Stable ubiquitin conjugation for biological interrogation of ubiquitinated tau repeat domain.

Protein semisynthesis approaches are key for gaining insights into the effects of post-translational modifications (PTMs) on the structure and function of modified proteins. Among PTMs, ubiquitination involves the conjugation of a small protein modifier to a substrate amino acid residue and is unique in controlling a variety of cellular processes. Interest has grown in understanding the role of ubiquitination in neurodegenerative conditions, including tauopathies. The latter are characterized by the accumulation of the intrinsically disordered protein tau in the form of neurofibrillary tangles in the brains of patients. The presence of ubiquitinated tau in the pathological aggregates suggests that ubiquitination might play a role in the formation of abnormal protein deposits. In this study, we developed a new strategy, based on dehydroalanine chemistry, to install wild type ubiquitin on a tau repeat domain construct with site-specificity. We optimized a three-step reaction which yielded a good amount of highly pure tau repeat domain ubiquitinated in position 353. The structural features of the conjugate were examined by circular dichroism and NMR spectroscopy. The ubiquitinated tau was challenged in a number of assays: fibrils formation under aggregating conditions in vitro, chemical stability upon exposure to a variety of biological media including cell extracts, and internalization into astrocytes. The results demonstrated the wide applicability of the new semisynthetic strategy for the investigation of ubiquitinated substrates in vitro or in cell, and in particular for studying if ubiquitination has a role in the molecular mechanisms that underlie the aberrant transition of tau into pathological aggregates.

Glucocorticoid receptors modulate dendritic spine plasticity and microglia activity in an animal model of Alzheimer's disease.

Chronic exposure to high circulating levels of glucocorticoids (GCs) may be a key risk factor for Alzheimer's Disease (AD) development and progression. In addition, hyper-activation of glucocorticoid receptors (GRs) induces brain alterations comparable to those produced by AD. In transgenic mouse models of AD, GCs increase the production of the most important and typical hallmarks of this dementia such as: Aß40, Aß42 and tau protein (both the total tau and its hyperphosphorylated isoforms). Moreover, GCs in brain are pivotal regulators of dendritic spine turnover and microglia activity, two phenomena strongly altered in AD. Although it is well-established that GCs primes the neuroinflammatory response in the brain to some stimuli, it is unknown whether or how GRs modulates dendritic spine plasticity and microglia activity in AD. In this study, we evaluated, using combined Golgi Cox and immunofluorescence techniques, the role of GR agonists and antagonists on dendritic spine plasticity and microglia activation in hippocampus of 3xTg-AD mice. We found that dexamethasone, an agonist of GRs, was able to significantly reduce dendritic spine density and induced proliferation and activation of microglia in CA1 region of hippocampus of 3xTg-AD mice at 6 and 10 months of age. On the contrary, the treatment with mifepristone, an antagonist of GRs, strongly enhanced dendritic spine density, decreased microglia density and improved the behavioural performance of 3xTg-AD mice. Additionally, primary microglial cells in vitro were directly activated by dexamethasone. Together, these data demonstrate that stress exacerbates AD and promotes a rapid progression of the pathology acting on both neurons and glial cells, supporting an important pro-inflammatory role of GC within CNS in AD. Consequently, these results further strengthen the need to test clinical interventions that correct GCs dysregulation as promising therapeutic strategy to delay the onset and slow down the progression of AD.

Autism-associated 16p11.2 microdeletion impairs prefrontal functional connectivity in mouse and human.

Human genetic studies are rapidly identifying variants that increase risk for neurodevelopmental disorders. However, it remains unclear how specific mutations impact brain function and contribute to neuropsychiatric risk. Chromosome 16p11.2 deletion is one of the most common copy number variations in autism and related neurodevelopmental disorders. Using resting state functional MRI data from the Simons Variation in Individuals Project (VIP) database, we show that 16p11.2 deletion carriers exhibit impaired prefrontal connectivity, resulting in weaker long-range functional coupling with temporal-parietal regions. These functional changes are associated with socio-cognitive impairments. We also document that a mouse with the same genetic deficiency exhibits similarly diminished prefrontal connectivity, together with thalamo-prefrontal miswiring and reduced long-range functional synchronization. These results reveal a mechanistic link between specific genetic risk for neurodevelopmental disorders and long-range functional coupling, and suggest that deletion in 16p11.2 may lead to impaired socio-cognitive function via dysregulation of prefrontal connectivity.

