Repeating Spatial-Temporal Motifs of CA3 Activity Dependent on Engineered Inputs from Dentate Gyrus Neurons in Live Hippocampal Networks.

Anatomical and behavioral studies, and in vivo and slice electrophysiology of the hippocampus suggest specific functions of the dentate gyrus (DG) and the CA3 subregions, but the underlying activity dynamics and repeatability of information processing remains poorly understood. To approach this problem, we engineered separate living networks of the DG and CA3 neurons that develop connections through 51 tunnels for axonal communication. Growing these networks on top of an electrode array enabled us to determine whether the subregion dynamics were separable and repeatable. We found spontaneous development of polarized propagation of 80% of the activity in the native direction from DG to CA3 and different spike and burst dynamics for these subregions. Spatial-temporal differences emerged when the relationships of target CA3 activity were categorized with to the number and timing of inputs from the apposing network. Compared to times of CA3 activity when there was no recorded tunnel input, DG input led to CA3 activity bursts that were 7× more frequent, increased in amplitude and extended in temporal envelope. Logistic regression indicated that a high number of tunnel inputs predict CA3 activity with 90% sensitivity and 70% specificity. Compared to no tunnel input, patterns of >80% tunnel inputs from DG specified different patterns of first-to-fire neurons in the CA3 target well. Clustering dendrograms revealed repeating motifs of three or more patterns at up to 17 sites in CA3 that were importantly associated with specific spatial-temporal patterns of tunnel activity. The number of these motifs recorded in 3 min was significantly higher than shuffled spike activity and not seen above chance in control networks in which CA3 was apposed to CA3 or DG to DG. Together, these results demonstrate spontaneous input-dependent repeatable coding of distributed activity in CA3 networks driven by engineered inputs from DG networks. These functional configurations at measured times of activation (motifs) emerge from anatomically accurate feed-forward connections from DG through tunnels to CA3

Oscillatory and epileptiform activity in human and rodent cortical regions in vitro

Epilepsy is a chronic neurological disorder in which patients have spontaneous recurrent seizures. Approximately 50 million people worldwide live with epilepsy and of those ~30% fail to adequately respond to anti-epileptic drugs (AEDs), indicating a need for further research. In this study oscillatory and epileptiform activity was explored in the rodent piriform cortex (PC) in vitro, an underexplored brain region implicated in the development of epilepsy. PC gamma oscillations have been studied in both anaesthetised and awake rodents in vivo; however, to date they have not been reported in vitro. Extracellular field potential recordings were made in rodent PC brain slices prepared from 70-100g male Wistar rats in vitro. Application of kainic acid and carbachol reliably induced persistent gamma oscillations (30 – 40 Hz) in layer II of the PC. These oscillations were found to be pharmacologically similar to gamma oscillations previously found in other rodent brain regions in vitro, as they were dependent on GABAA receptors, AMPA receptors and gap junctions. Persistent oscillations were also induced and characterised for the first time in human neuronal tissue in vitro. Human brain slices were prepared from excised tissue from various brain regions (primarily temporal) from paediatric patients undergoing surgery to alleviate the symptoms of drug resistant epilepsy. As in the rodent PC, oscillations were induced by application of kainic acid and carbachol, however, these oscillations were found to be within the beta frequency range (12 – 30 Hz). Despite this difference in frequency band, these beta oscillations were pharmacologically similar to gamma oscillations found in the rodent PC. Seizure-like events (SLEs) were induced in brain slices prepared from 70-100g male Wistar rats via application of zero Mg2+ artificial cerebral spinal fluid (0[Mg]2+ aCSF). The properties of these SLEs were found to be similar between brain regions when recordings were performed in layer II of the anterior and posterior PC and lateral entorhinal cortex (LEC) and the stratum pyramidale of CA1. In the majority of recordings SLEs occurred in the PC before the LEC or CA1 and SLEs were displayed in the PC in a higher proportion of slices than the LEC. The sensitivity of these PC slices to 0[Mg]2+ aCSF was assessed at several stages (24 hours and 1 week (early latent), 4 weeks (mid latent) and 3 months+ (chronic period)) following the reduced intensity status epilepticus (SE) protocol for epilepsy induction compared to age-matched controls (AMCs). A decrease in excitability of the slices was observed in slices prepared from AMC animals with age, as the inter-event interval and latency to first SLE was observed to be longer in slices prepared from aged compared to young AMC animals. Slices prepared from SE animals maintained their youthful hyperexcitability with no difference in IEI or latency to first SLE observed in the early latent period compared to the chronic period. The pharmacoresistance (or sensitivity) of these SLEs to single and double AED challenge was evaluated. Differences in efficacy of the AEDs were found between SE and AMC in the mid-latent period; increased efficacy of Na+ channel modulating AEDs were found in slices prepared from SE compared to AMC animals. The proportion of slices that displayed pharmacoresistance of these SLEs to AEDs was found to be higher in slices prepared from young animals (early latent period and AMCs), and was similar to that found clinically in human patients. The pharmacoresistance of the SLEs to AEDs was lower in slices prepared from older animals (mid latent, chronic and AMCs) compared to young animals (early latent and AMCs). This age-dependent reduction in resistance likely reflects normal alterations in neuronal networks with ageing. SLEs induced in young control PC slices could be exploited as a new in vitro model of drug resistant epilepsy. Overall, oscillatory and epileptiform activity in the PC and human cortex in vitro could be further explored as tools to evaluate the efficacy and mechanism of action of newly developed AEDs, as well as to explore the networks involved in drug resistant epilepsy

