Topographic map refinement and synaptic strengthening of a sound localization circuit require spontaneous peripheral activity.

KEY POINTS: Loss of the calcium sensor otoferlin disrupts neurotransmission from inner hair cells. Central auditory nuclei are functionally denervated in otoferlin knockout mice (Otof KOs) via gene ablation confined to the periphery. We employed juvenile and young adult Otof KO mice (postnatal days (P)10-12 and P27-49) as a model for lacking spontaneous activity and deafness, respectively. We studied the impact of peripheral activity on synaptic refinement in the sound localization circuit from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO). MNTB in vivo recordings demonstrated drastically reduced spontaneous spiking and deafness in Otof KOs. Juvenile KOs showed impaired synapse elimination and strengthening, manifested by broader MNTB-LSO inputs, imprecise MNTB-LSO topography and weaker MNTB-LSO fibres. The impairments persisted into young adulthood. Further functional refinement after hearing onset was undetected in young adult wild-types. Collectively, activity deprivation confined to peripheral protein loss impairs functional MNTB-LSO refinement during a critical prehearing period. ABSTRACT: Circuit refinement is critical for the developing sound localization pathways in the auditory brainstem. In prehearing mice (hearing onset around postnatal day (P)12), spontaneous activity propagates from the periphery to central auditory nuclei. At the glycinergic projection from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) of neonatal mice, super-numerous MNTB fibres innervate a given LSO neuron. Between P4 and P9, MNTB fibres are functionally eliminated, whereas the remaining fibres are strengthened. Little is known about MNTB-LSO circuit refinement after P20. Moreover, MNTB-LSO refinement upon activity deprivation confined to the periphery is largely unexplored. This leaves a considerable knowledge gap, as deprivation often occurs in patients with congenital deafness, e.g. upon mutations in the otoferlin gene (OTOF). Here, we analysed juvenile (P10-12) and young adult (P27-49) otoferlin knockout (Otof KO) mice with respect to MNTB-LSO refinement. MNTB in vivo recordings revealed drastically reduced spontaneous activity and deafness in knockouts (KOs), confirming deprivation. As RNA sequencing revealed Otof absence in the MNTB and LSO of wild-types, Otof loss in KOs is specific to the periphery. Functional denervation impaired MNTB-LSO synapse elimination and strengthening, which was assessed by glutamate uncaging and electrical stimulation. Impaired elimination led to imprecise MNTB-LSO topography. Impaired strengthening was associated with lower quantal content per MNTB fibre. In young adult KOs, the MNTB-LSO circuit remained unrefined. Further functional refinement after P12 appeared absent in wild-types. Collectively, we provide novel insights into functional MNTB-LSO circuit maturation governed by a cochlea-specific protein. The central malfunctions in Otof KOs may have implications for patients with sensorineuronal hearing loss.

GluN3A expression restricts spine maturation via inhibition of GIT1/Rac1 signaling.

NMDA-type glutamate receptors (NMDARs) guide the activity-dependent remodeling of excitatory synapses and associated dendritic spines during critical periods of postnatal brain development. Whereas mature NMDARs composed of GluN1 and GluN2 subunits mediate synapse plasticity and promote spine growth and stabilization, juvenile NMDARs containing GluN3A subunits are thought to inhibit these processes via yet unknown mechanisms. Here, we report that GluN3A binds G protein-coupled receptor kinase-interacting protein (GIT1), a postsynaptic scaffold that assembles actin regulatory complexes, including the Rac1 guanine nucleotide exchange factor ßPIX, to promote Rac1 activation in spines. Binding to GluN3A limits the synaptic localization of GIT1 and its ability to complex ßPIX, leading to decreased Rac1 activation and reduced spine density and size in primary cultured neurons. Conversely, knocking out GluN3A favors the formation of GIT1/ßPIX complexes and increases the activation of Rac1 and its main effector p21-activated kinase. We further show that binding of GluN3A to GIT1 is regulated by synaptic activity, a response that might restrict the negative regulatory effects of GluN3A on actin signaling to inactive synapses. Our results identify inhibition of Rac1/p21-activated kinase actin signaling pathways as an activity-dependent mechanism mediating the inhibitory effects of GluN3A on spine morphogenesis.

Developmental Impairments of Synaptic Refinement in the Thalamus of a Mouse Model of Fragile X Syndrome.