Activity-dependent vs. neurotrophic modulation of acetylcholine receptor expression: Evidence from rat soleus and extensor digitorum longus muscles confirms the exclusive role of activity.

Evoked electrical muscle activity suppresses the transcription of mRNAs for acetylcholine receptors in extrajunctional myonuclei. Muscle denervation or disuse releases such inhibition and extrajunctional receptors appear. However, in soleus muscles paralysed with nerve-applied tetrodotoxin, a restricted perijunctional region has been described where myonuclei remain inhibited, a finding attributed to nerve-derived trophic factor(s). Here, we reinvestigate extrajunctional acetylcholine receptor expression in soleus and extensor digitorum longus muscles up to 90 days after denervation or up to 20 days of disuse, to clarify the role of trophic factors, if any. The perijunctional region of soleus muscles strongly expressed acetylcholine receptors during the first 2-3 weeks of denervation. After 2-3 months, this expression had disappeared. No perijunctional expression was seen after paralysis by tetrodotoxin or botulinum toxin A. In contrast, the extensor digitorum longus never displayed suppressed perijunctional acetylcholine receptor expression after any treatment, suggesting that it is an intrinsic property of soleus muscles. Soleus denervation only transiently removed the suppression, and its presence in long-term denervated soleus muscles contradicts any contribution from nerve-derived trophic factor(s). In conclusion, our results confirm that evoked electrical activity is the physiological factor controlling the expression of acetylcholine receptors in the entire extrajunctional membrane of skeletal muscles.

Electrophysiological evaluation of cystic fibrosis conductance transmembrane regulator (CFTR) expression in human monocytes.

BACKGROUND: Cystic fibrosis is caused by mutations of CFTR gene, a protein kinase A-activated anion channel, and is associated to a persistent and excessive chronic lung inflammation, suggesting functional alterations of immune cells. Leukocytes express detectable levels of CFTR but the molecule has not been fully characterized in these cells. METHODS: Freshly isolated monocytes from healthy individuals and CF patients were assessed by protein expression, single cell electrophysiological and membrane depolarization assays. RESULTS: We recorded chloride currents by patch clamp in healthy monocytes, after the administration of a CFTR stimulus. Currents were sensitive to a specific blocker of the CFTR channel, CFTRinh-172 and were absent in CF monocytes. Next, we evaluated the effects of ex vivo exposure of monocytes from cystic fibrosis patients carrying the F508del mutation to a chemical corrector, Vertex-325. We found an increase in CFTR expression by confocal microscopy and a recovery of CFTR function by both patch clamp and single cell fluorescence analysis. CONCLUSIONS: We confirm the expression of functional CFTR in human monocytes and demonstrate that blood monocytes can represent an adequate source of primary cells to assess new therapies and define diagnosis of CF. GENERAL SIGNIFICANCE: Tests to evaluate CFTR functional abnormalities in CF disease might greatly benefit from the availability of a convenient source of primary cells. This electrophysiological study promotes the use of monocytes as a minimally invasive tool to study and monitor CFTR function in individual patients.

Effective delivery of recombinant proteins to rod photoreceptors via lipid nanovesicles.

The potential of liposomes to deliver functional proteins in retinal photoreceptors and modulate their physiological response was investigated by two experimental approaches. First, we treated isolated mouse retinas with liposomes encapsulating either recoverin, an important endogenous protein operating in visual phototransduction, or antibodies against recoverin. We then intravitrally injected in vivo liposomes encapsulating either rhodamin B or recoverin and we investigated the distribution in retina sections by confocal microscopy. The content of liposomes was found to be released in higher amount in the photoreceptor layer than in the other regions of the retina and the functional effects of the release were in line with the current model of phototransduction. Our study sets the basis for quantitative investigations aimed at assessing the potential of intraocular protein delivery via biocompatible nanovesicles, with promising implications for the treatment of retinal diseases affecting the photoreceptor layer.

HOMECAT: consensus homologs mapping for interspecific knowledge transfer and functional genomic data integration.

