IF du Neurocentre

57 publications

* equal contribution
Les IF indiqués ont été collectés par le Web of Sciences en Juin 2021

13/07/2021 | Cell Rep   IF 9.4
Probing the polarity of spontaneous perisomatic GABAergic synaptic transmission in the mouse CA3 circuit in vivo.
Dubanet O, Ferreira Gomes Da Silva A, Frick A, Hirase H, Beyeler A, Leinekugel X

The hypothesis that reversed, excitatory GABA may be involved in various brain pathologies, including epileptogenesis, is appealing but controversial because of the technical difficulty of probing endogenous GABAergic synaptic function in vivo. We overcome this challenge by non-invasive extracellular recording of neuronal firing responses to optogenetically evoked and spontaneously occurring inhibitory perisomatic GABAergic field potentials, generated by individual parvalbumin interneurons on their target pyramidal cells. Our direct probing of GABAergic transmission suggests a rather anecdotal participation of excitatory GABA in two specific models of epileptogenesis in the mouse CA3 circuit in vivo, even though this does not preclude its expression in other brain areas or pathological conditions. Our approach allows the detection of distinct alterations of inhibition during spontaneous activity in vivo, with high sensitivity. It represents a promising tool for the investigation of excitatory GABA in different pathological conditions that may affect the hippocampal circuit.

03/05/2021 | Int J Mol Sci   IF 5.9
A Proteomic Approach for Systematic Mapping of Substrates of Human Deubiquitinating Enzymes.
Ramirez J, Prieto G, Olazabal-Herrero A, Borràs E, Fernandez-Vigo E, Alduntzin U, Osinalde N, Beaskoetxea J, Lectez B, Aloria K, Rodriguez JA, Paradela A, Sabidó E, Muñoz J, Corrales F, Arizmendi JM, Mayor U

The human genome contains nearly 100 deubiquitinating enzymes (DUBs) responsible for removing ubiquitin moieties from a large variety of substrates. Which DUBs are responsible for targeting which substrates remain mostly unknown. Here we implement the (bio)Ub approach to identify DUB substrates in a systematic manner, combining gene silencing and proteomics analyses. Silencing of individual DUB enzymes is used to reduce their ubiquitin deconjugating activity, leading to an increase of the ubiquitination of their substrates, which can then be isolated and identified. We report here quantitative proteomic data of the putative substrates of 5 human DUBs. Furthermore, we have built a novel interactive database of DUB substrates to provide easy access to our data and collect DUB proteome data from other groups as a reference resource in the DUB substrates research field.

25/08/2020 | Proc Natl Acad Sci U S A   IF 9.4
One size does not fit all in Drosophila olfactory habituation.
Semelidou O, Skoulakis EMC

Autism Spectrum Disorders (ASD) are characterised by deficits in social interactions and repetitive behaviours. Multiple ASD-associated mutations have been identified in the Shank family of proteins that play a critical role in the structure and plasticity of glutamatergic synapses, leading to impaired synapse function and the presentation of ASD-associated behavioural deficits in mice. Shank proteins are highly regulated by zinc, where zinc binds the Shank SAM domain to drive synaptic protein recruitment and synaptic maturation. Here we have examined the influence of maternal dietary zinc supplementation during pregnancy and lactation on the development of ASD-associated behavioural and synaptic changes in the offspring Shank3 knockout (Shank3(-/-)) mice. Behavioural and electrophysiological experiments were performed in juvenile and adult Shank3(-/-) and wildtype littermate control mice born from mothers fed control (30 ppm, ppm) or supplemented (150 ppm) dietary zinc. We observed that the supplemented maternal zinc diet prevented ASD-associated deficits in social interaction and normalised anxiety behaviours in Shank3(-/-) offspring mice. These effects were maintained into adulthood. Repetitive grooming was also prevented in adult Shank3(-/-) offspring mice. At the synaptic level, maternal zinc supplementation altered postsynaptic NMDA receptor-mediated currents and presynaptic function at glutamatergic synapses onto medium spiny neurons in the cortico-striatal pathway of the Shank3(-/-) offspring mice. These data show that increased maternal dietary zinc during pregnancy and lactation can alter the development of ASD-associated changes at the synaptic and the behavioural levels, and that zinc supplementation from the beginning of brain development can prevent ASD-associated deficits in Shank3(-/-) mice long term.

02/2020 | Neurosci Biobehav Rev   IF 8.3
What we can learn from a genetic rodent model about autism.
Mohrle D, Fernandez M, Penagarikano O, Frick A, Allman B, Schmid S

Autism spectrum disorders (ASD) are complex neurodevelopmental disorders that are caused by genetic and/or environmental impacts, often probably by the interaction of both. They are characterised by deficits in social communication and interaction and by restricted and repetitive behaviours and interests from early childhood on, causing significant impairment. While it is clear that no animal model captures the full complexity of ASD in humans, genetic models are extremely useful for studying specific symptoms associated with ASD and the underlying cellular and molecular mechanisms. In this review we summarize the behavioral paradigms used in rodents to model ASD symptoms as they are listed in the DSM-5. We then review existing genetic rodent models with disruptions in ASD candidate genes, and we map their phenotypes onto these behavioural paradigms. The goal of this review is to give a comprehensive overview on how ASD symptoms can be studied in animal models and to give guidance for which animal models are appropriate to study specific symptom clusters.

2020 | front immunol   IF 5.1
CD8+ T Cell-Mediated Mechanisms Contribute to the Progression of Neurocognitive Impairment in Both Multiple Sclerosis and Alzheimer's Disease?
Stojic-Vukanic Z*, Hadzibegovic S*, Nicole O, Nacka-Aleksic M, Lestarevic S, Leposavic G

Neurocognitive impairment (NCI) is one of the most relevant clinical manifestations of multiple sclerosis (MS). The profile of NCI and the structural and functional changes in the brain structures relevant for cognition in MS share some similarities to those in Alzheimer's disease (AD), the most common cause of neurocognitive disorders. Additionally, despite clear etiopathological differences between MS and AD, an accumulation of effector/memory CD8+ T cells and CD8+ tissue-resident memory T (Trm) cells in cognitively relevant brain structures of MS/AD patients, and higher frequency of effector/memory CD8+ T cells re-expressing CD45RA (TEMRA) with high capacity to secrete cytotoxic molecules and proinflammatory cytokines in their blood, were found. Thus, an active pathogenetic role of CD8+ T cells in the progression of MS and AD may be assumed. In this mini-review, findings supporting the putative role of CD8+ T cells in the pathogenesis of MS and AD are displayed, and putative mechanisms underlying their pathogenetic action are discussed. A special effort was made to identify the gaps in the current knowledge about the role of CD8+ T cells in the development of NCI to 'catalyze' translational research leading to new feasible therapeutic interventions.

2020 | PLoS ONE   IF 2.7
A simultaneous optical and electrical in-vitro neuronal recording system to evaluate microelectrode performance.
Aqrawe Z, Patel N, Vyas Y, Bansal M, Montgomery J, Travas-Sejdic J, Svirskis D

OBJECTIVES: In this paper, we aim to detail the setup of a high spatio-temporal resolution, electrical recording system utilising planar microelectrode arrays with simultaneous optical imaging suitable for evaluating microelectrode performance with a proposed 'performance factor' metric. METHODS: Techniques that would facilitate low noise electrical recordings were coupled with voltage sensitive dyes and neuronal activity was recorded both electrically via a customised amplification system and optically via a high speed CMOS camera. This technique was applied to characterise microelectrode recording performance of gold and poly(3,4-ethylenedioxythiophene)/polystyrene sulfonate (PEDOT/PSS) coated electrodes through traditional signal to noise (SNR) calculations as well as the proposed performance factor. RESULTS: Neuronal activity was simultaneously recorded using both electrical and optical techniques and this activity was confirmed via tetrodotoxin application to inhibit action potential firing. PEDOT/PSS outperformed gold using both measurements, however, the performance factor metric estimated a 3 fold improvement in signal transduction when compared to gold, whereas SNR estimated an 8 fold improvement when compared to gold. CONCLUSION: The design and functionality of a system to record from neurons both electrically, through microelectrode arrays, and optically via voltage sensitive dyes was successfully achieved. SIGNIFICANCE: The high spatiotemporal resolution of both electrical and optical methods will allow for an array of applications such as improved detection of subthreshold synaptic events, validation of spike sorting algorithms and a provides a robust evaluation of extracellular microelectrode performance.

