Spine Pruning Research Articles

Altered hippocampal dendritic spine maturation after hypoxia-induced seizures in neonatal rats

Cognitive comorbidities often follow early-life seizures (ELS), especially in the setting of autism and other neurodevelopmental syndromes. However, there is an incomplete understanding of whether neuronal and synaptic development are concomitantly dysregulated. We have previously shown that hypoxia-induced seizures (HS) in postnatal day (P)10 rats increase acute and later-life hippocampal glutamatergic neurotransmission and spontaneous recurrent seizures, and impair cognition and behavior. As dendritic spines critically regulate synaptic function, we hypothesized that ELS can induce developmentally specific changes in dendritic spine maturation. At intervals during one month following HS in P10 rats, we assessed dendritic spine development on pyramidal neurons in the stratum radiatum of hippocampal area CA1. Compared to control rats in which spine density significantly decreased from P10 to early adulthood (P38), post-seizure rats failed to show a developmental decrease in spine density, and spines from P38 post-seizure rats appeared more immature-shaped (long, thin). In addition, compared to P38 control rats, post-seizure P38 rats expressed significantly more synaptic PSD-95, a marker of mature synapses. These changes were preceded by a transient increase in hippocampal expression of cofilin phosphorylated at Ser3, representing a decrease in cofilin activity. These results suggest that early-life seizures may impair normal dendritic spine maturation and pruning in CA1 during development, resulting in an excess of less efficient synapses, via activity-dependent modification of actin-regulating proteins such as cofilin. Given that multiple neurodevelopmental disorders show similar failures in developmental spine pruning, the current findings may represent a deficit in structural plasticity that could be a component of a mechanism leading to later-life cognitive consequences associated with early-life seizures.

Role of mTOR-Regulated Autophagy in Synaptic Plasticity Related Proteins Downregulation and the Reference Memory Deficits Induced by Anesthesia/Surgery in Aged Mice.

Postoperative cognitive dysfunction increases mortality and morbidity in perioperative patients and has become a major concern for patients and caregivers. Previous studies demonstrated that synaptic plasticity is closely related to cognitive function, anesthesia and surgery inhibit synaptic function. In central nervous system, autophagy is vital to synaptic plasticity, homeostasis of synapticproteins, synapse elimination, spine pruning, proper axon guidance, and when dysregulated, is associated with behavioral and memory functions disorders. The mammalian target of rapamycin (mTOR) negatively regulates the process of autophagy. This study aimed to explore whether rapamycin can ameliorate anesthesia/surgery-induced cognitive deficits by inhibiting mTOR, activating autophagy and rising synaptic plasticity-related proteins in the hippocampus. Aged C57BL/6J mice were used to establish POCD models with exploratory laparotomy under isoflurane anesthesia. The Morris Water Maze (MWM) was used to measure reference memory after anesthesia and surgery. The levels of mTOR phosphorylation (p-mTOR), Beclin-1 and LC3-II were examined on postoperative days 1, 3 and 7 by western blotting. The levels of synaptophysin (SYN) and postsynaptic density protein 95 (PSD-95) in the hippocampus were also examined by western blotting. Here we showed that anesthesia/surgery impaired reference memory and induced the activation of mTOR, decreased the expression of autophagy-related proteins such as Beclin-1 and LC3-II. A corresponding decline in the expression of neuronal/synaptic, plasticity-related proteins such as SYN and PSD-95 was also observed. Pretreating mice with rapamycin inhibited the activation of mTOR and restored autophagy function, also increased the expression of SYN and PSD-95. Furthermore, anesthesia/surgery-induced learning and memory deficits were also reversed by rapamycin pretreatment. In conclusion, anesthesia/surgery induced mTOR hyperactivation and autophagy impairments, and then reduced the levels of SYN and PSD-95 in the hippocampus. An mTOR inhibitor, rapamycin, ameliorated anesthesia/surgery-related cognitive impairments by inhibiting the mTOR activity, inducing activation of autophagy, enhancing SYN and PSD-95 expression.

Semaphorin3F Drives Dendritic Spine Pruning Through Rho-GTPase Signaling.

