A tailored approach to the management of post-haemorrhagic hydrocephalus.

PURPOSE: Neuro-endoscopic lavage (NEL) is an increasingly popular intervention for intraventricular haemorrhage (IVH) and post-haemorrhagic hydrocephalus (PHH), with considerable variation in technique dependent on clinician and clinical circumstances. Whilst efforts to standardise the technique are ongoing, this work describes a tertiary centre experience utilising NEL, highlighting potential caveats to standardisation. METHODS: A retrospective review of electronic case notes for patients undergoing temporising surgical intervention for IVH between 2012 and 2021 at our centre was performed. Data collected included (i) gestational age, (ii) aetiology of hydrocephalus, (iii) age at time of intervention, (iv) intervention performed, (v) need for permanent CSF diversion, (vi) 'surgical burden', i.e. number of procedures following primary intervention, and (vii) wound failure and infection rate. Data was handled in Microsoft Excel and statistical analysis SPSS v27.0 RESULTS: 49 neonates (n = 25 males) were included. Overall mean gestational age was 27 weeks and at intervention 35 + 3 weeks. IVH was the predominant cause of hydrocephalus (93.8%) and primary surgical interventions included insertion of a ventriculosubgaleal shunt (VSGS) in n = 41 (83.6%) patients, NEL in n = 6 (12.2%) patients and insertion of an EVD in n = 2 (4.1%). N = 9 (18.4%) patients underwent NEL at some point during the time interval reviewed; n = 4 (8.2%) received NEL monotherapy and n = 5 (10.2%) also received a VSGS. Rate of conversion to definitive CSF diversion between NEL (n = 8, 88.9%) and VSGS cohorts (n = 37, 92.5%) was not significantly different (p = 0.57), nor between NEL alone (n = 3, 75%) and NEL + VSGS (n = 5, 100%) (p = 0.44). None of the patients that underwent NEL monotherapy had any wound issues or CNS infection as a result of the initial intervention, compared to n = 3 (60%) of those that underwent NEL and implantation of VSGS (p = 0.1). CONCLUSION: Both NEL and VSGS are effective in temporising hydrocephalus in neonates, occasionally offering a definitive solution in and of themselves. The benefit of dual therapy however remains to be seen, with the addition of VSGS potentially increasing the risk of wound failure in an already vulnerable cohort.

Hydrocephalus in prematurity: does valve choice make a difference?

PURPOSE: Extremely premature neonates diagnosed with post-haemorrhagic hydrocephalus (PHH) are recognised to have particularly poor outcomes. This study assessed the impact of a number of variables on outcomes in this cohort, in particular the choice of shunt valve mechanism. METHODS: Electronic case notes were retrospectively reviewed of all premature neonates admitted to our centre for management of hydrocephalus between 2012 and 2021. Data included (i) gestational age, (ii) birth weight, (iii) hydrocephalus aetiology, (iv) surgical intervention, (v) shunt system, (vi) 'surgical burden' and (vii) wound failure and infection rate. Data was handled in Microsoft Excel and statistical analysis performed in SPSS v27.0 RESULTS: N = 53 premature hydrocephalic patients were identified (n = 28 (52.8%) female). Median gestational age at birth was 27 weeks (range: 23-36 + 6 weeks), with n = 35 extremely preterm patients and median birth weight of 1.9 kg (range: 0.8-3.6 kg). Total n = 99 programmable valves were implanted (n = 28 (28.3%) de novo, n = 71 (71.2%) revisions); n = 28 (28.3%) underwent n ≥ 1 pressure alterations, after which n = 21 (75%) patients had symptoms improve. In n = 8 patients exchanged from fixed to programmable valves, a mean reduction of 1.9 revisions per patient after exchange was observed (95%CI: 0.36-3.39, p = 0.02). Mean overall shunt survival was 39.5 weeks (95%CI: 30.6-48.5); 33.2 weeks (95%CI: 25.2-41.1) in programmable valves and 35.1 weeks (95%CI: 19.5-50.6) in fixed pressure (p = 0.22) with 12-month survival rates of 25.7% and 24.7%, respectively (p = 0.22). Shorter de novo shunt survival was associated with higher operation count overall (Pearson's R: - 0.54, 95%CI: - 0.72 to - 0.29, p < 0.01). Wound failure, gestational age and birth weight were significantly associated with shorter de novo shunt survival in a Cox regression proportional hazards model; gestational age had the greatest impact on shunt survival (Exp(B): 0.71, 95%CI: 0.63-0.81, p < 0.01). CONCLUSION: Hydrocephalus is especially challenging in extreme prematurity, with a shorter de novo shunt survival associated with higher number of future revisions. Programmable valves provide flexibility with regard to pressure setting, with the potential for fewer shunt revisions in this complex cohort.

