Дисертації з теми "CA3 pyramidal neurons"

It is well established that ethanol (EtOH), through the interaction with several membrane proteins, as well as intracellular pathways, is capable to modulate many neuronal function. Recent reports show that EtOH increases the firing rate of hippocampal GABAergic interneurons through the positive modulation of the hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels. This effect might be consistent with the increase of GABA release from presynaptic terminals observed in both CA1 and CA3 inhibitory synapses that leads the enhancement of the GABAergic system induced by EtOH. The activation of HCN produced an inward currents that are commonly called Ih. Ih play an important role for generating specific neuronal activities in different brain regions, including specific sub-regions of the hippocampal formation, such as CA1 and CA3 pyramidal neurons and hippocampal GABAergic interneurons. The main physiologic effect mediated by HCN-induced Ih is directed to the control of the neuronal resting membrane potential and action potential (AP) discharge as well as dampen synaptic integration. Since robust Ih are also present in CA3 glutamatergic neurons, I here investigated whether the action of EtOH in the control of CA3 excitability can be correlated with its possible direct interaction with these cation channels. For this purpose, patch-clamp experiments were performed in CA3 pyramidal neurons from hippocampal coronal slices obtained from male Sprague-Dawley rats. The data obtained demonstrated that EtOH is able to modulate Ih in biphasic manner depending on the concentrations used. Low EtOH concentrations enhanced Ih amplitude, while high reversibly reduced them. This biphasic action induced by EtOH reflects on firing rate and synaptic integration. In addition, in this reports it has been shown that EtOH modulates the function of HCN channels through interfering with the cAMP/AC/PKA intracellular pathways, an effect that is mimicked also by other endogenous compounds such as dopamine through D1 receptors activation. These data suggest that the HCN-mediated Ih currents in CA3 pyramidal neurons are sensitive to EtOH action, which at low or relevant concentrations is able to increase or reduce their function respectively. Altogether these data suggest a potential new mechanism of EtOH actions on hippocampal formation and may help to better understand the depressant central activity showed by this drug of abuse

In adulthood, mossy fibers (MFs), the axons of granule cells of the dentate gyrus (DG), release glutamate onto CA3 principal cells and interneurons. In contrast, during the first week of postnatal life MFs release -aminobutyric acid (GABA), which, at this early developmental stage exerts a depolarizing and excitatory action on targeted cells. The depolarizing action of GABA opens voltage-dependent calcium channels and NMDA receptors leading to calcium entry and activation of intracellular signaling pathways involved in several developmental processes, thus contributing to the refinement of neuronal connections and to the establishment of adult neuronal circuits. The release of GABA has been shown to be down regulated by several neurotransmitter receptors which would limit the enhanced excitability caused by the excitatory action of GABA. It is worth noting that the immature hippocampus exhibits spontaneous correlated activity, the so called giant depolarizing potentials or GDPs that act as coincident detector signals for enhancing synaptic activity, thus contributing to several developmental processes including synaptogenesis. GDPs render the immature hippocampus more prone to seizures. Here, I explored the molecular mechanisms underlying synaptic transmission and activity-dependent synaptic plasticity processes at immature GABAergic MF-CA3 synapses in wild-type rodents and in mice lacking the prion protein (Prnp0/0 mice). In the first paper, I studied the functional role of kainate receptors (KARs) in regulating GABA release from MF terminals. Presynaptic KARs regulate synaptic transmission in several brain areas and play a central role in modulating glutamate release at adult MF-CA3 synapses. I found that functional presynaptic GluK1 receptors are present on MF terminals where they down regulate GABA release. Thus, application of DNQX or UBP 302, a selective antagonist for GluK1 receptors, strongly increased the amplitude of MF-GABAA-mediated postsynaptic currents (GPSCs). This effect was associated with a decrease in failure rate and increase in PPR, indicating a presynaptic type of action. GluK1 receptors were found to be tonically activated by glutamate present in the extracellular space, since decreasing the extracellular concentration of glutamate with a glutamate scavenger system prevented their activation and mimicked the effects of KAR antagonists. The depressant effect of GluK1 on GABA release was dependent on pertussis toxin (PTx)-sensitive G protein-coupled kainate receptors since it was prevented when hippocampal slices were incubated in the presence of a solution containing PTx. This effect was presynaptic since application of UBP 302 to cells patched with an intracellular solution containing GDP S still potentiated synaptic responses. In addition, the depressant effect of GluK1 on GABA release was prevented by U73122, which selectively inhibits phospholipase C, downstream to G protein activation. Interestingly, U73122, enhanced the probability of GABA release, thus unveiling the ionotropic type of action of kainate receptors. In line with this, we found that GluK1 receptors enhanced MF excitability by directly depolarizing MF terminals via calcium-permeable cation channels. We also explored the possible involvement of GluK1 in spike time-dependent (STD) plasticity and we found that GluK1 dynamically regulate the direction of STD-plasticity, since the pharmacological block of this receptor shifted spike-time dependent potentiation into depression. The mechanisms underlying STD-LTD at immature MF-CA3 synapses have been investigated in detail in the second paper. STD-plasticity is a Hebbian form of learning which consists in bi-directional modifications of synaptic strength according to the temporal order of pre and postsynaptic spiking. Interestingly, we found that at immature mossy fibers (MF)-CA3 synapses, STD-LTD occurs regardless of the temporal order of stimulation (pre versus post or viceversa). However, as already mentioned, while STD-LTD induced by positive pairing (pre before post) could be shifted into STD-LTP after blocking presynaptic GluK1 receptors, STD-LTD induced by negative pairing (post before pre) relied on the activation of CB1 receptors. At P3 but not at P21, endocannabinoids released by the postsynaptic cell during spiking-induced membrane depolarization retrogradely activated CB1 receptors, probably expressed on MF terminals and persistently depressed GABA release in the rat hippocampus. Thus, bath application of selective CB1 receptor antagonists prevented STD-LTD. Pharmacological tools allow identifying anandamide as the endogenous ligand responsible of activity-dependent depressant effect. To further assess whether STD-LTD is dependent on the activation of CB1 receptors, similar experiments were performed on WT-littermates and CB1-KO mice. While in WT mice the pairing protocol produced a persistent depression of MF-GPSCs as in rats, in CB1-KO mice failed to induce LTD. Consistent with these data, in situ hybridization experiments revealed detectable levels of CB1 mRNA in the granule cell layer of P3 but not of P21mice. These experiments strongly suggest that at immature MF-CA3 synapses STD-LTD is mediated by CB1 receptors, probably transiently expressed, during a critical time window, on MF terminals. In the third paper, I studied synaptic transmission and activity dependent synaptic plasticity at immature MF-CA3 synapses in mice devoid of the prion protein (Prnp0/0). The prion protein (PrPC) is a conserved glycoprotein widely expressed in the brain and involved in several neuronal processes including neurotransmission. If converted to a conformationally altered form, PrPSc can cause neurodegenerative diseases, such as Creutzfeldt-Jakob disease in humans. Previous studies aimed at characterizing Prnp0/0 mice have revealed only mild behavioral changes, including an impaired spatial learning, accompanied by electrophysiological and biochemical alterations. Interestingly, PrPC is developmentally regulated and in the hippocampus its expression parallels the maturation of MF. Here, we tested the hypothesis that at immature (P3-P7) MF-CA3 synapses, PrPC interferes with synaptic plasticity processes. To this aim, the rising phase of Giant Depolarizing Potentials (GDPs), a hallmark of developmental networks, was used to stimulate granule cells in the dentate gyrus in such a way that GDPs were coincident with afferent inputs. In WT animals, the pairing procedure induced a persistent increase in amplitude of MF-GPSCs. In contrast, in Prnp0/0 mice, the same protocol produced a long-term depression (LTD). LTP was postsynaptic in origin and required the activation of cAMP-dependent PKA signaling while LTD was presynaptic and was reliant on G protein-coupled GluK1 receptor and protein lipase C downstream to G protein activation. In addition, at emerging CA3-CA1 synapses of PrPC-deficient mice, stimulation of Schaffer collateral failed to induce LTP, known to be PKA-dependent. Finally, we also found that LTD in Prnp0/0 mice was mediated by GluK1 receptors, since UBP 302 blocked its induction. These data suggest that in the immature hippocampus PrPC controls the direction of synaptic plasticity.

