J. Tiago Gonçalves

Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior

The dentate gyrus of the mammalian hippocampus continuously generates new neurons during adulthood. These adult-born neurons become functionally active and are thought to contribute to learning and memory, especially during their maturation phase, when they have extraordinary plasticity. In this Review, we discuss the molecular machinery involved in the generation of new neurons from a pool of adult neural stem cells and their integration into functional hippocampal circuits. We also summarize the potential functions of these newborn neurons in the adult brain, their contribution to behavior, and their relevance to disease.

Directly Reprogrammed Human Neurons Retain Aging-Associated Transcriptomic Signatures and Reveal Age-Related Nucleocytoplasmic Defects

Aging is a major risk factor for many human diseases, and in vitro generation of human neurons is an attractive approach for modeling aging-related brain disorders. However, modeling aging in differentiated human neurons has proved challenging. We generated neurons from human donors across a broad range of ages, either by iPSC-based reprogramming and differentiation or by direct conversion into induced neurons (iNs). While iPSCs and derived neurons did not retain aging-associated gene signatures, iNs displayed age-specific transcriptional profiles and revealed age-associated decreases in the nuclear transport receptor RanBP17. We detected an age-dependent loss of nucleocytoplasmic compartmentalization (NCC) in donor fibroblasts and corresponding iNs and found that reduced RanBP17 impaired NCC in young cells, while iPSC rejuvenation restored NCC in aged cells. These results show that iNs retain important aging-related signatures, thus allowing modeling of the aging process in vitro, and they identify impaired NCC as an important factor in human aging.

Internally mediated developmental desynchronization of neocortical network activity.

During neocortical development, neurons exhibit highly synchronized patterns of spontaneous activity, with correlated bursts of action potential firing dominating network activity. This early activity is eventually replaced by more sparse and decorrelated firing of cortical neurons, which modeling studies predict is a network state that is better suited for efficient neural coding. The precise time course and mechanisms of this crucial transition in cortical network activity have not been characterized in vivo. We used in vivo two-photon calcium imaging in combination with whole-cell recordings in both unanesthetized and anesthetized mice to monitor how spontaneous activity patterns in ensembles of layer 2/3 neurons of barrel cortex mature during postnatal development. We find that, as early as postnatal day 4, activity is highly synchronous within local clusters of neurons. At the end of the second postnatal week, neocortical networks undergo a transition to a much more desynchronized state that lacks a clear spatial structure. Strikingly, deprivation of sensory input from the periphery had no effect on the time course of this transition. Therefore, developmental desynchronization of spontaneous neuronal activity is a fundamental network transition in the neocortex that appears to be intrinsically generated.

Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing

In vivo two-photon calcium imaging would benefit from the use of multiple excitation beams to increase scanning speed, signal-to-noise ratio and field of view or to image different axial planes simultaneously. Using spatiotemporal multiplexing we circumvented light-scattering ambiguity inherent to deep-tissue multifocal two-photon microscopy. We demonstrate calcium imaging at multiple axial planes in the intact mouse brain to monitor network activity of ensembles of cortical neurons in three spatial dimensions.

Circuit level defects in the developing neocortex of fragile X mice

Subtle alterations in how cortical network dynamics are modulated by different behavioral states could disrupt normal brain function and underlie symptoms of neuropsychiatric disorders, including Fragile X syndrome (FXS). Using two-photon calcium imaging and electrophysiology, we recorded spontaneous neuronal ensemble activity in mouse somatosensory cortex. Unanesthetized Fmr1−/− mice exhibited abnormally high synchrony of neocortical network activity, especially during the first two postnatal weeks. Neuronal firing rates were threefold higher in Fmr1−/− mice than in wild-type mice during whole-cell recordings manifesting Up/Down states (slow-wave sleep, quiet wakefulness), probably as a result of a higher firing probability during Up states. Combined electroencephalography and calcium imaging experiments confirmed that neurons in mutant mice had abnormally high firing and synchrony during sleep. We conclude that cortical networks in FXS are hyperexcitable in a brain state–dependent manner during a critical period for experience-dependent plasticity. These state-dependent network defects could explain the intellectual, sleep and sensory integration dysfunctions associated with FXS.

