Nesprins and opposing microtubule motors generate a point force that drives directional nuclear motion in migrating neurons.

Nuclear migration of newly born neurons is essential for cortex formation in the brain. The nucleus is translocated by actin and microtubules, yet the actual force generated by the interplay of these cytoskeletons remains elusive. High-resolution time-lapse observation of migrating murine cerebellar granule cells revealed that the nucleus actively rotates along the direction of its translocation, independently of centrosome motion. Pharmacological and molecular perturbation indicated that spin torque is primarily generated by microtubule motors through the LINC complex in the absence of actomyosin contractility. In contrast to the prevailing view that microtubules are uniformly oriented around the nucleus, we observed that the perinuclear microtubule arrays are of mixed polarity and both cytoplasmic dynein complex and kinesin-1 are required for nuclear rotation. Kinesin-1 can exert a point force on the nuclear envelope via association with nesprins, and loss of kinesin-1 causes failure in neuronal migration in vivo Thus, microtubules steer the nucleus and drive its rotation and translocation via a dynamic, focal interaction of nesprins with kinesin-1 and dynein, and this is necessary for neuronal migration during brain development.

FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity.

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging.

Synergistic action of dendritic mitochondria and creatine kinase maintains ATP homeostasis and actin dynamics in growing neuronal dendrites.

The distribution of mitochondria within mature, differentiated neurons is clearly adapted to their regional physiological needs and can be perturbed under various pathological conditions, but the function of mitochondria in developing neurons has been less well studied. We have studied mitochondrial distribution within developing mouse cerebellar Purkinje cells and have found that active delivery of mitochondria into their dendrites is a prerequisite for proper dendritic outgrowth. Even when mitochondria in the Purkinje cell bodies are functioning normally, interrupting the transport of mitochondria into their dendrites severely disturbs dendritic growth. Additionally, we find that the growth of atrophic dendrites lacking mitochondria can be rescued by activating ATP-phosphocreatine exchange mediated by creatine kinase (CK). Conversely, inhibiting cytosolic CKs decreases dendritic ATP levels and also disrupts dendrite development. Mechanistically, this energy depletion appears to perturb normal actin dynamics and enhance the aggregation of cofilin within growing dendrites, reminiscent of what occurs in neurons overexpressing the dephosphorylated form of cofilin. These results suggest that local ATP synthesis by dendritic mitochondria and ATP-phosphocreatine exchange act synergistically to sustain the cytoskeletal dynamics necessary for dendritic development.

The preoptic area and dorsal habenula jointly support homeostatic navigation in larval zebrafish.

Animals must maintain physiological processes within an optimal temperature range despite changes in their environment. Through behavioral assays, whole-brain functional imaging, and neural ablations, we show that larval zebrafish, an ectothermic vertebrate, achieves thermoregulation through homeostatic navigation-non-directional and directional movements toward the temperature closest to its physiological setpoint. A brain-wide circuit encompassing several brain regions enables this behavior. We identified the preoptic area of the hypothalamus (PoA) as a key brain structure in triggering non-directional reorientation when thermal conditions are worsening. This result shows an evolutionary conserved role of the PoA as principal thermoregulator of the brain also in ectotherms. We further show that the habenula (Hb)-interpeduncular nucleus (IPN) circuit retains a short-term memory of the sensory history to support the generation of coherent directed movements even in the absence of continuous sensory cues. We finally provide evidence that this circuit may not be exclusive for temperature but may convey a more abstract representation of relative valence of physiologically meaningful stimuli regardless of their specific identity to enable homeostatic navigation.

Neural dynamics and architecture of the heading direction circuit in zebrafish.

Animals generate neural representations of their heading direction. Notably, in insects, heading direction is topographically represented by the activity of neurons in the central complex. Although head direction cells have been found in vertebrates, the connectivity that endows them with their properties is unknown. Using volumetric lightsheet imaging, we find a topographical representation of heading direction in a neuronal network in the zebrafish anterior hindbrain, where a sinusoidal bump of activity rotates following directional swims of the fish and is otherwise stable over many seconds. Electron microscopy reconstructions show that, although the cell bodies are located in a dorsal region, these neurons arborize in the interpeduncular nucleus, where reciprocal inhibitory connectivity stabilizes the ring attractor network that encodes heading. These neurons resemble those found in the fly central complex, showing that similar circuit architecture principles may underlie the representation of heading direction across the animal kingdom and paving the way to an unprecedented mechanistic understanding of these networks in vertebrates.

Dynamic Interaction Between Microtubules and the Nucleus Regulates Nuclear Movement During Neuronal Migration.

Fine structures of the mammalian brain are formed by neuronal migration during development. Newborn neurons migrate long distances from the germinal zone to individual sites of function by squeezing their largest cargo, the nucleus, through the crowded neural tissue. Nuclear translocation is thought to be orchestrated by microtubules, actin, and their associated motor proteins, dynein and myosin. However, where and how the cytoskeletal forces are converted to actual nuclear movement remains unclear. Using high-resolution confocal imaging of live migrating neurons, we demonstrated that microtubule-dependent forces are directly applied to the nucleus via the linker of nucleoskeleton and cytoskeleton complex, and that they induce dynamic nuclear movement, including translocation, rotation, and local peaking. Microtubules bind to small points on the nuclear envelope via the minus- and plus-oriented motor proteins, dynein and kinesin-1, and generate a point force independent of the actin-dependent force. Dynamic binding of microtubule motors might cause a continuously changing net force vector acting on the nucleus and results in a stochastic and inconsistent movement of the nucleus, which are seen in crowded neural tissues.

Differentiation of apical and basal dendrites in pyramidal cells and granule cells in dissociated hippocampal cultures.

Hippocampal pyramidal cells and dentate granule cells develop morphologically distinct dendritic arbors, yet also share some common features. Both cell types form a long apical dendrite which extends from the apex of the cell soma, while short basal dendrites are developed only in pyramidal cells. Using quantitative morphometric analyses of mouse hippocampal cultures, we evaluated the differences in dendritic arborization patterns between pyramidal and granule cells. Furthermore, we observed and described the final apical dendrite determination during dendritic polarization by time-lapse imaging. Pyramidal and granule cells in culture exhibited similar dendritic patterns with a single principal dendrite and several minor dendrites so that the cell types were not readily distinguished by appearance. While basal dendrites in granule cells are normally degraded by adulthood in vivo, cultured granule cells retained their minor dendrites. Asymmetric growth of a single principal dendrite harboring the Golgi was observed in both cell types soon after the onset of dendritic growth. Time-lapse imaging revealed that up until the second week in culture, final principal dendrite designation was not stabilized, but was frequently replaced by other minor dendrites. Before dendritic polarity was stabilized, the Golgi moved dynamically within the soma and was repeatedly repositioned at newly emerging principal dendrites. Our results suggest that polarized growth of the apical dendrite is regulated by cell intrinsic programs, while regression of basal dendrites requires cue(s) from the extracellular environment in the dentate gyrus. The apical dendrite designation is determined from among multiple growing dendrites of young developing neurons.