Revealing intact neuronal circuitry in centimeter-sized formalin-fixed paraffin-embedded brain.

Tissue-clearing and labeling techniques have revolutionized brain-wide imaging and analysis, yet their application to clinical formalin-fixed paraffin-embedded (FFPE) blocks remains challenging. We introduce HIF-Clear, a novel method for efficiently clearing and labeling centimeter-thick FFPE specimens using elevated temperature and concentrated detergents. HIF-Clear with multi-round immunolabeling reveals neuron circuitry regulating multiple neurotransmitter systems in a whole FFPE mouse brain and is able to be used as the evaluation of disease treatment efficiency. HIF-Clear also supports expansion microscopy and can be performed on a non-sectioned 15-year-old FFPE specimen, as well as a 3-month formalin-fixed mouse brain. Thus, HIF-Clear represents a feasible approach for researching archived FFPE specimens for future neuroscientific and 3D neuropathological analyses.

Neurosphere culture derived from aged hippocampal dentate gyrus.

The neurosphere assay is the gold standard for determining proliferative and differentiation potential of neural progenitor cells (NPCs) in neurogenesis studies 1-3 . While several in vitro assays have been developed to model the process of neurogenesis, they have predominantly used embryonic and early postnatal NPCs derived from the dentate gyrus (DG). A limitation of these approaches is that they do not provide insight into adult-born NPCs, which are modeled to affect hippocampal function and diseases later in life. Here, we show a novel free-floating neurosphere culture system using NPCs isolated from the DG of mature adult and aged mice. The protocol outlines detailed steps on the isolation, propagation, and maintenance of neurospheres from adult and aged (>12 months old) mouse brain and how to differentiate cultured neurospheres into neurons and astrocytes. Culturing adult and aged NPCs provides an important in vitro model to (1) investigate cellular and molecular properties of this unique cell population and (2) expand the understanding of plasticity in the adult and aging brain. This protocol requires ∼2 hours to complete dissection, dissociation and culture plating, while differentiation to neuronal and astrocytic lineages takes 9 days. By focusing on neurospheres obtained from animals at later ages this model facilitates investigation of important biological questions related to development and differentiation of hippocampal neurons generated throughout adult life.

Involvement of a BH3-only apoptosis sensitizer gene Blm-s in hippocampus-mediated mood control.

Mood disorders are an important public health issue and recent advances in genomic studies have indicated that molecules involved in neurodevelopment are causally related to mood disorders. BLM-s (BCL-2-like molecule, small transcript isoform), a BH3-only proapoptotic BCL-2 family member, mediates apoptosis of postmitotic immature neurons during embryonic cortical development, but its role in the adult brain is unknown. To better understand the physiological role of Blm-s gene in vivo, we generated a Blm-s-knockout (Blm-s-/-) mouse. The Blm-s-/- mice breed normally and exhibit grossly normal development. However, global depletion of Blm-s is highly associated with depression- and anxiety-related behaviors in adult mutant mice with intact learning and memory capacity. Functional magnetic resonance imaging of adult Blm-s-/- mice reveals reduced connectivity mainly in the ventral dentate gyrus (vDG) of the hippocampus with no alteration in the dorsal DG connectivity and in total hippocampal volume. At the cellular level, BLM-s is expressed in DG granule cells (GCs), and Blm-s-/- mice show reduced dendritic complexity and decreased spine density in mature GCs. Electrophysiology study uncovers that mature vGCs in adult Blm-s-/- DG are intrinsically more excitable. Interestingly, certain genetic variants of the human Blm homologue gene (VPS50) are significantly associated with depression traits from publicly resourced UK Biobank data. Taken together, BLM-s is required for the hippocampal mood control function. Loss of BLM-s causes abnormality in the electrophysiology and morphology of GCs and a disrupted vDG neural network, which could underlie Blm-s-null-associated anxiety and depression.

Age-Related Changes in Synaptic Plasticity Associated with Mossy Fiber Terminal Integration during Adult Neurogenesis.

