Sculpting neural circuits by axon and dendrite pruning.

The assembly of functional neural circuits requires the combined action of progressive and regressive events. Regressive events encompass a variety of inhibitory developmental processes, including axon and dendrite pruning, which facilitate the removal of exuberant neuronal connections. Most axon pruning involves the removal of axons that had already made synaptic connections; thus, axon pruning is tightly associated with synapse elimination. In many instances, these developmental processes are regulated by the interplay between neurons and glial cells that act instructively during neural remodeling. Owing to the importance of axon and dendritic pruning, these remodeling events require precise spatial and temporal control, and this is achieved by a range of distinct molecular mechanisms. Disruption of these mechanisms results in abnormal pruning, which has been linked to brain dysfunction. Therefore, understanding the mechanisms of axon and dendritic pruning will be instrumental in advancing our knowledge of neural disease and mental disorders.

Temporal Dynamics and Neuronal Specificity of Grin3a Expression in the Mouse Forebrain.

GluN3A subunits endow N-Methyl-D-Aspartate receptors (NMDARs) with unique biophysical, trafficking, and signaling properties. GluN3A-NMDARs are typically expressed during postnatal development, when they are thought to gate the refinement of neural circuits by inhibiting synapse maturation, and stabilization. Recent work suggests that GluN3A also operates in adult brains to control a variety of behaviors, yet a full spatiotemporal characterization of GluN3A expression is lacking. Here, we conducted a systematic analysis of Grin3a (gene encoding mouse GluN3A) mRNA expression in the mouse brain by combining high-sensitivity colorimetric and fluorescence in situ hybridization with labeling for neuronal subtypes. We find that, while Grin3a mRNA expression peaks postnatally, significant levels are retained into adulthood in specific brain regions such as the amygdala, medial habenula, association cortices, and high-order thalamic nuclei. The time-course of emergence and down-regulation of Grin3a expression varies across brain region, cortical layer of residence, and sensory modality, in a pattern that correlates with previously reported hierarchical gradients of brain maturation and functional specialization. Grin3a is expressed in both excitatory and inhibitory neurons, with strong mRNA levels being a distinguishing feature of somatostatin interneurons. Our study provides a comprehensive map of Grin3a distribution across the murine lifespan and paves the way for dissecting the diverse functions of GluN3A in health and disease.

Position-independent functional refinement within the vagus motor topographic map.

Motor neurons in the central nervous system often lie in a continuous topographic map, where neurons that innervate different body parts are spatially intermingled. This is the case for the efferent neurons of the vagus nerve, which innervate diverse muscle and organ targets in the head and viscera for brain-body communication. It remains elusive how neighboring motor neurons with different fixed peripheral axon targets develop the separate somatodendritic (input) connectivity they need to generate spatially precise body control. Here, we show that vagus motor neurons in the zebrafish indeed generate spatially appropriate peripheral responses to focal sensory stimulation even when they are transplanted into ectopic positions within the topographic map, indicating that circuit refinement occurs after the establishment of coarse topography. Refinement depends on motor neuron synaptic transmission, suggesting that an experience-dependent periphery-to-brain feedback mechanism establishes specific input connectivity among intermingled motor populations.

Short-Term Dendritic Dynamics of Neonatal Cortical Neurons Revealed by In Vivo Imaging with Improved Spatiotemporal Resolution.

Individual neurons in sensory cortices exhibit specific receptive fields based on their dendritic patterns. These dendritic morphologies are established and refined during the neonatal period through activity-dependent plasticity. This process can be visualized using two-photon in vivo time-lapse imaging, but sufficient spatiotemporal resolution is essential. We previously examined dendritic patterning from spiny stellate (SS) neurons, the major type of layer 4 (L4) neurons, in the mouse primary somatosensory cortex (barrel cortex), where mature dendrites display a strong orientation bias toward the barrel center. Longitudinal imaging at 8 h intervals revealed the long-term dynamics by which SS neurons acquire this unique dendritic pattern. However, the spatiotemporal resolution was insufficient to detect the more rapid changes in SS neuron dendrite morphology during the critical neonatal period. In the current study, we imaged neonatal L4 neurons hourly for 8 h and improved the spatial resolution by uniform cell surface labeling. The improved spatiotemporal resolution allowed detection of precise changes in dendrite morphology and revealed aspects of short-term dendritic dynamics unique to the neonatal period. Basal dendrites of barrel cortex L4 neurons were highly dynamic. In particular, both barrel-inner and barrel-outer dendrites (trees and branches) emerged/elongated and disappeared/retracted at similarly high frequencies, suggesting that SS neurons acquire biased dendrite patterns through rapid trial-and-error emergence, elongation, elimination, and retraction of dendritic trees and branches. We also found correlations between morphology and behavior (elongation/retraction) of dendritic tips. Thus, the current study revealed short-term dynamics and related features of cortical neuron dendrites during refinement.

