Interaction of STDP and metaplasticity in modelling heterosynaptic plasticity.

Although neuroscientists have still not found a comprehensive mechanism to underlie learning and memory, many investigations suggest that long term potentiation (LTP) and long term depression (LTD) are involved in establishment of learning and memory. As a consequence of certain neural activity, neurons need to modulate the activity of the synapse or the properties of ion channels, therefore, they use a mechanism called homeostatic plasticity to balance their activity and control their firing rate. Two forms of plasticity phenomena that are necessary for plasticity regulation are homosynaptic and heterosynaptic plasticity. In the dentate granule cell, induction of homosynaptic LTP in the activated pathway is accompanied by heterosynaptic LTD in the inactivated pathway. Because, the dentate granule cell shows changes in synaptic strengths, we used this cell to test the following hypotheses. The first hypothesis we propose is, with plasticity and metaplasticity models introduced in this thesis, and the modification of an average postsynaptic spike, we can reproduce homosynaptic LTP and concurrent heterosynaptic LTD. The second hypothesis is the metaplasticity generated after a high frequency stimulation (HFS) reduces the level of synaptic plasticity caused by a second HFS. To test these hypotheses we use computer simulation and combine the nearest-neighbor spike time dependent plasticity (STDP) and metaplasticity rules accompanied with noisy spontaneous activity and the nine compartmental model of a granule cell. For this study we use the experimental data from Abraham et al.(2001), Abraham et al. (2007) and Bowden et al. (2012). With the method mentioned above our model is able to reproduce homosynaptic LTP in the activated pathway and heterosynaptic LTD in the neighboring inactivated pathway. We also show, due to the metaplasticity effects of the plasticity generated from the first HFS, the same magnitude of LTP and LTD will not occur in both pathways during the second HFS. Our finding supports the assertion that the combination of our metaplasticity and nearest-neighbor STDP rules can be a reliable choice to reproduce synaptic plasticity in the dentate granule cell neuron. Our investigation also supports the idea that metaplasticity modulates synaptic plasticity and prevents the synapse from extreme increases, therefore, the same magnitude of synaptic plasticity will not occur during the second stimulation

Timing for hippocampal synaptic plasticity

The timing of a spike with afferent excitation has been proposed to influence the direction of synaptic plasticity. I hypothesize that positive excitation-spike (ES)- Pairing— generating a synaptic excitation before a spike— results in long-term potentiation (LTP), while the opposite (negative ES-Pairing) results in long-term depression (LTD) in vivo. Extracellular potentials were recorded in the hippocampal CA1 region in urethane-anesthetized rats. Basal dendritic excitation was evoked by subthreshold stratum oriens stimulation while stratum radiatum stimulation evoked a spike that invaded the basal dendrites. ES-Pairing (50 times at 5 Hz) at -10, 0 and +10, +20 ES Intervals resulted in a significant potentiation of the slope of the basal excitatory sink for 2 hr compared to controls. Pairing at -20 ms ES Interval did not result in significant potentiation compared to controls. Thus, dendritic excitation occurring within a short time window of a spike results in LTP in vivo

IST Austria Thesis

CA3 pyramidal neurons are thought to pay a key role in memory storage and pattern completion by activity-dependent synaptic plasticity between CA3-CA3 recurrent excitatory synapses. To examine the induction rules of synaptic plasticity at CA3-CA3 synapses, we performed whole-cell patch-clamp recordings in acute hippocampal slices from rats (postnatal 21-24 days) at room temperature. Compound excitatory postsynaptic potentials (ESPSs) were recorded by tract stimulation in stratum oriens in the presence of 10 µM gabazine. High-frequency stimulation (HFS) induced N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP). Although LTP by HFS did not requier postsynaptic spikes, it was blocked by Na+-channel blockers suggesting that local active processes (e.g.) dendritic spikes) may contribute to LTP induction without requirement of a somatic action potential (AP). We next examined the properties of spike timing-dependent plasticity (STDP) at CA3-CA3 synapses. Unexpectedly, low-frequency pairing of EPSPs and backpropagated action potentialy (bAPs) induced LTP, independent of temporal order. The STDP curve was symmetric and broad, with a half-width of ~150 ms. Consistent with these specific STDP induction properties, post-presynaptic sequences led to a supralinear summation of spine [Ca2+] transients. Furthermore, in autoassociative network models, storage and recall was substantially more robust with symmetric than with asymmetric STDP rules. In conclusion, we found associative forms of LTP at CA3-CA3 recurrent collateral synapses with distinct induction rules. LTP induced by HFS may be associated with dendritic spikes. In contrast, low frequency pairing of pre- and postsynaptic activity induced LTP only if EPSP-AP were temporally very close. Together, these induction mechanisms of synaptiic plasticity may contribute to memory storage in the CA3-CA3 microcircuit at different ranges of activity