2020 | front neurosci   IF 3.7
Hippocampal Deficits in Amyloid-beta-Related Rodent Models of Alzheimer's Disease.
Vyas Y, Montgomery JM, Cheyne JE

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is the most common cause of dementia. Symptoms of AD include memory loss, disorientation, mood and behavior changes, confusion, unfounded suspicions, and eventually, difficulty speaking, swallowing, and walking. These symptoms are caused by neuronal degeneration and cell loss that begins in the hippocampus, and later in disease progression spreading to the rest of the brain. While there are some medications that alleviate initial symptoms, there are currently no treatments that stop disease progression. Hippocampal deficits in amyloid-beta-related rodent models of AD have revealed synaptic, behavioral and circuit-level defects. These changes in synaptic function, plasticity, neuronal excitability, brain connectivity, and excitation/inhibition imbalance all have profound effects on circuit function, which in turn could exacerbate disease progression. Despite, the wealth of studies on AD pathology we don't yet have a complete understanding of hippocampal deficits in AD. With the increasing development of in vivo recording techniques in awake and freely moving animals, future studies will extend our current knowledge of the mechanisms underpinning how hippocampal function is altered in AD, and aid in progression of treatment strategies that prevent and/or delay AD symptoms.

SHANK3 is a postsynaptic structural protein localized at excitatory glutamatergic synapses in which deletions and mutations have been implicated in patients with autism spectrum disorders (ASD). The expression of Shank3 ASD mutations causes impairments in ionotropic glutamate receptor-mediated synaptic responses in neurons, which is thought to underlie ASD-related behaviors, thereby indicating glutamatergic synaptopathy as one of the major pathogenic mechanisms. However, little is known about the functional consequences of ASD-associated mutations in Shank3 on another important set of glutamate receptors, group I metabotropic glutamate receptors (mGluRs). Here, we further assessed how Shank3 mutations identified in patients with ASD (one de novo InsG mutation and two inherited point mutations, R87C and R375C) disrupt group I mGluR (mGluR1 and mGluR5) expression and function. To identify potential isoform-specific deficits induced by ASD-associated Shank3 mutations on group I mGluRs, we surface immunolabeled mGluR1 and mGluR5 independently. We also induced mGluR-dependent synaptic plasticity (R,S-3,5-dihydroxyphenylglycine [DHPG]-induced long-term depression [LTD]) as well as N-methyl-D-aspartate receptor (NMDAR)-dependent LTD. ASD-associated mutations in Shank3 differentially interfered with the ability of cultured hippocampal neurons to express mGluR5 and mGluR1 at synapses. Intriguingly, all ASD Shank3 mutations impaired mGluR-dependent LTD without altering NMDAR-dependent LTD. Our data show that the specific perturbation in mGluR-dependent synaptic plasticity occurs in neurons expressing ASD-associated Shank3 mutations, which may underpin synaptic dysfunction and subsequent behavioral deficits in ASD.

22/05/2019 | J Neurosci   IF 6.1
Hippocampal Mossy Fibers Synapses in CA3 Pyramidal Cells Are Altered at an Early Stage in a Mouse Model of Alzheimer's Disease.
Viana da Silva S, Zhang P, Haberl MG, Labrousse V, Grosjean N, Blanchet C, Frick A, Mulle C

Early Alzheimer's disease (AD) affects the brain non-uniformly, causing hippocampal memory deficits long before wide-spread brain degeneration becomes evident. Here we addressed whether mossy fiber inputs from the dentate gyrus onto CA3 principal cells are affected in an AD mouse model before amyloid beta plaque deposition. We recorded from CA3 pyramidal cells in a slice preparation from 6-month-old male APP/PS1 mice, and studied synaptic properties and intrinsic excitability. In parallel we performed a morphometric analysis of mossy fiber synapses following viral based labeling and 3D-reconstruction. We found that the basal structural and functional properties as well as presynaptic short-term plasticity at mossy fiber synapses are unaltered at 6 months in APP/PS1 mice. However, transient potentiation of synaptic transmission mediated by activity-dependent release of lipids was abolished. Whereas the presynaptic form of mossy fiber long-term potentiation (LTP) was not affected, the postsynaptic LTP of NMDAR-EPSCs was reduced. In addition, we also report an impairment in feedforward inhibition in CA3 pyramidal cells. This study, together with our previous work describing deficits at CA3-CA3 synapses, provides evidence that early AD affects synapses in a projection-dependent manner at the level of a single neuronal population.SIGNIFICANCE STATEMENT Because loss of episodic memory is considered the cognitive hallmark of Alzheimer's disease (AD), it is important to study whether synaptic circuits involved in the encoding of episodic memory are compromised in AD mouse models. Here we probe alterations in the synaptic connections between the dentate gyrus and CA3, which are thought to be critical for enabling episodic memories to be formed and stored in CA3. We found that forms of synaptic plasticity specific to these synaptic connections are markedly impaired at an early stage in a mouse model of AD, before deposition of beta amyloid plaques. Together with previous work describing deficits at CA3-CA3 synapses, we provide evidence that early AD affects synapses in an input-dependent manner within a single neuronal population.

02/01/2019 | J Cell Sci   IF 4.5
The mitochondrial phosphatidylserine decarboxylase Psd1 is involved in nitrogen starvation-induced mitophagy in yeast.
Vigie P, Cougouilles E, Bhatia-Kissova I, Salin B, Blancard C, Camougrand N

Mitophagy, the selective degradation of mitochondria by autophagy, is a central process that is essential for the maintenance of cell homeostasis. It is implicated in the clearance of superfluous or damaged mitochondria and requires specific proteins and regulators to perform. In yeast, Atg32, an outer mitochondrial membrane protein, interacts with the ubiquitin-like Atg8 protein, promoting the recruitment of mitochondria to the phagophore and their sequestration within autophagosomes. Atg8 is anchored to the phagophore and autophagosome membranes thanks to a phosphatidylethanolamine tail. In Saccharomyces cerevisiae, several phosphatidylethanolamine synthesis pathways have been characterized, but their contribution to autophagy and mitophagy are unknown. Through different approaches, we show that Psd1, the mitochondrial phosphatidylserine decarboxylase, is involved in mitophagy induction only after nitrogen starvation, whereas Psd2, which is located in vacuole, Golgi and endosome membranes, is required preferentially for mitophagy induction in the stationary phase of growth but also to a lesser extent for nitrogen starvation-induced mitophagy. Our results suggest that the mitophagy defect observed in Deltapsd1 yeast cells after nitrogen starvation may be due to a failure of Atg8 recruitment to mitochondria.This article has an associated First Person interview with the first author of the paper.

2019 | front mol neurosci   IF 3.7
Peripheral Delta Opioid Receptors Mediate Formoterol Anti-allodynic Effect in a Mouse Model of Neuropathic Pain.
Ceredig RA, Pierre F, Doridot S, Alduntzin U, Hener P, Salvat E, Yalcin I, Gaveriaux-Ruff C, Barrot M, Massotte D

Neuropathic pain is a challenging condition for which current therapies often remain unsatisfactory. Chronic administration of beta2 adrenergic agonists, including formoterol currently used to treat asthma and chronic obstructive pulmonary disease, alleviates mechanical allodynia in the sciatic nerve cuff model of neuropathic pain. The limited clinical data currently available also suggest that formoterol would be a suitable candidate for drug repurposing. The antiallodynic action of beta2 adrenergic agonists is known to require activation of the delta-opioid (DOP) receptor but better knowledge of the molecular mechanisms involved is necessary. Using a mouse line in which DOP receptors were selectively ablated in neurons expressing Nav1.8 sodium channels (DOP cKO), we showed that these DOP peripheral receptors were necessary for the antiallodynic action of the beta2 adrenergic agonist formoterol in the cuff model. Using a knock-in mouse line expressing a fluorescent version of the DOP receptor fused with the enhanced green fluorescent protein (DOPeGFP), we established in a previous study, that mechanical allodynia is associated with a smaller percentage of DOPeGFP positive small peptidergic sensory neurons in dorsal root ganglia (DRG), with a reduced density of DOPeGFP positive free nerve endings in the skin and with increased DOPeGFP expression at the cell surface. Here, we showed that the density of DOPeGFP positive free nerve endings in the skin is partially restored and no increase in DOPeGFP translocation to the plasma membrane is observed in mice in which mechanical pain is alleviated upon chronic oral administration of formoterol. This study, therefore, extends our previous results by confirming that changes in the mechanical threshold are associated with changes in peripheral DOP profile. It also highlights the common impact on DOP receptors between serotonin noradrenaline reuptake inhibitors such as duloxetine and the beta2 mimetic formoterol.