Les neurones pyramidaux de la région CA3 de l'hippocampe présentent une diversité morphologique, physiologique, biochimique, mais aussi fonctionnelle. Une partie des caractéristiques des neurones étant acquise pendant le développement, nous avons formulé l'hypothèse que la diversité morpho-fonctionnelle des neurones pyramidaux serait déterminée aux stades embryonnaires. Pour tester cette hypothèse, nous avons utilisé des souris transgéniques pour lesquelles l'expression d'un marqueur fluorescent (GFP) est conditionnée par la date de neurogenèse des neurones glutamatergiques. Nous avons enregistré l'activité des neurones en imagerie calcique et montré que les neurones pyramidaux nés le plus tôt déchargent pendant la phase d'initiation des activités épileptiformes générées par le blocage pharmacologique de la transmission GABAergique rapide. De plus, nous montrons que ces neurones précoces possèdent des propriétés morpho-physiologiques distinctes. Enfin, nous montrons que la stimulation de neurones pyramidaux nés tôt peut générer des activités épileptiformes à des stades immatures lorsqu'ils sont stimulés en groupe, et à des stades juvéniles lorsqu'ils sont stimulés individuellement. Ainsi nous démontrons qu'il existe un lien entre la date de neurogenèse et les propriétés morpho-fonctionnelles des neurones pyramidaux de CA3<br>There is increasing evidence that CA3 pyramidal cells are biochemically, electrophysiologically, morphologically and functionally diverse. As most of these properties are acquired during development, we hypothesized that the heterogeneity of the morphofunctionnal properties of pyramidal cells could be determined at the early stages of life. To test this hypothesis, we used a transgenic mouse line in which we glutamatergic cells are labelled with GFP according to their birth date. Using calcium imaging, we recorded multineuron activity in hippocampal slices and show that early generated pyramidal neurons fire during the build-up phase of epileptiform activities generated in the absence of fast GABAergic transmission. Moreover, we show that early generated pyramidal neurons display distinct morpho-physiological properties. Finally, we demonstrate that early generated neurons can generate epileptiform activities when stimulated as assemblies at immature stages, and when stimulated individually at juvenile stages. Thus we suggest a link between the date of birth and the morpho-functional properties of CA3 pyramidal neurons

Le neurone est une cellule polarisée qui se divise en deux compartiments spécialisés : le compartiment somato-dendritique et le compartiment axonal. Généralement, le premier reçoit l'information en provenance d'autres neurones et le second génère un message en sortie lorsque la somme des entrées dépasse une valeur seuil au segment initial de l'axone. Ce signal de nature discrète appelé potentiel d'action (PA) est propagé activement jusqu'à la terminaison synaptique où il déclenche la transmission chimique de l'information. Cependant, la fonction axonale ne se résume pas à la simple transmission des séquences de PA à l'image d'un câble de télégraphe. L'axone est également capable de transmettre des variations continues de signaux électriques infraliminaires dit analogues et les combiner avec l'information digitale véhiculée par le PA. J'ai consacré la majorité de mon travail de thèse à l'étude de ce nouvel aspect de la fonction axonale dans le cadre de la transmission synaptique entre les neurones pyramidaux au sein du réseau excitateur CA3 de l'hippocampe de rat. Les résultats obtenus à partir d'enregistrements de paires de neurones pendant ma thèse mettent en évidence deux sortes de signalisation analogue et digitale qui aboutissent à la facilitation de la transmission synaptique. La facilitation analogue-digitale (FAD) a été observée lors d'une dépolarisation prolongée, mais également à la suite d'une hyperpolarisation transitoire au niveau du corps cellulaire. Ce sont deux versants d'une même plasticité à court-terme qui découle de l'état biophysique des canaux ioniques sensibles au voltage étant à l'origine du PA<br>The neuron is a polarised cell divided into two specialized compartments: the somato-dendritic and the axonal compartment. Generally, the first one receives information arriving from other neurones and the second generates an output message, when the sum of inputs exceeds a threshold value at the axon initial segment. This all-or-none signal, called the action potential (AP) is propagated actively to the synaptic terminal where it triggers chemical transmission of information. However, axonal function is not limited to transmission of AP sequences like a telegraph cable. The axon is also capable of transmitting continuously changing sub-threshold electric signals called analogue signals and to combine them with the digital information carried by the AP. I devoted the majority of my thesis work to the study of these novel aspects of axonal function in the framework of synaptic transmission between pyramidal neurons in the CA3 excitatory network of the rat hippocampus. The results obtained through paired recordings brought to light two kinds of analogue and digital signalling that lead to a facilitation of synaptic transmission. Analogue-digital facilitation (ADF) was observed during prolonged presynaptic depolarization and also after a transient hyperpolarization of the neuronal cell body. These are two sides of the same form of short-term synaptic plasticity depending on the biophysical state of voltage gated ion channels responsible for AP generation. The first variant of ADF induced by depolarization (ADFD) is due to AP broadening and involves Kv1 potassium channels