Slow Inhibition and Inhibitory Recruitment in the Hippocampal Dentate Gyrus

L’hippocampe joue un rôle central dans la navigation spatiale, la mémoire et l’organisation spatio-temporelle des souvenirs. Ces fonctions sont maintenues par la capacité du gyrus denté (GD) de séparation des patrons d'activité neuronales. Le GD est situé à l’entrée de la formation hippocampique où il reconnaît la présence de nouveaux motifs parmi la densité de signaux afférant arrivant par la voie entorhinale (voie perforante). Le codage parcimonieux est la marque distinctive du GD. Ce type de codage est le résultat de la faible excitabilité intrinsèque des cellules granulaires (CGs) en combinaison avec une inhibition locale prédominante. En particulier, l’inhibition de type « feedforward » ou circuit inhibiteur antérograde, est engagée par la voie perforante en même temps que les CGs. Ainsi les interneurones du circuit antérograde fournissent des signaux GABAergique aux CGs de manière presque simultanée qu’elles reçoivent les signaux glutamatergiques. Cette thèse est centrée sur l’étude des interactions entre ces signaux excitateurs de la voie entorhinale et les signaux inhibiteurs provenant des interneurones résidant dans le GD et ceci dans le contexte du codage parcimonieux et le patron de décharge en rafale caractéristique des cellules granulaires. Nous avons adressé les relations entre les projections entorhinales et le réseau inhibitoire antérograde du GD en faisant des enregistrements électrophysiologiques des CG pendant que la voie perforante est stimulée de manière électrique ou optogénétique. Nous avons découvert un nouvel mécanisme d’inhibition qui apparait à délais dans les CGs suite à une stimulation dans les fréquences gamma. Ce mécanisme induit une hyperpolarisation de longue durée (HLD) et d’une amplitude prononce. Cette longue hyperpolarisation est particulièrement prolongée et dépasse la durée d’autres types d’inhibition transitoire lente décrits chez les CGs. L’induction de HLD crée une fenêtre temporaire de faible excitabilité suite à laquelle le patron de décharge des CGs et l’intégration d’autres signaux excitateurs sont altérés de manière transitoire. Nous avons donc conclu que l’activité inhibitrice antérograde joue un rôle central dans les processus de codage dans le GD. Cependant, alors qu’il existe une multitude d’études décrivant les interneurones qui font partie de ce circuit inhibiteur, la question de comment ces cellules sont recrutées par la voie entorhinale reste quelque peu explorée. Pour apprendre plus à ce sujet, nous avons enregistré des interneurones résidant iii dans la couche moléculaire du GD tout en stimulant la voie perforante de manière optogénétique. Cette méthode de stimulation nous a permis d’induire la libération de glutamate endogène des terminales entorhinales et ainsi d’observer le recrutement purement synaptique d’interneurones. De manière surprenante, les résultats de cette expérience démontrent un faible taux d’activation des interneurones, accompagné d’un tout aussi faible nombre total de potentiels d’action émis en réponse à la stimulation même à haute fréquence. Ce constat semble contre-intuitif étant donné qu’en générale on assume qu’une forte activité inhibitrice est requise pour le maintien du codage parcimonieux. Tout de même, l’analyse des patrons de décharge des interneurones qui ont été activés a fait ressortir la prééminence de trois grands types: décharge précoce, retardée ou régulière par rapport le début des pulses lumineux. Les résultats obtenus durant cette thèse mettent la lumière sur l’important conséquences fonctionnelles des interactions synaptique et polysynaptique de nature transitoire dans les réseaux neuronaux. Nous aimerions aussi souligner l’effet prononcé de l’inhibition à court terme du type prolongée sur l’excitabilité des neurones et leurs capacités d’émettre des potentiels d’action. De plus que cet effet est encore plus prononcé dans le cas de HLD dont la durée dépasse souvent la seconde et altère l’intégration d’autres signaux arrivants simultanément. Donc on croit que les effets de HLD se traduisent au niveau du réseaux neuronal du GD comme une composante cruciale pour le codage parcimonieux. En effet, ce type de codage semble être la marque distinctive de cette région étant donné que nous avons aussi observé un faible niveau d’activation chez les interneurones. Cependant, le manque d’activité accrue du réseau inhibiteur antérograde peut être compensé par le maintien d’un gradient GABAergique constant à travers le GD via l’alternance des trois modes de décharges des interneurones. En conclusion, il semble que le codage parcimonieux dans le GD peut être préservé même en absence d’activité soutenue du réseau inhibiteur antérograde et ceci grâce à des mécanismes alternatives d’inhibition prolongée à court terme.The hippocampus is implicated in spatial navigation, the generation and recall of memories, as well as their spatio-temporal organization. These functions are supported by the processes of pattern separation that occurs in the dentate gyrus (DG). Situated at the entry of the hippocampal formation, the DG is well placed to detect and sort novelty patterns amongst the high-density excitatory signals that arrive via the entorhinal cortex (EC). A hallmark of the DG is sparse encoding that is enabled by a combination of low intrinsic excitability of the principal cells and local inhibition. Feedforward inhibition (FFI) is recruited directly by the EC and simultaneously with the granule cells (GCs). Therefore, FFI provides fast GABA release and shapes input integration at the millisecond time scale. This thesis aimed to investigate the interplay of entorhinal excitatory signals with GCs and interneurons, from the FFI in the DG, in the framework of sparse encoding and GC’s characteristic burst firing. We addressed the long-range excitation – local inhibitory network interactions using electrophysiological recordings of GCs – while applying an electrical or optogenetic stimulation of the perforant path (PP) in the DG. We discovered and described a novel delayed-onset inhibitory post synaptic potential (IPSP) in GCs, following PP stimulation in the gamma frequency range. Most importantly, the IPSP was characterized by a large amplitude and prolonged decay, outlasting previously described slow inhibitory events in GCs. The long-lasting hyperpolarization (LLH) caused by the slow IPSPs generates a low excitability time window, alters the GCs firing pattern, and interferes with other stimuli that arrive simultaneously. FFI is therefore a key player in the computational processes that occurs in the DG. However, while many studies have been dedicated to the description of the various types of the interneurons from the FFI, the question of how these cells are synaptically recruited by the EC remains not entirely elucidated. We tackled this problem by recording from interneurons in the DG molecular layer during PP-specific optogenetic stimulation. Light-driven activation of the EC terminals enabled a purely synaptic recruitment of interneurons via endogenous glutamate release. We found that this method of stimulation recruits only a subset of interneurons. In addition, the total number of action potentials (AP) was surprisingly low even at high frequency stimulation. This result is counterintuitive, as strong and persistent inhibitory signals are assumed to restrict GC v activation and maintain sparseness. However, amongst the early firing interneurons, late and regular spiking patterns were clearly distinguishable. Interestingly, some interneurons expressed LLH similar to the GCs, arguing that it could be a commonly used mechanism for regulation of excitability across the hippocampal network. In summary, we show that slow inhibition can result in a prolonged hyperpolarization that significantly alters concurrent input’s integration. We believe that these interactions contribute to important computational processes such as sparse encoding. Interestingly, sparseness seems to be the hallmark of the DG, as we observed a rather low activation of the interneuron network as well. However, the alternating firing of ML-INs could compensate the lack of persistent activity by the continuous GABA release across the DG. Taken together these results offer an insight into a mechanism of feedforward inhibition serving as a sparse neural code generator in the DG