Habituation is the process that enables salience filtering, precipitating perceptual changes that alter the value of environmental stimuli. To discern the neuronal circuits underlying habituation to brief inconsequential stimuli, we developed a novel olfactory habituation paradigm, identifying two distinct phases of the response that engage distinct neuronal circuits. Responsiveness to the continuous odor stimulus is maintained initially, a phase we term habituation latency and requires Rutabaga Adenylyl-Cyclase-depended neurotransmission from GABAergic Antennal Lobe Interneurons and activation of excitatory Projection Neurons (PNs) and the Mushroom Bodies. In contrast, habituation depends on the inhibitory PNs of the middle Antenno-Cerebral Track, requires inner Antenno-Cerebral Track PN activation and defines a temporally distinct phase. Collectively, our data support the involvement of Lateral Horn excitatory and inhibitory stimulation in habituation. These results provide essential cellular substrates for future analyses of the molecular mechanisms that govern the duration and transition between these distinct temporal habituation phases. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

09/2018 | Eur J Neurosci   IF 2.8
Peripheral delta opioid receptors mediate duloxetine antiallodynic effect in a mouse model of neuropathic pain.
Ceredig RA, Pierre F, Doridot S, Alduntzin U, Salvat E, Yalcin I, Gaveriaux-Ruff C, Barrot M, Massotte D

Peripheral delta opioid (DOP) receptors are essential for the antiallodynic effect of the tricyclic antidepressant nortriptyline. However, the population of DOP-expressing cells affected in neuropathic conditions or underlying the antiallodynic activity of antidepressants remains unknown. Using a mouse line in which DOP receptors were selectively ablated in cells expressing Nav1.8 sodium channels (DOP cKO), we established that these DOP peripheral receptors were mandatory for duloxetine to alleviate mechanical allodynia in a neuropathic pain model based on sciatic nerve cuffing. We then examined the impact of nerve cuffing and duloxetine treatment on DOP-positive populations using a knock-in mouse line expressing a fluorescent version of the DOP receptor fused with the enhanced green fluorescent protein (DOPeGFP). Eight weeks postsurgery, we observed a reduced proportion of DOPeGFP-positive small peptidergic sensory neurons (calcitonin gene-related peptide (CGRP) positive) in dorsal root ganglia and a lower density of DOPeGFP-positive free nerve endings in the skin. These changes were not present in nerve-injured mice chronically treated with oral duloxetine. In addition, increased DOPeGFP translocation to the plasma membrane was observed in neuropathic conditions but not in duloxetine-treated neuropathic mice, which may represent an additional level of control of the neuronal activity by DOP receptors. Our results therefore established a parallel between changes in the expression profile of peripheral DOP receptors and mechanical allodynia induced by sciatic nerve cuffing.

11/06/2018 | Sci Rep   IF 4.1
D5 dopamine receptors control glutamatergic AMPA transmission between the motor cortex and subthalamic nucleus.
Froux L, Le Bon-Jego M, Miguelez C, Normand E, Morin S, Fioramonti S, Barresi M, Frick A, Baufreton J, Taupignon A

Corticofugal fibers target the subthalamic nucleus (STN), a component nucleus of the basal ganglia, in addition to the striatum, their main input. The cortico-subthalamic, or hyperdirect, pathway, is thought to supplement the cortico-striatal pathways in order to interrupt/change planned actions. To explore the previously unknown properties of the neurons that project to the STN, retrograde and anterograde tools were used to specifically identify them in the motor cortex and selectively stimulate their synapses in the STN. The cortico-subthalamic neurons exhibited very little sag and fired an initial doublet followed by non-adapting action potentials. In the STN, AMPA/kainate synaptic currents had a voltage-dependent conductance, indicative of GluA2-lacking receptors and were partly inhibited by Naspm. AMPA transmission displayed short-term depression, with the exception of a limited bandpass in the 5 to 15 Hz range. AMPA synaptic currents were negatively controlled by dopamine D5 receptors. The reduction in synaptic strength was due to postsynaptic D5 receptors, mediated by a PKA-dependent pathway, but did not involve a modified rectification index. Our data indicated that dopamine, through post-synaptic D5 receptors, limited the cortical drive onto STN neurons in the normal brain.

18/04/2018 | Cereb Cortex   IF 6.3
Dysfunctional Autism Risk Genes Cause Circuit-Specific Connectivity Deficits With Distinct Developmental Trajectories
Zerbi Valerio, Giovanna D. Ielacqua, Marija Markicevic, Matthias Georg Haberl, Mark H. Ellisman, A-Bhaskaran A, Frick A, Markus Rudin, Nicole Wenderoth

Autism spectrum disorders (ASD) are a set of complex neurodevelopmental disorders for which there is currently no targeted therapeutic approach. It is thought that alterations of genes regulating migration and synapse formation during development affect neural circuit formation and result in aberrant connectivity within distinct circuits that underlie abnormal behaviors. However, it is unknown whether deviant developmental trajectories are circuit-specific for a given autism risk-gene. We used MRI to probe changes in functional and structural connectivity from childhood to adulthood in Fragile-X (Fmr1−/y) and contactin-associated (CNTNAP2−/−) knockout mice. Young Fmr1−/y mice (30 days postnatal) presented with a robust hypoconnectivity phenotype in corticocortico and corticostriatal circuits in areas associated with sensory information processing, which was maintained until adulthood. Conversely, only small differences in hippocampal and striatal areas were present during early postnatal development in CNTNAP2−/− mice, while major connectivity deficits in prefrontal and limbic pathways developed between adolescence and adulthood. These findings are supported by viral tracing and electron micrograph approaches and define 2 clearly distinct connectivity endophenotypes within the autism spectrum. We conclude that the genetic background of ASD strongly influences which circuits are most affected, the nature of the phenotype, and the developmental time course of the associated changes.

2018 | Front Cell Neurosci   IF 4.3
Dietary Zinc Supplementation Prevents Autism Related Behaviors and Striatal Synaptic Dysfunction in Shank3 Exon 13-16 Mutant Mice.
Fourie C, Vyas Y, Lee K, Jung Y, Garner CC, Montgomery JM

The SHANK family of synaptic proteins (SHANK1-3) are master regulators of the organizational structure of excitatory synapses in the brain. Mutations in SHANK1-3 are prevalent in patients with autism spectrum disorders (ASD), and loss of one copy of SHANK3 causes Phelan-McDermid Syndrome, a syndrome in which Autism occurs in >80% of cases. The synaptic stability of SHANK3 is highly regulated by zinc, driving the formation of postsynaptic protein complexes and increases in excitatory synaptic strength. As ASD-associated SHANK3 mutations retain responsiveness to zinc, here we investigated how increasing levels of dietary zinc could alter behavioral and synaptic deficits that occur with ASD. We performed behavioral testing together with cortico-striatal slice electrophysiology on a Shank3 (-/-) mouse model of ASD (Shank3 (ex13-1616-/-)), which displays ASD-related behaviors and structural and functional deficits at striatal synapses. We observed that 6 weeks of dietary zinc supplementation in Shank3 (ex13-16-/-) mice prevented ASD-related repetitive and anxiety behaviors and deficits in social novelty recognition. Dietary zinc supplementation also increased the recruitment of zinc sensitive SHANK2 to synapses, reduced synaptic transmission specifically through N-methyl-D-aspartate (NMDA)-type glutamate receptors, reversed the slowed decay tau of NMDA receptor (NMDAR)-mediated currents and occluded long term potentiation (LTP) at cortico-striatal synapses. These data suggest that alterations in NMDAR function underlie the lack of NMDAR-dependent cortico-striatal LTP and contribute to the reversal of ASD-related behaviors such as compulsive grooming. Our data reveal that dietary zinc alters neurological function from synapses to behavior, and identifies dietary zinc as a potential therapeutic agent in ASD.

24/10/2017 | Nat Commun   IF 12.1
Altered surface mGluR5 dynamics provoke synaptic NMDAR dysfunction and cognitive defects in Fmr1 knockout mice.
Aloisi E, Le Corf K, Dupuis J, Zhang P, Ginger M, Labrousse V, Spatuzza M, Georg Haberl M, Costa L, Shigemoto R, Tappe-Theodor A, Drago F, Vincenzo Piazza P, Mulle C, Groc L, Ciranna L, Catania MV, Frick A

Metabotropic glutamate receptor subtype 5 (mGluR5) is crucially implicated in the pathophysiology of Fragile X Syndrome (FXS); however, its dysfunction at the sub-cellular level, and related synaptic and cognitive phenotypes are unexplored. Here, we probed the consequences of mGluR5/Homer scaffold disruption for mGluR5 cell-surface mobility, synaptic N-methyl-D-aspartate receptor (NMDAR) function, and behavioral phenotypes in the second-generation Fmr1 knockout (KO) mouse. Using single-molecule tracking, we found that mGluR5 was significantly more mobile at synapses in hippocampal Fmr1 KO neurons, causing an increased synaptic surface co-clustering of mGluR5 and NMDAR. This correlated with a reduced amplitude of synaptic NMDAR currents, a lack of their mGluR5-activated long-term depression, and NMDAR/hippocampus dependent cognitive deficits. These synaptic and behavioral phenomena were reversed by knocking down Homer1a in Fmr1 KO mice. Our study provides a mechanistic link between changes of mGluR5 dynamics and pathological phenotypes of FXS, unveiling novel targets for mGluR5-based therapeutics.