MOTIVATION: Comparative studies are encouraged by the fast increase of data availability from the latest high-throughput techniques, in particular from functional genomic studies. Yet, the size of datasets, the challenge of complete orthologs findings and not last, the variety of identification formats, make information integration challenging. With HOMECAT, we aim to facilitate cross-species relationship identification and data mapping, by combining orthology predictions from several publicly available sources, a convenient interface for high-throughput data download and automatic identifier conversion into a Cytoscape plug-in, that provides both an integration with a large set of bioinformatics tools, as well as a user-friendly interface. AVAILABILITY: HOMECAT and the Supplementary Materials are freely available at http://www.cbmc.it/homecat/.

Detection of CFTR protein in human leukocytes by flow cytometry.

Leukocytes have previously been shown to express detectable levels of the protein cystic fibrosis transmembrane conductance regulator (CFTR). This study aims to evaluate the application of flow cytometric (FC) analysis to detect CFTR expression, and changes thereof, in these cells. Aliquots (200 µL) of peripheral whole blood from 12 healthy control volunteers (CTRLs), 12 carriers of a CFTR mutation (CFC), and 40 patients with cystic fibrosis (CF) carrying various combinations of CFTR mutations were incubated with specific fluorescent probes recognizing CFTR protein expressed on the plasma membrane of leukocytes. FC was applied to analyze CFTR expression in monocytes, lymphocytes, and polymorphonuclear (PMN) cells. CFTR protein was detected in monocytes and lymphocytes, whereas inconclusive results were obtained from the analysis of PMN cells. Mean fluorescence intensity (MFI) ratio value and %CFTR-positive cells above a selected threshold were the two parameters selected to quantify CFTR expression in cells. Lowest variability and the highest reproducibility were obtained when analyzing monocytes. ANOVA results indicated that both parameters were able to discriminate monocytes of healthy controls and CF individuals according to CFTR mutation classes with high accuracy. Significantly increased MFI ratio values were recorded in CFTR-defective cells that were also able to improve CFTR function after ex vivo treatment with PTC124 (Ataluren), an investigative drug designed to permit the ribosome to read through nonsense CFTR mutations. The method described is minimally invasive and may be used in the monitoring of responses to drugs whose efficacy can depend on increased CFTR protein expression levels. © 2014 International Society for Advancement of Cytometry.

Challenging the diagnosis of cystic fibrosis in a patient carrying the 186-8T/C allelic variant in the CF transmembrane conductance regulator gene.

BACKGROUND: This report describe for the first time a clinical case with a CFTR allelic variant 186-8T/C (c.54-8 T/C) in intron 1 of CFTR and underline the importance of applying a combination of genetic and functional tests to establish or exclude a diagnosis of Cystic Fibrosis. In this case the diagnostic algorithm proposed for CF has been successfully applied at our Center and previous CF diagnosis assigned in a different Center was not confirmed. CASE PRESENTATION: A 38 year-old Italian woman had been treated as affected by CF since 2010, following diagnosis based on sweat tests (reported values of 73 and 57 mEq/L) and a clinical history consistent with CF. No mutations were identified by first level of genetic analysis. Afterwards the patient referred to our center for assessing the relevance of these findings. The genetic variant 186-8T/C (c.54-8 T/C) in intron 1 of the CFTR gene was detected by sequencing. Low-level interstitial-alveolar infiltration was recorded by high-resolution computerized tomography. Lung function was normal and sputum and Broncho Alveolar Lavage cultures resulted bacteriologically negative. Sweat chloride levels was re-assessed and resulted with values of 57 and 35 mEq/L, with a borderline range between 40 and 60 mEq/L. Nasal Potential Difference measurements resulted in three reliable measurements consistent with a non-CF phenotype. Differential diagnosis with ciliary dyskinesia was excluded, as was exon 2 skipping of CFTR gene that might have caused a CFTR functional defect. Furthermore, single cell fluorescence analysis in response to cAMP agonists performed in patient's monocytes overlapped those obtained in healthy donors. CONCLUSION: We concluded that this patient was not affected by CF. This case highlights the need for referrals to highly specialized centers and the importance of combined functional and genetic tests in making a correct diagnosis. Moreover, we confirmed a correlation between NPD tracings and cell depolarization in monocytes providing a rationale for proposing the use of leukocytes as a potential support for CF diagnosis.