Oxytocin (OT) is a neuropeptide that exerts different peripheral and central actions. I aimed at characterizing the neuromodulatory effects of OT in the hippocampus. Electrophysiological experiments were performed on mouse brain slices using the whole-cell patch-clamp technique on pyramidal neurons (PYR) and GABAergic interneurons (INs) located in CA1 stratum pyramidale. The effect of TGOT (Thr4,Gly7-oxytocin), a selective OT receptor (OTR) agonist, was first evaluated on spontaneous inhibitory postsynaptic currents (sIPSC) recorded from PYRs in Otr+/+ mice. TGOT caused a significant decrease in the sIPSC interval and an increase in the sIPSC amplitude; it also caused an increase in the sIPSC time constant of decay: this suggests the involvement of GABAA receptors (GABAAR) located in a perisynaptic position that deactivate slower than synaptic receptors, generating slower sIPSCs. The TGOT-mediated effects were dependent on the activation of OTRs, being abolished by the OTR antagonist SSR126768A; furthermore, TGOT didn’t modulate sIPSCs in Otr-/- mice. Then, we recorded the miniature inhibitory postsynaptic currents (mIPSC), isolated by applying tetrodotoxin, a voltage-gated Na+ channel blocker, to prevent action potential firing in the presynaptic terminal. TGOT was not able to modulate the mIPSC interval, amplitude and kinetics of decay, indicating that the effects elicited by the agonist are dependent on the firing activity of the presynaptic neuron. After having clarified the action of TGOT on ‘phasic’ inhibitory transmission, elicited by synaptic and perisynaptic GABAARs, we enquire if the peptide could influence ‘tonic’ currents, mediated by extrasynaptic GABAARs. First, we demonstrated the presence of tonic currents by measuring the ‘baseline holding current’ required to clamp PYRs at a given potential, in control conditions and during the application of the GABAAR antagonist bicuculline: we observed an inward shift in the ‘baseline holding current’ in the presence of bicuculline, consistent with the abolition of tonic currents. Then, we found that TGOT was able to increase tonic currents, causing an outward shift in the ‘baseline holding current’. Subsequently, we tried to understand the source of that TGOT-mediated increased inhibition, finding that TGOT depolarized mainly the stuttering fast-spiking INs. The same depolarization was observed in the presence of synaptic blockers, suggesting that the effect is due to a direct binding to OTRs. Indeed, the perfusion of the OTR antagonist completely abolished the depolarization. We tried to investigate the ionic mechanism underlying the TGOT-induced depolarization. We tested the putative involvement of a Ca2+ current by using nifedipine, a selective L-type channel blocker. Actually, in the majority of INs examined, nifedipine was able to abolish the depolarization elicited by TGOT. Finally, we investigated the effect of TGOT on the membrane potential of PYRs. Most of them, examined at their spike threshold, became hyperpolarized by TGOT and their firing rate was significantly decreased. The hyperpolarizing response was completely abolished by the blockade of GABAARs, indicating that the effect requires the activation of extrasynaptic GABAARs that mediate a prolonged (or tonic) hyperpolarizing current. The TGOT-mediated hyperpolarization caused a reduction in cell excitability, altering the capability of PYRs to generate action potentials in response to depolarizing current steps. This was evident in the firing rate-to-injected current (F-I) relationship that was shifted to the right during perfusion of TGOT. The gain (i.e., the slope) of the curve was not influenced by TGOT. This behavior indicates an increase in tonically active inhibitory currents that lead to a persistent reduction in the input resistance and therefore in cell excitability.

Thesis (Ph.D.)--University of Toledo, 2007.<br>"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Elizabeth Tietz. Includes abstract. Title from title page of PDF document. Bibliography: pages 88-94, 130-136, 178-189, 218-266.

Homeostatic synaptic plasticity (HSP) regulates synaptic strength in response to changing neuronal firing patterns. This form of plasticity is defined by neurons’ ability to sense and over time integrate their level of firing activity, and to actively maintain it within a defined range. For instance, a compensatory increase in synaptic strength occurs when neuronal activity is chronically attenuated. However, the underpinning cellular mechanisms of this fundamental neural process remain poorly understood. We previously found that during activity deprivation, HSP leads to an increase in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor function as well as a shift in subunit composition from Ca2+-impermeable GluA2-containing AMPA receptors to Ca2+-permeable GluA2-lacking AMPA receptors not only at synapses, but also at extrasynaptic sites. Neurons therefore appear to be actively enhancing Ca2+ entry, possibly as a compensatory mechanism in response to a prolonged Ca2+ deficit. To test whether the homeostatic response may, at least in part, be mediated by internal Ca2+ stores, we depleted endoplasmic reticulum (ER) Ca2+ stores by using the Sarco/endoplasmic reticulum Ca2+ ATPases (SERCA) pump blocker cyclopiazonic acid (CPA) for a prolonged period. Interestingly, we have found that prolonged Ca2+-store depletion leads not only to an increase in synaptic strength per se, but also a cell-wide increase in synaptic Ca2+-permeable GluA2-lacking AMPARs. This increase in Ca2+ influx following periods of inactivity is conceptually highly reminiscent of a store-operated response, whereby cells re-establish their calcium levels following Ca2+ store depletion using cell surface Ca2+ channels. Our results suggest that neurons use synaptic receptors as means to regulate store Ca2+ levels, thus significantly expanding our understanding of the repertoire used by neurons to modulate cellular excitability.

The electrophysiological properties of somatic and dendritic membranes of CA1 pyramidal neurons were investigated using the rat in vitro hippocampal slice preparation. A comprehensive analysis of extracellular field potentials, current-source density (CSD) and intracellular activity has served to identify the site of origin of action potential (AP) discharge in CA1 pyramidal neurons. 1) Action potential discharge of CA1 pyramidal cells was evoked by suprathreshold stimulation of the alveus (antidromic) or afferent synaptic inputs in stratum oriens (SO) or stratum radiatum (SR). Laminar profiles of the "stimulus evoked" extracellular field potentials were recorded at 25µm intervals along the dendro-somatic axis of the pyramidal cell and a 1-dimensional CSD analysis applied. 2) The shortest latency population spike response and current sink was recorded in stratum pyramidale or the proximal stratum oriens, a region corresponding to somata and axon hillocks of CA1 pyramidal neurons. A biphasic positive/negative spike potential (current source/sink) was recorded in dendritic regions, with both components increasing in peak latency through the dendritic field with distance from the border of stratum pyramidale. 3) A comparative intracellular analysis of evoked activity in somatic and dendritic membranes revealed a basic similarity in the pattern of AP discharge at all levels of the dendro-somatic axis. Stimulation of the alveus, SO, or SR evoked a single spike while injection of depolarizing current evoked a repetitive train of spikes grouped for comparative purposes into three basic patterns of AP discharge. 4) Both current and stimulus evoked intracellular spikes displayed a progressive decline in amplitude and increase in halfwidth with distance from the border of stratum pyramidale. 5) The only consistent voltage threshold for intracellular spike discharge was found in the region of the cell body, with no apparent threshold for spike activation in dendritic locations. 6) Stimulus evoked intradendritic spikes were evoked beyond the peak of the population spike recorded in stratum pyramidale, and aligned with the biphasic extradendritic field potential shown through laminar profile analysis to conduct with increasing latency from the cell body layer. The evoked characteristics of action potential discharge in CA1 pyramidal cells are interpreted to indicate the initial generation of a spike in the region of the soma-axon hillock and a subsequent retrograde spike invasion of dendritic arborizations.<br>Medicine, Faculty of<br>Cellular and Physiological Sciences, Department of<br>Graduate