Dendritic spines of cortical pyramidal neurons are initially overproduced then remodeled substantially in the adolescent brain to achieve appropriate excitatory balance in mature circuits. Here we investigated the molecular mechanism of developmental spine pruning by Semaphorin 3F (Sema3F) and its holoreceptor complex, which consists of immunoglobulin-class adhesion molecule NrCAM, Neuropilin-2 (Npn2), and PlexinA3 (PlexA3) signaling subunits. Structure-function studies of the NrCAM-Npn2 interface showed that NrCAM stabilizes binding between Npn2 and PlexA3 necessary for Sema3F-induced spine pruning. Using a mouse neuronal culture system, we identified a dual signaling pathway for Sema3F-induced pruning, which involves activation of Tiam1-Rac1-PAK1-3 -LIMK1/2-Cofilin1 and RhoA-ROCK1/2-Myosin II in dendritic spines. Inhibitors of actin remodeling impaired spine collapse in the cortical neurons. Elucidation of these pathways expands our understanding of critical events that sculpt neuronal networks and may provide insight into how interruptions to these pathways could lead to spine dysgenesis in diseases such as autism, bipolar disorder, and schizophrenia.

Astrocyte glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex.

The uptake of glutamate by astrocytes actively shapes synaptic transmission, however its role in the development and plasticity of neuronal circuits remains poorly understood. The astrocytic glutamate transporter, GLT1 is the predominant source of glutamate clearance in the adult mouse cortex. Here, we examined the structural and functional development of the visual cortex in GLT1 heterozygous (HET) mice using two-photon microscopy, immunohistochemistry and slice electrophysiology. We find that though eye-specific thalamic axonal segregation is intact, binocular refinement in the primary visual cortex is disrupted. Eye-specific responses to visual stimuli in GLT1 HET mice show altered binocular matching, with abnormally high responses to ipsilateral compared to contralateral eye stimulation and a greater mismatch between preferred orientation selectivity of ipsilateral and contralateral eye responses. Furthermore, we observe an increase in dendritic spine density in the basal dendrites of layer 2/3 excitatory neurons suggesting aberrant spine pruning. Monocular deprivation induces atypical ocular dominance plasticity in GLT1 HET mice, with an unusual depression of ipsilateral open eye responses; however, this change in ipsilateral responses correlates well with an upregulation of GLT1 protein following monocular deprivation. These results demonstrate that a key function of astrocytic GLT1 function during development is the experience-dependent refinement of ipsilateral eye inputs relative to contralateral eye inputs in visual cortex.

Harnessing the paradoxical phenotypes of APOE ɛ2 and APOE ɛ4 to identify genetic modifiers in Alzheimer's disease.

The strongest genetic risk factor for idiopathic late‐onset Alzheimer's disease (LOAD) is apolipoprotein E (APOE) ɛ4, while the APOE ɛ2 allele is protective. However, there are paradoxical APOE ɛ4 carriers who remain disease‐free and APOE ɛ2 carriers with LOAD. We compared exomes of healthy APOE ɛ4 carriers and APOE ɛ2 Alzheimer's disease (AD) patients, prioritizing coding variants based on their predicted functional impact, and identified 216 genes with differential mutational load between these two populations. These candidate genes were significantly dysregulated in LOAD brains, and many modulated tau‐ or β42‐induced neurodegeneration in Drosophila. Variants in these genes were associated with AD risk, even in APOE ɛ3 homozygotes, showing robust predictive power for risk stratification. Network analyses revealed involvement of candidate genes in brain cell type‐specific pathways including synaptic biology, dendritic spine pruning and inflammation. These potential modifiers of LOAD may constitute novel biomarkers, provide potential therapeutic intervention avenues, and support applying this approach as larger whole exome sequencing cohorts become available.

Caspase inhibition rescues F1Fo ATP synthase dysfunction-mediated dendritic spine elimination