Contributions to models of single neuron computation in striatum and cortex

A deeper understanding is required of how a single neuron utilizes its nonlinear subcellular devices to generate complex neuronal dynamics. Two compartmental models of cortex and striatum are accurately formulated and firmly grounded in the experimental reality of electrophysiology to address the questions: how striatal projection neurons implement location-dependent dendritic integration to carry out association-based computation and how cortical pyramidal neurons strategically exploit the type and location of synaptic contacts to enrich its computational capacities.Neuronale Zellen transformieren kontinuierliche Signale in diskrete Zeitserien von Aktionspotentialen und kodieren damit Perzeptionen und interne Zustände. Kompartiment-Modelle werden formuliert von Nervenzellen im Kortex und Striatum, die elektrophysiologisch fundiert sind, um spezifische Fragen zu adressieren: i) Inwiefern implementieren Projektionen vom Striatum ortsabhängige dendritische Integration, um Assoziationens-basierte Berechnungen zu realisieren? ii) Inwiefern nutzen kortikale Zellen den Typ und den Ort, um die durch sie realisierten Berechnungen zu optimieren

Steep, Spatially Graded Recruitment of Feedback Inhibition by Sparse Dentate Granule Cell Activity

The dentate gyrus of the hippocampus is thought to subserve important physiological functions, such as 'pattern separation'. In chronic temporal lobe epilepsy, the dentate gyrus constitutes a strong inhibitory gate for the propagation of seizure activity into the hippocampus proper. Both examples are thought to depend critically on a steep recruitment of feedback inhibition by active dentate granule cells. Here, I used two complementary experimental approaches to quantitatively investigate the recruitment of feedback inhibition in the dentate gyrus. I showed that the activity of approximately 4% of granule cells suffices to recruit maximal feedback inhibition within the local circuit. Furthermore, the inhibition elicited by a local population of granule cells is distributed non-uniformly over the extent of the granule cell layer. Locally and remotely activated inhibition differ in several key aspects, namely their amplitude, recruitment, latency and kinetic properties. Finally, I show that net feedback inhibition facilitates during repetitive stimulation. Taken together, these data provide the first quantitative functional description of a canonical feedback inhibitory microcircuit motif. They establish that sparse granule cell activity, within the range observed in-vivo, steeply recruits spatially and temporally graded feedback inhibition

Electrophysiological and Morphological Characterization of Potentiated Synapses at the Micro and Nanoscale