Selective dorsal rhizotomy in non-ambulant children with cerebral palsy: a multi-center prospective study.

PURPOSE: Assess the effects of selective dorsal rhizotomy (SDR) on motor function and quality of life in children with a Gross Motor Function Classification System (GMFCS) level of IV or V (non-ambulatory). METHODS: This is a prospective, observational study in three tertiary neurosurgery units in England, UK, performing SDR on children aged 3-18 with spastic diplegic cerebral palsy, and a GMFCS level of IV or V, between 2012 and 2019. The primary outcome measure was the change in the 66-item Gross Motor Function Measure (GMFM-66) from baseline to 24 months after SDR, using a linear mixed effects model. Secondary outcomes included spasticity, bladder function, quality of life, and pain scores. RESULTS: Between 2012 and 2019, 144 children who satisfied these inclusion criteria underwent SDR. The mean age was 8.2 years. Fifty-two percent were female. Mean GMFM-66 score was available in 77 patients (53.5%) and in 39 patients (27.1%) at 24 months after SDR. The mean increase between baseline and 24 months post-SDR was 2.4 units (95% CI 1.7-3.1, p < 0.001, annual change 1.2 units). Of the 67 patients with a GMFM-66 measurement available, a documented increase in gross motor function was seen in 77.6% (n = 52). Of 101 patients with spasticity data available, mean Ashworth scale decreased after surgery (2.74 to 0.30). Of patients' pain scores, 60.7% (n = 34) improved, and 96.4% (n = 56) of patients' pain scores remained the same or improved. Bladder function improved in 30.9% of patients. CONCLUSIONS: SDR improved gross motor function and reduced pain in most patients at 24 months after surgery, although the improvement is less pronounced than in children with GMFCS levels II and III. SDR should be considered in non-ambulant patients.

Intracranial Empyemas in the COVID-19 Era: A New Phenomenon? A Paediatric Case Series and Review of the Literature.

INTRODUCTION: We present the largest series of paediatric intracranial empyemas occurring after COVID-19 infection to date, and discuss the potential implications of the pandemic on this neurosurgical pathology. METHODS: Patients admitted to our centre between January 2016 and December 2021 with a confirmed radiological diagnosis of intracranial empyema were retrospectively reviewed, excluding non-otorhinological source cases. Patients were grouped according to onset before or after onset of the COVID-19 pandemic and COVID-19 status. A literature review of all post-COVID-19 intracranial empyemas was performed. SPSS v27 was used for statistical analysis. RESULTS: Sixteen patients were diagnosed with intracranial empyema: n = 5 prior to 2020 and n = 11 after, resulting in an average annual incidence of 0.3% prior to onset of the pandemic and 1.2% thereafter. Of those diagnosed since the pandemic, 4 (25%) were confirmed to have COVID-19 on recent PCR test. Time from COVID-19 infection until empyema diagnosis ranged from 15 days to 8 weeks. Mean age for post-COVID-19 cases was 8.5 years (range: 7-10 years) compared to 11 years in non-COVID cases (range: 3-14 years). Streptococcus intermedius was grown in all cases of post-COVID-19 empyema, and 3 of 4 (75%) post-COVID-19 cases developed cerebral sinus thromboses, compared to 3 of 12 (25%) non-COVID-19 cases. All cases were discharged home with no residual deficit. CONCLUSION: Our post-COVID-19 intracranial empyema series demonstrates a greater proportion of cerebral sinus thromboses than non-COVID-19 cases, potentially reflecting the thrombogenic effects of COVID-19. Incidence of intracranial empyema at our centre has increased since the start of the pandemic, causes of which require further investigation and multicentre collaboration.