In der Großhirnrinde von Säugetieren befindet sich die Mehrheit erregender Synapsen auf Dornfortsätzen, kleinen dendritischen Ausbuchtungen, die in Größe und Form stark variieren. Die Auslösung aktivitätsabhängiger synaptischer Langzeitplastizität geht mit strukturellen Veränderungen dendritischer Dornen einher. Da das beugungsbegrenzte Auflösungsvermögen konventioneller Lichtmikroskope nicht ausreicht um die Morphologie der Dornen verlässlich zu untersuchen, stellte die Elektronenmikroskopie bisher das wichtigste bildgebende Verfahren zur Erforschung von struktureller Plastizität dar, blieb dabei jedoch auf die Betrachtung fixierter Gewebeproben beschränkt. Die Anwendung hochauflösender Laser-Raster-Mikroskopie mit Stimulierter-Emissions-Auslöschung hat es mir möglich gemacht, die Dynamik dendritischer Dornenmorphologie in lebenden Zellen zu studieren. Die N-Methyl-D-Aspartat-Rezeptor-abhängige Langzeitpotenzierung von Pyramidenzellen der Cornu-Ammonis Region 1 des Hippocampus bildete dabei den Mechanismus, welcher plastische Veränderungen hervorrief. Nach Potenzierung exzitatorischer Synapsen durch die lokale Ultraviolett-Photolyse von caged-Glutamat wurde ein starker, vorübergehender Anstieg des Anteils dendritischer Dornen mit sichelförmigen Köpfen und ein leichter, anhaltender Zuwachs an pilzförmigen Dornfortsätzen über einen Zeitraum von 50 Minuten beobachtet. Meine Untersuchungen ergänzen frühere Studien zur Wechselbeziehung zwischen synaptischer Potenzierung und struktureller Plastizität dendritischer Dornen und korrespondieren mit dem aktuellen Kenntnisstand der zu Grunde liegenden molekularen Mechanismen.<br>The majority of excitatory synapses in the cortex of mammalian brains is situated on dendritic spines, small protrusions, heterogeneous in size and shape. The induction of activity-dependent long-term synaptic plasticity has been associated with changes in the ultrastructure of spines, particularly in size, head shape and neck width. Since the dimensions of dendritic spines are at the border of the diffraction-limited resolving power of conventional light microscopes, until recently, electron microscopy on fixed tissue constituted the primary method for investigations on spine morphology. I have employed live cell stimulated emission depletion imaging to analyse spine motility and structural transitions in response to n-methyl-d-aspartate receptor dependent long-term potentiation over time at super-resolution in Cornu Ammonis area 1 pyramidal neurons of the hippocampus. Local induction of long-term potentiation via ultraviolet photolysis of caged glutamate facilitated a strong transient increase in the proportion of spines with curved heads and a subtle persistent growth in the amount of mushroom spines over a time course of 50 minutes. My findings reinforce previous investigations on the relation of synaptic potentiation and spine motility, and are in good agreement with the current knowledge of the molecular mechanisms underlying long-term plasticity.

The layer 4 neurones of the mammalian primary sensory neocortex comprise diverse functional components for the first stage of cortical sensory processing. Dual intracellular recordings of synaptically connected pairs of neurones with biocytin-filling were used to study intra-laminar layer 4 connections in adult cat and rat slices. Interestingly, all excitatory cells involved in intralaminar layer 4 connections were regular spiking despite burst firing cells comprising 37% of the population recorded. Neuronal morphology and synaptic properties were similar in both species, as were the probabilities of finding connections; 1 in 43 for pyramid to pyramid, 1 in 21 for pyramid to interneurone and 1 in 12 for interneurone to pyramid (cat and rat data combined). Pyramid to pyramid connections generated EPSPs 1.33 0.9mV in amplitude (mean SD), with rise times of 1.71 0.83ms and half width 14.67 7.1ms. All EPSPs recorded in excitatory cells and in parvalbumin immuno-positive interneurones exhibited depression, the second and subsequent EPSPs in trains being smaller in amplitude than the first. Fluctuation analysis indicated that this depression was presynaptically mediated. The interneuronal EPSPs recorded in this study, were briefer (rise times 0.63 0.26ms and half width 5.25 2.85ms) than those recorded in pyramidal cells. Two interneurones that were immuno-negative for parvalbumin received EPSPs that were facilitating, second and subsequent EPSPs in trains being significantly larger than the first. Again, fluctuation analysis indicated that this facilitation was presynaptically mediated. Possible branch point failure of axonal conduction, feed-forward inhibition and post-tetanic potentiation were observed at some excitatory connections in layer 4. In addition, novel evidence for electrical gap junctions between adult pyramidal cells was obtained in one dual recording in which current injections into an impaled layer 3 pyramidal cell elicited full action potentials and 'spikelets', both of which elicited EPSPs in a layer 5 pyramidal cell.

Epilepsy is a neurological disorder characterized by abnormal neuronal excitability which has been suggested to result from altered ion channel activity. Of particular interest are the hyperpolarization-activated cation (HCN) channels which have recently been shown to play an important role in temporal lobe epilepsy (TLE) development. To evaluate how HCN channels are modulated during TLE development, I obtained acute hippocampal slices from appropriate control and “epileptic” rats and recorded the hyperpolarization-activated cation current (Ih) from CA1 pyramidal neurons. My results showed that Ih was enhanced 24 hours after seizure-induction compared to controls but this effect was diminished 1week later. However, in chronically epileptic rats that had started to experience spontaneous seizures Ih was significantly reduced compared to controls, and the excitability of CA1 pyramidal neurons was profoundly increased. To elucidate whether Ih modulation per se affects CA1 pyramidal neuron excitability, I used a recently published conductance-based model of a reconstructed CA1 pyramidal neuron which I adapted to match my experimental conditions. My simulations showed that modifications in Ih influences CA1 pyramidal neuron excitability consistent with that observed in “epileptic” neurons. The simulations also suggested that alteration in other currents, such as the persistent sodium current, together with the reduction of Ih, is required to determine the hyperexcitability of CA1 pyramidal neurons from chronically epileptic rats. In conclusion, Ih can be differentially modulated in CA1 pyramidal neurons during TLE development. These changes are likely to significantly affect CA1 pyramidal cell excitability and may contribute to the process of epileptogenesis.