LOCAL SUPPLEMENTATION OF BRAINDERIVED NEUROTROPHIC FACTOR FOR THE TREATMENT OF NEURONAL DAMAGE.

The importance of nerve growth factors, especially brain-derived neurotrophic factor (BDNF) in the regulation of neuronal survival and plastic changes in morphology and function has been increasingly studied during the recent years. It has been proposed that the pathogenesis of some neurological diseases may be due to an alteration in neurotrophic factor and/or Trk receptor levels. The use of neurotrophic factors as therapeutic agents is a promising approach aimed at restoring and maintaining neuronal function in the central nervous system (CNS). This study is undertaken to develop a novel stem cell-based gene therapy to deliver neurotrophic factors to vulnerable regions of the CNS. Stem cell-based gene therapy is a potential delivery option by which cells are engineered to produce neurotrophic factors in vitro and then transplanted to the target area where neurotrophic factors are secreted to exert protective and/or restorative effects on the host tissue. A recently isolated mesodermal stem cell, mesoangioblast (MAB), has a high adhesin-dependent migratory capacity and may selectively cross the blood-brain barrier and home in the lesioned areas. Therefore, MABs provide an ideal cellular source for BDNF delivery. In this study, we generated a genetically modified mesoangioblast producing BDNF (MABs-BDNF). These engineered MABs maintained transgene expression and secretion of bioactive BDNF in time. We investigated the protective effects of MABs-BDNF in vitro using primary cultures and organotypic cultures of hippocampal slices. The viability of the cultured slices was assessed in several ways: fluorescein diacetate (FDA) hydrolysis assay, lactate dehydrogenase (LDH) release assay, immunohistochemistry for MAP2, immunoblot for neurofilament 68, and field potential recordings. Direct exposure of recombinant BDNF to primary cultured neurons and adult slices resulted in a concentration-dependant protective effect. The conditioned medium from MABs-BDNF highly promoted cell survival, while the conditioned medium from control cells (MABs) or an equivalent amount of rBDNF showed beneficial effects on cell survival to a lesser extent. The protective effects of MABs-BDNF were attenuated by adding either with the TrkB receptor blocker K252a or the BDNF scavenger TrkB-IgG.. This indicates that the conditioned medium from MABs-BDNF can foster the adult slice culture through secreting the engineered BDNF and unknown pro-survival factors produced intrinsically by MABs. The MABs-BDNF conditioned medium was optimal for retention of morphologic characteristics and viability in organotypic cultures from adult hippocampal slices. Moreover, MABs-BDNF were found to promote neurogenesis and glia proliferation. Treatment with the MABs-BDNF conditioned medium was found to increase the number of BrdU-labeled and BrdU/NeuN double labeled cells in the dentate gyrus of cultured slices. These in vitro findings demonstrate the beneficial effects of MABs-BDNF on neurons and provide a rationale for transplanting MABs-BDNF in the damaged brain as a therapeutic approach. Thus, we tested the transplantation of MABs-BDNF in an animal model of neuronal loss, the hippocampal sclerosis induced by status epilepticus. So far, we have not detected a deposition of MABs-BDNF in the epileptic brain after their systemic administration. Future experiments will aim at optimizing the transplanting conditions, changing the delivering routes, and assessing their therapeutic value in other neurological diseases associated with cell death. In terms of their prominent beneficial by-stander effects on neurons, MABs-BDNF hold substantial promise as therapeutic agents in the treatment of neurological diseases