Mouse hippocampus retains the capacity for neurogenesis throughout lifetime, but such plasticity decreases with age. Adult hippocampal neurogenesis (AHN) involves the birth, maturation, and synaptic integration of newborn granule cells (GCs) into preexisting hippocampal circuitry. While functional integration onto adult-born GCs has been extensively studied, maturation of efferent projections onto CA3 pyramidal cells is less understood, particularly in aged brain. Here, using combined light and reconstructive electron microscopy (EM), we describe the maturation of mossy fiber bouton (MFB) connectivity with CA3 pyramidal cells in young adult and aged mouse brain. We found mature synaptic contacts of newborn GCs were formed in both young and aged brains. However, the dynamics of their spatiotemporal development and the cellular process by which these cells functionally integrated over time were different. In young brain newborn GCs either formed independent nascent MFB synaptic contacts or replaced preexisting MFBs, but these contacts were pruned over time to a mature state. In aged brain only replacement of preexisting MFBs was observed and new contacts were without evidence of pruning. These data illustrate that functional synaptic integration of AHN occurs in young adult and aged brain, but with distinct dynamics. They suggest elimination of preexisting connectivity is required for the integration of adult-born GCs in aged brain.

Regulation of axon repulsion by MAX-1 SUMOylation and AP-3.

During neural development, growing axons express specific surface receptors in response to various environmental guidance cues. These axon guidance receptors are regulated through intracellular trafficking and degradation to enable navigating axons to reach their targets. In Caenorhabditis elegans, the UNC-5 receptor is necessary for dorsal migration of developing motor axons. We previously found that MAX-1 is required for UNC-5-mediated axon repulsion, but its mechanism of action remained unclear. Here, we demonstrate that UNC-5-mediated axon repulsion in C. elegans motor axons requires both max-1 SUMOylation and the AP-3 complex ß subunit gene, apb-3 Genetic interaction studies show that max-1 is SUMOylated by gei-17/PIAS1 and acts upstream of apb-3 Biochemical analysis suggests that constitutive interaction of MAX-1 and UNC-5 receptor is weakened by MAX-1 SUMOylation and by the presence of APB-3, a competitive interactor with UNC-5. Overexpression of APB-3 reroutes the trafficking of UNC-5 receptor into the lysosome for protein degradation. In vivo fluorescence recovery after photobleaching experiments shows that MAX-1 SUMOylation and APB-3 are required for proper trafficking of UNC-5 receptor in the axon. Our results demonstrate that SUMOylation of MAX-1 plays an important role in regulating AP-3-mediated trafficking and degradation of UNC-5 receptors during axon guidance.

Monocular enucleation alters retinal waves in the surviving eye.

BACKGROUND: Activity in neurons drives afferent competition that is critical for the refinement of nascent neural circuits. In ferrets, when an eye is lost in early development, surviving retinogeniculate afferents from the spared eye spread across the thalamus in a manner that is dependent on spontaneous retinal activity. However, how this spontaneous activity, also known as retinal waves, might dynamically regulate afferent terminal targeting remains unknown. METHODS: We recorded retinal waves from retinae ex vivo using multi-electrode arrays. Retinae came from ferrets who were binocular or who had one eye surgically removed at birth. Linear mixed effects models were used to investigate the effects of early monocular enucleation on retinal wave activity. RESULTS: When an eye is removed at birth, spontaneous bursts of action potentials by retinal ganglion cells (RGCs) in the surviving eye are shorter in duration. The shortening of RGC burst duration results in decreased pairwise RGC correlations across the retina and is associated with the retinal wave-dependent spread of retinogeniculate afferents previously reported in enucleates. CONCLUSION: Our findings show that removal of the competing eye modulates retinal waves and could underlie the dynamic regulation of competition-based refinement during retinogeniculate development.

Increasing Spontaneous Retinal Activity before Eye Opening Accelerates the Development of Geniculate Receptive Fields.

Visually evoked activity is necessary for the normal development of the visual system. However, little is known about the capacity for patterned spontaneous activity to drive the maturation of receptive fields before visual experience. Retinal waves provide instructive retinotopic information for the anatomical organization of the visual thalamus. To determine whether retinal waves also drive the maturation of functional responses, we increased the frequency of retinal waves pharmacologically in the ferret (Mustela putorius furo) during a period of retinogeniculate development before eye opening. The development of geniculate receptive fields after receiving these increased neural activities was measured using single-unit electrophysiology. We found that increased retinal waves accelerate the developmental reduction of geniculate receptive field sizes. This reduction is due to a decrease in receptive field center size rather than an increase in inhibitory surround strength. This work reveals an instructive role for patterned spontaneous activity in guiding the functional development of neural circuits.

Retinal waves regulate afferent terminal targeting in the early visual pathway.