Efficacy and safety of the Miethke programmable differential pressure valve (proGAV®2.0): a single-centre retrospective analysis.

PURPOSE: Achieving decompression without CSF over-drainage remains a challenge in hydrocephalus. Differential pressure valves are a popular treatment modality, with evidence suggesting that incorporation of gravitational units helps minimise over-drainage. This study seeks to describe the utility of the proGAV®2.0 programmable valve in a paediatric population. METHODS: Clinical records and imaging of all patients fitted with proGAV®2.0 valves and Miethke fixed-pressure valves between 2014 and 2019 at our tertiary centre were analysed. Patient demographics, indication for shunt and valve insertion/revision and time to shunt/valve revision were collected. Ventricular linear metrics (fronto-occipital horn ratio (FOHR) and fronto-occipital horn width ratio (FOHWR)) were collected pre- and post-valve insertion. Microsoft Excel and SPSS v24 were used for data collection and statistical analysis. RESULTS: Eighty-eight proGAV®2.0 valves were inserted in a population of 77 patients (n = 45 males (58%), mean age 5.1 years (IQR: 0.4-11.0 years)). A total of 102 Miethke fixed-pressure valves were inserted over the same time period. Median follow-up was 17.5 months (1.0-47.3). One (1.1%) proGAV®2.0 was revised due to over-drainage, compared to 2 (1.9%) fixed-pressure valves (p > 0.05). ProGAV®2.0 insertion resulted in a significant decrease in the mean number of revisions per patient per year (1.77 vs 0.25; p = 0.01). Overall shunt system survival with the proGAV®2.0 was 80.4% at 12 months, and mean time to revision was 37.1 months, compared to 31.0 months (95%CI: 25.7-36.3) and 58.3% in fixed-pressure valves (p < 0.01). Significant decreases were seen following proGAV®2.0 insertion in both FOHR and FOHWR, by 0.014 (95%CI: 0.006-0.023, p = 0.002) and 0.037 (95%CI: 0.005-0.069, p = 0.024) respectively. CONCLUSION: The proGAV®2.0 provides effective decompression of hydrocephalic patients, significantly reduces the number of valve revisions per patient and had a significantly greater mean time to revision than fixed-pressure valves.

Infant hydrocephalus: what valve first?

PURPOSE: To review the use of different valve types in infants with hydrocephalus, in doing so, determining whether an optimal valve choice exists for this patient cohort. METHODS: We conducted (1) a literature review for all studies describing valve types used (programmable vs. non-programmable, valve size, pressure) in infants (≤ 2 years) with hydrocephalus, (2) a review of data from the pivotal BASICS trial for infant patients and (3) a separate, institutional cohort study from Alder Hey Children's Hospital NHS Foundation Trust. The primary outcome was any revision not due to infection. RESULTS: The search identified 19 studies that were included in the review. Most did not identify a superior valve choice between programmable and non-programmable, small compared to ultra-small, and differential pressure compared to flow-regulating valves. Five studies investigated a single-valve type without a comparator group. The BASICS data identified 391 infants, with no statistically significant difference between gravitational and programmable subgroups. The institutional data from our tertiary referral centre did not reveal any significant difference in failure rate between valve subtypes. CONCLUSION: Our review highlights the challenges of valve selection in infant hydrocephalus, reiterating that the concept of an optimal valve choice in this group remains a controversial one. While the infant-hydrocephalic population is at high risk of valve failure, heterogeneity and a lack of direct comparison between valves in the literature limit our ability to draw meaningful conclusions. Data that does exist suggests at present that there is no difference in non-infective failure rate are increasing in number, with the British valve subtypes in infant hydrocephalus, supported by both the randomised trial and institutional data in this study.