Dendritic spine injury underlies synaptic failure in many neurological disorders. Mounting evidence suggests a mitochondrial pathway of local nonapoptotic caspase signaling in mediating spine pruning. However, it remains unclear whether this caspase signaling plays a key role in spine loss when severe mitochondrial functional defects are present. The answer to this question is critical especially for some pathological states, in which mitochondrial deficits are prominent and difficult to fix. F1Fo ATP synthase is a pivotal mitochondrial enzyme and the dysfunction of this enzyme involves in diseases with spinopathy. Here, we inhibited F1Fo ATP synthase function in primary cultured hippocampal neurons by using non-lethal oligomycin A treatment. Oligomycin A induced mitochondrial defects including collapsed mitochondrial membrane potential, dissipated ATP production, and elevated reactive oxygen species (ROS) production. In addition, dendritic mitochondria underwent increased fragmentation and reduced positioning to dendritic spines along with increased caspase 3 cleavage in dendritic shaft and spines in response to oligomycin A. Concurring with these dendritic mitochondrial changes, oligomycin A-insulted neurons displayed spine loss and altered spine architecture. Such oligomycin A-mediated changes in dendritic spines were substantially prevented by the inhibition of caspase activation by using a pan-caspase inhibitor, quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh). Of note, the administration of Q-VD-OPh showed no protective effect on oligomycin A-induced mitochondrial dysfunction. Our findings suggest a pivotal role of caspase 3 signaling in mediating spine injury and the modulation of caspase 3 activation may benefit neurons from spine loss in diseases, at least, in those with F1Fo ATP synthase defects.

Role of mTOR-regulated autophagy in spine pruning defects and memory impairments induced by binge-like ethanol treatment in adolescent mice.

Adolescence is a brain maturation developmental period during which remodeling and changes in synaptic plasticity and neural connectivity take place in some brain regions. Different mechanism participates in adolescent brain maturation, including autophagy that plays a role in synaptic development and plasticity. Alcohol is a neurotoxic compound and its abuse in adolescence induces neuroinflammation, synaptic and myelin alterations, neural damage and behavioral impairments. Changes in synaptic plasticity and its regulation by mTOR have also been suggested to play a role in the behavioral dysfunction of binge ethanol drinking in adolescence. Therefore, by considering the critical role of mTOR in both autophagy and synaptic plasticity in the developing brain, the present study aims to evaluate whether binge ethanol treatment in adolescence would induce dysfunctions in synaptic plasticity and cognitive functions and if mTOR inhibition with rapamycin is capable of restoring both effects. Using C57BL/6 adolescent female and male mice (PND30) treated with ethanol (3 g/kg) on two consecutive days at 48‐hour intervals over 2 weeks, we show that binge ethanol treatment alters the density and morphology of dendritic spines, effects that are associated with learning and memory impairments and changes in the levels of both transcription factor CREB phosphorylation and miRNAs. Rapamycin administration (3 mg/kg) prior to ethanol administration restores ethanol‐induced changes in both plasticity and behavior dysfunctions in adolescent mice. These results support the critical role of mTOR/autophagy dysfunctions in the dendritic spines alterations and cognitive alterations induced by binge alcohol in adolescence.

The endogenous neuronal complement inhibitor SRPX2 protects against complement-mediated synapse elimination during development.

Complement-mediated synapse elimination has emerged as an important process in both brain development and neurological diseases, but whether neurons express complement inhibitors that protect synapses against complement-mediated synapse elimination remains unknown. Here, we show that the sushi domain protein SRPX2 is a neuronally expressed complement inhibitor that regulates complement-dependent synapse elimination. SRPX2 directly binds to C1q and blocks its activity, and SRPX2−/Y mice show increased C3 deposition and microglial synapse engulfment. They also show a transient decrease in synapse numbers and increase in retinogeniculate axon segregation in the lateral geniculate nucleus. In the somatosensory cortex, SRPX2−/Y mice show decreased thalamocortical synapse numbers and increased spine pruning. Dual C3−/−;SRPX2−/Y mice exhibit phenotypes associated with C3−/− mice rather than SRPX2−/Y mice, indicating that C3 is necessary for SRPX2’s effect on synapse elimination. Together, these results show that SRPX2 protects synapses against complement-mediated elimination in both the thalamus and the cortex.

Sex and Pubertal Status Influence Dendritic Spine Density on Frontal Corticostriatal Projection Neurons in Mice

In humans, nonhuman primates, and rodents, the frontal cortices exhibit grey matter thinning and dendritic spine pruning that extends into adolescence. This maturation is believed to support higher cognition but may also confer psychiatric vulnerability during adolescence. Currently, little is known about how specific cell types in the frontal cortex mature or whether puberty plays a role in the maturation of some cell types but not others. Here, we used mice to characterize the spatial topography and adolescent development of cross-corticostriatal (cSTR) neurons that project through the corpus collosum to the dorsomedial striatum. We found that apical spine density on cSTR neurons in the medial prefrontal cortex decreased significantly between late juvenile (P29) and young adult time points (P60), with females exhibiting higher spine density than males at both ages. Adult males castrated prior to puberty onset had higher spine density compared to sham controls. Adult females ovariectomized before puberty onset showed greater variance in spine density measures on cSTR cells compared to controls, but their mean spine density did not significantly differ from sham controls. Our findings reveal that these cSTR neurons, a subtype of the broader class of intratelencephalic-type neurons, exhibit significant sex differences and suggest that spine pruning on cSTR neurons is regulated by puberty in male mice.