Spike timing during oscillation has been suggested to play an important role in hippocampal processing. However, how the hippocampal network and the individual neurons interact to precisely control spike timing when they receive synaptic inputs from two major excitatory input pathways - Schaffer collateral and perforant path - during natural network oscillation is yet unknown. Investigation of spike timing control mechanism would shed light on how the local STDP learning rule could be influenced by different cortical inputs during theta oscillation. Here I used whole-cell path-clamp recording of CAl pyramidal neurons in vitro and dynamic clamp to simulate in vivo-like theta frequency oscillation at the soma to characterise the spike timing responses of CAl pyramidal neurons to Schaffer collateral and perforant path inputs during theta oscillation and present them as phase response curves (PRCs), Analysis of PRCs revealed that postsynaptic spike times could not only be advanced but also be delayed depending on the timing of excitatory inputs relative to the oscillation. Such control of spike timing during theta oscillation was dependent on the synaptic weight of the input and the frequency of the oscillation. Ih and GABAB receptor-mediated inhibition were identified as an intrinsic and synaptic mechanism, respectively, underlying spike time delay during oscillation. Activation of both Ih and GABAi3 receptor-mediated inhibition by perforant path stimulation contributed to greater spike time delay compared to that with Schaf.:fer collateral input stimulation which was only mediated by Ih. Such different spike timing characteristics were important in STDP induction at the Schaffer collateral-CAl pyramidal cell synapse, Depending on the timing of the perforant path activation during theta oscillation, perforant path input could control the timing of the postsynaptic spike during STDP induction which could reverse the sign of the synaptic modification, Thus, during natural network oscillation with multiple synaptic inputs active, timing of the heterosynaptic inputs from entorhinal cortex to the hippocampus could control the outcome of the homosynaptic plasticity in the CAL These results may have implications for how the external information could be encoded and stored in the hippocampal network.

Integrins are cell adhesion heterodimers that mediate cell-cell and cell-extracellular matrix (ECM) interactions. They regulate various cellular functions in the central nervous system such as the localization of ion channels, calcium (Ca2+) homeostasis and synaptic transmission. Impairment of these functions can lead to neurodevelopment disorders such as Autism Spectrum Disorders (ASD). Small-conductance Ca2+-activated K+ channels (SK channels) are responsible for the medium afterhyperpolarization (mAHP) current (mIAHP) that regulates excitability and firing pattern in many neurons. I investigated firing properties of layer V pyramidal neurons of the medial prefrontal cortex (mPFC) in WT and Itgb3, the murine gene encoding the β3 integrin subunit, KO mice. I could distinguish two populations of pyramidal neurons in layer V (LV): extratelencephalic (ET) and intratelencephalic (IT). By using electrophysiological recordings and pharmacology, I identify a mIAHP in both types of neurons and in both genotypes, albeit with different characteristics: mIAHP is larger and faster in ET than in IT neurons; in the Itgb3 KO, the mIAHP is smaller in ET neurons, as compared to WT. Furthermore, the SK channel-specific blocker apamin affects differently the firing pattern of ET and IT neurons; it increases adaptation in ET neurons, while having no significant effect in IT neurons. To complement the electrophysiological results, I used viral retrograde labelling to investigate expression of β3 integrin and SK channel in both neuronal types. Altogether, my findings indicate that β3 integrin regulates the mIAHP and the firing properties of layer V pyramidal neurons, although to different degrees in ET and IT neurons. These differences might mirror the diverse genetic, anatomy and function of the two neuron subpopulations, allowing them to respond differently to external perturbations that can promote neurodevelopment disorders.

Dans l'hippocampe, les processus de mémoire et d'apprentissage dépendent fortement de l'inhibition GABAergique, qui est fournis par une population hétérogène d'interneurones (INs) via l'activation de sous-types spécifiques de récepteurs GABA. La sous-unité alpha5-GABAAR (α5-GABAAR) est fortement exprimée dans l'hippocampe de la souris, du singe et du cerveau humain. Il a été rapporté que, dans les cellules pyramidales CA1, cette sous-unité est principalement localisée sur les sites extrasynaptiques, où elle est responsable de la génération de la conductance inhibitrice tonique. Si la sous-unité α5-GABAAR peut être ciblée sur des types spécifiques de synapses dans des types cellulaires distincts reste inconnue. En utilisant l'immunohistochimie dans des coupes d'hippocampe de souris, nous avons étudié l'expression spécifique de la sous-unité α5-GABAAR dans les cellules et les synapses de l’oriens/alveus de le région CA1. Nos résultats démontrent que la sous-unité α5-GABAAR est principalement exprimée dans les INs positifs à la somatostatine. De plus, la densité de sous-unité était plus élevée dans les dendrites proximales et diminuait avec la distance par rapport au soma, ce qui correspond à une diminution de la densité des synapses inhibitrices dépendant de la distance. De plus, l'α5-GABAAR ciblait les synapses formées par les entrées exprimant le peptide intestinal vasoactif (VIP+) et la calrétinine (CR+) et, dans une moindre mesure, celles produites par les projections exprimant de la parvalbumine (PV+). En résumé, nos résultats montrent que la sous-unité α5-GABAAR présente une expression spécifique à la cellule et à la synapse dans l'hippocampe CA1. Comme la sous-unité α5-GABAAR a été impliquée dans plusieurs maladies, comprenant la maladie d'Alzheimer et le syndrome de Down, les nouvelles connaissances sur la localisation de l'α5-GABAAR seront importantes pour le développement de la thérapie cellulaire spécifique.<br>In the hippocampus, memory and learning processes are highly dependent on the GABAergic inhibition, which is provided by a heterogeneous population of interneurons (INs) via activation of specific sub-types of GABA receptors. The alpha5-GABAAR subunit (α5-GABAAR) is highly expressed in the hippocampus of the mouse, monkey and human brain. It has been reported that, in the CA1 pyramidal cells, this subunit is predominantly located at extrasynaptic sites, where it is responsible for generation of tonic inhibitory conductance. Whether the α5-GABAAR subunit can be targeted to specific types of synapses in distinct cell types remains unknown. Using immunohistochemistry and electophysiological approach in mouse hippocampal slices, we studied the cell- and synapse-specific expression of the α5-GABAAR subunit in the CA1 oriens/alveus INs. Our results demonstrate that the α5-GABAAR subunit is mainly expressed in the somatostatin-positive INs. In addition, the subunit density was higher in proximal dendrites and declined with distance from the soma, consistent with a distance-dependent decrease in the density of inhibitory synapses. Furthermore, the α5-GABAAR was targeted to synapses made by the vasoactive intestinal peptide (VIP+)- and calretinin (CR+)-expressing inputs and to a lesser extent to those made by the parvalbumin-positive (PV+) projections. In summary, our results show that the α5-GABAAR subunit exhibits a cell- and input-specific expression in the CA1 hippocampus. As the α5-GABAAR subunit has been implicated in several diseases, including Alzheimer’s disease and Down syndrome, the new insights into the α5-GABAAR localization will be important for the development of cell- and site-specific therapy.