Elfn1-Induced Constitutive Activation of mGluR7 Determines Frequency-Dependent Recruitment of Somatostatin Interneurons.

Excitatory synapses onto somatostatin (SOM) interneurons show robust short-term facilitation. This hallmark feature of SOM interneurons arises from a low initial release probability that regulates the recruitment of interneurons in response to trains of action potentials. Previous work has shown that Elfn1 (extracellular leucine rich repeat and fibronectin Type III domain containing 1) is necessary to generate facilitating synapses onto SOM neurons by recruitment of two separate presynaptic components: mGluR7 (metabotropic glutamate receptor 7) and GluK2-KARs (kainate receptors containing glutamate receptor, ionotropic, kainate 2). Here, we identify how a transsynaptic interaction between Elfn1 and mGluR7 constitutively reduces initial release probability onto mouse cortical SOM neurons. Elfn1 produces glutamate-independent activation of mGluR7 via presynaptic clustering, resulting in a divergence from the canonical "autoreceptor" role of Type III mGluRs, and substantially altering synaptic pharmacology. This structurally induced determination of initial release probability is present at both layer 2/3 and layer 5 synapses. In layer 2/3 SOM neurons, synaptic facilitation in response to spike trains is also dependent on presynaptic GluK2-KARs. In contrast, layer 5 SOM neurons do not exhibit presynaptic GluK2-KAR activity at baseline and show reduced facilitation. GluK2-KAR engagement at synapses onto layer 5 SOM neurons can be induced by calmodulin activation, suggesting that synaptic function can be dynamically regulated. Thus, synaptic facilitation onto SOM interneurons is mediated both by constitutive mGluR7 recruitment by Elfn1 and regulated GluK2-KAR recruitment, which determines the extent of interneuron recruitment in different cortical layers.SIGNIFICANCE STATEMENT This study identifies a novel mechanism for generating constitutive GPCR activity through a transsynaptic Elfn1/mGluR7 structural interaction. The resulting tonic suppression of synaptic release probability deviates from canonical autoreceptor function. Constitutive suppression delays the activation of somatostatin interneurons in circuits, necessitating high-frequency activity for somatostatin interneuron recruitment. Furthermore, variations in the synaptic proteome generate layer-specific differences in facilitation at pyr → SOM synapses. The presence of GluK2 kainate receptors in L2/3 enhances synaptic transmission during prolonged activity. Thus, layer-specific synaptic properties onto somatostatin interneurons are mediated by both constitutive mGluR7 recruitment and regulated GluK2 kainate receptor recruitment, revealing a mechanism that generates diversity in physiological responses of interneurons.

Synaptic activity suppresses expression of neurogenic differentiation factor 2 in an NMDA receptor-dependent manner.

Neurogenic differentiation factor 2 (NeuroD2) is a highly expressed transcription factor in the developing central nervous system. In newborn neurons, NeuroD2-mediated gene expression promotes differentiation, maturation, and survival. In addition to these early, cell-intrinsic developmental processes, NeuroD2 in postmitotic neurons also regulates synapse growth and ion channel expression to control excitability. While NeuroD2 transactivation can be induced in an activity-dependent manner, little is known about how expression of NeuroD2 itself is regulated. Using genome-wide, mRNA-based microarray analysis, we found that NeuroD2 is actually one of hundreds of genes whose mRNA levels are suppressed by synaptic activity, in a manner dependent upon N-methyl d-aspartate receptor (NMDAR) activation. We confirmed this observation both in vitro and in vivo and provide evidence that this happens at the level of transcription and not mRNA stability. Our experiments further indicate that suppression of NeuroD2 message by NMDARs likely involves both CaMKII and MAPK but not voltage-gated calcium channels, in contrast to its mechanism of transactivation. We predict from these data that NMDARs may transduce information about the level of synaptic activity a developing neuron receives, to down-regulate NeuroD2 and allow proper maturation of cortical circuits by suppressing expression of neurite and synaptic growth promoting gene products.

The transcription factor NeuroD2 coordinates synaptic innervation and cell intrinsic properties to control excitability of cortical pyramidal neurons.