Increased Expression of Autophagy-Related Genes in Alzheimer's Disease-Type 2 Diabetes Mellitus Comorbidity Models in Cells.

The association between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) has been extensively demonstrated, but despite this, the pathophysiological mechanisms underlying it are still unknown. In previous work, we discovered a central role for the autophagy pathway in the common alterations observed between AD and T2DM. In this study, we further investigate the role of genes belonging to this pathway, measuring their mRNA expression and protein levels in 3xTg-AD transgenic mice, an animal model of AD. Moreover, primary mouse cortical neurons derived from this model and the human H4Swe cell line were used as cellular models of insulin resistance in AD brains. Hippocampal mRNA expression showed significantly different levels for Atg16L1, Atg16L2, GabarapL1, GabarapL2, and Sqstm1 genes at different ages of 3xTg-AD mice. Significantly elevated expression of Atg16L1, Atg16L2, and GabarapL1 was also observed in H4Swe cell cultures, in the presence of insulin resistance. Gene expression analysis confirmed that Atg16L1 was significantly increased in cultures from transgenic mice when insulin resistance was induced. Taken together, these results emphasise the association of the autophagy pathway in AD-T2DM co-morbidity, providing new evidence about the pathophysiology of both diseases and their mutual interaction.

Integrative transcriptomic and metabolic analyses of the mammalian hibernating brain identifies a key role for succinate dehydrogenase in ischemic tolerance.

Ischemic stroke results in a loss of tissue homeostasis and integrity, the underlying pathobiology of which stems primarily from the depletion of cellular energy stores and perturbation of available metabolites 1 . Hibernation in thirteen-lined ground squirrels (TLGS), Ictidomys tridecemlineatus , provides a natural model of ischemic tolerance as these mammals undergo prolonged periods of critically low cerebral blood flow without evidence of central nervous system (CNS) damage 2 . Studying the complex interplay of genes and metabolites that unfolds during hibernation may provide novel insights into key regulators of cellular homeostasis during brain ischemia. Herein, we interrogated the molecular profiles of TLGS brains at different time points within the hibernation cycle via RNA sequencing coupled with untargeted metabolomics. We demonstrate that hibernation in TLGS leads to major changes in the expression of genes involved in oxidative phosphorylation and this is correlated with an accumulation of the tricarboxylic acid (TCA) cycle intermediates citrate, cis-aconitate, and α-ketoglutarate-αKG. Integration of the gene expression and metabolomics datasets led to the identification of succinate dehydrogenase (SDH) as the critical enzyme during hibernation, uncovering a break in the TCA cycle at that level. Accordingly, the SDH inhibitor dimethyl malonate (DMM) was able to rescue the effects of hypoxia on human neuronal cells in vitro and in mice subjected to permanent ischemic stroke in vivo . Our findings indicate that studying the regulation of the controlled metabolic depression that occurs in hibernating mammals may lead to novel therapeutic approaches capable of increasing ischemic tolerance in the CNS.

Mouse transgenic lines that selectively label Type I, Type IIA, and Types IIX+B skeletal muscle fibers.

Skeletal muscle fibers vary in contractile and metabolic properties. Four main fiber types are present in mammalian trunk and limb muscles; they are called I, IIA, IIX, and IIB, ranging from slowest- to fastest-contracting. Individual muscles contain stereotyped proportions of two or more fiber types. Fiber type is determined by a combination of nerve-dependent and -independent influences, leading to formation of "homogeneous motor units" in which all branches of a single motor neuron form synapses on fibers of a single type. Fiber type composition of muscles can be altered in adulthood by multiple factors including exercise, denervation, hormones, and aging. To facilitate analysis of muscle development, plasticity, and innervation, we generated transgenic mouse lines in which Type I, Type IIA, and Type IIX+B fibers can be selectively labeled with distinguishable fluorophores. We demonstrate their use for motor unit reconstruction and live imaging of nerve-dependent alterations in fiber type.

Genetic perturbation of postsynaptic activity regulates synapse elimination in developing cerebellum.