While somatosensory over-reactivity is a common feature of autism spectrum disorders such as fragile X syndrome (FXS), the thalamic mechanisms underlying this remain unclear. Here, we found that the developmental elimination of synapses formed between the principal nucleus of V (PrV) and the ventral posterior medial nucleus (VPm) of the somatosensory system was delayed in fragile X mental retardation 1 gene knockout (Fmr1 KO) mice, while the developmental strengthening of these synapses was disrupted. Immunohistochemistry showed excessive VGluT2 puncta in mutants at P12-13, but not at P7-8 or P15-16, confirming a delay in somatic pruning of PrV-VPm synapses. Impaired synaptic function was associated with a reduction in the frequency of quantal AMPA events, as well as developmental deficits in presynaptic vesicle size and density. Our results uncovered the developmental impairment of thalamic relay synapses in Fmr1 KO mice and suggest that a thalamic contribution to the somatosensory over-reactivity in FXS should be considered.

Stimulus-dependent synaptic plasticity underlies neuronal circuitry refinement in the mouse primary visual cortex.

Perceptual learning improves our ability to interpret sensory stimuli present in our environment through experience. Despite its importance, the underlying mechanisms that enable perceptual learning in our sensory cortices are still not fully understood. In this study, we used in vivo two-photon imaging to investigate the functional and structural changes induced by visual stimulation in the mouse primary visual cortex (V1). Our results demonstrate that repeated stimulation leads to a refinement of V1 circuitry by decreasing the number of responsive neurons while potentiating their response. At the synaptic level, we observe a reduction in the number of dendritic spines and an overall increase in spine AMPA receptor levels in the same subset of neurons. In addition, visual stimulation induces synaptic potentiation in neighboring spines within individual dendrites. These findings provide insights into the mechanisms of synaptic plasticity underlying information processing in the neocortex.

Autophagy-Mediated Synaptic Refinement and Auditory Neural Pruning Contribute to Ribbon Synaptic Maturity in the Developing Cochlea.

In rodents, massive initial synapses are formed in the auditory peripheral nervous system at the early postnatal stage, and one of the major phenomena is that the number of afferent synapses in the cochlea is significantly reduced in the duration of development. This raises the hypothesis that the number of cochlear ribbon synapses are dramatically changed with hearing development and maturation. In this study, several tracers identifying activities of autophagy were applied to estimate the level of autophagy activity in the process of ribbon synapse development in mice; further, changes in the synaptic number and spiral ganglion nerve (SGN) fibers were quantitatively measured. We found robust expression of LC3B and lysosomal-associated membrane protein 1 as well as LysoTracker in or near inner hair cells and cochlear ribbon synapses in the early stage of postnatal development. Moreover, we found a significant loss in the intensity of SGN fibers at ribbon synaptic development and hearing onset. Thus, this study demonstrates that ribbon synaptic refinement and SGN fibers pruning are closely associated with the morphological and functional maturation of ribbon synapses and that synaptic refinement and SGN fiber pruning are regulated by the robust activities of autophagy in the earlier stages of auditory development.

Phosphatidylserine, inflammation, and central nervous system diseases.

Phosphatidylserine (PS) is an anionic phospholipid in the eukaryotic membrane and is abundant in the brain. Accumulated studies have revealed that PS is involved in the multiple functions of the brain, such as activation of membrane signaling pathways, neuroinflammation, neurotransmission, and synaptic refinement. Those functions of PS are related to central nervous system (CNS) diseases. In this review, we discuss the metabolism of PS, the anti-inflammation function of PS in the brain; the alterations of PS in different CNS diseases, and the possibility of PS to serve as a therapeutic agent for diseases. Clinical studies have showed that PS has no side effects and is well tolerated. Therefore, PS and PS liposome could be a promising supplementation for these neurodegenerative and neurodevelopmental diseases.

Sensory Experience Engages Microglia to Shape Neural Connectivity through a Non-Phagocytic Mechanism.

Sensory experience remodels neural circuits in the early postnatal brain through mechanisms that remain to be elucidated. Applying a new method of ultrastructural analysis to the retinogeniculate circuit, we find that visual experience alters the number and structure of synapses between the retina and the thalamus. These changes require vision-dependent transcription of the receptor Fn14 in thalamic relay neurons and the induction of its ligand TWEAK in microglia. Fn14 functions to increase the number of bulbous spine-associated synapses at retinogeniculate connections, likely contributing to the strengthening of the circuit that occurs in response to visual experience. However, at retinogeniculate connections near TWEAK-expressing microglia, TWEAK signals via Fn14 to restrict the number of bulbous spines on relay neurons, leading to the elimination of a subset of connections. Thus, TWEAK and Fn14 represent an intercellular signaling axis through which microglia shape retinogeniculate connectivity in response to sensory experience.