Si les connaissances relatives au vieillissement du systeme nerveux central ont considerablement progresse au cours des dernieres annees, il reste a determiner si l'une ou l'autre des alterations, apparaissant au cours du temps, est plus determinante dans le dysfonctionnement neuronal. Nous nous sommes interesses aux modifications des proprietes des neurones pyramidaux du champ ca1 de l'hippocampe survenant au cours du temps, et plus precisement a la contribution relative des atteintes cholinergique et de l'homeostasie calcique grace a deux modeles animaux (denervation cholinergique induit par une immunotoxine, la 192 igg-saporine et deficit calcique induit chez des souris transgeniques n'exprimant plus une proteine liant le calcium, la calbindine ou cabp). Nous avons montre chez l'animal age une diminution de l'excitabilite membranaire independante des atteintes cholinergique ou calcique. Une diminution des reponses synaptiques inhibitrices mediees par les recepteurs gaba#b etaient egalement observee mais qui n'etait pas retrouvee chez les souris deficientes en cabp. Ceci indique que la perte de la calbindine observee chez l'animal age ne peut rendre compte a elle seule de l'atteinte des reponses gaba#b comme suggere dans la litterature. Les reponses glutamatergiques non-nmda etaient deprimees du fait d'une diminution de la liberation du glutamate tandis que les reponses nmda demeuraient peu affectees malgre une baisse de densite, suggerant l'intervention de phenomenes de compensation. Enfin, aucune modification des mecanismes de plasticite synaptique n'etait observee chez ces animaux. L'absence d'innervation cholinergique chez les rats traites par la saporine entrainait une augmentation des reponses glutamatergiques sans modification significative de la liberation du glutamate et du nombre de ces recepteurs. Ceci suggere un controle tonique inhibiteur de l'acetylcholine (ach) sur les recepteurs glutamatergiques et plus specifiquement sur ceux de type nmda. Par ailleurs, l'absence d'alteration de la plasticite synaptique chez ces animaux remet en question une participation significative de l'ach dans cette propriete neuronale. Parallelement, nous avons montre chez les souris deficientes en cabp l'atteinte de proprietes membranaires dependante du calcium, suggerant une regulation des canaux calciques par cette proteine. D'autre part, l'absence de cabp entrainait une alteration des reponses glutamatergiques qui pourrait refleter une augmentation de la densite des recepteurs non-nmda et une inactivation plus rapide des recepteurs nmda. Enfin, un deficit des potentialisation a long terme etait observe suggerant, pour la premiere fois la participation de la calbindine dans les mecanismes de plasticite synaptique. Ainsi grace a cette etude, nous avons pu non seulement preciser le role de l'ach et de la calbindine dans la fonction neuronale, mais aussi preciser la participation relative des atteintes cholinergique et calcique dans les alterations liees au vieillissement cerebral.

Le neurone est une cellule hautement spécialisée qui permet, par des impulsions électriques appelées potentiel d’action (PA) d’assurer la communication neuronale de manière rapide et efficace vers les autres neurones du cerveau. L’axone occupe une place privilégiée dans la genèse du PA. En effet, une région spécialisée de l’axone appelé segment initial de l’axone (SIA) concentre les protéines canaux qui sont à l’origine du PA, les canaux sodium.Le sujet de cette thèse a pour objet d’identifier les facteurs géométriques et électriques contrôlant le seuil du PA. Par une approche essentiellement électrophysiologique couplée à la modélisation, nous identifions ici pour la première fois l’importance de la résistance axiale de l’axone, des canaux sodium et de certains canaux potassium dans le seuil du PA mesuré au corps cellulaire. Cette étude devrait permettre d’affiner et de valider les modèles de seuil du PA en apportant une meilleure compréhension de l’excitabilité neuronale<br>The neuron is a highly specialized cell which permits, thanks to electrical impulsion called action potential (AP), to ensure the neuronal communication in a quick and efficient manner towards the other neurons of the brain. The axon takes a privileged place in AP genesis. Indeed, a specified region of the axon, called the axon initial segment (AIS) concentrates channel proteins that are at the origin of the AP, the sodium channels.The subject of this thesis aims to identify the geometrical and electrical factors controlling the threshold of AP. Essentially using an electrophysiological approach coupled with modeling, we identify for the first time here the importance of the axial resistance of the axon, the sodium channels, and some of the potassium channels in the threshold of AP measured in the cell body. This study should permit to refine and validate models of AP threshold by bringing a better understanding of neuronal excitability