In many parts of the vertebrate nervous system, synaptic connections are remodeled during early postnatal life. Neural activity plays an important role in regulating one such rearrangement, synapse elimination, in the developing neuromuscular system, but there is little direct evidence on roles of pre- or postsynaptic activity in regulating synapse elimination in the developing brain. To address this issue, we expressed a chloride channel-yellow fluorescent protein fusion in cerebellar Purkinje cells (PCs) of transgenic mice to decrease their excitability. We then assessed elimination of supernumerary climbing fiber inputs to PCs. Individual PCs are innervated by multiple climbing fibers at birth; all but one are eliminated during the first three postnatal weeks in wild-type mice, but multiple innervation persists for at least three months in the transgenic mice. The normal redistribution of climbing fiber synapses from PC somata to proximal dendrites was also blunted in transgenics. These results show that normal electrical activity of the postsynaptic cell is required for it to attain a mature innervation pattern.

Early Application of Ipsilateral Cathodal-tDCS in a Mouse Model of Brain Ischemia Results in Functional Improvement and Perilesional Microglia Modulation.

Early stroke therapeutic approaches rely on limited options, further characterized by a narrow therapeutic time window. In this context, the application of transcranial direct current stimulation (tDCS) in the acute phases after brain ischemia is emerging as a promising non-invasive tool. Despite the wide clinical application of tDCS, the cellular mechanisms underlying its positive effects are still poorly understood. Here, we explored the effects of cathodal tDCS (C-tDCS) 6 h after focal forelimb M1 ischemia in Cx3CR1GFP/+ mice. C-tDCS improved motor functionality of the affected forelimb, as assessed by the cylinder and foot-fault tests at 48 h, though not changing the ischemic volume. In parallel, histological analysis showed that motor recovery is associated with decreased microglial cell density in the area surrounding the ischemic core, while astrocytes were not affected. Deeper analysis of microglia morphology within the perilesional area revealed a shift toward a more ramified healthier state, with increased processes' complexity and a less phagocytic anti-inflammatory activity. Taken together, our findings suggest a positive role for early C-tDCS after ischemia, which is able to modulate microglia phenotype and morphology in parallel to motor recovery.

In vivo morphological alterations of TAMs during KCa3.1 inhibition-by using in vivo two-photon time-lapse technology.

Tumor associated macrophages (TAMs) are the mostprevalent cells recruited in the tumor microenvironment (TME). Once recruited, TAMs acquire a pro-tumor phenotype characterized by a typical morphology: ameboid in the tumor core and with larger soma and thick branches in the tumor periphery. Targeting TAMs by reverting them to an anti-tumor phenotype is a promising strategy for cancer immunotherapy. Taking advantage of Cx3cr1GFP/WT heterozygous mice implanted with murine glioma GL261-RFP cells we investigated the role of Ca2+-activated K+ channel (KCa3.1) on the phenotypic shift of TAMs at the late stage of glioma growth through in vivo two-photon imaging. We demonstrated that TAMs respond promptly to KCa3.1 inhibition using a selective inhibitor of the channel (TRAM-34) in a time-dependent manner by boosting ramified projections attributable to a less hypertrophic phenotype in the tumor core. We also revealed a selective effect of drug treatment by reducing both glioma cells and TAMs in the tumor core with no interference with surrounding cells. Taken together, our data indicate a TRAM-34-dependent progressive morphological transformation of TAMs toward a ramified and anti-tumor phenotype, suggesting that the timing of KCa3.1 inhibition is a key point to allow beneficial effects on TAMs.

High-frequency rTMS modulates emotional behaviors and structural plasticity in layers II/III and V of the mPFC.

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive neuromodulation technique, and it has been increasingly used as a nonpharmacological intervention for the treatment of various neurological and neuropsychiatric diseases, including depression. In humans, rTMS over the prefrontal cortex is used to induce modulation of the neural circuitry that regulates emotions, cognition, and depressive symptoms. However, the underlying mechanisms are still unknown. In this study, we investigated the effects of a short (5-day) treatment with high-frequency (HF) rTMS (15 Hz) on emotional behavior and prefrontal cortex morphological plasticity in mice. Mice that had undergone HF-rTMS showed an anti-depressant-like activity as evidenced by decreased immobility time in both the Tail Suspension Test and the Forced Swim Test along with increased spine density in both layer II/III and layer V apical and basal dendrites. Furthermore, dendritic complexity assessed by Sholl analysis revealed increased arborization in the apical portions of both layers, but no modifications in the basal dendrites branching. Overall, these results indicate that the antidepressant-like activity of HF-rTMS is paralleled by structural remodeling in the medial prefrontal cortex.