Early Sensory Loss Alters the Dendritic Branching and Spine Density of Supragranular Pyramidal Neurons in Rodent Primary Sensory Cortices.

Multisensory integration in primary auditory (A1), visual (V1), and somatosensory cortex (S1) is substantially mediated by their direct interconnections and by thalamic inputs across the sensory modalities. We have previously shown in rodents (Mongolian gerbils) that during postnatal development, the anatomical and functional strengths of these crossmodal and also of sensory matched connections are determined by early auditory, somatosensory, and visual experience. Because supragranular layer III pyramidal neurons are major targets of corticocortical and thalamocortical connections, we investigated in this follow-up study how the loss of early sensory experience changes their dendritic morphology. Gerbils were sensory deprived early in development by either bilateral sciatic nerve transection at postnatal day (P) 5, ototoxic inner hair cell damage at P10, or eye enucleation at P10. Sholl and branch order analyses of Golgi-stained layer III pyramidal neurons at P28, which demarcates the end of the sensory critical period in this species, revealed that visual and somatosensory deprivation leads to a general increase of apical and basal dendritic branching in A1, V1, and S1. In contrast, dendritic branching, particularly of apical dendrites, decreased in all three areas following auditory deprivation. Generally, the number of spines, and consequently spine density, along the apical and basal dendrites decreased in both sensory deprived and non-deprived cortical areas. Therefore, we conclude that the loss of early sensory experience induces a refinement of corticocortical crossmodal and other cortical and thalamic connections by pruning of dendritic spines at the end of the critical period. Based on present and previous own results and on findings from the literature, we propose a scenario for multisensory development following early sensory loss.

Corticosterone-mediated microglia activation affects dendritic spine plasticity and motor learning functions in minimal hepatic encephalopathy

Minimal hepatic encephalopathy (MHE) is characterized as cognitive deficits including memory and learning dysfunctions after liver injuries or hepatic diseases. Our understandings of neurological mechanisms of MHE-associated cognitive syndromes, however, are far from complete. In the current study we generated a mouse MHE model by repetitive administrations of thioacetamide (TAA), which induced hyperammonemia plus elevated proinflammatory cytokines in both the general circulation and motor cortex. MHE mice presented prominent motor learning deficits, which were associated with excess dendritic spine pruning in the motor cortex under 2-photon in vivo microscopy. The pharmaceutical blockade of glucocorticoid receptor or suppression of its biosynthesis further rescued motor learning deficits and synaptic protein loss. Moreover, MHE mice presented microglial activation, which can be alleviated after glucocorticoid pathway inhibition. In sum, our data demonstrates corticosterone-induced microglial activation, synaptic over-pruning and motor learning impairments in MHE, providing new insights for MHE pathogenesis and potential targets of clinical interventions.

Caspase-2 promotes AMPA receptor internalization and cognitive flexibility via mTORC2-AKT-GSK3\u03b2 signaling

Caspase-2 is the most evolutionarily conserved member in the caspase family of proteases and is constitutively expressed in most cell types including neurons; however, its physiological function remains largely unknown. Here we report that caspase-2 plays a critical role in synaptic plasticity and cognitive flexibility. We found that caspase-2 deficiency led to deficits in dendritic spine pruning, internalization of AMPA receptors and long-term depression. Our results indicate that caspase-2 degrades Rictor, a key mTOR complex 2 (mTORC2) component, to inhibit Akt activation, which leads to enhancement of the GSK3β activity and thereby long-term depression. Furthermore, we found that mice lacking caspase-2 displayed elevated levels of anxiety, impairment in reversal water maze learning, and little memory loss over time. These results not only uncover a caspase-2–mTORC2–Akt–GSK3β signaling pathway, but also suggest that caspase-2 is important for memory erasing and normal behaviors by regulating synaptic number and transmission.