Current models of retinogeniculate development have proposed that connectivity between the retina and the dorsal lateral geniculate nucleus (dLGN) is established by gradients of axon guidance molecules, to allow initial coarse connections, and by competitive Hebbian-like processes, to drive eye-specific segregation and refine retinotopy. Here we show that when intereye competition is eliminated by monocular enucleation, blocking cholinergic stage II retinal waves disrupts the intraeye competition-mediated expansion of the retinogeniculate projection and results in the permanent disorganization of its laminae. This disruption of stage II retinal waves also causes long-term impacts on receptive field size and fine-scale retinotopy in the dLGN. Our results reveal a novel role for stage II retinal waves in regulating retinogeniculate afferent terminal targeting by way of intraeye competition, allowing for correct laminar patterning and the even allocation of synaptic territory. These findings should contribute to answering questions regarding the role of neural activity in guiding the establishment of neural circuits.

Epibatidine blocks eye-specific segregation in ferret dorsal lateral geniculate nucleus during stage III retinal waves.

The segregation and maintenance of eye-specific inputs in the dorsal lateral geniculate nucleus (dLGN) during early postnatal development requires the patterned spontaneous activity of retinal waves. In contrast to the development of the mouse, ferret eye-specific segregation is not complete at the start of stage III glutamatergic retinal waves, and the remaining overlap is limited to the C/C1 lamina of the dLGN. To investigate the role of patterned spontaneous activity in this late segregation, we disrupted retinal waves pharmacologically for 5 day windows from postnatal day (P) 10 to P25. Multi-electrode array recordings of the retina in vitro reveal that the cholinergic agonist epibatidine disrupts correlated retinal activity during stage III waves. Epibatidine also prevents the segregation of eye-specific inputs in vivo during that period. Our results reveal a novel role for cholinergic influence on stage III retinal waves as an instructive signal for the continued segregation of eye-specific inputs in the ferret dLGN.

Eye-specific retinogeniculate segregation proceeds normally following disruption of patterned spontaneous retinal activity.

BACKGROUND: Spontaneous retinal activity (SRA) is important during eye-specific segregation within the dorsal lateral geniculate nucleus (dLGN), but the feature(s) of activity critical for retinogeniculate refinement are controversial. Pharmacologically or genetically manipulating cholinergic signaling during SRA perturbs correlated retinal ganglion cell (RGC) spiking and disrupts eye-specific retinofugal refinement in vivo, consistent with an instructive role for SRA during visual system development. Paradoxically, ablating the starburst amacrine cells (SACs) that generate cholinergic spontaneous activity disrupts correlated RGC firing without impacting retinal activity levels or eye-specific segregation in the dLGN. Such experiments suggest that patterned SRA during retinal waves is not critical for eye-specific refinement and instead, normal activity levels are permissive for retinogeniculate development. Here we revisit the effects of ablating the cholinergic network during eye-specific segregation and show that SAC ablation disrupts, but does not eliminate, retinal waves with no concomitant impact on normal eye-specific segregation in the dLGN. RESULTS: We induced SAC ablation in postnatal ferret pups beginning at birth by intraocular injection of a novel immunotoxin selective for the ferret vesicular acetylcholine transporter (Ferret VAChT-Sap). Through dual-patch whole-cell and multi-electrode array recording we found that SAC ablation altered SRA patterns and led to significantly smaller retinal waves compared with controls. Despite these defects, eye-specific segregation was normal. Further, interocular competition for target territory in the dLGN proceeded in cases where SAC ablation was asymmetric in the two eyes. CONCLUSIONS: Our data demonstrate normal eye-specific retinogeniculate development despite significant abnormalities in patterned SRA. Comparing our current results with earlier studies suggests that defects in retinal wave size, absolute levels of SRA, correlations between RGC pairs, RGC burst frequency, high frequency RGC firing during bursts, and the number of spikes per RGC burst are each uncorrelated with abnormalities in eye-specific segregation in the dLGN. An increase in the fraction of asynchronous spikes occurring outside of bursts and waves correlates with eye-specific segregation defects in studies reported to date. These findings highlight the relative importance of different features of SRA while providing additional constraints for computational models of Hebbian plasticity mechanisms in the developing visual system.

Blm-s, a BH3-only protein enriched in postmitotic immature neurons, is transcriptionally upregulated by p53 during DNA damage.

Programmed cell death is a pivotal process that regulates neuronal number during development. Key regulators of this process are members of the BCL-2 family. Using mRNA differential display, we identified a Bcl-2 family gene, Blm-s (Bcl-2-like molecule, short form), enriched in postmitotic neurons of the developing cerebral cortex. BLM-s functions as a BH3-only apoptosis sensitizer/derepressor and causes BAX-dependent mitochondria-mediated apoptosis by selectively binding to prosurvival BCL-2 or MCL-1. When challenged with γ-irradiation that produces DNA double-strand breaks (DSBs), Blm-s is transcriptionally upregulated in postmitotic immature neurons with concurrently increased apoptosis. RNAi-mediated depletion of Blm-s protects immature neurons from irradiation-induced apoptosis. Furthermore, Blm-s is a direct target gene of p53 and AP1 via the ataxia telangiectasia mutated (ATM)- and c-Jun N-terminal kinase (JNK)-signaling pathways activated by DSBs. Thus, BLM-s is likely an apoptosis sensor activated by DSBs accumulating in postmitotic immature neurons.