KEY POINTS: Synaptic excitation and inhibition must be properly balanced in individual neurons and neuronal networks to allow proper brain function. Disrupting this balance may lead to autism spectral disorders and epilepsy. We show the basic helix-loop-helix transcription factor NeuroD2 promotes inhibitory synaptic drive but also decreases cell-intrinsic neuronal excitability of cortical pyramidal neurons both in vitro and in vivo. We identify two genes potentially downstream of NeuroD2-mediated transcription that regulate these parameters: gastrin-releasing peptide and the small conductance, calcium-activated potassium channel, SK2. Our results reveal an important function for NeuroD2 in balancing synaptic neurotransmission and intrinsic excitability. Our results offer insight into how synaptic innervation and intrinsic excitability are coordinated during cortical development. ABSTRACT: Synaptic excitation and inhibition must be properly balanced in individual neurons and neuronal networks for proper brain function. Disruption of this balance during development may lead to autism spectral disorders and epilepsy. Synaptic excitation is counterbalanced by synaptic inhibition but also by attenuation of cell-intrinsic neuronal excitability. To maintain proper excitation levels during development, neurons must sense activity over time and regulate the expression of genes that control these parameters. While this is a critical process, little is known about the transcription factors involved in coordinating gene expression to control excitatory/inhibitory synaptic balance. We show here that the basic helix-loop-helix transcription factor NeuroD2 promotes inhibitory synaptic drive but also decreases cell-intrinsic neuronal excitability of cortical pyramidal neurons both in vitro and in vivo as shown by ex vivo analysis of a NeuroD2 knockout mouse. Using microarray analysis and comparing wild-type and NeuroD2 knockout cortical networks, we identified two potential gene targets of NeuroD2 that contribute to these processes: gastrin-releasing peptide (GRP) and the small conductance, calcium-activated potassium channel, SK2. We found that the GRP receptor antagonist RC-3059 and the SK2 specific blocker apamin partially reversed the effects of increased NeuroD2 expression on inhibitory synaptic drive and action potential repolarization, respectively. Our results reveal an important function for NeuroD2 in balancing synaptic neurotransmission and intrinsic excitability and offer insight into how these processes are coordinated during cortical development.

The Rac1 inhibitor NSC23766 suppresses CREB signaling by targeting NMDA receptor function.

NMDA receptor signaling plays a complex role in CREB activation and CREB-mediated gene transcription, depending on the subcellular location of NMDA receptors, as well as how strongly they are activated. However, it is not known whether Rac1, the prototype of Rac GTPase, plays a role in neuronal CREB activation induced by NMDA receptor signaling. Here, we report that NSC23766, a widely used specific Rac1 inhibitor, inhibits basal CREB phosphorylation at S133 (pCREB) and antagonizes changes in pCREB levels induced by NMDA bath application in rat cortical neurons. Unexpectedly, we found that NSC23766 affects the levels of neuronal pCREB in a Rac1-independent manner. Instead, our results indicate that NSC23766 can directly regulate NMDA receptors as indicated by their strong effects on both exogenous and synaptically evoked NMDA receptor-mediated currents in mouse and rat neurons, respectively. Our findings strongly suggest that Rac1 does not affect pCREB signaling in cortical neurons and reveal that NSC23766 could be a novel NMDA receptor antagonist.

Selective and brain-penetrant HCN1 inhibitors reveal links between synaptic integration, cortical function, and working memory.

Hyperpolarization-activated and cyclic-nucleotide-gated 1 (HCN1) ion channels are proposed to be critical for cognitive function through regulation of synaptic integration. However, resolving the precise role of HCN1 in neurophysiology and exploiting its therapeutic potential has been hampered by minimally selective antagonists with poor potency and limited in vivo efficiency. Using automated electrophysiology in a small-molecule library screen and chemical optimization, we identified a primary carboxamide series of potent and selective HCN1 inhibitors with a distinct mode of action. In cognition-relevant brain circuits, selective inhibition of native HCN1 produced on-target effects, including enhanced excitatory postsynaptic potential summation, while administration of a selective HCN1 inhibitor to rats recovered decrement working memory. Unlike prior non-selective HCN antagonists, selective HCN1 inhibition did not alter cardiac physiology in human atrial cardiomyocytes or in rats. Collectively, selective HCN1 inhibitors described herein unmask HCN1 as a potential target for the treatment of cognitive dysfunction in brain disorders.