2012/2013Col termine generale di “plasticità sinaptica” si intendono tutti i meccanismi che stanno alla base della capacità del sistema nervoso di plasmarsi a seguito della sua maturazione e a fronte di stimoli esterni. Variazioni nella forma e nelle dimensioni oltre che l’instaurazione di nuove sinapsi o l’eliminazione di altre (sinaptogenesi) sono i meccanismi che regolano la plasticità sinaptica. Il sistema nervoso centrale è in grado di mettere in atto fenomeni di plasticità sinaptica in grado di modificarne la struttura e la funzionalità sia a corto che a lungo termine. Uno dei più studiati meccanismi cellulari alla base della memoria e dell’apprendimento è il potenziamento a lungo termine (Long Term Potentiation – LTP), una forma di plasticità neuronale che porta a un incremento dell’efficienza della trasmissione sinaptica durevole nel tempo. A livello cellulare, l’LTP aumenta la capacità di due neuroni di comunicare attraverso le sinapsi. Il meccanismo molecolare alla base di tale aumento dell’efficienza della trasmissione sinaptica non è univocamente stabilito, questo in parte è dovuto al fatto che l’LTP è determinato da diversi meccanismi che variano in base alla specie e alla regione del cervello in cui viene indotto. Una volta innescato, l’LTP conduce a varie modificazioni postsinaptiche, tra cui sintesi di nuovi recettori, nascita di nuove sinapsi (in particolare a livello del recettore glutamatergico NMDA) e cambiamenti a livello delle spine dendritiche (Engert and Bonhoeffer, 1999). Ragionevolmente, per indurre potenziamento a lungo termine è necessario che la membrana postsinaptica sia depolarizzata nell’intervallo di tempo in cui il terminale presinaptico libera glutammato: la depolarizzazione rimuove il blocco degli ioni magnesio dai recettori NMDA consentendo il passaggio (oltre al sodio e al potassio) anche agli ioni calcio. Il calcio è l'elemento centrale del processo perché, una volta raggiunta una certa concentrazione nella cellula, è in grado di attivare un processo per cui i recettori AMPA presenti nella cellula vengono trasferiti sulla membrana e i recettori già presenti lasciano passare una maggiore quantità di ioni. La sinapsi risulta così rinforzata. Questa condizione è stata sperimentalmente dimostrata su campioni di fettine di ippocampo usando una stimolazione elettrica (tetanica) (Nishi et al., 2001). Dopo la stimolazione tetanica, il neurone bersaglio rafforzato dall’LTP è molto più responsivo e produce un aumento dell’ampiezza delle correnti eccitatorie post-sinaptiche (Excitatory Post Synaptic Currents – EPSC) che perdura nel tempo. Questo comportamento trova spiegazione in una modificazione delle spine dendritiche sia nella forma, sia nel numero e dimensione. L’attività del mio dottorato di ricerca è stata condotta nell’ambito del progetto NanoMosquito, il cui scopo prinicipale consiste nell’indurre fenomeni di plasticità neuronale in cellule dissociate d’ippocampo di ratto e, successivamente, nel caratterizzare le mutazioni funzionali (tramite la tecnica elettrofisiologica del patch-clamping) e morfologiche, in scala micro e nanometrica, utilizzando tecniche quali la microscopia confocale e la microscopia a forza atomica (Atomic Force Microscopy – AFM). Diverse stimolazioni sono state testate per carcare di capire quali potessero indurre potenziamento della rete. Studi di plasticità vengono condotti in genere su fettine organotipiche, ma queste rendono impossibile studiare i cambiamenti che avvengono a livello delle spine dendritiche con tecniche in scala nanometrica, quali l’AFM. Diversi protocolli di stimolazione (treni a bassa frequenza, theta burst) sono stati utilizzati in esperimenti a doppio patch (due elettrodi usati in simultanea) su due cellule neuronali vicinali. Questo tipo di stimolazione ha portato però solo a un numero limitato di sinapsi potenziate e per questo motive abbiamo deciso di uitlizzare una particolare forma di plasticità sinaptica che prende il nome di Spike-Timing Dependent Plasticity (STDP). In questo tipo di plasticità il preciso ordine temporale tra i potenziali d’azione presinaptici e postsinaptici determina i cambiamenti che avverrano a livello della sinapsi stessa; per ottenere un potenziamento a livello del contatto sinaptico, il potenziale d’azione a livello postsinaptico deve seguire la depolarizzazione a livello presinaptico in una finestra temporale che va dai 5 ai 20 millisecondi (Bi and Poo, 1998). Anche in questo caso, monitorando successivamente l’ampiezza delle EPSCs, solo poche sinpasi andavano incontro a plasticità e il meccanismo che sta alla base di questo deve essere ancora