Close Homolog of L1 Regulates Dendritic Spine Density in the Mouse Cerebral Cortex Through Semaphorin 3B.

Dendritic spines in the developing mammalian neocortex are initially overproduced and then eliminated during adolescence to achieve appropriate levels of excitation in mature networks. We show here that the L1 family cell adhesion molecule Close Homolog of L1 (CHL1) and secreted repellent ligand Semaphorin 3B (Sema3B) function together to induce dendritic spine pruning in developing cortical pyramidal neurons. Loss of CHL1 in null mutant mice in both genders resulted in increased spine density and a greater proportion of immature spines on apical dendrites in the prefrontal and visual cortex. Electron microscopy showed that excitatory spine synapses with postsynaptic densities were increased in the CHL1-null cortex, and electrophysiological recording in prefrontal slices from mutant mice revealed deficiencies in excitatory synaptic transmission. Mechanistically, Sema3B protein induced elimination of spines on apical dendrites of cortical neurons cultured from wild-type but not CHL1-null embryos. Sema3B was secreted by the cortical neuron cultures, and its levels increased when cells were treated with the GABA antagonist gabazine. In vivo CHL1 was coexpressed with Sema3B in pyramidal neuron subpopulations and formed a complex with Sema3B receptor subunits Neuropilin-2 and PlexinA4. CHL1 and NrCAM, a closely related L1 adhesion molecule, localized primarily to distinct spines and promoted spine elimination to Sema3B or Sema3F, respectively. These results support a new concept in which selective spine elimination is achieved through different secreted semaphorins and L1 family adhesion molecules to sculpt functional neural circuits during postnatal maturation.SIGNIFICANCE STATEMENT Dendritic spines in the mammalian neocortex are initially overproduced and then pruned in adolescent life through unclear mechanisms to sculpt maturing cortical circuits. Here, we show that spine and excitatory synapse density of pyramidal neurons in the developing neocortex is regulated by the L1 adhesion molecule, Close Homolog of L1 (CHL1). CHL1 mediated spine pruning in response to the secreted repellent ligand Semaphorin 3B and associated with receptor subunits Neuropilin-2 and PlexinA4. CHL1 and related L1 adhesion molecule NrCAM localized to distinct spines, and promoted spine elimination to Semaphorin 3B and -3F, respectively. These results support a new concept in which selective elimination of individual spines and nascent synapses can be achieved through the action of distinct secreted semaphorins and L1 adhesion molecules.

Delayed Spine Pruning of Direct Pathway Spiny Projection Neurons in a Mouse Model of Parkinson's Disease.

In animal models of Parkinson’s disease (PD), principal striatal spiny projection neurons (SPNs) lose axospinous synapses. However, there has been a disagreement about whether this loss is restricted to a specific type of SPN or not, as some studies have reported pruning in both direct pathway SPNs and indirect pathway SPNs, while others have found this pruning to be restricted to indirect pathway SPNs. One possible explanation for the discrepancy is the period between the induction of the parkinsonian state and the assessment of spine loss. To test this hypothesis, transgenic mice were subjected to unilateral 6-hydroxydopamine (6-OHDA) lesions of nigrostriatal dopaminergic neurons and then direct pathway SPNs examined in ex vivo brain slices using two photon laser scanning microscopy either one or 2 months afterwards. These studies revealed that 1 month after the lesion, there was no loss of spines in direct pathway SPNs. However, 2 months after the lesion, spine loss was significant in direct pathway SPNs. In addition to reconciling the existing literature on the impact of the parkinsonian state on axospinous synapse elimination in SPNs, our results suggest that the delayed spine loss in direct pathway SPNs is not driven by homeostatic mechanisms [as posited for indirect pathway (iSPNs)], but rather by network pathophysiology.