An improved and simplified protocol to combine Golgi-Cox staining with immunofluorescence and transmission electron microscopy techniques.

Approaches utilizing multiple analysis techniques on a single sample are highly desirable in research, especially to reduce the number of animals and obtain the maximum information. Golgi-Cox staining is a widely used method for characterizing axon and dendritic morphology and several attempts to combine this technique with immunofluorescence and transmission electron microscopy have been proposed. With few exceptions, most of the protocols were characterized by a high degree of complexity and low reproducibility. Here we show a simplified procedure of perfusion, fixation and staining of brain tissues that allows Golgi-Cox staining, immunofluorescence and transmission electron microscopy in the same sample, to obtain high-quality images with a low-cost procedure. The main novelty in this protocol is the possibility of performing Golgi-Cox staining after the perfusion and post-fixation of brain tissue with a buffered solution containing, not only formaldehyde, but also glutaraldehyde. This renders the tissue suitable for electron microscopy, but it is also compatible with immunofluorescence staining. This combined protocol can be used in most neuroscience laboratories as it does not require special equipment and skills. This protocol will be useful in a broad range of neuroscience topics to study morphological changes during brain development and plasticity in physiological and pathological conditions.

High-frequency repetitive transcranial magnetic stimulation enhances layer II/III morphological dendritic plasticity in mouse primary motor cortex.

High-frequency repeated transcranial magnetic stimulation (HF-rTMS) is a safe non-invasive neuromodulatory technique and there is a body of evidence shows that it can modulate plasticity in different brain areas. One of the most interesting application of HF-rTMS is the modulation of plasticity in primary motor cortex (M1) to promote recovery after brain injuries. However, the underlying mechanism by which HF-rTMS modulates motor cortex plasticity remain to be investigated. In this study, we investigated the effects of HF-rTMS treatment on morphological plasticity of pyramidal neurons in layer II/III (L2/3) of the primary motor cortex in mice. Our results show that the treatment did not increase anxiety in mice in the open field test and the elevated plus-maze test. Treated mice displayed increased total spine density in apical and basal dendrites, with a predominance of thin spines. The treatment also increased dendritic complexity, as assessed by Sholl analysis at both apical and basal dendrites. Collectively, the results show that HF-rTMS induced remarkable changes in dendritic complexity in primary motor cortex L2/3 connections which may strengthen corticocortical connections increasing integration of information across cortical areas. The data support the use of HF-rTMS as a circuit-targeting neuromodulation strategy.

Therapeutic targeting of Lyn kinase to treat chorea-acanthocytosis.

Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a-/- mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, γ-synuclein and phospho-tau proteins in Vps13a-/- basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a-/- Lyn-/- showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a-/- hematological and neurological phenotypes, improving autophagy and preventing neuroinflammation. Our data support the proposal to repurpose nilotinib as new therapeutic option for ChAc patients.

Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition.

Synaptic activity drives synaptic rearrangement in the vertebrate nervous system; indeed, this appears to be a main way in which experience shapes neural connectivity. One rearrangement that occurs in many parts of the nervous system during early postnatal life is a competitive process called 'synapse elimination'. At the neuromuscular junction, where synapse elimination has been analysed in detail, muscle fibres are initially innervated by multiple axons, then all but one are withdrawn and the 'winner' enlarges. In support of the idea that synapse elimination is activity dependent, it is slowed or speeded when total neuromuscular activity is decreased or increased, respectively. However, most hypotheses about synaptic rearrangement postulate that change depends less on total activity than on the relative activity of the competitors. Intuitively, it seems that the input best able to excite its postsynaptic target would be most likely to win the competition, but some theories and results make other predictions. Here we use a genetic method to selectively inhibit neurotransmission from one of two inputs to a single target cell. We show that more powerful inputs are strongly favoured competitors during synapse elimination.