Target-derived cues promote local differentiation of axons into nerve terminals at sites of synaptic contact. Using clustering of synaptic vesicles in cultured neurons as an assay, we purified putative target-derived presynaptic organizing molecules from mouse brain and identified FGF22 as a major active species. FGF7 and FGF10, the closest relatives of FGF22, share this activity; other FGFs have distinct effects. FGF22 is expressed by cerebellar granule cells during the period when they receive synapses. Its receptor, FGFR2, is expressed by pontine and vestibular neurons when their axons (mossy fibers) are making synapses on granule cells. Neutralization of FGF7, -10, and -22 inhibits presynaptic differentiation of mossy fibers at sites of contact with granule cells in vivo. Inactivation of FGFR2 has similar effects. These results indicate that FGF22 and its relatives are presynaptic organizing molecules in the mammalian brain and suggest new functions for this family of signaling molecules.

Various growth factors regulate synapse development and neurogenesis, and are essential for brain function. Changes in growth factor signaling are implicated in many neuropsychiatric disorders such as depression, autism and epilepsy. We have previously identified that fibroblast growth factor 22 (FGF22) is critical for excitatory synapse formation in several brain regions including the hippocampus. Mice with a genetic deletion of FGF22 (FGF22 null mice) have fewer excitatory synapses in the hippocampus. We have further found that as a behavioral consequence, FGF22 null mice show a depression-like behavior phenotype such as increased passive stress-coping behavior and anhedonia, without any changes in motor, anxiety, or social cognitive tests, suggesting that FGF22 is specifically important for affective behavior. Thus, addressing the precise roles of FGF22 in the brain will help understand how synaptogenic growth factors regulate affective behavior. In the hippocampus, FGF22 is expressed mainly by CA3 pyramidal neurons, but also by a subset of dentate granule cells. We find that in addition to synapse formation, FGF22 also contributes to neurogenesis in the dentate gyrus: FGF22 null mice show decreased dentate neurogenesis. To understand the cell type-specific roles of FGF22, we generated and analyzed CA3-specific FGF22 knockout mice (FGF22-CA3KO). We show that FGF22-CA3KO mice have reduced excitatory synapses on CA3 pyramidal neurons, but do not show changes in dentate neurogenesis. Behaviorally, FGF22-CA3KO mice still show increased immobility and decreased latency to float in the forced swim test and decreased preference for sucrose in the sucrose preference test, which are suggestive of a depressive-like phenotype similar to FGF22 null mice. These results demonstrate that: (i) CA3-derived FGF22 serves as a target-derived excitatory synaptic organizer in CA3 in vivo; (ii) FGF22 plays important roles in dentate neurogenesis, but CA3-derived FGF22 is not involved in neurogenesis; and (iii) a depression-like phenotype can result from FGF22 inactivation selectively in CA3 pyramidal neurons. Our results link the role of CA3-derived FGF22 in synapse development, and not in neurogenesis, to affective behavior.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities.

Huntington's disease (HD) is a devastating monogenic neurodegenerative disease characterized by early, selective pathology in the basal ganglia despite the ubiquitous expression of mutant huntingtin. The molecular mechanisms underlying this region-specific neuronal degeneration and how these relate to the development of early cognitive phenotypes are poorly understood. Here we show that there is selective loss of synaptic connections between the cortex and striatum in postmortem tissue from patients with HD that is associated with the increased activation and localization of complement proteins, innate immune molecules, to these synaptic elements. We also found that levels of these secreted innate immune molecules are elevated in the cerebrospinal fluid of premanifest HD patients and correlate with established measures of disease burden.In preclinical genetic models of HD, we show that complement proteins mediate the selective elimination of corticostriatal synapses at an early stage in disease pathogenesis, marking them for removal by microglia, the brain's resident macrophage population. This process requires mutant huntingtin to be expressed in both cortical and striatal neurons. Inhibition of this complement-dependent elimination mechanism through administration of a therapeutically relevant C1q function-blocking antibody or genetic ablation of a complement receptor on microglia prevented synapse loss, increased excitatory input to the striatum and rescued the early development of visual discrimination learning and cognitive flexibility deficits in these models. Together, our findings implicate microglia and the complement cascade in the selective, early degeneration of corticostriatal synapses and the development of cognitive deficits in presymptomatic HD; they also provide new preclinical data to support complement as a therapeutic target for early intervention.

Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression-related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression-related behaviour. The NR2A subunit of the NMDA receptor is tyrosine-phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr-1325-Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock-in mice show antidepressant-like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock-in mice, DARPP-32 phosphorylation at Thr 34, which is important for the regulation of depression-related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src-induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor-mediated downstream signalling to modulate depression-related behaviour.

