An epigenetic barrier sets the timing of human neuronal maturation.

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.

Combined small-molecule treatment accelerates maturation of human pluripotent stem cell-derived neurons.

The maturation of human pluripotent stem cell (hPSC)-derived neurons mimics the protracted timing of human brain development, extending over months to years for reaching adult-like function. Prolonged in vitro maturation presents a major challenge to stem cell-based applications in modeling and treating neurological disease. Therefore, we designed a high-content imaging assay based on morphological and functional readouts in hPSC-derived cortical neurons which identified multiple compounds that drive neuronal maturation including inhibitors of lysine-specific demethylase 1 and disruptor of telomerase-like 1 and activators of calcium-dependent transcription. A cocktail of four factors, GSK2879552, EPZ-5676, N-methyl-D-aspartate and Bay K 8644, collectively termed GENtoniK, triggered maturation across all parameters tested, including synaptic density, electrophysiology and transcriptomics. Maturation effects were further validated in cortical organoids, spinal motoneurons and non-neural lineages including melanocytes and pancreatic ß-cells. The effects on maturation observed across a broad range of hPSC-derived cell types indicate that some of the mechanisms controlling the timing of human maturation might be shared across lineages.

Projection-Targeted Photopharmacology Reveals Distinct Anxiolytic Roles for Presynaptic mGluR2 in Prefrontal- and Insula-Amygdala Synapses.

Dissecting how membrane receptors regulate neural circuit function is critical for deciphering basic principles of neuromodulation and mechanisms of therapeutic drug action. Classical pharmacological and genetic approaches are not well-equipped to untangle the roles of specific receptor populations, especially in long-range projections which coordinate communication between brain regions. Here we use viral tracing, electrophysiological, optogenetic, and photopharmacological approaches to determine how presynaptic metabotropic glutamate receptor 2 (mGluR2) activation in the basolateral amygdala (BLA) alters anxiety-related behavior. We find that mGluR2-expressing neurons from the ventromedial prefrontal cortex (vmPFC) and posterior insular cortex (pIC) preferentially target distinct cell types and subregions of the BLA to regulate different forms of avoidant behavior. Using projection-specific photopharmacological activation, we find that mGluR2-mediated presynaptic inhibition of vmPFC-BLA, but not pIC-BLA, connections can produce long-lasting decreases in spatial avoidance. In contrast, presynaptic inhibition of pIC-BLA connections decreased social avoidance, novelty-induced hypophagia, and increased exploratory behavior without impairing working memory, establishing this projection as a novel target for the treatment of anxiety disorders. Overall, this work reveals new aspects of BLA neuromodulation with therapeutic implications while establishing a powerful approach for optical mapping of drug action via photopharmacology.

Transcriptional maintenance of cortical somatostatin interneuron subtype identity during migration.

Although cardinal cortical interneuron identity is established upon cell-cycle exit, it remains unclear whether specific interneuron subtypes are pre-established, and if so, how their identity is maintained prior to circuit integration. We conditionally removed Sox6 (Sox6-cKO) in migrating somatostatin (Sst+) interneurons and assessed the effects on their mature identity. In adolescent mice, five of eight molecular Sst+ subtypes were nearly absent in the Sox6-cKO cortex without a reduction in cell number. Sox6-cKO cells displayed electrophysiological maturity and expressed genes enriched within the broad class of Sst+ interneurons. Furthermore, we could infer subtype identity prior to cortical integration (embryonic day 18.5), suggesting that the loss in subtype was due to disrupted subtype maintenance. Conversely, Sox6 removal at postnatal day 7 did not disrupt marker expression in the mature cortex. Therefore, Sox6 is necessary during migration for maintenance of Sst+ subtype identity, indicating that subtype maintenance requires active transcriptional programs.

Structural and compositional diversity in the kainate receptor family.

The kainate receptors (KARs) are members of the ionotropic glutamate receptor family and assemble into tetramers from a pool of five subunit types (GluK1-5). Each subunit confers distinct functional properties to a receptor, but the compositional and stoichiometric diversity of KAR tetramers is not well understood. To address this, we first solve the structure of the GluK1 homomer, which enables a systematic assessment of structural compatibility among KAR subunits. Next, we analyze single-cell RNA sequencing data, which reveal extreme diversity in the combinations of two or more KAR subunits co-expressed within the same cell. We then investigate the composition of individual receptor complexes using single-molecule fluorescence techniques and find that di-heteromers assembled from GluK1, GluK2, or GluK3 can form with all possible stoichiometries, while GluK1/K5, GluK2/K5, and GluK3/K5 can form 3:1 or 2:2 complexes. Finally, using three-color single-molecule imaging, we discover that KARs can form tri- and tetra-heteromers.