Experience-dependent structural rearrangements of synaptic connectivity in the adult central nervous system

The functioning of the brain critically relies on its capacity to adapt and respond to its environment. The brain’s ability to change in response to experience is called plasticity and underlies principal brain functions, such as learning and memory. My thesis work investigated the ability of the brain to structurally remodel upon altered experiences, and changes that occur during normal aging. Furthermore, I addressed what might be the molecular mechanisms regulating such remodeling. I will therefore start by introducing the term of experience-dependent plasticity and exemplify the brain’s capacity to adapt to changes in experience and usage. I will then attempt to describe mechanisms of experience-dependent plasticity on the functional, molecular and structural level. Furthermore, I will discuss the impact of age and life-style on the brain’s capacity for plasticity. Finally, I will close the introduction by outlining the function and anatomy of the brain region that was the main subject of our investigations, namely the hippocampus, and specifically the mossy fiber pathwa

The spontaneous activity of organotypic and dissociated networks

In the absence of external stimuli, the nervous system exhibits a spontaneous electrical activity whose functions are not fully understood, and that represents the background noise of brain operations. In vitro models have long represented a simple and useful tool for studying the basic properties of neurons and networks. This study provides a detailed characterization of spontaneous activity of neuronal networks in different in vitro models. In particular, it clarifies the role of the extra-cellular environment and of the intrinsic architecture in shaping the spontaneous activity of networks by means of calcium imaging techniques. The results presented within this study come from three experimental works, each one addressing a particular feature of the network model: \u2022 Chemical composition of the extra-cellular environment: a comparison of dissociated hippocampal cultures grown in three different culturing media revealed that the use of an astrocyte-conditioned medium improves significantly the frequency and synchronization of neuronal signaling. \u2022 Mechanical and topographical properties of the extra-cellular environment: the design of a hybrid micro-nano substrate for dissociated hippocampal cultures revealed that nano-scaled patterns provide an improved artificial extra-cellular matrix for obtaining neuronal networks with a frequent spontaneous signaling. \u2022 Network architecture: synchronized events called Global Up states - involving the totality of neurons in the network - are observed in both organotypic and dissociated neurons; the duration of Global Up states increases by increasing the complexity of the network, while their frequency decreases. Simulations with simplified models of single- and multilayered networks confirm the experimental data. Taken together, these results show that the spontaneous synchronous activity of neurons is a result of their intrinsic biophysical properties, arising also after disruption of the original network architecture. However, dissociated neurons show different levels of synchrony depending on the chemical and topographical composition of the surrounding artificial extra-cellular matrix. Moreover, the specific architecture of the network and its single- or multilayered composition has an influence on the frequency and duration of spontaneous events, suggesting a potential explanation for the diversity of oscillatory rhythms observed in the brain

NLRP3 inflammasome as a target to reduce epileptiform activity in organotypic slices