determinato. Al contrario, il Brain Derived Neurotrophic Factor (BDNF), membro della famiglia delle neurotrofine e abbondantemente espresso nel sistema nervoso centrale (SNC), sta emergendo come un importante mediatore nella sopravvivenza, sviluppo e funzione dei neuroni (Lu, 2003). Colture embrionali dissociate di ippocampo sono state per la prima volta trattate cronicamente con BDNF promuovendo la formazione di nuove sinapsi, sia a livello eccitatorio che inibitorio, con conseguente aumento dell’attività spontanea dell’intera rete. Il BDNF inoltre si pensa induca modificazioni morfologiche sia nella complessità dell’albero dendritico che nel promuovere la crescita delle terminazioni assonali (Vicario-Abejon et al., 1998). Registrazioni elettrofisiologiche sono state effettuate per monitare l’attività spontanea della rete: nel dettaglio sono state misurate le EPSC e le IPSC tra neuroni incubati in BDNF e campioni di controllo mentre registrazioni doppie sono state effettuate per confrontare la percentuale di accoppiamento. Abbiamo così visto come il BDNF rafforzi l’attività sinaptica della rete e aumenti il numero di connessioni sinaptiche eccitatorie. Registrazioni paired-pulse ed esperimenti di imaging con FM1-43 hanno invece dimostrato come il BDNF induca anche delle modificazioni nella probabilità di rilascio vescicolare, in quanto, anche in questo caso l’ampiezza della risposta risulta aumentata nelle colture incubate. Marcando i neruoni (β-tubulin III) abbiamo visto anche come il BDNF aumenti la sopravvivenza neuronale, sopratutto a carico delle cellule piramidali, riconosciute dalla loro forma. Inoltre, eseprimenti condottti su cellule transfettate con cds-BDNF hanno confermato ulteriormente i nostril dati su come il BDNF aumenti la trasmissione sinpatica. La caratteristica comune di tutti questi diversi approcci è stata quella di indurre modifiche funzionali nelle connessioni sinaptiche eccitatorie. Successivamente l'induzione della plasticità sinaptica, la microscopia a scansione sarà utilizzata per seguire in tempo reale i cambiamenti morfologici delle sinapsi.The brain is programmed to drive behaviour by exactly wiring the appropriate neuronal circuits. Wiring and rewiring of neuronal circuits widely depends on the orchestrated changes in the strengths of synaptic contacts. For many years, neuroscientists believed that neurogenesis - the generation of new neurons – and establishment of new neuronal connections was restricted to early brain development (Segal et al, 2005). New findings have challenged this view and currently many neuroscientists believe that the capacity for circuitry rearrangement is maintained throughout life. However the mechanisms that controls plasticity in the adult brain are still not entirely clear. The connection between neurons is named synapse. The synapse is the most fundamental unit of information transmission in the nervous system. Information storage, including all forms of memory and behavioural adaptation, are believed to come out from changes in neuronal transmission, both in the short-term and the long-term, a property known as synaptic plasticity. Synaptic plasticity is a highly regulated process, refers to all the mechanisms that underlie the ability of the nervous system to adapt to external stimuli. Variations in the shape and size as well as establishment of new synapses or the elimination of others (synaptogenesis) are the mechanisms that regulate synaptic plasticity. Thus, understanding the mechanisms underlying synaptic plasticity may help to apprehend general learning and memory processes. Changes in synaptic plasticity are achieved by changes in inhibitory or excitatory neurotransmission or both. This thesis deals with the modulation of excitatory neurotransmission. The principal excitatory neurotransmitter in the brain is glutamate. The regulation of glutamate-mediated excitatory neurotransmission has been shown to play a critical role in many aspects of synaptic plasticity. One of the most studied cellular mechanisms is the long-term potentiation (LTP), a form of synaptic plasticity that leads to an increase in the efficiency of synaptic transmission (Engert et al., 1999). The induction of LTP is classically achieved by tetanic stimulation but it is also possible to induce chemically a long-term potentiation of the synaptic efficacy, thus enhancing a larger number of synapses compared to electrical stimulation. The work of this thesis has been conducted in the wider framework of the NanoMosquito project, whose major aim was to combine electrophysiological measurements, scanning probe microscopy (AFM-Atomic