Postnatal Sox6 Regulates Synaptic Function of Cortical Parvalbumin-Expressing Neurons.

Cortical parvalbumin-expressing (Pvalb+) neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. This class of inhibitory neurons undergoes extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. While several transcription factors, such as Nkx2-1, Lhx6, and Sox6, are known to be necessary for the differentiation of progenitors into Pvalb+ neurons, which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons' innervation and synaptic function remains largely unknown. Because Sox6 is continuously expressed in Pvalb+ neurons until adulthood, we used conditional knock-out strategies to investigate its putative role in the postnatal maturation and synaptic function of cortical Pvalb+ neurons in mice of both sexes. We found that early postnatal loss of Sox6 in Pvalb+ neurons leads to failure of synaptic bouton growth, whereas later removal in mature Pvalb+ neurons in the adult causes shrinkage of already established synaptic boutons. Paired recordings between Pvalb+ neurons and pyramidal neurons revealed reduced release probability and increased failure rate of Pvalb+ neurons' synaptic output. Furthermore, Pvalb+ neurons lacking Sox6 display reduced expression of full-length tropomyosin-receptor kinase B (TrkB), a key modulator of GABAergic transmission. Once re-expressed in neurons lacking Sox6, TrkB was sufficient to rescue the morphologic synaptic phenotype. Finally, we showed that Sox6 mRNA levels were increased by motor training. Our data thus suggest a constitutive role for Sox6 in the maintenance of synaptic output from Pvalb+ neurons into adulthood.SIGNIFICANCE STATEMENT Cortical parvalbumin-expressing (Pvalb+) inhibitory neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. These inhibitory neurons undergo extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. However, it remains largely unknown which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons. Here, we show that the transcription factor Sox6 cell-autonomously regulates the synaptic maintenance and output of Pvalb+ neurons until adulthood, leaving unaffected other maturational features of this neuronal population.

A fine-tuned azobenzene for enhanced photopharmacology in vivo.

Despite the power of photopharmacology for interrogating signaling proteins, many photopharmacological systems are limited by their efficiency, speed, or spectral properties. Here, we screen a library of azobenzene photoswitches and identify a urea-substituted "azobenzene-400" core that offers bistable switching between cis and trans with improved kinetics, light sensitivity, and a red-shift. We then focus on the metabotropic glutamate receptors (mGluRs), neuromodulatory receptors that are major pharmacological targets. Synthesis of "BGAG12,400," a photoswitchable orthogonal, remotely tethered ligand (PORTL), enables highly efficient, rapid optical agonism following conjugation to SNAP-tagged mGluR2 and permits robust optical control of mGluR1 and mGluR5 signaling. We then produce fluorophore-conjugated branched PORTLs to enable dual imaging and manipulation of mGluRs and highlight their power in ex vivo slice and in vivo behavioral experiments in the mouse prefrontal cortex. Finally, we demonstrate the generalizability of our strategy by developing an improved soluble, photoswitchable pore blocker for potassium channels.

Defining the Homo- and Heterodimerization Propensities of Metabotropic Glutamate Receptors.

The eight metabotropic glutamate receptors (mGluRs) serve critical modulatory roles throughout the nervous system. The molecular diversity of mGluRs is thought to be further expanded by the formation of heterodimers, but the co-expression of mGluR subtypes at the cellular level and the relative propensities of heterodimer formation are not well known. Here, we analyze single-cell RNA sequencing data and find that cortical pyramidal cells express multiple mGluR subtypes with distinct profiles for different receptor combinations. We then develop quantitative, fluorescence-based assays to define the relative homo- and heterodimer propensities across group-I, -II, and -III mGluRs. We find a strong preference for heterodimerization in a number of cases, including mGluR2 with mGluR3, which we confirm in frontal cortex using in situ RNA hybridization and co-immunoprecipitation. Together, our findings support the biological relevance of mGluR heterodimerization and highlight the complex landscape of mGluR populations in the brain.

ALK4 coordinates extracellular and intrinsic signals to regulate development of cortical somatostatin interneurons.