Tese de mestrado, Neurociências, Universidade de Lisboa, Faculdade de Medicina, 2018A epilepsia é uma doença do foro neurológico caracterizada pela predisposição duradoura para gerar crises epilépticas. Cada crise é uma perturbação transitória da atividade neuronal que se torna síncrona e excessiva, interrompendo, momentaneamente, a função normal do cérebro. Calcula-se que, em Portugal, 1 em cada 200 pessoas tenha esta disfunção do sistema neurológico. Não existe cura para a epilepsia, no entanto, existem fármacos antiepilépticos (FAEs) que previnem a ocorrência de crises epilépticas. Os FAEs são prescritos de acordo com o tipo de epilepsia e com os fatores individuais de cada pessoa. Por vezes são necessárias várias tentativas e/ou politerapia para encontrar a medicamentação adequada para controlar as crises. Contudo nem todas as pessoas reagem aos FAEs. Estima-se que 30-40% das pessoas em todo o mundo sofrem de epilepsia refratária, ou seja, não têm a doença controlada. Uma vez que a epilepsia refratária pode ser incapacitante em algumas vertentes pessoais e profissionais e existe um elevado número de pessoas a sofrer desta doença, é necessário uma melhor compreensão da epilepsia e do processo de epileptogénese. Este processo é responsável por tornar um cérebro normal num cérebro com atividade neuronal anormal. Além disso, também é imprescindível testar novas terapias antiepileptogénicas que possibilitem uma melhor qualidade de vida a estes doentes. A maioria dos FAEs modula os mecanismos excitatórios e inibitórios relacionados direta ou indiretamente com a neurotransmissão. No entanto, tem havido um crescente interesse no estudo de moléculas/ fármacos anti-inflamatórios como terapias antiepileptogénicas. Além dos neurónios, as células gliais, ou seja, os astrócitos e a microglia, têm um papel importante no processo de epileptogénese. Estas células gliais estão envolvidas no processo de neuroinflamação produzindo diversas citocinas e outras moléculas que irão potenciar a inflamação. Atualmente sabe-se que existe um circuito positivo entre a epilepsia e a inflamação. A atividade epiléptica promove a libertação de fatores inflamatórios e a inflamação, por sua vez, potencia a atividade epiléptica. Uma das citocinas pro-inflamatórias mais estudada no âmbito da epilepsia é a interleucina-1β (IL-1β). Estudos recentes demonstraram que a expressão desta molécula está aumentada em modelos animais de epilepsia, bem como em pacientes com esta patologia. A IL-1β é produzida por um complexo multiproteico associado à imunidade inata denominado de inflamassoma NLRP3. Este complexo é ativado na presença de agentes patogénicos ou perigosos ao organismo, tais como os lipopolissacarídeos (LPS) e a adenosina trifosfato (ATP). Os LPS são moléculas que estão presentes na membrana exterior de bactérias gram-negativas, enquanto o ATP é uma molécula que transporta energia e é essencial às células. Ao ser ativado, o inflamassoma NLRP3 promove a clivagem da pro-IL-1β (forma inativa) em IL-1β, através da ativação da capase-1. Na forma ativa, a IL-1β sai da célula e promove a inflamação nas células vizinhas. Diversos estudos têm-se focado na modulação do mecanismo de ação desta interleucina, através de anticorpos anti-IL-1β ou através de antagonistas do seu recetor. Medicamentos que contêm substâncias como Anacinra (antagonista do recetor humano da interleucina-1) ou Canacinumab (anticorpo anti-IL-1β monoclonal totalmente humanizado) têm sido amplamente utilizados no tratamento de doenças relacionadas com o inflamassoma NLRP3, tal como a artrite reumatóide. No entanto, até 2016 ainda nenhum destes medicamentos tinha sido testado em doentes epilépticos. Casos clínicos reportados recentemente têm demonstrado a eficácia destes medicamentos em doentes com epilepsia refratária. Até então, apenas se tinham realizado estudos com substâncias similares em modelos animais. Adicionalmente, também foram desenvolvidos estudos para inibir a caspase-1, que tiveram sucesso na supressão de atividade epiléptica em modelos animais, mas não foram aprovados nos ensaios clínicos, ficando apenas na fase II. O presente estudo teve como principal objetivo modular a ativação do inflamassoma NLRP3 e avaliar o impacto na atividade epiléptica de fatias organotípicas. As fatias organotípicas são um ótimo modelo ex vivo, uma vez que preservam a arquitetura tridimensional, as conexões neuronais e as interações entre os neurónios e células gliais, por longos períodos. Além disso, também permitem testar potenciais fármacos de modo mais rápido, menos doloroso e menos dispendioso do que aconteceria em modelos animais. Inicialmente foi estabelecido o modelo de ativação do inflamassoma NLRP3 em fatias organotípicas de hipocampo e córtex preparadas a partir dos cérebros de ratos Sprague-Dawley com 6/7 dias de vida, por exposição a diferentes concentrações de LPS (5, 10 e 20ng/mL) durante 3 e 6h ou a LPS (10ng/mL) e ATP (1mM) simultaneamente. Após uma pré-incubação apenas com LPS durante 3h, o ATP foi co-incubado com o LPS durante mais 3h. De modo a escolher a melhor condição de ativação do inflamassoma, estudou-se a expressão proteica da αII-espectrina, para avaliar a morte celular, de componentes do inflamassoma (NLRP3 e ASC), e de marcadores das células gliais (Iba1 e GFAP). Adicionalmente foram quantificados, no tecido e no meio de cultura, os níveis de duas citocinas pro-inflamatórias: a IL-1β e os Fatores de Necrose Tumoral Alfa (TNF-α). Verificou-se que a exposição das fatias organotípicas ao LPS/ATP promoveu a diminuição de necrose, bem como a potenciação da libertação de citocinas para o meio extracelular. Uma vez que estes processos são característicos da ativação do inflamassoma, optou-se por utilizar esta condição. Posteriormente