Sema3E/plexin-D1 mediated epithelial-to-mesenchymal transition in ovarian endometrioid cancer.

Cancer cells often employ developmental cues for advantageous growth and metastasis. Here, we report that an axon guidance molecule, Sema3E, is highly expressed in human high-grade ovarian endometrioid carcinoma, but not low-grade or other ovarian epithelial tumors, and facilitates tumor progression. Unlike its known angiogenic activity, Sema3E acted through Plexin-D1 receptors to augment cell migratory ability and concomitant epithelial-to-mesenchymal transition (EMT). Sema3E-induced EMT in ovarian endometrioid cancer cells was dependent on nuclear localization of Snail1 through activation of phosphatidylinositol-3-kinase and ERK/MAPK. RNAi-mediated knockdown of Sema3E, Plexin-D1 or Snail1 in Sema3E-expressing tumor cells resulted in compromised cell motility, concurrent reversion of EMT and diminished nuclear localization of Snail1. By contrast, forced retention of Snail1 within the nucleus of Sema3E-negative tumor cells induced EMT and enhanced cell motility. These results show that in addition to the angiogenic effects of Sema3E on tumor vascular endothelium, an EMT strategy could be exploited by Sema3E/Plexin-D1 signaling in tumor cells to promote cellular invasion/migration.

Disrupted-in-Schizophrenia 1-mediated axon guidance involves TRIO-RAC-PAK small GTPase pathway signaling.

Defects in neuronal connectivity of the brain are well documented among schizophrenia patients. Although the schizophrenia susceptibility gene Disrupted-in-Schizophrenia 1 (DISC1) has been implicated in various neurodevelopmental processes, its role in regulating axonal connections remains elusive. Here, a heterologous DISC1 transgenic system in the relatively simple and well-characterized Caenorhabditis elegans motor neurons has been established to investigate whether DISC1 regulates axon guidance during development. Transgenic DISC1 in C. elegans motor neurons is enriched in the migrating growth cones and causes guidance defects of their growing axons. The abnormal axonal phenotypes induced by DISC1 are similar to those by gain-of-function rac genes. In vivo genetic interaction studies revealed that the UNC-73/TRIO-RAC-PAK signaling pathway is activated by ectopic DISC1 in C. elegans motor axons. Using in vitro GST pull-down and coimmunoprecipitation assays, we found that DISC1 binds specifically to the amino half of spectrin repeats of TRIO, thereby preventing TRIO's amino half of spectrin repeats from interacting with its first guanine nucleotide exchange factor (GEF) domain, GEF1, and facilitating the recruitment of RAC1 to TRIO. In cultured mammalian cells, RAC1 is activated by increased TRIO's GEF activity when DISC1 is present. These results together indicate that the TRIO-RAC-PAK signaling pathway can be exploited and modulated by DISC1 to regulate axonal connectivity in the developing brain.

Emergence of lamina-specific retinal ganglion cell connectivity by axon arbor retraction and synapse elimination.

Throughout the nervous system, neurons restrict their connections to specific depths or "layers" of their targets to constrain the type and number of synapses they make. Despite the importance of lamina-specific synaptic connectivity, the mechanisms that give rise to this feature in mammals remain poorly understood. Here we examined the cellular events underlying the formation of lamina-specific retinal ganglion cell (RGC) axonal projections to the superior colliculus (SC) of the mouse. By combining a genetically encoded marker of a defined RGC subtype (OFF-αRGCs) with serial immunoelectron microscopy, we resolved the ultrastructure of axon terminals fated for laminar stabilization versus those fated for removal. We found that OFF-αRGCs form synapses across the full depth of the retinorecipient SC before undergoing lamina-specific arbor retraction and synapse elimination to arrive at their mature, restricted pattern of connectivity. Interestingly, we did not observe evidence of axon degeneration or glia-induced synapse engulfment during this process. These findings indicate that lamina-specific visual connections are generated through the selective stabilization of correctly targeted axon arbors and suggest that the decision to maintain or eliminate an axonal projection reflects the molecular compatibility of presynaptic and postsynaptic neurons at a given laminar depth.