In the developing hippocampus, fibroblast growth factor (FGF) 22 promotes the formation of excitatory presynaptic terminals. Remarkably, FGF22 knockout (KO) mice show resistance to generalized seizures in adults as assessed by chemical kindling, a model that is widely used to study epileptogenesis (Terauchi et al., 2010). Repeated injections of low dose pentylenetetrazol (PTZ) induce generalized seizures ("kindled") in wild type (WT) mice. With additional PTZ injections, FGF22KO mice do show moderate seizures, but they do not kindle. Thus, analyses of how FGF22 impacts seizure susceptibility will contribute to the better understanding of the molecular and cellular mechanisms of epileptogenesis. To decipher the roles of FGF22 in the seizure phenotype, we examine four pathophysiological changes in the hippocampus associated with epileptogenesis: enhancement of dentate neurogenesis, hilar ectopic dentate granule cells (DGCs), increase in hilar cell death, and formation of mossy fiber sprouting (MFS). Dentate neurogenesis is enhanced, hilar ectopic DGCs appeared, and hilar cell death is increased in PTZ-kindled WT mice relative to PBS-injected WT mice. Even in WT mice with fewer PTZ injections, which showed only mild seizures (so were not kindled), neurogenesis, hilar ectopic DGCs, and hilar cell death are increased, suggesting that mild seizures are enough to induce these changes in WT mice. In contrast, PTZ-injected FGF22KO mice do not show these changes despite having moderate seizures: neurogenesis is rather suppressed, hilar ectopic DGCs do not appear, and hilar cell death is unchanged in PTZ-injected FGF22KO mice relative to PBS-injected FGF22KO mice. These results indicate that FGF22 plays important roles in controlling neurogenesis, ectopic migration of DGCs, and hilar cell death after seizures, which may contribute to the generalized seizure-resistant phenotype of FGF22KO mice and suggests a possibility that inhibition of FGF22 may alleviate epileptogenesis.

Phosphorylation of neural proteins in response to a diverse array of external stimuli is one of the main mechanisms underlying dynamic changes in neural circuitry. The NR2B subunit of the NMDA receptor is tyrosine-phosphorylated in the brain, with Tyr-1472 its major phosphorylation site. Here, we generate mice with a knockin mutation of the Tyr-1472 site to phenylalanine (Y1472F) and show that Tyr-1472 phosphorylation is essential for fear learning and amygdaloid synaptic plasticity. The knockin mice show impaired fear-related learning and reduced amygdaloid long-term potentiation. NMDA receptor-mediated CaMKII signaling is impaired in YF/YF mice. Electron microscopic analyses reveal that the Y1472F mutant of the NR2B subunit shows improper localization at synapses in the amygdala. We thus identify Tyr-1472 phosphorylation as a key mediator of fear learning and amygdaloid synaptic plasticity.

Functional synapse formation requires tight coordination between pre- and post-synaptic termini. Previous studies have shown that postsynaptic expression of heparan sulfate proteoglycan syndecan-2 (SDC2) induces dendritic spinogenesis. Those SDC2-induced dendritic spines are frequently associated with presynaptic termini. However, how postsynaptic SDC2 accelerates maturation of corresponding presynaptic termini is unknown. Because fibroblast growth factor 22 (FGF22), a heparan sulfate binding growth factor, has been shown to act as a presynaptic organizer released from the postsynaptic site, it seems possible that postsynaptic SDC2 presents FGF22 to the presynaptic FGF receptor to promote presynaptic differentiation. Here, we show that postsynaptic SDC2 uses its ectodomain to interact with and facilitate dendritic filopodial targeting of FGF22, triggering presynaptic maturation. Since SDC2 also enhances filopodial targeting of NMDAR via interaction with the CASK-mLIN7-MINT1 adaptor complex, presynaptic maturation promoted by FGF22 further feeds back to activate NMDAR at corresponding postsynaptic sites through increased neurotransmitter release and, consequently, promotes the dendritic filopodia-spines (F-S) transition. Meanwhile, via regulation of the KIF17 motor, CaMKII (activated by the NMDAR pathway) may further facilitate FGF22 targeting to dendritic filopodia that receive presynaptic stimulation. Our study suggests a positive feedback that promotes the coordination of postsynaptic and presynaptic differentiation.

The SNARE-mediated vesicular transport pathway plays major roles in synaptic remodeling associated with formation of long-term memories, but the mechanisms that regulate this pathway during memory acquisition are not fully understood. Here we identify miRNAs that are up-regulated in the rodent hippocampus upon contextual fear-conditioning and identify the vesicular transport and synaptogenesis pathways as the major targets of the fear-induced miRNAs. We demonstrate that miR-153, a member of this group, inhibits the expression of key components of the vesicular transport machinery, and down-regulates Glutamate receptor A1 trafficking and neurotransmitter release. MiR-153 expression is specifically induced during LTP induction in hippocampal slices and its knockdown in the hippocampus of adult mice results in enhanced fear memory. Our results suggest that miR-153, and possibly other fear-induced miRNAs, act as components of a negative feedback loop that blocks neuronal hyperactivity at least partly through the inhibition of the vesicular transport pathway.