Although the role of transcription factors in fate specification of cortical interneurons is well established, how these interact with extracellular signals to regulate interneuron development is poorly understood. Here we show that the activin receptor ALK4 is a key regulator of the specification of somatostatin interneurons. Mice lacking ALK4 in GABAergic neurons of the medial ganglionic eminence (MGE) showed marked deficits in distinct subpopulations of somatostatin interneurons from early postnatal stages of cortical development. Specific losses were observed among distinct subtypes of somatostatin+/Reelin+ double-positive cells, including Hpse+ layer IV cells targeting parvalbumin+ interneurons, leading to quantitative alterations in the inhibitory circuitry of this layer. Activin-mediated ALK4 signaling in MGE cells induced interaction of Smad2 with SATB1, a transcription factor critical for somatostatin interneuron development, and promoted SATB1 nuclear translocation and repositioning within the somatostatin gene promoter. These results indicate that intrinsic transcriptional programs interact with extracellular signals present in the environment of MGE cells to regulate cortical interneuron specification.

Diversity of Interneurons in the Dorsal Striatum Revealed by Single-Cell RNA Sequencing and PatchSeq.

Striatal locally projecting neurons, or interneurons, act on nearby circuits and shape functional output to the rest of the basal ganglia. We performed single-cell RNA sequencing of striatal cells enriching for interneurons. We find seven discrete interneuron types, six of which are GABAergic. In addition to providing specific markers for the populations previously described, including those expressing Sst/Npy, Th, Npy without Sst, and Chat, we identify two small populations of cells expressing Cck with or without Vip. Surprisingly, the Pvalb-expressing cells do not constitute a discrete cluster but rather are part of a larger group of cells expressing Pthlh with a spatial gradient of Pvalb expression. Using PatchSeq, we show that Pthlh cells exhibit a continuum of electrophysiological properties correlated with expression of Pvalb. Furthermore, we find significant molecular differences that correlate with differences in electrophysiological properties between Pvalb-expressing cells of the striatum and those of the cortex.

Transcriptional Convergence of Oligodendrocyte Lineage Progenitors during Development.

Pdgfra+ oligodendrocyte precursor cells (OPCs) arise in distinct specification waves during embryogenesis in the central nervous system (CNS). It is unclear whether there is a correlation between these waves and different oligodendrocyte (OL) states at adult stages. Here, we present bulk and single-cell transcriptomics resources providing insights on how transitions between these states occur. We found that post-natal OPCs from brain and spinal cord present similar transcriptional signatures. Moreover, post-natal OPC progeny of E13.5 Pdgfra+ cells present electrophysiological and transcriptional profiles similar to OPCs derived from subsequent specification waves, indicating that Pdgfra+ pre-OPCs rewire their transcriptional network during development. Single-cell RNA-seq and lineage tracing indicates that a subset of E13.5 Pdgfra+ cells originates cells of the pericyte lineage. Thus, our results indicate that embryonic Pdgfra+ cells in the CNS give rise to distinct post-natal cell lineages, including OPCs with convergent transcriptional profiles in different CNS regions.

Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq.

The mammalian cerebral cortex supports cognitive functions such as sensorimotor integration, memory, and social behaviors. Normal brain function relies on a diverse set of differentiated cell types, including neurons, glia, and vasculature. Here, we have used large-scale single-cell RNA sequencing (RNA-seq) to classify cells in the mouse somatosensory cortex and hippocampal CA1 region. We found 47 molecularly distinct subclasses, comprising all known major cell types in the cortex. We identified numerous marker genes, which allowed alignment with known cell types, morphology, and location. We found a layer I interneuron expressing Pax6 and a distinct postmitotic oligodendrocyte subclass marked by Itpr2. Across the diversity of cortical cell types, transcription factors formed a complex, layered regulatory code, suggesting a mechanism for the maintenance of adult cell type identity.

Automatic tracking of cells for video microscopy in patch clamp experiments.

BACKGROUND: Visualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. Here we created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons. METHODS: Neurons are first identified according to visual criteria (e.g. fluorescence protein expression, shape, viability etc.) and a transmission microscopy image Differential Interference Contrast (DIC) or Dodt contrast containing the cell used as a reference for the tracking algorithm. A fluorescence image can also be acquired later to be used as a mask (that can be overlaid on the target during live transmission video). As patch-clamp experiments require translating the microscope stage, we used pattern matching to track reference neurons in order to move the fluorescence mask to match the new position of the objective in relation to the sample. For the image processing we used the Open Source Computer Vision (OpenCV) library, including the Speeded-Up Robust Features (SURF) for tracking cells. The dataset of images (n = 720) was analyzed under normal conditions of acquisition and with influence of noise (defocusing and brightness). RESULTS: We validated the method in dissociated neuronal cultures and fresh brain slices expressing Enhanced Yellow Fluorescent Protein (eYFP) or Tandem Dimer Tomato (tdTomato) proteins, which considerably decreased the exposure to fluorescence excitation, thereby minimising photodamage. We also show that the neuron tracking can be used in differential interference contrast or Dodt contrast microscopy. CONCLUSION: The techniques of digital image processing used in this work are an important addition to the set of microscopy tools used in modern electrophysiology, specially in experiments with neuron cultures and brain slices.