The epithelial Na+ channel UNC-8 promotes an endocytic mechanism that recycles presynaptic components to new boutons in remodeling neurons.

Circuit refinement involves the formation of new presynaptic boutons as others are dismantled. Nascent presynaptic sites can incorporate material from recently eliminated synapses, but the recycling mechanisms remain elusive. In early-stage C. elegans larvae, the presynaptic boutons of GABAergic DD neurons are removed and new outputs established at alternative sites. Here, we show that developmentally regulated expression of the epithelial Na+ channel (ENaC) UNC-8 in remodeling DD neurons promotes a Ca2+ and actin-dependent mechanism, involving activity-dependent bulk endocytosis (ADBE), that recycles presynaptic material for reassembly at nascent DD synapses. ADBE normally functions in highly active neurons to accelerate local recycling of synaptic vesicles. In contrast, we find that an ADBE-like mechanism results in the distal recycling of synaptic material from old to new synapses. Thus, our findings suggest that a native mechanism (ADBE) can be repurposed to dismantle presynaptic terminals for reassembly at new, distant locations.

Inter-axonal molecular crosstalk via Lumican proteoglycan sculpts murine cervical corticospinal innervation by distinct subpopulations.

How CNS circuits sculpt their axonal arbors into spatially and functionally organized domains is not well understood. Segmental specificity of corticospinal connectivity is an exemplar for such regional specificity of many axon projections. Corticospinal neurons (CSN) innervate spinal and brainstem targets with segmental precision, controlling voluntary movement. Multiple molecularly distinct CSN subpopulations innervate the cervical cord for evolutionarily enhanced precision of forelimb movement. Evolutionarily newer CSNBC-lat exclusively innervate bulbar-cervical targets, while CSNmedial are heterogeneous; distinct subpopulations extend axons to either bulbar-cervical or thoraco-lumbar segments. We identify that Lumican controls balance of cervical innervation between CSNBC-lat and CSNmedial axons during development, which is maintained into maturity. Lumican, an extracellular proteoglycan expressed by CSNBC-lat, non-cell-autonomously suppresses cervical collateralization by multiple CSNmedial subpopulations. This inter-axonal molecular crosstalk between CSN subpopulations controls murine corticospinal circuitry refinement and forelimb dexterity. Such crosstalk is generalizable beyond the corticospinal system for evolutionary incorporation of new neuron populations into preexisting circuitry.

Dietary fat: a potent microglial influencer.

Poor nutrition, lack of exercise, and genetic predisposition all contribute to the growing epidemic of obesity. Overweight/obesity create an environment of chronic inflammation that leads to negative physiological and neurological outcomes, such as diabetes, cardiovascular disease, and anxiety/depression. While the whole body contributes to metabolic homeostasis, the neuroimmune system has recently emerged as a key regulator of metabolism. Microglia, the resident immune cells of the brain, respond both directly and indirectly to dietary fat, and the environment in which microglia develop contributes to their responsiveness later in life. Thus, high maternal weight during pregnancy may have consequences for microglial function in offspring. Here, we discuss the most recent findings on microglia signaling in overweight/obesity with a focus on perinatal programming.

Microglia and Sensitive Periods in Brain Development.

From embryonic neuronal migration to adolescent circuit refinement, the immune system plays an essential role throughout central nervous system (CNS) development. Immune signaling molecules serve as a common language between the immune system and CNS, allowing them to work together to modulate brain function both in health and disease. As the resident CNS macrophage, microglia comprise the majority of immune cells in the brain. Much like their peripheral counterparts, microglia survey their environment for pathology, clean up debris, and propagate inflammatory responses when necessary. Beyond this, recent studies have highlighted that microglia perform a number of complex tasks during neural development, from directing neuronal and axonal positioning to pruning synapses, receptors, and even whole cells. In this chapter, we discuss this literature within the framework that immune activation during discrete windows of neural development can profoundly impact brain function long-term, and thus the risk of neurodevelopmental and neuropsychiatric disorders. In this chapter, we review three sensitive developmental periods - embryonic wiring, early postnatal synaptic pruning, and adolescent circuit refinement - in order to highlight the diversity of functions that microglia perform in building a brain. In reviewing this literature, it becomes obvious that timing matters, perhaps more so than the nature of the immune activation itself; largely conserved patterns of microglial response to diverse insults result in different functional impacts depending on the stage of brain maturation at the time of the challenge.