Nitric oxide controls excitatory/inhibitory balance in the hypoglossal nucleus during early postnatal development.

Synaptic remodeling during early postnatal development lies behind neuronal networks refinement and nervous system maturation. In particular, the respiratory system is immature at birth and is subjected to significant postnatal development. In this context, the excitatory/inhibitory balance dramatically changes in the respiratory-related hypoglossal nucleus (HN) during the 3 perinatal weeks. Since, development abnormalities of hypoglossal motor neurons (HMNs) are associated with sudden infant death syndrome and obstructive sleep apnea, deciphering molecular partners behind synaptic remodeling in the HN is of basic and clinical relevance. Interestingly, a transient expression of the neuronal isoform of nitric oxide (NO) synthase (NOS) occurs in HMNs at neonatal stage that disappears before postnatal day 21 (P21). NO, in turn, is a determining factor for synaptic refinement in several physiopathological conditions. Here, intracerebroventricular chronic administration (P7-P21) of the broad spectrum NOS inhibitor L-NAME (N(ω)-nitro-L-arginine methyl ester) differentially affected excitatory and inhibitory rearrangement during this neonatal interval in the rat. Whilst L-NAME led to a reduction in the number of excitatory structures, inhibitory synaptic puncta were increased at P21 in comparison to administration of the inactive stereoisomer D-NAME. Finally, L-NAME decreased levels of the phosphorylated form of myosin light chain in the nucleus, which is known to regulate the actomyosin contraction apparatus. These outcomes indicate that physiologically synthesized NO modulates excitatory/inhibitory balance during early postnatal development by acting as an anti-synaptotrophic and/or synaptotoxic factor for inhibitory synapses, and as a synaptotrophin for excitatory ones. The mechanism of action could rely on the modulation of the actomyosin contraction apparatus.

Circuit Reorganization Shapes the Developing Human Foveal Midget Connectome toward Single-Cone Resolution.

The human visual pathway is specialized for the perception of fine spatial detail. The neural circuitry that determines visual acuity begins in the retinal fovea, where the resolution afforded by a dense array of cone photoreceptors is preserved in the retinal output by a remarkable non-divergent circuit: cone → midget bipolar interneuron → midget ganglion cell (the "private line"). How the private line develops is unknown; it could involve early specification of extremely precise synaptic connections or, by contrast, emerge slowly in concordance with the gradual maturation of foveal architecture and visual sensitivity. To distinguish between these hypotheses, we reconstructed the midget circuitry in the fetal human fovea by serial electron microscopy. We discovered that the midget private line is sculpted by synaptic remodeling beginning early in fetal life, with midget bipolar cells contacting a single cone by mid-gestation and bipolar cell-ganglion cell connectivity undergoing a more protracted period of refinement.

Removing 4E-BP Enables Synapses to Refine without Postsynaptic Activity.

Throughout the developing nervous system, considerable synaptic re-organization takes place as postsynaptic neurons extend dendrites and incoming axons refine their synapses, strengthening some and eliminating others. It is well accepted that these processes rely on synaptic activity; however, the mechanisms that lead to this developmental reorganization are not fully understood. Here, we explore the regulation of cap-dependent translation, a mechanism known to play a role in synaptic growth and plasticity. Using sympathetic ganglia in α3 nicotinic acetylcholine receptor (nAChR)-knockout (KO) mice, we establish that electrophysiologically silent synapses between preganglionic axons and postsynaptic sympathetic neurons do not refine, and the growth of dendrites and the targeting of synapses on postsynaptic neurons are impaired. Remarkably, genetically removing 4E-BP, a suppressor of cap-dependent translation, from these α3 nAChR-KO mice largely restores these features. We conclude that synaptic connections can re-organize and refine without postsynaptic activity during post-natal development when 4E-BP-regulated cap-dependent translation is enhanced.

Visual Experience-Dependent Expression of Fn14 Is Required for Retinogeniculate Refinement.