Neurodevelopmental role for VGLUT2 in pyramidal neuron plasticity, dendritic refinement, and in spatial learning.

The level and integrity of glutamate transmission during critical periods of postnatal development plays an important role in the refinement of pyramidal neuron dendritic arbor, synaptic plasticity, and cognition. Presently, it is not clear how excitatory transmission via the two predominant isoforms of the vesicular glutamate transporter (VGLUT1 and VGLUT2) participate in this process. To assess a neurodevelopmental role for VGLUT2 in pyramidal neuron maturation, we generated recombinant VGLUT2 knock-out mice and inactivated VGLUT2 throughout development using Emx1-Cre(+/+) knock-in mice. We show that VGLUT2 deficiency in corticolimbic circuits results in reduced evoked glutamate transmission, release probability, and LTD at hippocampal CA3-CA1 synapses during a formative developmental period (postnatal days 11-14). In adults, we find a marked reduction in the amount of dendritic arbor across the span of the dendritic tree of CA1 pyramidal neurons and reduced long-term potentiation and levels of synaptic markers spinophilin and VGLUT1. Loss of dendritic arbor is accompanied by corresponding reductions in the number of dendritic spines, suggesting widespread alterations in synaptic connectivity. Conditional VGLUT2 knock-out mice exhibit increased open-field exploratory activity yet impaired spatial learning and memory, endophenotypes similar to those of NMDA receptor knock-down mice. Remarkably, the impairment in learning can be partially restored by selectively increasing NMDA receptor-mediated glutamate transmission in adult mice by prolonged treatment with d-serine and a d-amino acid oxidase inhibitor. Our data indicate that VGLUT2 expression is pivotal to the proper development of mature pyramidal neuronal architecture and plasticity, and that such glutamatergic deficiency leads to cognitive malfunction as observed in several neurodevelopmental psychiatric disorders.

Within-Mice Comparison of Microdialysis and Fiber Photometry-Recorded Dopamine Biosensor during Amphetamine Response.

A fundamental concept in neuroscience is the transmission of information between neurons via neurotransmitters, -modulators, and -peptides. For the past decades, the gold standard for measuring neurochemicals in awake animals has been microdialysis (MD). The emergence of genetically encoded fluorescence-based biosensors, as well as in vivo optical techniques such as fiber photometry (FP), has introduced technologically distinct means of measuring neurotransmission. To directly compare MD and FP, we performed concurrent within-animal recordings of extracellular dopamine (DA) in the dorsal striatum (DS) before and after administration of amphetamine in awake, freely behaving mice expressing the dopamine sensor dLight1.3b. We show that despite temporal differences, MD- and FP-based readouts of DA correlate well within mice. Down-sampling of FP data showed temporal correlation to MD data, with less variance observed using FP. We also present evidence that DA fluctuations periodically reach low levels, and naïve animals have rapid, predrug DA dynamics measured with FP that correlate to the subsequent pharmacodynamics of amphetamine as measured with MD and FP.

Improvement of sensory deficits in fragile X mice by increasing cortical interneuron activity after the critical period.

Changes in the function of inhibitory interneurons (INs) during cortical development could contribute to the pathophysiology of neurodevelopmental disorders. Using all-optical in vivo approaches, we find that parvalbumin (PV) INs and their immature precursors are hypoactive and transiently decoupled from excitatory neurons in postnatal mouse somatosensory cortex (S1) of Fmr1 KO mice, a model of fragile X syndrome (FXS). This leads to a loss of parvalbumin INs (PV-INs) in both mice and humans with FXS. Increasing the activity of future PV-INs in neonatal Fmr1 KO mice restores PV-IN density and ameliorates transcriptional dysregulation in S1, but not circuit dysfunction. Critically, administering an allosteric modulator of Kv3.1 channels after the S1 critical period does rescue circuit dynamics and tactile defensiveness. Symptoms in FXS and related disorders could be mitigated by targeting PV-INs.