Assembly and maintenance of GABAergic and Glycinergic circuits in the mammalian nervous system.

Inhibition in the central nervous systems (CNS) is mediated by two neurotransmitters: gamma-aminobutyric acid (GABA) and glycine. Inhibitory synapses are generally GABAergic or glycinergic, although there are synapses that co-release both neurotransmitter types. Compared to excitatory circuits, much less is known about the cellular and molecular mechanisms that regulate synaptic partner selection and wiring patterns of inhibitory circuits. Recent work, however, has begun to fill this gap in knowledge, providing deeper insight into whether GABAergic and glycinergic circuit assembly and maintenance rely on common or distinct mechanisms. Here we summarize and contrast the developmental mechanisms that regulate the selection of synaptic partners, and that promote the formation, refinement, maturation and maintenance of GABAergic and glycinergic synapses and their respective wiring patterns. We highlight how some parts of the CNS demonstrate developmental changes in the type of inhibitory transmitter or receptor composition at their inhibitory synapses. We also consider how perturbation of the development or maintenance of one type of inhibitory connection affects other inhibitory synapse types in the same circuit. Mechanistic insight into the development and maintenance of GABAergic and glycinergic inputs, and inputs that co-release both these neurotransmitters could help formulate comprehensive therapeutic strategies for treating disorders of synaptic inhibition.

Hox2 Genes Are Required for Tonotopic Map Precision and Sound Discrimination in the Mouse Auditory Brainstem.

Tonotopy is a hallmark of auditory pathways and provides the basis for sound discrimination. Little is known about the involvement of transcription factors in brainstem cochlear neurons orchestrating the tonotopic precision of pre-synaptic input. We found that in the absence of Hoxa2 and Hoxb2 function in Atoh1-derived glutamatergic bushy cells of the anterior ventral cochlear nucleus, broad input topography and sound transmission were largely preserved. However, fine-scale synaptic refinement and sharpening of isofrequency bands of cochlear neuron activation upon pure tone stimulation were impaired in Hox2 mutants, resulting in defective sound-frequency discrimination in behavioral tests. These results establish a role for Hox factors in tonotopic refinement of connectivity and in ensuring the precision of sound transmission in the mammalian auditory circuit.

Neuronal remodeling in retinal circuit assembly, disassembly, and reassembly.

Developing neuronal circuits often undergo a period of refinement to eliminate aberrant synaptic connections. Inappropriate connections can also form among surviving neurons during neuronal degeneration. The laminar organization of the vertebrate retina enables synaptic reorganization to be readily identified. Synaptic rearrangements are shown to help sculpt developing retinal circuits, although the mechanisms involved remain debated. Structural changes in retinal diseases can also lead to functional rewiring. This poses a major challenge to retinal repair because it may be necessary to untangle the miswired connections before reconnecting with proper synaptic partners. Here, we review our current understanding of the mechanisms that underlie circuit remodeling during retinal development, and discuss how alterations in connectivity during damage could impede circuit repair.

Competition from newborn granule cells does not drive axonal retraction of silenced old granule cells in the adult hippocampus.

In the developing nervous system synaptic refinement, typified by the neuromuscular junction where supernumerary connections are eliminated by axon retraction leaving the postsynaptic target innervated by a single dominant input, critically regulates neuronal circuit formation. Whether such competition-based pruning continues in established circuits of mature animals remains unknown. This question is particularly relevant in the context of adult neurogenesis where newborn cells must integrate into preexisting circuits, and thus, potentially compete with functionally mature synapses to gain access to their postsynaptic targets. The hippocampus plays an important role in memory formation/retrieval and the dentate gyrus (DG) subfield exhibits continued neurogenesis into adulthood. Therefore, this region contains both mature granule cells (old GCs) and immature recently born GCs that are generated throughout adult life (young GCs), providing a neurogenic niche model to examine the role of competition in synaptic refinement. Recent work from an independent group in developing animals indicated that embryonically/early postnatal generated GCs placed at a competitive disadvantage by selective expression of tetanus toxin (TeTX) to prevent synaptic release rapidly retracted their axons, and that this retraction was driven by competition from newborn GCs lacking TeTX. In contrast, following 3-6 months of selective TeTX expression in old GCs of adult mice we did not observe any evidence of axon retraction. Indeed ultrastructural analyses indicated that the terminals of silenced GCs even maintained synaptic contact with their postsynaptic targets. Furthermore, we did not detect any significant differences in the electrophysiological properties between old GCs in control and TeTX conditions. Thus, our data demonstrate a remarkable stability in the face of a relatively prolonged period of altered synaptic competition between two populations of neurons within the adult brain.