foi verificado o efeito da ativação do inflamassoma, pelo LPS/ATP, nas células gliais e na atividade epileptiforme. Através da técnica de imunohistoquímica foi possível observar a migração da microglia em direção ao meio de cultura. Na presença de LPS/ATP, a secção da fatia mais próxima do meio de cultura apresentava maior densidade celular, relativamente ao controlo, e maior ativação da microglia. Estas características correspondem a um processo de microgliose. Uma vez que existia elevada produção de IL-1β e microgliose nas fatias expostas a LPS/ATP antecipou-se um aumento na atividade epileptiforme nestas fatias. No entanto, tal não foi observado. As fatias expostas aos ativadores do inflamassoma apresentaram um número de bursts por fatia e características intrínsecas dos bursts semelhantes aos observados em fatias controlo. Os bursts são conjuntos de atividade neuronal excessiva que representam a fase ictal (correspondente à crise epiléptica). Após uma avaliação cuidada verificou-se que as fatias controlo, não sujeitas à adição de fármacos, exibiam uma elevada atividade epileptiforme. As fatias denominadas controlo neste estudo sofreram apenas uma mudança do meio de cultura, processo este que já havia sido descrito por diversos estudos. Durante os primeiros 14 dias in vitro, as fatias foram cultivadas num meio que continha soro de cavalo (meio Opti-MEM). Como este soro não é quimicamente definido, ou seja, varia de lote para lote, o meio de cultura foi alterado para um meio sem soro (meio Neurobasal A) no dia antes da adição dos fármacos. No entanto, verificou-se que as fatias controlo apresentavam claramente mais atividade epiléptica, que as fatias cultivadas sempre em meio Opti-MEM. Posteriormente foi avaliado o impacto da inibição do inflamassoma NLRP3 na produção da IL-1β e na microgliose, uma vez que tinham sido potenciadas na presença de LPS/ATP. De modo a inibir o inflamassoma, utilizou-se um inibidor seletivo deste complexo multiproteico denominado de MCC950 (10μM). Na presença dos ativadores do inflamassoma, o MCC950 não foi capaz de reverter a libertação da citocina pro-inflamatória nem a microgliose. Por estudos electrofisiológicos verificou-se que na presença dos ativadores do inflamassoma, o MCC950 não foi capaz de reverter a atividade neuronal excessiva. No entanto, quando o MCC950 foi incubado sozinho, observou-se uma clara diminuição da atividade ictal nas fatias organotípicas. Conclui-se que a atividade epileptiforme induzida nas fatias organotípicas pela mudança do meio de cultura era dependente do inflamassoma, dado que foi revertida por incubação com o seu inibidor. Em suma, os nossos resultados demonstram que o inflamassoma NLRP3 está relacionado com a indução e potenciação da atividade epileptiforme. Adicionalmente, também sugerem que o MCC950 é um potencial agente terapêutico para a epilepsia e que o inflamassoma NLRP3 deve continuar a ser meticulosamente estudado como potencial alvo terapêutico para a epilepsia.Extensive evidence has supported the involvement of neuroinflammation in epileptic seizures. Recently, analysis of serum blood samples of epileptic patients revealed an increased production of several pro-inflammatory cytokines, namely interleukin-1β (IL-1β).This protein is produced by NLRP3 inflammasome, a cytosolic multiprotein complex involved in innate immune response. The canonic activation of NLRP3 inflammasome involves a priming signal (as lipopolysaccharides – LPS), which upregulates the expression of NLRP3 and pro-IL1β; and an activating signal (as adenosine triphosphate – ATP), which promotes the assembly of the complex. The goals of this study were to enhance epileptiform activity of organotypic slices through LPS/ATP – activated NLRP3 inflammasome and to reduce this activity by selectively inhibiting the inflammasome. Organotypic cortex-hippocampus slices of Sprague-Dawley rats with 6/7 days were exposed to different LPS concentrations (5, 10, 20ng/mL) at 3 or 6h or LPS (10ng/mL) plus ATP (1mM) to choose the best condition to activate the NLRP3 inflammasome. In order to inhibit the inflammasome, a selective inhibitor, MCC950 (10μM), was added 1h before co-incubation with LPS and ATP. Negative controls with each compound alone were carried out in some assays. Regarding inflammasome activation, LPS and ATP together decreased necrosis (assessed by ratio SBDP120/α-IISpectrin) and potentiated the release of pro-inflammatory cytokines (interleukin-1β (IL-1β) and tumor necrosis factor α). These events are characteristics of NLRP3 inflammasome activation. Expression of inflammasome components (NLRP3 e ASC) and glial cells markers (Iba1 e GFAP) were also evaluated, but did not show differences. In LPS/ATP presence, slices presented microgliosis in the layers near the culture medium. However, they depicted a similar epileptiform activity when compared with control slices, which were not exposed to drugs. In this study was possible to verify that control slices, that only underwent culture medium exchange, had an exacerbated synchronous neuronal activity, when compared with slices that did not undergo this process. In slices treated with MCC950 in the presence of LPS/ATP, neither IL-1β release nor microgliosis were reversed by MCC950. Moreover, NLRP3 inflammasome inhibitor did not affect epileptiform activity in the presence of LPS/ATP. Nevertheless, when MCC950 was incubated alone, the epileptiform activity was dramatically reduced. That is, MCC950 reversed the epileptiform activity induced by medium exchange, suggesting that this process involved the NLRP3 inflammasome. Our findings demonstrate the important role of NLRP3 inflammasome in the promotion of epileptiform activity and begin to unravel a potential target for antiepileptic therapy