10/10/2017 | Proc Natl Acad Sci U S A   IF 9.7
Drk-mediated signaling to Rho kinase is required for anesthesia-resistant memory in Drosophila.
Kotoula V, Moressis A, Semelidou O, Skoulakis EMC

Anesthesia-resistant memory (ARM) was described decades ago, but the mechanisms that underlie this protein synthesis-independent form of consolidated memory in Drosophila remain poorly understood. Whether the several signaling molecules, receptors, and synaptic proteins currently implicated in ARM operate in one or more pathways and how they function in the process remain unclear. We present evidence that Drk, the Drosophila ortholog of the adaptor protein Grb2, is essential for ARM within adult mushroom body neurons. Significantly, Drk signals engage the Rho kinase Drok, implicating dynamic cytoskeletal changes in ARM, and this is supported by reduced F-actin in the mutants and after pharmacological inhibition of Drok. Interestingly, Drk-Drok signaling appears independent of the function of Radish (Rsh), a protein long implicated in ARM, suggesting that the process involves at least two distinct molecular pathways. Based on these results, we propose that signaling pathways involved in structural plasticity likely underlie this form of translation-independent memory.

19/07/2017 | Neuropsychopharmacology   IF 6.4
Potential Involvement of Impaired BKCa Channel Function in Sensory Defensiveness and Some Behavioral Disturbances Induced by Unfamiliar Environment in a Mouse Model of Fragile X Syndrome.
Carreno-Munoz MI, Martins F, Medrano MC, Aloisi E, Pietropaolo S, Dechaud C, Subashi E, Bony G, Ginger M, Moujahid A, Frick A, Leinekugel X

In fragile X syndrome (FXS), sensory hypersensitivity and impaired habituation is thought to result in attention overload and various behavioral abnormalities in reaction to the excessive and remanent salience of environment features that would normally be ignored. This phenomenon, termed sensory defensiveness, has been proposed as the potential cause of hyperactivity, hyperarousal, and negative reactions to changes in routine that are often deleterious for FXS patients. However, the lack of tools for manipulating sensory hypersensitivity has not allowed the experimental testing required to evaluate the relevance of this hypothesis. Recent work has shown that BMS-204352, a BKCa channel agonist, was efficient to reverse cortical hyperexcitability and related sensory hypersensitivity in the Fmr1-KO mouse model of FXS. In the present study, we report that exposing Fmr1-KO mice to novel or unfamiliar environments resulted in multiple behavioral perturbations, such as hyperactivity, impaired nest building and excessive grooming of the back. Reversing sensory hypersensitivity with the BKCa channel agonist BMS-204352 prevented these behavioral abnormalities in Fmr1-KO mice. These results are in support of the sensory defensiveness hypothesis, and confirm BKCa as a potentially relevant molecular target for the development of drug medication against FXS/ASD.Neuropsychopharmacology advance online publication, 16 August 2017; doi:10.1038/npp.2017.149.

07/06/2017 | autism res   IF 3.8
Behavioral abnormalities in the Fmr1-KO2 mouse model of fragile X syndrome: The relevance of early life phases.
Gaudissard J*, Ginger M*, Premoli M, Memo M, Frick A*, Pietropaolo S*

Fragile X syndrome (FXS) is a developmental disorder caused by a mutation in the X-linked FMR1 gene, coding for the FMRP protein which is largely involved in synaptic function. FXS patients present several behavioral abnormalities, including hyperactivity, anxiety, sensory hyper-responsiveness, and cognitive deficits. Autistic symptoms, e.g., altered social interaction and communication, are also often observed: FXS is indeed the most common monogenic cause of autism. Mouse models of FXS are therefore of great interest for research on both FXS and autistic pathologies. The Fmr1-KO2 mouse line is the most recent FXS model, widely used for brain studies; surprisingly, little is known about the face validity of this model, i.e., its FXS-like behavioral phenotype. Furthermore, no data are available for the age-related expression of the pathological phenotypes in this mouse line, a critical issue for modelling neurodevelopmental disorders. Here we performed an extensive behavioral characterization of the KO2 model at infancy, adolescent and adult ages. Hyperactivity, altered emotionality, sensory hyper-responsiveness and memory deficits were already present in KO mice at adolescence and remained evident at adulthood. Alterations in social behaviors were instead observed only in young KO animals: during the first 2 weeks of life, KOs emitted longer ultrasonic vocalizations compared to their WT littermates and as adolescents they displayed more aggressive behaviors towards a conspecific. These results strongly support the face validity of the KO2 mouse as a model for FXS, at the same time demonstrating that its ability to recapitulate social autistic-relevant phenotypes depends on early testing ages. Autism Res 2017. (c) 2017 International Society for Autism Research, Wiley Periodicals, Inc.

10/04/2017 | Nat Neurosci   IF 17.8
Abnormal wiring of CCK+ basket cells disrupts spatial information coding.
Del Pino I, Brotons-Mas JR, Marques-Smith A, Marighetto A, Frick A, Marin O, Rico B

The function of cortical GABAergic interneurons is largely determined by their integration into specific neural circuits, but the mechanisms controlling the wiring of these cells remain largely unknown. This is particularly true for a major population of basket cells that express the neuropeptide cholecystokinin (CCK). Here we found that the tyrosine kinase receptor ErbB4 was required for the normal integration into cortical circuits of basket cells expressing CCK and vesicular glutamate transporter 3 (VGlut3). The number of inhibitory synapses made by CCK+VGlut3+ basket cells and the inhibitory drive they exerted on pyramidal cells were reduced in conditional mice lacking ErbB4. Developmental disruption of the connectivity of these cells diminished the power of theta oscillations during exploratory behavior, disrupted spatial coding by place cells, and caused selective alterations in spatial learning and memory in adult mice. These results suggest that normal integration of CCK+ basket cells in cortical networks is key to support spatial coding in the hippocampus.

07/02/2017 | Proc Natl Acad Sci U S A   IF 9.7
Interference of the complex between NCS-1 and Ric8a with phenothiazines regulates synaptic function and is an approach for fragile X syndrome.
Mansilla A, Chaves-Sanjuan A, Campillo NE, Semelidou O, Martinez-Gonzalez L, Infantes L, Gonzalez-Rubio JM, Gil C, Conde S, Skoulakis EM, Ferrus A, Martinez A, Sanchez-Barrena MJ

The protein complex formed by the Ca(2+) sensor neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor protein Ric8a coregulates synapse number and probability of neurotransmitter release, emerging as a potential therapeutic target for diseases affecting synapses, such as fragile X syndrome (FXS), the most common heritable autism disorder. Using crystallographic data and the virtual screening of a chemical library, we identified a set of heterocyclic small molecules as potential inhibitors of the NCS-1/Ric8a interaction. The aminophenothiazine FD44 interferes with NCS-1/Ric8a binding, and it restores normal synapse number and associative learning in a Drosophila FXS model. The synaptic effects elicited by FD44 feeding are consistent with the genetic manipulation of NCS-1. The crystal structure of NCS-1 bound to FD44 and the structure-function studies performed with structurally close analogs explain the FD44 specificity and the mechanism of inhibition, in which the small molecule stabilizes a mobile C-terminal helix inside a hydrophobic crevice of NCS-1 to impede Ric8a interaction. Our study shows the drugability of the NCS-1/Ric8a interface and uncovers a suitable region in NCS-1 for development of additional drugs of potential use on FXS and related synaptic disorders.

2017 | Methods Mol Biol
Dual Anterograde and Retrograde Viral Tracing of Reciprocal Connectivity.
Haberl MG, Ginger M, Frick A

Current large-scale approaches in neuroscience aim to unravel the complete connectivity map of specific neuronal circuits, or even the entire brain. This emerging research discipline has been termed connectomics. Recombinant glycoprotein-deleted rabies virus (RABV G) has become an important tool for the investigation of neuronal connectivity in the brains of a variety of species. Neuronal infection with even a single RABV G particle results in high-level transgene expression, revealing the fine-detailed morphology of all neuronal features-including dendritic spines, axonal processes, and boutons-on a brain-wide scale. This labeling is eminently suitable for subsequent post-hoc morphological analysis, such as semiautomated reconstruction in 3D. Here we describe the use of a recently developed anterograde RABV G variant together with a retrograde RABV G for the investigation of projections both to, and from, a particular brain region. In addition to the automated reconstruction of a dendritic tree, we also give as an example the volume measurements of axonal boutons following RABV G-mediated fluorescent marker expression. In conclusion RABV G variants expressing a combination of markers and/or tools for stimulating/monitoring neuronal activity, used together with genetic or behavioral animal models, promise important insights in the structure-function relationship of neural circuits.

