Although the physiological effects of BDNF have been examined in detail, target proteins which mediate its actions remain largely unknown. Here, we report that BDNF stimulates the expression of tissue-type plasminogen activator (tPA) in primary cultures of cortical neurons in a time- and concentration-dependent manner. Among the other members of the neurotrophin family, neurotrophin-4 (NT-4) and to a lesser extent neurotrophin-3 (NT-3) also increased tPA mRNA expression, while nerve growth factor (NGF) was devoid of any effect. Induction of tPA expression by BDNF is accompanied by an increase in the proteolytic activity of tPA associated with cortical neurons and a release of tPA into the extracellular space. Release of tPA induced by BDNF depends on extracellular Ca2+ since it is markedly reduced in the presence of ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). Up-regulation of tPA expression by BDNF is followed by the induction of plasminogen activator inhibitor 2 (PAI-2), an inhibitor of tPA. Together these results suggest that activation of tPA by BDNF may contribute to structural changes associated with neuronal development or synaptic plasticity.