On the Dynamics of the Spontaneous Activity in Neuronal Networks

Most neuronal networks, even in the absence of external stimuli, produce spontaneous bursts of spikes separated by periods of reduced activity. The origin and functional role of these neuronal events are still unclear. The present work shows that the spontaneous activity of two very different networks, intact leech ganglia and dissociated cultures of rat hippocampal neurons, share several features. Indeed, in both networks: i) the inter-spike intervals distribution of the spontaneous firing of single neurons is either regular or periodic or bursting, with the fraction of bursting neurons depending on the network activity; ii) bursts of spontaneous spikes have the same broad distributions of size and duration; iii) the degree of correlated activity increases with the bin width, and the power spectrum of the network firing rate has a 1/f behavior at low frequencies, indicating the existence of long-range temporal correlations; iv) the activity of excitatory synaptic pathways mediated by NMDA receptors is necessary for the onset of the long-range correlations and for the presence of large bursts; v) blockage of inhibitory synaptic pathways mediated by GABA(A) receptors causes instead an increase in the correlation among neurons and leads to a burst distribution composed only of very small and very large bursts. These results suggest that the spontaneous electrical activity in neuronal networks with different architectures and functions can have very similar properties and common dynamics

Model-free reconstruction of neuronal network connectivity from calcium imaging signals