Layer 5 (L5) is a major neocortical output layer containing L5A slender-tufted (L5A-st) and L5B thick-tufted (L5B-tt) pyramidal neurons. These neuron types differ in their in vivo firing patterns, connectivity and dendritic morphology amongst other features, reflecting their specific functional role within the neocortical circuits. Here, we asked whether the active properties of the basal dendrites that receive the great majority of synaptic inputs within L5 differ between these two pyramidal neuron classes. To quantify their active properties, we measured the efficacy with which action potential (AP) firing patterns backpropagate along the basal dendrites by measuring the accompanying calcium transients using two-photon laser scanning microscopy in rat somatosensory cortex slices. For these measurements we used both 'artificial' three-AP patterns and more complex physiological AP patterns that were previously recorded in anesthetized rats in L5A-st and L5B-tt neurons in response to whisker stimulation. We show that AP patterns with relatively few APs (3APs) evoke a calcium response in L5B-tt, but not L5A-st, that is dependent on the temporal pattern of the three APs. With more complex in vivo recorded AP patterns, the average calcium response was similar in the proximal dendrites but with a decay along dendrites (measured up to 100 mum) of L5B-tt but not L5A-st neurons. Interestingly however, the whisker evoked AP patterns-although very different for the two cell types-evoke similar calcium responses. In conclusion, although the effectiveness with which different AP patterns evoke calcium transients vary between L5A-st and L5B-tt cell, the calcium influx appears to be tuned such that whisker-evoked calcium transients are within the same dynamic range for both cell types.

17/06/2016 | Nat Commun   IF 11.3
Early synaptic deficits in the APP/PS1 mouse model of Alzheimer's disease involve neuronal adenosine A2A receptors.
Viana da Silva S, Haberl MG, Zhang P, Bethge P, Lemos C, Goncalves N, Gorlewicz A, Malezieux M, Goncalves FQ, Grosjean N, Blanchet C, Frick A, Nagerl UV, Cunha RA, Mulle C

Synaptic plasticity in the autoassociative network of recurrent connections among hippocampal CA3 pyramidal cells is thought to enable the storage of episodic memory. Impaired episodic memory is an early manifestation of cognitive deficits in Alzheimer's disease (AD). In the APP/PS1 mouse model of AD amyloidosis, we show that associative long-term synaptic potentiation (LTP) is abolished in CA3 pyramidal cells at an early stage. This is caused by activation of upregulated neuronal adenosine A2A receptors (A2AR) rather than by dysregulation of NMDAR signalling or altered dendritic spine morphology. Neutralization of A2AR by acute pharmacological inhibition, or downregulation driven by shRNA interference in a single postsynaptic neuron restore associative CA3 LTP. Accordingly, treatment with A2AR antagonists reverts one-trial memory deficits. These results provide mechanistic support to encourage testing the therapeutic efficacy of A2AR antagonists in early AD patients.

Post-learning hippocampal sharp wave-ripples (SWRs) generated during slow wave sleep are thought to play a crucial role in memory formation. While in Alzheimer's disease, abnormal hippocampal oscillations have been reported, the functional contribution of SWRs to the typically observed spatial memory impairments remains unclear. These impairments have been related to degenerative synaptic changes produced by soluble amyloid beta oligomers (Abetaos) which, surprisingly, seem to spare the SWR dynamics during routine behavior. To unravel a potential effect of Abetaos on SWRs in cognitively-challenged animals, we submitted vehicle- and Abetao-injected mice to spatial recognition memory testing. While capable of forming short-term recognition memory, Abeta mice exhibited faster forgetting, suggesting successful encoding but an inability to adequately stabilize and/or retrieve previously acquired information. Without prior cognitive requirements, similar properties of SWRs were observed in both groups. In contrast, when cognitively challenged, the post-encoding and -recognition peaks in SWR occurrence observed in controls were abolished in Abeta mice, indicating impaired hippocampal processing of spatial information. These results point to a crucial involvement of SWRs in spatial memory formation and identify the Abeta-induced impairment in SWRs dynamics as a disruptive mechanism responsible for the spatial memory deficits associated with Alzheimer's disease.

11/2015 | sci adv
Structural-functional connectivity deficits of neocortical circuits in the Fmr1 (-/y) mouse model of autism.
Haberl MG, Zerbi V, Veltien A, Ginger M, Heerschap A, Frick A

Fragile X syndrome (FXS), the most common inherited form of intellectual disability disorder and a frequent cause of autism spectrum disorder (ASD), is characterized by a high prevalence of sensory symptoms. Perturbations in the anatomical connectivity of neocortical circuits resulting in their functional defects have been hypothesized to contribute to the underlying etiology of these disorders. We tested this idea by probing alterations in the functional and structural connectivity of both local and long-ranging neocortical circuits in the Fmr1 (-/y) mouse model of FXS. To achieve this, we combined in vivo ultrahigh-field diffusion tensor magnetic resonance imaging (MRI), functional MRI, and viral tracing approaches in adult mice. Our results show an anatomical hyperconnectivity phenotype for the primary visual cortex (V1), but a disproportional low connectivity of V1 with other neocortical regions. These structural data are supported by defects in the structural integrity of the subcortical white matter in the anterior and posterior forebrain. These anatomical alterations might contribute to the observed functional decoupling across neocortical regions. We therefore identify FXS as a 'connectopathy,' providing a translational model for understanding sensory processing defects and functional decoupling of neocortical areas in FXS and ASD.

10/11/2014 | Nat Neurosci   IF 15
Dendritic channelopathies contribute to neocortical and sensory hyperexcitability in Fmr1 mice.
Zhang Y*, Bonnan A*, Bony G*, Ferezou I, Pietropaolo S, Ginger M, Sans N, Rossier J, Oostra B, Lemasson G, Frick A

Hypersensitivity in response to sensory stimuli and neocortical hyperexcitability are prominent features of Fragile X Syndrome (FXS) and autism spectrum disorders, but little is known about the dendritic mechanisms underlying these phenomena. We found that the primary somatosensory neocortex (S1) was hyperexcited in response to tactile sensory stimulation in Fmr1-/y mice. This correlated with neuronal and dendritic hyperexcitability of S1 pyramidal neurons, which affect all major aspects of neuronal computation, from the integration of synaptic input to the generation of action potential output. Using dendritic electrophysiological recordings, calcium imaging, pharmacology, biochemistry and a computer model, we found that this defect was, at least in part, attributable to the reduction and dysfunction of dendritic h- and BKCa channels. We pharmacologically rescued several core hyperexcitability phenomena by targeting BKCa channels. Our results provide strong evidence pointing to the utility of BKCa channel openers for the treatment of the sensory hypersensitivity aspects of FXS.

Dendritic spines are basic units of neuronal information processing and their structure is closely reflected in their function. Defects in synaptic development are common in neurodevelopmental disorders, making detailed knowledge of age-dependent changes in spine morphology essential for understanding disease mechanisms. However, little is known about the functionally important fine-morphological structures, such as spine necks, due to the limited spatial resolution of conventional light microscopy. Using stimulated emission depletion microscopy (STED), we examined spine morphology at the nanoscale during normal development in mice, and tested the hypothesis that it is impaired in a mouse model of fragile X syndrome (FXS). In contrast to common belief, we find that, in normal development, spine heads become smaller, while their necks become wider and shorter, indicating that synapse compartmentalization decreases substantially with age. In the mouse model of FXS, this developmental trajectory is largely intact, with only subtle differences that are dependent on age and brain region. Together, our findings challenge current dogmas of both normal spine development as well as spine dysgenesis in FXS, highlighting the importance of super-resolution imaging approaches for elucidating structure-function relationships of dendritic spines.

11/04/2014 | Brain Struct Funct   IF 4.6
An anterograde rabies virus vector for high-resolution large-scale reconstruction of 3D neuron morphology.
Haberl MG*, Viana da Silva S*, Guest JM, Ginger M, Ghanem A, Mulle C, Oberlaender M, Conzelmann KK, Frick A

Glycoprotein-deleted rabies virus (RABV G) is a powerful tool for the analysis of neural circuits. Here, we demonstrate the utility of an anterograde RABV G variant for novel neuroanatomical approaches involving either bulk or sparse neuronal populations. This technology exploits the unique features of RABV G vectors, namely autonomous, rapid high-level expression of transgenes, and limited cytotoxicity. Our vector permits the unambiguous long-range and fine-scale tracing of the entire axonal arbor of individual neurons throughout the brain. Notably, this level of labeling can be achieved following infection with a single viral particle. The vector is effective over a range of ages (>14 months) aiding the studies of neurodegenerative disorders or aging, and infects numerous cell types in all brain regions tested. Lastly, it can also be readily combined with retrograde RABV G variants. Together with other modern technologies, this tool provides new possibilities for the investigation of the anatomy and physiology of neural circuits.