Tese de mestrado. Biologia Humana e Ambiente. Universidade de Lisboa, Faculdade de Ciências, 2010<br>Stress is one of the primary factors leading to many disorders, including depression, one of the most prevalent psychiatric disorders. Additionally, it has been well documented that hippocampal plasticity is vulnerable to the effects of stress and these effects are often sexually differentiated. Women are twice as likely as men to experience stress-related disorders during the lifespan. In fact, a growing number of women experience psychological stress, such as depression and anxiety, during pregnancy and the postpartum period. This maternal stress may have detrimental effects on maternal mood and maternal care of offspring. In turn, recent research has documented a significant impact of pregnancy and motherhood on hippocampus plasticity in the mother. However, very little research has focused the impact of stress during gestation on the neurobiology of mother. Therefore, the present study investigated how stress affects dendritic morphology of CA3 pyramidal neurons in the hippocampus of pregnant females, and whether these effects differ from those in virgin females. Age-matched pregnant and virgin female Wistar rats were divided into two conditions: 1) Stress and 2) Control. Females in the stress condition were restrained for 1 hour/day for 2 weeks, beginning on gestation day 8 and at matched time-points in virgin females. Females were sacrificed the day after the last restraint session, prior to giving birth, and the brains were processed using Golgi impregnation technique. The results obtained show that repeated restraint stress results in dendritic atrophy in the apical region of CA3 pyramidal neurons in both pregnant and virgin females. Moreover, pregnant females resulted in less complex CA3 pyramidal neurons compared to virgin females. Stress had no effect on weight gain in virgin and pregnant, or litter characteristics and sex of fetuses in pregnant females. These factors were also not associated with CA3 dendritic morphology. Further work is needed to determine how restraint stress affects dendritic morphology in other regions of the hippocampus.<br>O quotidiano é preenchido por diversos episódios stressantes que podem representar uma grande ameaça ao bem-estar físico e emocional. De facto, o stress é um dos principais factores que leva a diversos transtornos, incluindo depressão, um dos transtornos psiquiátricos mais prevalentes. Assim, para lidar adequadamente com situações de stress, ajustes fisiológicos ou estratégias comportamentais são de extrema importância e são normalmente acompanhadas pela activação da resposta ao stress, com a intenção de manter ou alcançar a homeostase interna. Uma activação e desactivação da resposta ao stress bem sucedidas são, então, vitais para a sobrevivência. A resposta ao stress é coordenada pelo cérebro, que interpreta as experiências como ameaçadoras ou não e, de acordo com a situação, determina as respostas comportamentais e psicológicas. Portanto, quando uma ameaça real ou percebida ocorre, a resposta ao stress é activada no cérebro e envolve a libertação de hormonas pelo sistema nervoso simpático e pelo eixo hipotálamo-pituitária-adrenal (HPA). Os glucocorticóides (GC), cortisol nos humanos e corticosterona em roedores, desempenham um papel central na mediação de aspectos essenciais à resposta ao stress e retorno à homeostase. A duração do stress também está implicada nesta resposta neuronal, sendo que uma duração prolongada por mais de uma semana acarreta efeitos mais profundos ao nível dos neurónios. O hipocampo, constituído principalmente pelas regiões do cornu ammonis (CA) e pelo giro denteado (DG), para além de desempenhar um papel essencial na aprendizagem e memória, tem também a função de regulação de “feedback” negativo da resposta ao stress através do eixo HPA. A grande concentração de receptores de GC na formação hipocampal sugere que os efeitos desta hormona no hipocampo sejam directos, tornando esta área do cérebro particularmente sensível ao stress e aos GC. De facto, tem sido bem documentado que a plasticidade do hipocampo é vulnerável aos efeitos do stress, através de níveis elevados de GC, causando alterações estruturais e funcionais no hipocampo. Os neurónios piramidais da região CA3 do hipocampo são particularmente sensíveis ao efeito do stress crónico, apresentando remodelação dendrítica. Sendo que esta região está envolvida na formação de memórias e processamento espacial, é interessante que eventos stressantes repetitivos resultem em atrofia dos neurónios piramidais CA3, caracterizada pela redução da complexidade dendrítica e do comprimento dendrítico total em machos, o que igualmente afecta a função do hipocampo, incluindo perda de memória espacial. Esta remodelação dendrítica pode ter duas interpretações: uma resposta mal adaptada, com a retracção dendrítica a contribuir para uma maior vulnerabilidade do hipocampo a outros eventos, como doenças, e factores stressantes crónicos, ou uma resposta compensatória para protecção contra efeitos neurotóxicos. É também importante ter em consideração que estes efeitos do stress são muitas vezes sexualmente diferenciados. A propensão para desenvolver transtornos relacionados com stress é estimada em duas vezes mais para mulheres em relação aos homens, durante a vida. Esta tendência é marcada pelo envolvimento das hormonas gonadais femininas, progesterona e estradiol, e a sua acção no eixo HPA. Tendo em consideração que, para além de desempenharem um papel chave no desenvolvimento diferencial do cérebro, estas hormonas estão também envolvidas na formação da plasticidade cerebral nos principais centros emocionais e podem exercer um papel importante na modulação da resposta ao stress, é cada vez mais reconhecida uma ligação entre género e transtornos relacionados com stress, com as discrepâncias entre géneros atribuídas ao efeito das hormonas gonadais. O ciclo reprodutivo da mulher está intimamente relacionado com os níveis de GC, com elevada libertação desta hormona e elevada sensibilidade ao stress durante a fase folicular do ciclo menstrual bem como da fase proestro do ciclo estral em roedores, quando os níveis de estrogénio estão elevados. Assim, uma potencial combinação de GC e hormonas gonadais pode levar a uma maior incidência de transtornos relacionados com stress em fêmeas. De facto, um número crescente de mulheres sofre stress psicológico, como depressão e ansiedade, durante a gravidez e o período pós-parto. Por outro lado, pesquisas recentes têm documentado um impacto significativo da gravidez e maternidade na plasticidade do hipocampo da mãe. Este impacto pode estar relacionado com o envolvimento do hipocampo nas importantes adaptações hormonais, neurológicas e comportamentais necessárias na mãe para assegurar a sobrevivência da prole, na transição para a maternidade. A placenta, os ovários e o feto contribuem para as flutuações dramáticas de hormonas esteróides e peptídicas que ocorrem durante a gravidez e o período pós-parto e são importantes para a indução do circuito maternal e o inicio dos comportamentos maternos. Além disso, visto os efeitos que as hormonas esteróides têm nas propriedades estruturais do hipocampo, estas flutuações hormonais no período reprodutivo podem ter também um impacto na plasticidade desta área do cérebro. O stress e os níveis de GC têm também um impacto na mãe. Apesar das alterações normais nos níveis de GC serem importantes para diversos aspectos da maternidade, o stress durante a gestação leva ao aumento da concentração basal de GC e pode ter efeitos prejudiciais sobre o humor materno e os cuidados maternos da prole. No entanto, pouca pesquisa tem focado o impacto do stress durante a gestação sobre a neurobiologia da mãe. Assim, o presente estudo investigou o efeito do stress sobre a morfologia dendrítica dos neurónios piramidais da região CA3 do hipocampo de fêmeas grávidas e se, estes efeitos, diferem em fêmeas virgens. Ratos Wistar fêmeas, grávidas e virgens de idades correspondentes, foram divididos em duas condições: Stress e Controlo. As fêmeas na condição de stress foram contidas em caixas de contenção uma hora/dia durante duas semanas, começando no oitavo dia de gestação e em tempos correspondentes em fêmeas virgens. As fêmeas foram sacrificadas no dia a seguir à última sessão de contenção, antes do parto. O útero das fêmeas grávidas foi dissecado para permitir a contagem dos fetos, tendo também em conta o seu sexo. Os cérebros foram processados usando a técnica de impregnação de Golgi, que consiste numa impregnação metálica e permite detectar as árvores dendríticas e as espinhas dendríticas. Para a análise da morfologia dendrítica, seis células piramidais CA3 por cada cérebro foram escolhidas e o número de pontos de ramificação, bem como o comprimento total da árvore dendrítica, foram avaliados separadamente para a região apical e basal. A distribuição e complexidade das dendrites foram analisadas recorrendo à contagem das intersecções das dendrites com círculos concêntricos equidistantes (análise de Sholl). Os resultados obtidos mostraram que as fêmeas grávidas e virgens, na condição de stress, tiveram atrofia dendrítica significativa na região apical dos neurónios piramidais CA3, em comparação com as fêmeas controlo. Para além disso, as fêmeas grávidas apresentaram neurónios piramidais CA3 significativamente menos complexos, em comparação com as fêmeas virgens. O stress não teve efeito sobre o peso em virgens e grávidas, nem afectou as características das ninhadas. Este estudo forneceu novas evidências de que o stress e a gravidez têm um impacto na morfologia dendrítica dos neurónios piramidais CA3. Pesquisa futura irá avaliar a morfologia dendrítica e a densidade das espinhas dentríticas na região CA1 e DG bem como o possível papel do stress e da maternidade no desempenho de tarefas dependentes do hipocampo na fêmea adulta.<br>Fundação para a Ciência e a Tecnologia (FCT); Centro de Biologia Ambiental (CBA), Portugal