Force Microscopy) and fluorescence microscopy in order to develop new generation neurophysiological tool to understand neuronal plasticity at the nanoscale. Studies of synaptic plasticity are often carried out in slices of hippocampus, but these prevent to study change in nanoscale with a surface-microscopy technique such is AFM: dissociated hippocampal neurons lend themselves well for this purpose. Understanding in detail the mechanism of action of these processes may be of critical importance not only for a detailed view of memory related processes but also in the case of some diseases: being able to control synaptic plasticity may help to restore a functional connectivity lost, for example, in the case of brain lesions. The first part of this thesis handles the setting of an electrophysiological stimulation to induce neuronal plasticity, starting from the stimulations trains usually performed in hippocampal slices, such as slow frequency stimulation and theta burst. Long-term synaptic modifications can be induced also by a particular form of synaptic plasticity named Spike-Timing Dependent Plasticity (STDP) where the precise timing and the order of presynaptic and postsynaptic action potentials determine the magnitude and the direction of the changes in synaptic strength (Bi and Poo, 1998). I have tested trains of with a delay of 5, 10 and 20 milliseconds between pre- and postsynaptic neuron. By monitoring the amplitude and frequency of the EPSCs, responses varied from no changes to potentiation but just in a small sample of coupled neurons where we measured a strong increase in the amplitude and frequency of spontaneous EPSCs after the stimulation. The cellular basis that gives rise to the induction of such synaptic modifications remains to be determined. On the other hand, BDNF ability to mediate activity-dependent modifications in synaptic strength (Bolton et al., 2000; Vicario-Abejón et al., 1998) has recently received considerable attention; in particular the acute BDNF effects on excitatory synapses have been the object of an increasing amount of studies. On the contrary, the role of BDNF in regulating long-lasting changes in synaptic function is comparably less investigated and may have large impact on post injury alteration of synaptic networks and neuronal rescue. To address this issue, during my PhD, I studied the long-term (chronic) effects of BDNF on AMPA receptor mediated excitatory synaptic transmission and on neuronal survival in vitro. Dissociated rat (P2-P3) hippocampal cultures were chronically treated (4 days) with BDNF between 4 and 8 days in vitro (DIV). Single and dual patch-clamp recordings in whole-cell configuration were used to monitor spontaneous and evoked post synaptic currents (IPSCs and EPSCs) in hippocampal network grown in culture for 8-10 DIV. Excitatory PSCs (EPSC) were identified by their kinetic (fast decay τ) and pharmacology (CNQX sensitivity). EPSCs recorded from BDNF-treated cultures show a strong increase in their mean frequency and amplitude when compared to controls untreated sister cultures. In the presence of TTX, miniature excitatory PSCs (mEPSCs) in BDNF treated networks still displayed an increase in both frequency and amplitude. In BDNF-treated cultures pair recordings showed an increased probability of finding coupled pairs. Paired pulse (20 Hz) experiments and FM1-43 fluorescence imaging suggested that BDNF treatment increased the probability of release. Immunofluorescence (β-tubulin III) visualization of neurons allowed to quantify neuronal density and showed that BDNF mediated an increase (40%) in neuronal survival, when compared to controls, together with an increase in the pyramidal neuron/interneuron ratio (0.33 for BDNF, 0.19 for controls). Additionally, neuronal cells were transfected with different BDNF-GFP expressing vectors to gain insights in the specific molecular mechanisms involved in long term BDNF effects on synapses. However the common feature of all these functional modifications is in the direction of a pronounced potentiation of excitatory synaptic connections. Subsequently to the induction of synaptic plasticity, scanning probe microscopy would be used to follow in real time morphological changes of synapses undergoing potentiation or neuronal processes development with submicrometrical resolution in all 3 dimensions. Final goal of the entire project, whereof this thesis is the fundamental initial step, will be the development of new paradigms to evaluate and induce synaptic plasticity on specific synapses to govern in a controlled way neuronal outgrowth and synaptogenesis.XXVI Ciclo198