Cellular compartments that cannot be biochemically isolated are challenging to characterize. Here we demonstrate the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, we mapped the proteomes of two of the most common excitatory and inhibitory synaptic clefts in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed us to identify Mdga2 as a potential specificity factor influencing Neuroligin-2's recruitment of presynaptic neurotransmitters at inhibitory synapses.

Communication between pre- and postsynaptic cells promotes the initial organization of synaptic specializations, but subsequent synaptic stabilization requires transcriptional regulation. Here we show that fibroblast growth factor 22 (FGF22), a target-derived presynaptic organizer in the mouse hippocampus, induces the expression of insulin-like growth factor 2 (IGF2) for the stabilization of presynaptic terminals. FGF22 is released from CA3 pyramidal neurons and organizes the differentiation of excitatory nerve terminals formed onto them. Local application of FGF22 on the axons of dentate granule cells (DGCs), which are presynaptic to CA3 pyramidal neurons, induces IGF2 in the DGCs. IGF2, in turn, localizes to DGC presynaptic terminals and stabilizes them in an activity-dependent manner. IGF2 application rescues presynaptic defects of Fgf22(-/-) cultures. IGF2 is dispensable for the initial presynaptic differentiation, but is required for the following presynaptic stabilization both in vitro and in vivo. These results reveal a novel feedback signal that is critical for the activity-dependent stabilization of presynaptic terminals in the mammalian hippocampus.

Anxiety disorders are a highly prevalent and disabling class of psychiatric disorders. There is growing evidence implicating the glutamate system in the pathophysiology and treatment of anxiety disorders, though the molecular mechanism by which the glutamate system regulates anxiety-like behavior remains unclear.

During cortical synaptic development, thalamic axons must establish synaptic connections despite the presence of the more abundant intracortical projections. How thalamocortical synapses are formed and maintained in this competitive environment is unknown. Here, we show that astrocyte-secreted protein hevin is required for normal thalamocortical synaptic connectivity in the mouse cortex. Absence of hevin results in a profound, long-lasting reduction in thalamocortical synapses accompanied by a transient increase in intracortical excitatory connections. Three-dimensional reconstructions of cortical neurons from serial section electron microscopy (ssEM) revealed that, during early postnatal development, dendritic spines often receive multiple excitatory inputs. Immuno-EM and confocal analyses revealed that majority of the spines with multiple excitatory contacts (SMECs) receive simultaneous thalamic and cortical inputs. Proportion of SMECs diminishes as the brain develops, but SMECs remain abundant in Hevin-null mice. These findings reveal that, through secretion of hevin, astrocytes control an important developmental synaptic refinement process at dendritic spines.

The remodeling of axonal circuits after injury requires the formation of new synaptic contacts to enable functional recovery. Which molecular signals initiate such axonal and synaptic reorganisation in the adult central nervous system is currently unknown. Here, we identify FGF22 as a key regulator of circuit remodeling in the injured spinal cord. We show that FGF22 is produced by spinal relay neurons, while its main receptors FGFR1 and FGFR2 are expressed by cortical projection neurons. FGF22 deficiency or the targeted deletion of FGFR1 and FGFR2 in the hindlimb motor cortex limits the formation of new synapses between corticospinal collaterals and relay neurons, delays their molecular maturation, and impedes functional recovery in a mouse model of spinal cord injury. These results establish FGF22 as a synaptogenic mediator in the adult nervous system and a crucial regulator of synapse formation and maturation during post-injury remodeling in the spinal cord.

The time of day strongly influences adaptive behaviors like long-term memory, but the correlating synaptic and molecular mechanisms remain unclear. The circadian clock comprises a canonical transcription-translation feedback loop (TTFL) strictly dependent on the BMAL1 transcription factor. We report that BMAL1 rhythmically localizes to hippocampal synapses in a manner dependent on its phosphorylation at Ser42 [pBMAL1(S42)]. pBMAL1(S42) regulates the autophosphorylation of synaptic CaMKIIα and circadian rhythms of CaMKIIα-dependent molecular interactions and LTP but not global rest/activity behavior. Therefore, our results suggest a model in which repurposing of the clock protein BMAL1 to synapses locally gates the circadian timing of plasticity.