OLM interneurons differentially modulate CA3 and entorhinal inputs to hippocampal CA1 neurons.

The vast diversity of GABAergic interneurons is believed to endow hippocampal microcircuits with the required flexibility for memory encoding and retrieval. However, dissection of the functional roles of defined interneuron types has been hampered by the lack of cell-specific tools. We identified a precise molecular marker for a population of hippocampal GABAergic interneurons known as oriens lacunosum-moleculare (OLM) cells. By combining transgenic mice and optogenetic tools, we found that OLM cells are important for gating the information flow in CA1, facilitating the transmission of intrahippocampal information (from CA3) while reducing the influence of extrahippocampal inputs (from the entorhinal cortex). Furthermore, we found that OLM cells were interconnected by gap junctions, received direct cholinergic inputs from subcortical afferents and accounted for the effect of nicotine on synaptic plasticity of the Schaffer collateral pathway. Our results suggest that acetylcholine acting through OLM cells can control the mnemonic processes executed by the hippocampus.

Sex differences in aversive memory in rats: possible role of extinction and reactive emotional factors.

Studies usually show better spatial learning in males and stronger emotional memory in females. Spatial memory differences could relate to diverse strategies, while dissimilar stress reactions could cause emotional memory differences. We compared male and female rats in two emotional (classical emotional conditioning and aversive discrimination memory) and two emotionally "neutral" tasks: (1) plus-maze discriminative avoidance, containing two open and two enclosed arms, one of which presenting aversive stimuli (light/noise). No differences were found in learning, retrieving, or basal emotional levels, while only male rats presented extinction of the task; (2) contextual fear conditioning--a cage was paired to mild foot shocks. Upon reexposure, freezing behavior was decreased in females; (3) spontaneous alternation--the animals were expected to alternate among the arms of a four-arm maze. No differences between genders were found and (4) open-field habituation was addressed in an arena which the rats were allowed to explore for 10 min. Habituation was similar between genders. Differences were found only in tasks with strong emotional contexts, where different fear responses and stress effects could be determinant. The lack of extinction of discriminative avoidance by females points out to stronger consolidation and/or impaired extinction of aversive memories.

A rodent model of appetitive discrimination with concomitant evaluation of anxiety-like behavior.

The plus-maze discriminative avoidance paradigm has been used to study the relationship between aversive memory and anxiety. The present study aims to verify if the elevated plus-maze can provide information about appetitive memory and anxiety-like behavior, through a task motivated by food reward. Animals were allowed to explore an elevated plus-maze and received reinforcement in one of the enclosed arms. In a test session performed 24h later, in the absence of reward, rats showed preference for the previously rewarded enclosed arm over the neutral enclosed arm. The administration of diazepam and pentylenetetrazole before training induced, respectively, anxiolytic and anxiogenic effects (as evaluated by open-arm exploration). Both drugs induced amnestic effects, i.e., lack of preference for the rewarded arm in the test session. The results suggest that appetitive memory can be influenced by anxiety levels as well. The plus-maze appetitive discrimination task seems to be a useful model to investigate the relationship between memory and anxiety.

Memory impairment induced by low doses of reserpine in rats: possible relationship with emotional processing deficits in Parkinson disease.

We have recently verified that the monoamine-depleting drug reserpine--at doses that do not modify motor function--impairs memory in a rodent model of aversive discrimination. In this study, the effects of reserpine (0.1-0.5 mg/kg) on the performance of rats in object recognition, spatial working memory (spontaneous alternation) and emotional memory (contextual freezing conditioning) tasks were investigated. While object recognition and spontaneous alternation behavior were not affected by reserpine treatment, contextual fear conditioning was impaired. Together with previous studies, these results suggest that low doses of reserpine would preferentially induce deficits in tasks involved with emotional contexts. Possible relationships with cognitive and emotional processing deficits in Parkinson disease are discussed.