Storage of spatiotemporal input sequences in dendrites of pyramidal neurons

Plastic changes in neurons are widely considered to underpin the formation and maintenance of memory. The mechanisms of induction and expression of plasticity are, therefore, crucial to our understanding of the capacity of information storage that neurons possess. Using two-photon glutamate uncaging and whole-cell electrophysiological recordings, I demonstrate that dendrites of neurons are capable of preferentially storing specific spatiotemporal sequences, and describe the physiological properties of this new form of plasticity. Such plastic changes are dependent on Ca2+ influx through NMDA receptors, which is consistent with previous reports regarding induction of potentiation. Using two-photon Ca2+ imaging, I demonstrate that spatiotemporal plasticity is a result of a distinct homogeneous spatial increase in Ca2+ influx of different spatiotemporal sequences. Using the NEURON simulation environment, I used my experimental findings to perform simulations of synaptic plasticity rules. I found that homogeneous increases in synaptic strength across the dendrite can result in the spatiotemporal plasticity that I empirically observed. Moreover, I employed a genetic optimization algorithm and parallelized simulations to show that such changes are within physiological parameters observed in cortical neurons. My PhD therefore describes a novel form of plasticity, and proposes that dendrites are capable of more extensive information storage than was previously assumed

Neuromodulation of Spike-Timing-Dependent Plasticity: Past, Present, and Future.

Spike-timing-dependent synaptic plasticity (STDP) is a leading cellular model for behavioral learning and memory with rich computational properties. However, the relationship between the millisecond-precision spike timing required for STDP and the much slower timescales of behavioral learning is not well understood. Neuromodulation offers an attractive mechanism to connect these different timescales, and there is now strong experimental evidence that STDP is under neuromodulatory control by acetylcholine, monoamines, and other signaling molecules. Here, we review neuromodulation of STDP, the underlying mechanisms, functional implications, and possible involvement in brain disorders.BBSR

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