Striatum-projecting prefrontal cortex neurons support working memory maintenance.

Neurons in the medial prefrontal cortex (mPFC) are functionally linked to working memory (WM) but how distinct projection pathways contribute to WM remains unclear. Based on optical recordings, optogenetic perturbations, and pharmacological interventions in male mice, we report here that dorsomedial striatum (dmStr)-projecting mPFC neurons are essential for WM maintenance, but not encoding or retrieval, in a T-maze spatial memory task. Fiber photometry of GCaMP6m-labeled mPFC→dmStr neurons revealed strongest activity during the maintenance period, and optogenetic inhibition of these neurons impaired performance only when applied during this period. Conversely, enhancing mPFC→dmStr pathway activity-via pharmacological suppression of HCN1 or by optogenetic activation during the maintenance period-alleviated WM impairment induced by NMDA receptor blockade. Moreover, cellular-resolution miniscope imaging revealed that >50% of mPFC→dmStr neurons are active during WM maintenance and that this subpopulation is distinct from neurons active during encoding and retrieval. In all task periods, neuronal sequences were evident. Striatum-projecting mPFC neurons thus critically contribute to spatial WM maintenance.

Activity-dependent ubiquitination of GluA1 mediates a distinct AMPA receptor endocytosis and sorting pathway.

The accurate trafficking of AMPA receptors (AMPARs) to and from the synapse is a critical component of learning and memory in the brain, whereas dysfunction of AMPAR trafficking is hypothesized to be an underlying mechanism of Alzheimer's disease. Previous work has shown that ubiquitination of integral membrane proteins is a common posttranslational modification used to mediate endocytosis and endocytic sorting of surface proteins in eukaryotic cells. Here we report that mammalian AMPARs become ubiquitinated in response to their activation. Using a mutant of GluA1 that is unable to be ubiquitinated at lysines on its C-terminus, we demonstrate that ubiquitination is required for internalization of surface AMPARs and their trafficking to the lysosome in response to the AMPAR agonist AMPA but not for internalization of AMPARs in response to the NMDA receptor agonist NMDA. Through overexpression or RNA interference-mediated knockdown, we identify that a specific E3 ligase, Nedd4-1 (neural-precursor cell-expressed developmentally downregulated gene 4-1), is necessary for this process. Finally, we show that ubiquitination of GluA1 by Nedd4-1 becomes more prevalent as neurons mature. Together, these data show that ubiquitination of GluA1-containing AMPARs by Nedd4-1 mediates their endocytosis and trafficking to the lysosome. Furthermore, these results provide insight into how hippocampal neurons regulate AMPAR trafficking and degradation with high specificity in response to differing neuronal signaling cues and suggest that changes to this pathway may occur as neurons mature.

Identification of Natural Antisense Transcripts in Mouse Brain and Their Association With Autism Spectrum Disorder Risk Genes.

Genome-wide sequencing technologies have greatly contributed to our understanding of the genetic basis of neurodevelopmental disorders such as autism spectrum disorder (ASD). Interestingly, a number of ASD-related genes express natural antisense transcripts (NATs). In some cases, these NATs have been shown to play a regulatory role in sense strand gene expression and thus contribute to brain function. However, a detailed study examining the transcriptional relationship between ASD-related genes and their NAT partners is lacking. We performed strand-specific, deep RNA sequencing to profile expression of sense and antisense reads with a focus on 100 ASD-related genes in medial prefrontal cortex (mPFC) and striatum across mouse post-natal development (P7, P14, and P56). Using de novo transcriptome assembly, we generated a comprehensive long non-coding RNA (lncRNA) transcriptome. We conducted BLAST analyses to compare the resultant transcripts with the human genome and identified transcripts with high sequence similarity and coverage. We assembled 32861 de novo antisense transcripts mapped to 12182 genes, of which 1018 are annotated by Ensembl as lncRNA. We validated the expression of a subset of selected ASD-related transcripts by PCR, including Syngap1 and Cntnap2. Our analyses revealed that more than 70% (72/100) of the examined ASD-related genes have one or more expressed antisense transcripts, suggesting more ASD-related genes than previously thought could be subject to NAT-mediated regulation in mice. We found that expression levels of antisense contigs were mostly positively correlated with their cognate coding sense strand RNA transcripts across developmental age. A small fraction of the examined transcripts showed brain region specific enrichment, indicating possible circuit-specific roles. Our BLAST analyses identified 110 of 271 ASD-related de novo transcripts with >90% identity to the human genome at >90% coverage. These findings, which include an assembled de novo antisense transcriptome, contribute to the understanding of NAT regulation of ASD-related genes in mice and can guide NAT-mediated gene regulation strategies in preclinical investigations toward the ultimate goal of developing novel therapeutic targets for ASD.