Dendritic spines are basic units of neuronal information processing and their structure is closely reflected in their function. Defects in synaptic development are common in neurodevelopmental disorders, making detailed knowledge of age-dependent changes in spine morphology essential for understanding disease mechanisms. However, little is known about the functionally important fine- morphological structures, such as spine necks, due to the limited spatial resolution of conventional light microscopy. Using stimulated emission depletion microscopy (STED), we examined spine morphology at the nanoscale during normal development in mice, and tested the hypothesis that it is impaired in a mouse model of fragile X syndrome (FXS). In contrast to common belief, we find that, in normal development, spine heads become smaller, while their necks become wider and shorter, indicating that synapse compartmentalization decreases substantially with age. In the mouse model of FXS, this developmental trajectory is largely intact, with only subtle differences that are dependent on age and brain region. Together, our findings challenge current dogmas of both normal spine development as well as spine dysgenesis in FXS, highlighting the importance of super-resolution imaging approaches for elucidating structure–function relationships of dendritic spines.

Most neuron types possess elaborate dendritic arbors that receive and integrate excitatory and inhibitory inputs from numerous other neurons to give rise to cell-type specific firing patterns. The computational properties of these dendrites are therefore crucial for neuronal information processing, and are strongly determined by the expression of many types of voltage-gated ion channels in their membrane. The dendritic distribution patterns of these ion channels are characteristic for each ion channel type, are dependent on the neuronal identity, and can be modified in a plastic or pathophysiological manner. We present a method that enables us to semi-automatically map and quantify in 3D the expression levels of specific ion channel types across the entire dendritic arbor. To achieve this, standard immunohistochemistry was combined with reconstruction and quantification procedures for the localization and relative distribution of ion channels with respect to dendritic morphology. This method can, in principle, be applied to any fluorescent signal, including fluorescently tagged membrane proteins, RNAs, or intracellular signaling molecules.

2013 | Front Neural Circuits
Revealing the secrets of neuronal circuits with recombinant rabies virus technology.
Ginger M*, Haberl M*, Conzelmann KK, Schwarz MK, Frick A

An understanding of how the brain processes information requires knowledge of the architecture of its underlying neuronal circuits, as well as insights into the relationship between architecture and physiological function. A range of sophisticated tools is needed to acquire this knowledge, and recombinant rabies virus (RABV) is becoming an increasingly important part of this essential toolbox. RABV has been recognized for years for its properties as a synapse-specific trans-neuronal tracer. A novel genetically modified variant now enables the investigation of specific monosynaptic connections. This technology, in combination with other genetic, physiological, optical, and computational tools, has enormous potential for the visualization of neuronal circuits, and for monitoring and manipulating their activity. Here we will summarize the latest developments in this fast moving field and provide a perspective for the use of this technology for the dissection of neuronal circuit structure and function in the normal and diseased brain.

Loss of the RNA-binding fragile X protein [fragile X mental retardation protein (FMRP)] results in a spectrum of cognitive deficits, the fragile X syndrome (FXS), while aging individuals with decreased protein levels present with a subset of these symptoms and tremor. The broad range of behavioral deficits likely reflects the ubiquitous distribution and multiple functions of the protein. FMRP loss is expected to affect multiple neuronal proteins and intracellular signaling pathways, whose identity and interactions are essential in understanding and ameliorating FXS symptoms. We used heterozygous mutants and targeted RNA interference-mediated abrogation in Drosophila to uncover molecular pathways affected by FMRP reduction. We present evidence that FMRP loss results in excess metabotropic glutamate receptor (mGluR) activity, attributable at least in part to elevation of the protein in affected neurons. Using high-resolution behavioral, genetic, and biochemical analyses, we present evidence that excess mGluR upon FMRP attenuation is linked to the cAMP decrement reported in patients and models, and underlies olfactory associative learning and memory deficits. Furthermore, our data indicate positive transcriptional regulation of the fly fmr1 gene by cAMP, via protein kinase A, likely through the transcription factor CREB. Because the human Fmr1 gene also contains CREB binding sites, the interaction of mGluR excess and cAMP signaling defects we present suggests novel combinatorial pharmaceutical approaches to symptom amelioration upon FMRP attenuation.

2012 | Nat Commun   IF 10
Uncoupling of the endocannabinoid signalling complex in a mouse model of fragile X syndrome.
Jung KM , Sepers M , Henstridge CM , Lassalle O , Neuhofer D , Martin H , Ginger M , Frick A , DiPatrizio NV , Mackie K , Katona I , Piomelli D , Manzoni OJ

Fragile X syndrome, the most commonly known genetic cause of autism, is due to loss of the fragile X mental retardation protein, which regulates signal transduction at metabotropic glutamate receptor-5 in the brain. Fragile X mental retardation protein deletion in mice enhances metabotropic glutamate receptor-5-dependent long-term depression in the hippocampus and cerebellum. Here we show that a distinct type of metabotropic glutamate receptor-5-dependent long-term depression at excitatory synapses of the ventral striatum and prefrontal cortex, which is mediated by the endocannabinoid 2-arachidonoyl-sn-glycerol, is absent in fragile X mental retardation protein-null mice. In these mutants, the macromolecular complex that links metabotropic glutamate receptor-5 to the 2-arachidonoyl-sn-glycerol-producing enzyme, diacylglycerol lipase-alpha (endocannabinoid signalosome), is disrupted and metabotropic glutamate receptor-5-dependent 2-arachidonoyl-sn-glycerol formation is compromised. These changes are accompanied by impaired endocannabinoid-dependent long-term depression. Pharmacological enhancement of 2-arachidonoyl-sn-glycerol signalling normalizes this synaptic defect and corrects behavioural abnormalities in fragile X mental retardation protein-deficient mice. The results identify the endocannabinoid signalosome as a molecular substrate for fragile X syndrome, which might be targeted by therapy.

10/2010 | Cereb Cortex   IF 6.5
Cell type-specific thalamic innervation in a column of rat vibrissal cortex.
Meyer HS, Wimmer VC, Hemberger M, Bruno RM, de Kock CP, Frick A, Sakmann B, Helmstaedter M

This is the concluding article in a series of 3 studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). We used viral synaptophysin-enhanced green fluorescent protein expression in thalamic neurons and reconstructions of biocytin-labeled cortical neurons in TC slices to quantify the number and distribution of boutons from the ventral posterior medial (VPM) and posteromedial (POm) nuclei potentially innervating dendritic arbors of excitatory neurons located in layers (L)2-6 of a cortical column in rat somatosensory cortex. We found that 1) all types of excitatory neurons potentially receive substantial TC input (90-580 boutons per neuron); 2) pyramidal neurons in L3-L6 receive dual TC input from both VPM and POm that is potentially of equal magnitude for thick-tufted L5 pyramidal neurons (ca. 300 boutons each from VPM and POm); 3) L3, L4, and L5 pyramidal neurons have multiple (2-4) subcellular TC innervation domains that match the dendritic compartments of pyramidal cells; and 4) a subtype of thick-tufted L5 pyramidal neurons has an additional VPM innervation domain in L4. The multiple subcellular TC innervation domains of L5 pyramidal neurons may partly explain their specific action potential patterns observed in vivo. We conclude that the substantial potential TC innervation of all excitatory neuron types in a cortical column constitutes an anatomical basis for the initial near-simultaneous representation of a sensory stimulus in different neuron types.

Pyramidal neurons of layer 5A are a major neocortical output type and clearly distinguished from layer 5B pyramidal neurons with respect to morphology, in vivo firing patterns, and connectivity; yet knowledge of their dendritic properties is scant. We used a combination of whole-cell recordings and Ca(2+) imaging techniques in vitro to explore the specific dendritic signaling role of physiological action potential patterns recorded in vivo in layer 5A pyramidal neurons of the whisker-related 'barrel cortex'. Our data provide evidence that the temporal structure of physiological action potential patterns is crucial for an effective invasion of the main apical dendrites up to the major branch point. Both the critical frequency enabling action potential trains to invade efficiently and the dendritic calcium profile changed during postnatal development. In contrast to the main apical dendrite, the more passive properties of the short basal and apical tuft dendrites prevented an efficient back-propagation. Various Ca(2+) channel types contributed to the enhanced calcium signals during high-frequency firing activity, whereas A-type K(+) and BK(Ca) channels strongly suppressed it. Our data support models in which the interaction of synaptic input with action potential output is a function of the timing, rate and pattern of action potentials, and dendritic location.