Sensory experience influences the establishment of neural connectivity through molecular mechanisms that remain unclear. Here, we employ single-nucleus RNA sequencing to investigate the contribution of sensory-driven gene expression to synaptic refinement in the dorsal lateral geniculate nucleus of the thalamus, a region of the brain that processes visual information. We find that visual experience induces the expression of the cytokine receptor Fn14 in excitatory thalamocortical neurons. By combining electrophysiological and structural techniques, we show that Fn14 is dispensable for early phases of refinement mediated by spontaneous activity but that Fn14 is essential for refinement during a later, experience-dependent period of development. Refinement deficits in mice lacking Fn14 are associated with functionally weaker and structurally smaller retinogeniculate inputs, indicating that Fn14 mediates both functional and anatomical rearrangements in response to sensory experience. These findings identify Fn14 as a molecular link between sensory-driven gene expression and vision-sensitive refinement in the brain.

Glutamatergic Retinal Waves.

Spontaneous activity patterns propagate through many parts of the developing nervous system and shape the wiring of emerging circuits. Prior to vision, waves of activity originating in the retina propagate through the lateral geniculate nucleus (LGN) of the thalamus to primary visual cortex (V1). Retinal waves have been shown to instruct the wiring of ganglion cell axons in LGN and of thalamocortical axons in V1 via correlation-based plasticity rules. Across species, retinal waves mature in three stereotypic stages (I-III), in which distinct circuit mechanisms give rise to unique activity patterns that serve specific functions in visual system refinement. Here, I review insights into the patterns, mechanisms, and functions of stage III retinal waves, which rely on glutamatergic signaling. As glutamatergic waves spread across the retina, neighboring ganglion cells with opposite light responses (ON vs. OFF) are activated sequentially. Recent studies identified lateral excitatory networks in the inner retina that generate and propagate glutamatergic waves, and vertical inhibitory networks that desynchronize the activity of ON and OFF cells in the wavefront. Stage III wave activity patterns may help segregate axons of ON and OFF ganglion cells in the LGN, and could contribute to the emergence of orientation selectivity in V1.

Investigation of Developmental Changes of Convergence Ratios of MNTB-LSO Synapses in Circling Mice / 대한해부학회지

The developmental changes of convergence ratios of medial nucleus of trapezoid body (MNTB) axons to single lateral superior olive (LSO) neuron were investigated using voltage clamp technique in homologous (cir/cir) circling mice, animal model for the congenital deafness with autosomal recessive inheritance. As the developmental reduction of convergence ratio reported in rats indicates the presence of synaptic refinement, we aimed to find out whether the similar reduction of convergence ratio also occurs in circling mice. Heterologous (+/cir) mice were used as control and mice younger than postnatal (P) day 4 or older than P9 were used. The convergence ratios of MNTB axons to single LSO neuron were 29.16+/-2.7 (n=12, P9) in homologous (cir/cir) mice, while they were 37.89+/-3.8 (n=9, P9) in heterozygous (+/cir) mice. The significant changes were observed only in heterozygous (+/cir) mice, which indicated that synaptic refinement of MNTBLSO synapses occurs in heterozygous (+/cir) mice, not in homozygous (cir/cir) mice. Considering homologous (cir/cir) mice being animal model for the congenital deafness, our data might contribute to the understanding of developmental changes of brain stem auditory circuits of congenitally deaf patients.

Tetanus-induced LTD of Developing MNTB-LSO Synapses in Rat is Dependent on Postsynaptic Ca2+

Because synaptic refinement of medial nucleus of trapezoid body (MNTB) - lateral superior olive (LSO) synapses is most active during the first postnatal week and the long term depression (LTD) has been suggested as one of its mechanisms, LTD of MNTB-LSO synapses was investigated in neonatal rat brain stem slices with the whole cell voltage clamp technique. In Mg2+ free condition, tetanus (10 stimuli at 10 Hz for 2 min) in the current clamp mode induced a robust LTD of isolated D, L-APV-sensitive postsynaptic currents (PSCs) for more than 30 min (n=6, 2.4+/-0.4% of the control), while isolated CNQX-sensitive PSCs were not suppressed (n=6, 95.3+/-1.6%). Tetanus also elicited similar LTD in the isolated GABAergic/glycinergic PSCs (n=5, 3.6+/-0.5%) and mixed PSCs (GABAergic/glycinergic/glutamatergic) (n=4, 2.2+/-0.7%). However, such a strong LTD was not observed in the mixed PSCs when 10 mM EGTA was added in the internal solution (n=10), indicating that postsynaptic Ca2+ rise is needed for the strong LTD. This robust LTD might contribute to the active synaptic refinement occurring during the first postnatal week.