Regulation of thalamocortical patterning and synaptic maturation by NeuroD2.

During cortical development, both activity-dependent and genetically determined mechanisms are required to establish proper neuronal connectivity. While activity-dependent transcription may link the two processes, specific transcription factors that mediate such a process have not been identified. We identified the basic helix-loop-helix (bHLH) transcription factor Neurogenic Differentiation 2 (NeuroD2) in a screen for calcium-regulated transcription factors and report that it is required for the proper development of thalamocortical connections. In neuroD2 null mice, thalamocortical axon terminals fail to segregate in the somatosensory cortex, and the postsynaptic barrel organization is disrupted. Additionally, synaptic transmission is defective at thalamocortical synapses in neuroD2 null mice. Total excitatory synaptic currents are reduced in layer IV in the knockouts, and the relative contribution of AMPA and NMDA receptor-mediated currents to evoked responses is decreased. These observations indicate that NeuroD2 plays a critical role in regulating synaptic maturation and the patterning of thalamocortical connections.

Krüppel-like factor 9 is necessary for late-phase neuronal maturation in the developing dentate gyrus and during adult hippocampal neurogenesis.

The dentate gyrus (DG) is modified throughout life by integration of new adult-born neurons. Similarities in neuronal maturation during DG development and adult hippocampal neurogenesis suggest that genetically encoded intrinsic regulatory mechanisms underlying these temporally distinct processes are conserved and reused. Here, we identify a novel transcriptional regulator of dentate granule neuron maturation, Krüppel-like factor 9 (Klf-9). We show that Klf-9 expression is induced by neuronal activity and as dentate granule neurons functionally integrate in the developing and adult DG. During development, dentate granule neurons lacking Klf-9 show delayed maturation as reflected by altered expression of early-phase markers, dendritic spine formation, and electrophysiological properties. Adult Klf-9-null mice exhibit normal stem cell proliferation and cell fate specification in the DG but show impaired differentiation of adult-born neurons and decreased neurogenesis-dependent synaptic plasticity. Behavioral analysis of Klf-9-null mice revealed a subtle increase in anxiety-like behavior and an impairment in contextual fear discrimination learning. Thus, Klf-9 is necessary for late-phase maturation of dentate granule neurons both in DG development and during adult hippocampal neurogenesis. Klf-9-dependent neuronal maturation may therefore represent a candidate regulatory mechanism underlying these temporally distinct processes.

Regulation of AMPA receptor recruitment at developing synapses.

Fast synaptic current at most excitatory synapses in the brain is carried by AMPA and NMDA subtypes of ionotropic glutamate receptors (AMPARs and NMDARs). During development there is an increase in the ratio of AMPAR- to NMDAR-mediated current at these synapses. Recent studies indicate that NMDAR signaling early in development negatively regulates AMPAR expression and function at multiple levels, which likely accounts for the small AMPAR current at developing synapses. This contrasts with the positive role of NMDAR signaling in recruiting AMPARs to synapses during long-term potentiation in the adult brain. Thus, NMDARs exert differential effects on the recruitment of AMPA receptors to synapses depending on the developmental state of the neural circuit.