One of the most common types of epilepsy in adults is temporal lobe epilepsy. Temporal lobe epilepsy is often resistant to pharmacological treatment, requiring urgent understanding of its molecular and cellular mechanisms. It is generally accepted that an imbalance between excitatory and inhibitory inputs is related to epileptogenesis. We have recently identified that fibroblast growth factor (FGF) 7 is critical for inhibitory synapse formation in the developing hippocampus. Remarkably, FGF7 knockout mice are prone to epileptic seizures induced by chemical kindling (Terauchi et al., 2010). Here we show that FGF7 knockout mice exhibit epileptogenesis-related changes in the hippocampus even without kindling induction. FGF7 knockout mice show mossy fiber sprouting and enhanced dentate neurogenesis by 2 months of age, without apparent spontaneous seizures. These results suggest that FGF7-deficiency impairs inhibitory synapse formation, which results in mossy fiber sprouting and enhanced neurogenesis during development, making FGF7 knockout mice vulnerable to epilepsy.

Formation of appropriate synaptic connections is critical for proper functioning of the brain. After initial synaptic differentiation, active synapses are stabilized by neural activity-dependent signals to establish functional synaptic connections. However, the molecular mechanisms underlying activity-dependent synapse maturation remain to be elucidated. Here we show that activity-dependent ectodomain shedding of signal regulatory protein-α (SIRPα) mediates presynaptic maturation. Two target-derived molecules, fibroblast growth factor 22 and SIRPα, sequentially organize the glutamatergic presynaptic terminals during the initial synaptic differentiation and synapse maturation stages, respectively, in the mouse hippocampus. SIRPα drives presynaptic maturation in an activity-dependent fashion. Remarkably, neural activity cleaves the extracellular domain of SIRPα, and the shed ectodomain in turn promotes the maturation of the presynaptic terminal. This process involves calcium/calmodulin-dependent protein kinase, matrix metalloproteinases and the presynaptic receptor CD47. Finally, SIRPα-dependent synapse maturation has an impact on synaptic function and plasticity. Thus, ectodomain shedding of SIRPα is an activity-dependent trans-synaptic mechanism for the maturation of functional synapses.

At least three forms of signaling between pre- and postsynaptic partners are necessary during synapse formation. First, "targeting" signals instruct presynaptic axons to recognize and adhere to the correct portion of a postsynaptic target cell. Second, trans-synaptic "organizing" signals induce differentiation in their synaptic partner so that each side of the synapse is specialized for synaptic transmission. Finally, in many regions of the nervous system an excess of synapses are initially formed, therefore "refinement" signals must either stabilize or destabilize the synapse to reinforce or eliminate connections, respectively. Because of both their importance in processing visual information and their accessibility, retinogeniculate synapses have served as a model for studying synaptic development. Molecular signals that drive retinogeniculate "targeting" and "refinement" have been identified, however, little is known about what "organizing" cues are necessary for the differentiation of retinal axons into presynaptic terminals. To identify such "organizing" cues, we used microarray analysis to assess whether any target-derived "synaptic organizers" were enriched in the mouse dorsal lateral geniculate nucleus (dLGN) during retinogeniculate synapse formation. One candidate "organizing" molecule enriched in perinatal dLGN was FGF22, a secreted cue that induces the formation of excitatory nerve terminals in muscle, hippocampus, and cerebellum. In FGF22 knockout mice, the development of retinal terminals in dLGN was impaired. Thus, FGF22 is an important "organizing" cue for the timely development of retinogeniculate synapses.

To establish functional neural circuits in the brain, synaptic connections are refined by neural activity during development, where active connections are maintained and inactive ones are eliminated. However, the molecular signals that regulate synapse refinement remain to be elucidated. When we inactivate a subset of neurons in the mouse cingulate cortex, their callosal connections are eliminated through activity-dependent competition. Using this system, we identify JAK2 tyrosine kinase as a key regulator of inactive synapse elimination. We show that JAK2 is necessary and sufficient for elimination of inactive connections; JAK2 is activated at inactive synapses in response to signals from other active synapses; STAT1, a substrate of JAK2, mediates inactive synapse elimination; JAK2 signaling is critical for physiological refinement of synapses during normal development; and JAK2 regulates synapse refinement in multiple brain regions. We propose that JAK2 is an activity-dependent switch that serves as a determinant of inactive synapse elimination.

The visual system is the best system to study activity-dependent sensory circuit development. The connections from the retina to the dorsal lateral geniculate nucleus, the retinogeniculate connections, undergo extensive remodeling during early postnatal life. Thus, techniques that allow the expression of transgenes early in the developing retina are essential to study visual system development. Here, we describe a protocol to express genes-of-interest in the developing mouse retina via in utero intraocular adeno-associated virus injections. For complete details on the use and execution of this protocol, please refer to Yasuda et al. (2021).