Rho-kinase inhibition has antidepressant-like efficacy and expedites dendritic spine pruning in adolescent mice

Adolescence represents a critical period of neurodevelopment, defined by structural and synaptic pruning within the prefrontal cortex. While characteristic of typical development, this structural instability may open a window of vulnerability to developing neuropsychiatric disorders, including depression. Thus, therapeutic interventions that support or expedite neural remodeling in adolescence may be advantageous. Here, we inhibited the neuronally-expressed cytoskeletal regulatory factor Rho-kinase (ROCK), focusing primarily on the clinically-viable ROCK inhibitor fasudil. ROCK inhibition had rapid antidepressant-like effects in adolescent mice, and its efficacy was comparable to ketamine and fluoxetine. It also modified levels of the antidepressant-related signaling factors, tropomyosin/tyrosine receptor kinase B and Akt, as well as the postsynaptic marker PSD-95, in the ventromedial prefrontal cortex (vmPFC). Meanwhile, adolescent-typical dendritic spine pruning on excitatory pyramidal neurons in the vmPFC was expedited. Further, vmPFC-specific shRNA-mediated reduction of ROCK2, the dominant ROCK isoform in the brain, had antidepressant-like consequences. We cautiously suggest that ROCK inhibitors may have therapeutic potential for adolescent-onset depression.

Neurocan Inhibits Semaphorin 3F Induced Dendritic Spine Remodeling Through NrCAM in Cortical Neurons.

Neurocan is a chondroitin sulfate proteoglycan present in perineuronal nets, which are associated with closure of the critical period of synaptic plasticity. During postnatal development of the neocortex dendritic spines on pyramidal neurons are initially overproduced; later they are pruned to achieve an appropriate balance of excitatory to inhibitory synapses. Little is understood about how spine pruning is terminated upon maturation. NrCAM (Neuron-glial related cell adhesion molecule) was found to mediate spine pruning as a subunit of the receptor complex for the repellent ligand Semaphorin 3F (Sema3F). As shown here in the postnatal mouse frontal and visual neocortex, Neurocan was localized at both light and electron microscopic level to the cell surface of cortical pyramidal neurons and was adjacent to neuronal processes and dendritic spines. Sema3F-induced spine elimination was inhibited by Neurocan in cortical neuron cultures. Neurocan also blocked Sema3F-induced morphological retraction in COS-7 cells, which was mediated through NrCAM and other subunits of the Sema3F holoreceptor, Neuropilin-2, and PlexinA3. Cell binding and ELISA assays demonstrated an association of Neurocan with NrCAM. Glycosaminoglycan chain interactions of Neurocan were required for inhibition of Sema3F-induced spine elimination, but the C-terminal sushi domain was dispensable. These results describe a novel mechanism wherein Neurocan inhibits NrCAM/Sema3F-induced spine elimination.

Adolescent exposure to \u03949-tetrahydrocannabinol alters the transcriptional trajectory and dendritic architecture of prefrontal pyramidal neurons

Neuronal circuits within the prefrontal cortex (PFC) mediate higher cognitive functions and emotional regulation that are disrupted in psychiatric disorders. The PFC undergoes significant maturation during adolescence, a period when cannabis use in humans has been linked to subsequent vulnerability to psychiatric disorders such as addiction and schizophrenia. Here, we investigated in a rat model the effects of adolescent exposure to Δ9-tetrahydrocannabinol (THC), a psychoactive component of cannabis, on the morphological architecture and transcriptional profile of layer III pyramidal neurons—using cell type- and layer-specific high-resolution microscopy, laser capture microdissection and next-generation RNA-sequencing. The results confirmed known normal expansions in basal dendritic arborization and dendritic spine pruning during the transition from late adolescence to early adulthood that were accompanied by differential expression of gene networks associated with neurodevelopment in control animals. In contrast, THC exposure disrupted the normal developmental process by inducing premature pruning of dendritic spines and allostatic atrophy of dendritic arborization in early adulthood. Surprisingly, there was minimal overlap of the developmental transcriptomes between THC- and vehicle-exposed rats. THC altered functional gene networks related to cell morphogenesis, dendritic development, and cytoskeleton organization. Marked developmental network disturbances were evident for epigenetic regulators with enhanced co-expression of chromatin- and dendrite-related genes in THC-treated animals. Dysregulated PFC co-expression networks common to both the THC-treated animals and patients with schizophrenia were enriched for cytoskeletal and neurite development. Overall, adolescent THC exposure altered the morphological and transcriptional trajectory of PFC pyramidal neurons, which could enhance vulnerability to psychiatric disorders.

Vocal practice regulates singing activity-dependent genes underlying age-independent vocal learning in songbirds.