An in vivo model of functional and vascularized human brain organoids

Differentiation of human pluripotent stem cells to small brain-like structures known as brain organoids offers an unprecedented opportunity to model human brain development and disease. To provide a vascularized and functional in vivo model of brain organoids, we established a method for transplanting human brain organoids into the adult mouse brain. Organoid grafts showed progressive neuronal differentiation and maturation, gliogenesis, integration of microglia, and growth of axons to multiple regions of the host brain. In vivo two-photon imaging demonstrated functional neuronal networks and blood vessels in the grafts. Finally, in vivo extracellular recording combined with optogenetics revealed intragraft neuronal activity and suggested graft-to-host functional synaptic connectivity. This combination of human neural organoids and an in vivo physiological environment in the animal brain may facilitate disease modeling under physiological conditions.

In vivo imaging of dendritic pruning in dentate granule cells

We longitudinally imaged the developing dendrites of adult-born mouse dentate granule cells (DGCs) in vivo and found that they underwent over-branching and pruning. Exposure to an enriched environment and constraint of dendritic growth by disrupting Wnt signaling led to increased branch addition and accelerated growth, which were, however, counteracted by earlier and more extensive pruning. Our results indicate that pruning is regulated in a homeostatic fashion to oppose excessive branching and promote a similar dendrite structure in DGCs.

Calmodulin interaction with hEAG1 visualized by FRET microscopy.

Background Ca2+-mediated regulation of ion channels provides a link between intracellular signaling pathways and membrane electrical activity. Intracellular Ca2+ inhibits the voltage-gated potassium channel EAG1 through the direct binding of calmodulin (CaM). Three CaM binding sites (BD-C1: 674-683, BD-C2: 711-721, BD-N: 151-165) have been identified in a peptide screen and were proposed to mediate binding. The participation of the three sites in CaM binding to the native channel, however, remains unclear. Methodology/Principal Findings Here we studied the binding of Ca2+/CaM to the EAG channel by visualizing the interaction between YFP-labeled CaM and Cerulean-labeled hEAG1 in mammalian cells by FRET. The results of our cellular approach substantiate that two CaM binding sites are predominantly involved; the high-affinity 1-8-14 based CaM binding domain in the N-terminus and the second C-terminal binding domain BD-C2. Mutations at these sites completely abolished CaM binding to hEAG1. Conclusions/Significance We demonstrated that the BD-N and BD-C2 binding domains are sufficient for CaM binding to the native channel, and, therefore, that BD-C1 is unable to bind CaM independently.

Beyond excitation/inhibition imbalance in multidimensional models of neural circuit changes in brain disorders

A leading theory holds that neurodevelopmental brain disorders arise from imbalances in excitatory and inhibitory (E/I) brain circuitry. However, it is unclear whether this one-dimensional model is rich enough to capture the multiple neural circuit alterations underlying brain disorders. Here, we combined computational simulations with analysis of in vivo two-photon Ca2+ imaging data from somatosensory cortex of Fmr1 knock-out (KO) mice, a model of Fragile-X Syndrome, to test the E/I imbalance theory. We found that: (1) The E/I imbalance model cannot account for joint alterations in the observed neural firing rates and correlations; (2) Neural circuit function is vastly more sensitive to changes in some cellular components over others; (3) The direction of circuit alterations in Fmr1 KO mice changes across development. These findings suggest that the basic E/I imbalance model should be updated to higher dimensional models that can better capture the multidimensional computational functions of neural circuits.

Erratum: An in vivo model of functional and vascularized human brain organoids

Nat. Biotechnol. 36, 432–441 (2018); published online 16 April 2018; corrected after print 18 July 2018 In the version of this article initially published, credit for part of Figure 4a was omitted. The image of a lens and microscope stage was originally published elsewhere. The error has been corrected in the HTML and PDF versions of the article.