15/02/2009 | J Physiol
Synaptic ionotropic glutamate receptors and plasticity are developmentally altered in the CA1 field of Fmr1 knockout mice
Pilpel Y, Kolleker A, Berberich S, Ginger M, Frick A, Mientjes E, Oostra B A, Seeburg P H

Fragile X syndrome is one of the most common forms of mental retardation, yet little is known about the physiological mechanisms causing the disease. In this study, we probed the ionotropic glutamate receptor content in synapses of hippocampal CA1 pyramidal neurons in a mouse model for fragile X (Fmr1 KO2). We found that Fmr1 KO2 mice display a significantly lower AMPA to NMDA ratio than wild-type mice at 2 weeks of postnatal development but not at 6-7 weeks of age. This ratio difference at 2 weeks postnatally is caused by down-regulation of the AMPA and up-regulation of the NMDA receptor components. In correlation with these changes, the induction of NMDA receptor-dependent long-term potentiation following a low-frequency pairing protocol is increased in Fmr1 KO2 mice at this developmental stage but not later in maturation. We propose that ionotropic glutamate receptors, as well as potentiation, are altered at a critical time point for hippocampal network development, causing long-term changes. Associated learning and memory deficits would contribute to the fragile X mental retardation phenotype.

15/01/2009 | J Physiol
Kinase-dependent modification of dendritic excitability after long-term potentiation
Rosenkranz J A, Frick A, Johnston D

Patterns of presynaptic activity properly timed with postsynaptic action potential output can not only increase the strength of synaptic inputs but can also increase the excitability of dendritic branches of adult CA1 pyramidal neurons. Here, we examined the role of protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) in the enhancement of dendritic excitability that occurs during theta-burst pairing of presynaptic and postsynaptic firing activity. Using dendritic and somatic whole-cell recordings in rat hippocampal slices, we measured the increase in the amplitude of back-propagating action potentials in the apical dendrite that occurs in parallel with long-term potentiation (LTP) of synaptic inputs. We found that inhibition of the MAPK pathway prevents this enhancement of dendritic excitability using either a weak or strong LTP induction protocol, while synaptic LTP can still be induced by the strong protocol. Both forms of plasticity are blocked by inhibition of PKA and occluded by interfering with cAMP degradation, consistent with a PKA-mediated increase in MAPK activity following induction of LTP. This provides a signalling mechanism for plasticity of dendritic excitability that occurs during neuronal activity and demonstrates the necessity of MAPK activation. Furthermore, this study uncovers an additional contribution of kinase activation to plasticity that may occur during learning.

Layer 5 (L5) of somatosensory cortex is a major gateway for projections to intra- and subcortical brain regions. This layer is further divided into 5A and 5B characterized by relatively separate afferent and efferent connections. Little is known about the organization of connections within L5A of neocortical columns. We therefore used paired recordings to probe the anatomy and physiology of monosynaptic connections between L5A pyramidal neurons within the barrel columns of somatosensory cortex in acute slices of approximately 3-week-old rats. Post hoc reconstruction and calculation of the axodendritic overlap of pre- and postsynaptic neurons, together with identification of putative synaptic contacts (3.5 per connection), indicated a preferred innervation domain in the proximal dendritic region. Synaptic transmission was reliable (failure rate <2%) and had a low variability (coefficient of variation of 0.3). Unitary excitatory postsynaptic potential (EPSP) amplitudes varied 30-fold with a mean of 1.2 mV and displayed depression over a wide range of frequencies (2-100 Hz) during bursts of presynaptic firing. A single L5A pyramidal neuron was estimated to target approximately 270 other pyramidal neurons within the same layer of its home barrel column, suggesting a mechanism of feed-forward excitation by which synchronized single action potentials are efficiently transmitted within L5A of juvenile cortex.

The probability of synaptic transmitter release determines the spread of excitation and the possible range of computations at unitary connections. To investigate whether synaptic properties between neocortical pyramidal neurons change during the assembly period of cortical circuits, whole-cell voltage recordings were made simultaneously from two layer 5A (L5A) pyramidal neurons within the cortical columns of rat barrel cortex. We found that synaptic transmission between L5A pyramidal neurons is very reliable between 2 and 3 weeks of postnatal development with a mean unitary EPSP amplitude of approximately 1.2 mV, but becomes less efficient and fails more frequently in the more mature cortex of approximately 4 weeks of age with a mean unitary EPSP amplitude of 0.65 mV. Coefficient of variation and failure rate increase as the unitary EPSP amplitude decreases during development. The paired-pulse ratio (PPR) of synaptic efficacy at 10 Hz changes from 0.7 to 1.04. Despite the overall increase in PPR, short-term plasticity displays a large variability at 4 weeks, ranging from strong depression to strong facilitation (PPR, range 0.6-2.1), suggesting the potential for use-dependent modifications at this intracortical synapse. In conclusion, the transmitter release probability at the L5A-L5A connection is developmentally regulated in such a way that in juvenile animals excitation by single action potentials is efficiently transmitted, whereas in the more mature cortex synapses might be endowed with a diversity of filtering characteristics.

22/11/2006 | J Neurosci
Deletion of Kv4.2 gene eliminates dendritic A-type K+ current and enhances induction of long-term potentiation in hippocampal CA1 pyramidal neurons
Chen X, Yuan L L, Zhao C, Birnbaum S G, Frick A, Jung W E, Schwarz T L, Sweatt J D, Johnston D

Dendritic, backpropagating action potentials (bAPs) facilitate the induction of Hebbian long-term potentiation (LTP). Although bAPs in distal dendrites of hippocampal CA1 pyramidal neurons are attenuated when propagating from the soma, their amplitude can be increased greatly via downregulation of dendritic A-type K+ currents. The channels that underlie these currents thus may represent a key regulatory component of the signaling pathways that lead to synaptic plasticity. We directly tested this hypothesis by using Kv4.2 knock-out mice. Deletion of the Kv4.2 gene and a loss of Kv4.2 protein resulted in a specific and near-complete elimination of A-type K+ currents from the apical dendrites of CA1 pyramidal neurons. The absence of dendritic Kv4.2-encoded A-type K+ currents led to an increase of bAP amplitude and an increase of concurrent Ca2+ influx. Furthermore, CA1 pyramidal neurons lacking dendritic A-type K+ currents from Kv4.2 knock-out mice exhibited a lower threshold than those of wild-type littermates for LTP induction with the use of a theta burst pairing protocol. LTP triggered with the use of a saturating protocol, on the other hand, remained indistinguishable between Kv4.2 knock-out and wild-type neurons. Our results support the hypothesis that dendritic A-type K+ channels, composed of Kv4.2 subunits, regulate action potential backpropagation and the induction of specific forms of synaptic plasticity.

11/04/2006 | Proc Natl Acad Sci U S A
A noncoding RNA is a potential marker of cell fate during mammary gland development
Ginger M R, Shore A N, Contreras A, Rijnkels M, Miller J, Gonzalez-Rimbau M F, Rosen J M

PINC is a large, alternatively spliced, developmentally regulated, noncoding RNA expressed in the regressed terminal ductal lobular unit-like structures of the parous mammary gland. Previous studies have shown that this population of cells possesses not only progenitor-like qualities (the ability to proliferate and repopulate a mammary gland) and the ability to survive developmentally programmed cell death but also the inhibition of carcinogen-induced proliferation. Here we report that PINC expression is temporally and spatially regulated in response to developmental stimuli in vivo and that PINC RNA is localized to distinct foci in either the nucleus or the cytoplasm in a cell-cycle-specific manner. Loss-of-function experiments suggest that PINC performs dual roles in cell survival and regulation of cell-cycle progression, suggesting that PINC may contribute to the developmentally mediated changes previously observed in the terminal ductal lobular unit-like structures of the parous gland. This is one of the first reports describing the functional properties of a large, developmentally regulated, mammalian, noncoding RNA.

01/07/2005 | J Neurobiol
Plasticity of dendritic excitability
Frick A, Johnston D

Dendrites are equipped with a plethora of voltage-gated ion channels that greatly enrich the computational and storage capacity of neurons. The excitability of dendrites and dendritic function display plasticity under diverse circumstances such as neuromodulation, adaptation, learning and memory, trauma, or disorders. This adaptability arises from alterations in the biophysical properties or the expression levels of voltage-gated ion channels-induced by the activity of neurotransmitters, neuromodulators, and second-messenger cascades. In this review we discuss how this plasticity of dendritic excitability could alter information transfer and processing within dendrites, neurons, and neural networks under physiological and pathological conditions.