The development of highly complex vocal skill, like human language and bird songs, is underlain by learning. Vocal learning, even when occurring in adulthood, is thought to largely depend on a sensitive/critical period during postnatal development, and learned vocal patterns emerge gradually as the long-term consequence of vocal practice during this critical period. In this scenario, it is presumed that the effect of vocal practice is thus mainly limited by the intrinsic timing of age-dependent maturation factors that close the critical period and reduce neural plasticity. However, an alternative, as-yet untested hypothesis is that vocal practice itself, independently of age, regulates vocal learning plasticity. Here, we explicitly discriminate between the influences of age and vocal practice using a songbird model system. We prevented zebra finches from singing during the critical period of sensorimotor learning by reversible postural manipulation. This enabled to us to separate lifelong vocal experience from the effects of age. The singing-prevented birds produced juvenile-like immature song and retained sufficient ability to acquire a tutored song even at adulthood when allowed to sing freely. Genome-wide gene expression network analysis revealed that this adult vocal plasticity was accompanied by an intense induction of singing activity-dependent genes, similar to that observed in juvenile birds, rather than of age-dependent genes. The transcriptional changes of activity-dependent genes occurred in the vocal motor robust nucleus of the arcopallium (RA) projection neurons that play a critical role in the production of song phonology. These gene expression changes were accompanied by neuroanatomical changes: dendritic spine pruning in RA projection neurons. These results show that self-motivated practice itself changes the expression dynamics of activity-dependent genes associated with vocal learning plasticity and that this process is not tightly linked to age-dependent maturational factors.

Postnatal Ablation of Synaptic Retinoic Acid Signaling Impairs Cortical Information Processing and Sensory Discrimination in Mice.

Retinoic acid (RA) and its receptors (RARs) are well established essential transcriptional regulators during embryonic development. Recent findings in cultured neurons identified an independent and critical post-transcriptional role of RA and RARα in the homeostatic regulation of excitatory and inhibitory synaptic transmission in mature neurons. However, the functional relevance of synaptic RA signaling in vivo has not been established. Here, using somatosensory cortex as a model system and the RARα conditional knock-out mouse as a tool, we applied multiple genetic manipulations to delete RARα postnatally in specific populations of cortical neurons, and asked whether synaptic RA signaling observed in cultured neurons is involved in cortical information processing in vivo Indeed, conditional ablation of RARα in mice via a CaMKIIα-Cre or a layer 5-Cre driver line or via somatosensory cortex-specific viral expression of Cre-recombinase impaired whisker-dependent texture discrimination, suggesting a critical requirement of RARα expression in L5 pyramidal neurons of somatosensory cortex for normal tactile sensory processing. Transcranial two-photon imaging revealed a significant increase in dendritic spine elimination on apical dendrites of somatosensory cortical layer 5 pyramidal neurons in these mice. Interestingly, the enhancement of spine elimination is whisker experience-dependent as whisker trimming rescued the spine elimination phenotype. Additionally, experiencing an enriched environment improved texture discrimination in RARα-deficient mice and reduced excessive spine pruning. Thus, RA signaling is essential for normal experience-dependent cortical circuit remodeling and sensory processing.SIGNIFICANCE STATEMENT The importance of synaptic RA signaling has been demonstrated in in vitro studies. However, whether RA signaling mediated by RARα contributes to neural circuit functions in vivo remains largely unknown. In this study, using a RARα conditional knock-out mouse, we performed multiple regional/cell-type-specific manipulation of RARα expression in the postnatal brain, and show that RARα signaling contributes to normal whisker-dependent texture discrimination as well as regulating spine dynamics of apical dendrites from layer (L5) pyramidal neurons in S1. Deletion of RARα in excitatory neurons in the forebrain induces elevated spine elimination and impaired sensory discrimination. Our study provides novel insights into the role of RARα signaling in cortical processing and experience-dependent spine maturation.

Regulation and Roles of Autophagy at Synapses.

Genetic studies have demonstrated that conditional ablation of core autophagy genes in the neural lineage leads to progressive neurodegeneration, indicating that this catabolic pathway is indispensable for neuronal maintenance. However, accumulating evidence also indicates that autophagy is not merely a housekeeping process. Instead, autophagy may be dynamically regulated in different neuronal compartments and dictate the turnover of selected cargo in a time- and space-dependent manner and thus contribute to specialized neuronal functions. Here, we review the mechanisms regulating autophagy in neurons and discuss recent findings on the role of autophagy in synaptic morphology and function. Moreover, we discuss relevant questions that remain open and highlight forthcoming issues in the field of neuronal autophagy.