Guidance molecules in axon pruning and cell death.

Axon pruning and neuronal cell death constitute two major regressive events that enable the establishment of fully mature brain architecture and connectivity. Although the cellular mechanisms for these two events are thought to be distinct, recent evidence has indicated the direct involvement of axon guidance molecules, including semaphorins, netrins, and ephrins, in controlling both processes. Here, we review how axon guidance cues regulate regressive events in paradigmatic models of neural development, from early control of apoptosis of neural progenitors, to later maintenance of neuronal survival and stereotyped pruning of axonal branches. These new findings are also discussed in the context of neural diseases and the potential links between axon pruning and degeneration.

Functions of axon guidance molecules in synapse formation.

Axon guidance and synapse formation are important developmental events for establishing a functional neuronal circuitry. These two related cellular processes occur in a coordinated fashion but previous studies from multiple model organisms seemed to suggest that axon guidance and synapse formation are mediated by distinct molecular cues. Thus, axon guidance molecules are responsible for guiding the navigating axon toward its target area, while other adhesion or ligand-receptor molecules specify the synapse formation within the target area. However, accumulative evidence has shown that axon guidance molecules can regulate the localization and formation of pre-synaptic and post-synaptic components during synapse formation. These results demonstrate a role for axon guidance molecules in synapse formation and provide insight into how axon guidance and synapse formation are coordinated at the molecular level.

Deal breaker: semaphorin and specificity in the spinal stretch reflex circuit.

Stretch reflex circuits are a prime example of wiring specificity in the vertebrate spinal cord. Homonymous sensory afferents and motoneurons typically form monosynaptic connections, while neurons innervating antagonistic or unrelated muscles do not. Pecho-Vrieseling et al. now show that the semaphorin Sema3E and its receptor Plexin-D1 prevent monosynaptic connectivity in the cutaneous maximus muscle stretch reflex circuit.

A RAC/CDC-42-independent GIT/PIX/PAK signaling pathway mediates cell migration in C. elegans.

P21 activated kinase (PAK), PAK interacting exchange factor (PIX), and G protein coupled receptor kinase interactor (GIT) compose a highly conserved signaling module controlling cell migrations, immune system signaling, and the formation of the mammalian nervous system. Traditionally, this signaling module is thought to facilitate the function of RAC and CDC-42 GTPases by allowing for the recruitment of a GTPase effector (PAK), a GTPase activator (PIX), and a scaffolding protein (GIT) as a regulated signaling unit to specific subcellular locations. Instead, we report here that this signaling module functions independently of RAC/CDC-42 GTPases in vivo to control the cell shape and migration of the distal tip cells (DTCs) during morphogenesis of the Caenorhabditis elegans gonad. In addition, this RAC/CDC-42-independent PAK pathway functions in parallel to a classical GTPase/PAK pathway to control the guidance aspect of DTC migration. Among the C. elegans PAKs, only PAK-1 functions in the GIT/PIX/PAK pathway independently of RAC/CDC42 GTPases, while both PAK-1 and MAX-2 are redundantly utilized in the GTPase/PAK pathway. Both RAC/CDC42-dependent and -independent PAK pathways function with the integrin receptors, suggesting that signaling through integrins can control the morphology, movement, and guidance of DTC through discrete pathways. Collectively, our results define a new signaling capacity for the GIT/PIX/PAK module that is likely to be conserved in vertebrates and demonstrate that PAK family members, which are redundantly utilized as GTPase effectors, can act non-redundantly in pathways independent of these GTPases.

Development of hippocampal mossy fiber synaptic outputs by new neurons in the adult brain.

New neurons are continuously generated in restricted regions of the adult mammalian brain. Although these adult-born neurons have been shown to receive synaptic inputs, little is known about their synaptic outputs. Using retrovirus-mediated birth-dating and labeling in combination with serial section electron microscopic reconstruction, we report that mossy fiber en passant boutons of adult-born dentate granule cells form initial synaptic contacts with CA3 pyramidal cells within 2 weeks after their birth and reach morphologic maturity within 8 weeks in the adult hippocampus. Knockdown of Disrupted-in-Schizophrenia-1 (DISC1) in newborn granule cells leads to defects in axonal targeting and development of synaptic outputs in the adult brain. Together with previous reports of synaptic inputs, these results demonstrate that adult-born neurons are fully integrated into the existing neuronal circuitry. Our results also indicate a role for DISC1 in presynaptic development and may have implications for the etiology of schizophrenia and related mental disorders.