The propagation and integration of signals in the dendrites of pyramidal neurons is regulated, in part, by the distribution and biophysical properties of voltage-gated ion channels. It is thus possible that any modification of these channels in a specific part of the dendritic tree might locally alter these signaling processes. Using dendritic and somatic whole-cell recordings, combined with calcium imaging in rat hippocampal slices, we found that the induction of long-term potentiation (LTP) was accompanied by a local increase in dendritic excitability that was dependent on the activation of NMDA receptors. These changes favored the back-propagation of action potentials into this dendritic region with a subsequent boost in the Ca(2+) influx. Dendritic cell-attached patch recordings revealed a hyperpolarized shift in the inactivation curve of transient, A-type K(+) currents that can account for the enhanced excitability. These results suggest an important mechanism associated with LTP for shaping signal processing and controlling dendritic function.

10/2003 | J Neurophysiol
A modified Sindbis vector for prolonged gene expression in neurons.
Jeromin A, Yuan LL, Frick A, Pfaffinger P, Johnston D

Sindbis viruses have been widely used in neurobiology to express a variety of genes in cultured neurons, in cultured slices, and in vivo. They provide fast onset and high levels of expression of foreign genes, but the expression is limited to a short time window due to a shut-off of host protein synthesis. We have used a mutation in an essential gene (nsP2) of the life cycle of Sindbis, which allows the functional analysis of changes in protein expression for >/=6 days after infection. This Sindbis mutant (nsP2) was used to express enhanced green fluorescent protein (EGFP) in hippocampal neurons in culture and in vivo without any sign of toxicity, based on two-photon imaging and electrophysiology. In addition, the EGFP mutant virus can be injected in vivo to visualize spines and other details of neuronal structure. The Sindbis mutant described here provides an improved tool in neurobiology with reduced cytotoxicity and a prolonged time window of expression for novel applications in imaging and behavior. In addition, the use of this vector for the functional expression of mammalian voltage-gated ion channels in organotypic slices is demonstrated.

29/04/2003 | Philos Trans R Soc Lond B Biol Sci
Active dendrites, potassium channels and synaptic plasticity.
Johnston D, Christie BR, Frick A, Gray R, Hoffman DA, Schexnayder LK, Watanabe S, Yuan LL

The dendrites of CA1 pyramidal neurons in the hippocampus express numerous types of voltage-gated ion channel, but the distributions or densities of many of these channels are very non-uniform. Sodium channels in the dendrites are responsible for action potential (AP) propagation from the axon into the dendrites (back-propagation); calcium channels are responsible for local changes in dendritic calcium concentrations following back-propagating APs and synaptic potentials; and potassium channels help regulate overall dendritic excitability. Several lines of evidence are presented here to suggest that back-propagating APs, when coincident with excitatory synaptic input, can lead to the induction of either long-term depression (LTD) or long-term potentiation (LTP). The induction of LTD or LTP is correlated with the magnitude of the rise in intracellular calcium. When brief bursts of synaptic potentials are paired with postsynaptic APs in a theta-burst pairing paradigm, the induction of LTP is dependent on the invasion of the AP into the dendritic tree. The amplitude of the AP in the dendrites is dependent, in part, on the activity of a transient, A-type potassium channel that is expressed at high density in the dendrites and correlates with the induction of the LTP. Furthermore, during the expression phase of the LTP, there are local changes in dendritic excitability that may result from modulation of the functioning of this transient potassium channel. The results support the view that the active properties of dendrites play important roles in synaptic integration and synaptic plasticity of these neurons.

15/04/2003 | J Neurosci
Normalization of Ca2+ signals by small oblique dendrites of CA1 pyramidal neurons.
Frick A, Magee J, Koester HJ, Migliore M, Johnston D

Oblique dendrites of CA1 pyramidal neurons predominate in stratum radiatum and receive approximately 80% of the synaptic input from Schaffer collaterals. Despite this fact, most of our understanding of dendritic signal processing in these neurons comes from studies of the main apical dendrite. Using a combination of Ca2+ imaging and whole-cell recording techniques in rat hippocampal slices, we found that the properties of the oblique dendrites differ markedly from those of the main dendrites. These different properties tend to equalize the Ca2+ rise from single action potentials as they backpropagate into the oblique dendrites from the main trunk. Evidence suggests that this normalization of Ca2+ signals results from a higher density of a transient, A-type K+ current [I(K(A))] in the oblique versus the main dendrites. The higher density of I(K(A)) may have important implications for our understanding of synaptic integration and plasticity in these structures.

12/2001 | Ann N Y Acad Sci
Mechanisms of hormonal prevention of breast cancer.
Medina D, Sivaraman L, Hilsenbeck SG, Conneely O, Ginger M, Rosen J, Omalle BW

Reproductive history is a consistent risk factor for human breast cancer. Epidemiological studies have repeatedly demonstrated that early age of first pregnancy is a strong protective factor against breast cancer and provides a physiologically operative model to achieve a practical mode of prevention. In rodents, the effects of full-term pregnancy can be mimicked by a three-week exposure to low doses of estrogen and progesterone. Neither hormone alone is sufficient to induce protection. The cellular and molecular mechanisms that underlie hormone-induced refractoriness are largely unresolved. Our recent studies have demonstrated that an early cellular response that is altered in hormone-treated mammary cells is the initial proliferative burst induced by the chemical carcinogen methylnitrosourea. The decrease in proliferation is also accompanied by a decrease in the ability of estrogen receptor-positive cells to proliferate. RNA expression of several mammary cell-cycle-related genes is not altered in hormone-treated mice; however, immunohistochemical assays demonstrate that the protein level and nuclear compartmentalization of the p53 tumor suppressor gene are markedly upregulated as a consequence of hormone treatment. These results support the hypothesis that hormone stimulation, at a critical period in mammary development, results in cells with persistent changes in the intracellular regulatory loops governing proliferation and response to DNA damage. A corollary to this hypothesis is that the genes affected by estrogen and progesterone are independent of alveolar differentiation-specific genes. Suppressive subtractive hybridization-PCR methods have identified several genes that are differentially expressed as a consequence of prior estrogen and progesterone treatment. Future experiments are aimed at determining the mechanisms of hormone-induced upregulation of p53 protein expression as part of the overall goal of identifying and functionally characterizing the genes responsible for the refractory phenotype.

Apical dendrites of layer V cortical pyramidal neurons are a major target for glutamatergic synaptic inputs from cortical and subcortical brain regions. Because innervation from these regions is somewhat laminar along the dendrites, knowing the distribution of glutamate receptors on the apical dendrites is of prime importance for understanding the function of neural circuits in the neocortex. To examine this issue, we used infrared-guided laser stimulation combined with whole cell recordings to quantify the spatial distribution of glutamate receptors along the apical dendrites of layer V pyramidal neurons. Focally applied (<10 microm) flash photolysis of caged glutamate on the soma and along the apical dendrite revealed a highly nonuniform distribution of glutamate responsivity. Up to four membrane areas (extent 22 microm) of enhanced glutamate responsivity (hot spots) were detected on the dendrites with the amplitude and integral of glutamate-evoked responses at hot spots being three times larger than responses evoked at neighboring sites. We found no association of these physiological hot spots with dendritic branch points. It appeared that the larger responses evoked at hot spots resulted from an increase in activation of both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors and not a recruitment of voltage-activated sodium or calcium conductances. Stimulation of hot spots did, however, facilitate the triggering of both Na+ spikes and Ca(2+) spikes, suggesting that hot spots may serve as dendritic initiation zones for regenerative spikes.

01/10/1999 | Science
Precisely localized LTD in the neocortex revealed by infrared-guided laser stimulation.
Dodt H, Eder M, Frick A, Zieglgansberger W

In a direct approach to elucidate the origin of long-term depression (LTD), glutamate was applied onto dendrites of neurons in rat neocortical slices. An infrared-guided laser stimulation was used to release glutamate from caged glutamate in the focal spot of an ultraviolet laser. A burst of light flashes caused an LTD-like depression of glutamate receptor responses, which was highly confined to the region of 'tetanic' stimulation (<10 micrometers). A similar depression of glutamate receptor responses was observed during LTD of synaptic transmission. A spatially highly specific postsynaptic mechanism can account for the LTD induced by glutamate release.

11/1998 | Eur J Neurosci
NMDA and AMPA receptors on neocortical neurons are differentially distributed.
Dodt HU, Frick A, Kampe K, Zieglgansberger W

The distribution of glutamate receptor subtypes on the surface of neurons is highly relevant for synaptic activation and signal processing in the neocortex. As a novel approach we have used infra-red videomicroscopy in combination with photostimulation or microiontophoresis in brain slices of rat neocortex to map the distribution of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on pyramidal neurons of layer V. Both modes of application revealed a spatially distinct distribution of glutamate receptor subtypes: the soma and the proximal dendrite of neurons are highly sensitive to NMDA, whereas the more distal parts of the dendrite are more sensitive to AMPA. An implication is that NMDA receptors near the soma might regulate the amplification of synaptic signals resulting from AMPA receptor activation on remote dendritic sites.