A systematic assessment of global neural network connectivity through direct electrophysiological assays has remained technically unfeasible even in dissociated neuronal cultures. We introduce an improved algorithmic approach based on Transfer Entropy to reconstruct approximations to network structural connectivities from network activity monitored through calcium fluorescence imaging. Based on information theory, our method requires no prior assumptions on the statistics of neuronal firing and neuronal connections. The performance of our algorithm is benchmarked on surrogate time-series of calcium fluorescence generated by the simulated dynamics of a network with known ground-truth topology. We find that the effective network topology revealed by Transfer Entropy depends qualitatively on the time-dependent dynamic state of the network (e.g., bursting or non-bursting). We thus demonstrate how conditioning with respect to the global mean activity improves the performance of our method. [...] Compared to other reconstruction strategies such as cross-correlation or Granger Causality methods, our method based on improved Transfer Entropy is remarkably more accurate. In particular, it provides a good reconstruction of the network clustering coefficient, allowing to discriminate between weakly or strongly clustered topologies, whereas on the other hand an approach based on cross-correlations would invariantly detect artificially high levels of clustering. Finally, we present the applicability of our method to real recordings of in vitro cortical cultures. We demonstrate that these networks are characterized by an elevated level of clustering compared to a random graph (although not extreme) and by a markedly non-local connectivity.Comment: 54 pages, 8 figures (+9 supplementary figures), 1 table; submitted for publicatio

A pre-docking source for the power-law behavior of spontaneous quantal release: application to the analysis of LTP

In neurons, power-law behavior with different scaling exponents has been reported at many different levels, including fluctuations in membrane potentials, synaptic transmission up to neuronal network dynamics. Unfortunately in most cases the source of this nonlinear feature remains controversial. Here we have analyzed the dynamics of spontaneous quantal release at hippocampal synapses and characterized their power-law behavior. While in control conditions a fractal exponent greater than zero was rarely observed, its value was greatly increased by α-latrotoxin (α-LTX), a potent stimulator of spontaneous release, known to act at the very last step of vesicle fusion. Based on computer modeling, we confirmed that at an increase in fusion probability would unmask a pre-docking phenomenon with 1/f structure, where α estimated from the release series appears to sense the increase in release probability independently from the number of active sites. In the simplest scenario the pre-docking 1/f process could coincide with the Brownian diffusion of synaptic vesicles. Interestingly, when the effect of long-term potentiation (LTP) was tested, a ∼200% long-lasting increase in quantal frequency was accompanied by a significant increase in the scaling exponent. The similarity between the action of LTP and of α-LTX suggests an increased contribution of high release probability sites following the induction of LTP. In conclusion, our results indicate that the source of the synaptic powerlaw behavior arises before synaptic vesicles dock to the active zone and that the fractal exponent α is capable of sensing a change in release probability independently from the number of active sites or synapses. © 2015 Lamanna, Signorini, Cerutti and Malgaroli

Endocannabinoid signaling modulates neurons of the pedunculopontine nucleus (PPN) via astrocytes.

The pedunculopontine nucleus (PPN) is known as the cholinergic part of the reticular activating system (RAS) and it plays an important role in transitions of slow-wave sleep to REM sleep and wakefulness. Although both exogenous and endocannabinoids affect sleep, the mechanism of endocannabinoid neuromodulation has not been characterized at cellular level in the PPN. In this paper, we demonstrate that both neurons and glial cells from the PPN respond to cannabinoid type 1 (CB1) receptor agonists. The neuronal response can be depolarization or hyperpolarization, while astrocytes exhibit more frequent calcium waves. All these effects are absent in CB1 gene-deficient mice. Blockade of the fast synaptic neurotransmission or neuronal action potential firing does not change the effect on the neuronal membrane potential significantly, while inhibition of astrocytic calcium waves by thapsigargin diminishes the response. Inhibition of group I metabotropic glutamate receptors (mGluRs) abolishes hyperpolarization, whereas blockade of group II mGluRs prevents depolarization. Initially active neurons and glial cells display weaker responses partially due to the increased endocannabinoid tone in their environment. Taken together, we propose that cannabinoid receptor stimulation modulates PPN neuronal activity in the following manner: active neurons may elicit calcium waves in astrocytes via endogenous CB1 receptor agonists. Astrocytes in turn release glutamate that activates different metabotropic glutamate receptors of neurons and modulate PPN neuronal activity