Intricate interactions among constituent components are defining hallmarks of biological systems and sculpt physiology across different scales spanning gene networks to behavioural repertoires. Whereas interactions among channels and receptors define neuronal physiology, interactions among different cells specify the characteristic features of network physiology. From a single-neuron perspective, it is now evident that the somato-dendritic plasma membrane of hippocampus pyramidal neurons is endowed with several voltage-gated ion channels (VGICs) with varying biophysical properties and sub cellular expression profiles. Structural and physiological interactions among these channels define generation and propagation of electrical signals, thereby transforming neuronal dendrites to actively excitable membrane endowed with complex computational capabilities. In parallel to this complex network of plasma membrane channels is an elegantly placed continuous intraneuronal membrane of the endoplasmic reticulum (ER) that runs throughout the neuronal morphology. Akin to the plasma membrane, the ER is also endowed with a variety of channels and receptors, prominent among them being the inositol trisphosphate (InsP3) receptors (InsP3Rs) and ryanodine receptors (RyR), both of which are calcium release channels. Physiological interactions among these receptors transform the ER into a calcium excitable membrane, capable of active propagation of calcium waves and of spatiotemporal integration of neuronal signals. Thus, a neuron is endowed with two continuously parallel excitable membranes that actively participate in the bidirectional flow of intraneuronal information, through interactions among different channels and receptors on either membrane. Although the interactions among sets of channels and receptors present individually on either membrane are very well characterized, our understanding of cross-membrane interactions among channels and receptors across these two membranes has been very limited. Recent literature has emphasized the critical nature of such cross-membrane interactions and the several physiological roles played by such interactions. Such cross-channel interactions include ER depletion-induced signaling involving store-operated calcium channels, generation and propagation of calcium waves through interactions between plasma membrane and ER membrane receptors, and the plasticity of plasma membrane VGICs and receptors induced by ER Ca2+. Such tight interactions between these two membranes have highlighted several roles of the ER in the integration of intraneuronal information, in regulating signalling microdomains and in regulating the downstream signaling pathways that are regulated by these Ca2+ signals. Yet, our understanding about the functional interactions between the ion channels and receptors present on either of these membranes is very limited from the perspective of the combinatorial possibilities that encompass the span of channels and receptors across these two membranes. In this context, the first part of this thesis deals with two specific instances of such cross-membrane functional interactions, presented as two subparts with each probing different direction of impact. Specifically, whereas the first of these subparts deals with the impact of plasma membrane VGICs on the physiology of ER receptors, the second subpart presents an instance of the effect of ER receptor activation on plasma membrane VGIC. In the first subpart of the thesis, we establish a novel role for the A-type potassium current in regulating the release of calcium through inositol triphosphate receptors (InsP3R) that reside on the endoplasmic reticulum (ER) of hippocampus pyramidal neurons. Specifically, the A-type potassium current has been implicated in the regulation of several physiological processes including the regulation of calcium influx through voltage-gated calcium channels (VGCCs). Given the dependence of InsP3R open probability on cytosolic calcium concentration ([Ca2+]c) we asked if this regulation of calcium influx by A-type potassium current could translate into the regulation of release of calcium through InsP3Rs by the A-type potassium current. To answer this, we constructed morphologically realistic, conductance-based neuronal models equipped with kinetic schemes that govern several calcium signalling modules and pathways, and constrained the distributions and properties of constitutive components by experimental measurements from these neurons. Employing these models, we establish a bell-shaped dependence of calcium release through InsP3Rs on the density of A-type potassium current, during the propagation of an intraneuronal calcium wave initiated through established protocols. Exploring the sensitivities of calcium wave initiation and propagation to several underlying parameters, we found that ER calcium release critically depends on dendrite diameter and wave initiation occurred at branch points as a consequence of high surface area to volume ratio of oblique dendrites. Further, analogous to the role of A-type potassium channels in regulating spike latency, we found that an increase in the density of A-type potassium channels led to increases in the latency and the temporal spread of a propagating calcium wave. Next, we incorporated kinetic models for the metabotropic glutamate receptor (miler) signalling components and a calcium-controlled plasticity rule into our model and demonstrate that the presence of mGluRs induced a leftward shift in a BCM-like synaptic plasticity profile. Finally, we show that the A-type potassium current could regulate the relative contribution of ER calcium to synaptic plasticity induced either through 900 pulses of various stimulus frequencies or through theta burst stimulation. These results establish a novel form of interaction between active dendrites and the ER membrane and suggest that A-type K+ channels are ideally placed for inhibiting the suppression of InsP3Rs in thin-caliber dendrites. Furthermore, they uncover a powerful mechanism that could regulate biophysical/biochemical signal integration and steer the spatiotemporal spread of signalling micro domains through changes in dendritic excitability. In the second subpart, we turned our focus to the role of calcium released through InsP3Rs in regulating the properties of VGICs present on the plasma membrane, thereby altering neuronal intrinsic properties that are dependent on these VGICs. Specifically, the synaptic plasticity literature has focused on establishing necessity and sufficiency as two essential and distinct features in causally relating a signalling molecule to plasticity induction, an approach that has been surprisingly lacking in the intrinsic plasticity literature. Here, we complemented the recently established necessity of inositol trisphosphate (InsP3) receptors (InsP3R) in a form of intrinsic plasticity by asking if ER InsP3R activation was sufficient to induce plasticity in intrinsic properties of hippocampus neurons. To do this, we employed whole-cell patch-clamp recordings to infuse D-myo-InsP3, the endogenous ligand for InsP3Rs, into hippocampus pyramidal neurons and assessed the impact of InsP3R activation on neuronal intrinsic properties. We found that such activation reduced input resistance, maximal impedance amplitude and temporal summation, but increased resonance frequency, resonance strength, sag ratio, and impedance phase lead of hippocampus pyramidal neurons. Strikingly, the magnitude of plasticity in all these measurements was dependent upon [InsP3], emphasizing the graded dependence of such plasticity on InsP3R activation. Mechanistically, we found that this InsP3-induced plasticity depended on hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. Moreover, this calcium-dependent form of plasticity was critically reliant on the release of calcium through InsP3Rs, the influx of calcium through N-methyl-D -aspartate receptors and voltage-gated calcium channels, and on the protein kinase A pathway. These results delineate a causal role for InsP3Rs in graded adaptation of neuronal response dynamics through changes in plasma membrane ion channels, thereby revealing novel regulatory roles for the endoplasmic reticulum in neural coding and homeostasis. Whereas the first part of the thesis dealt with bidirectional interactions between ER membrane and plasma membrane channels/receptors within a neuron, second part focuses on cross-cellular interactions, specifically between ER membrane on astrocytes and dendritic plasma membrane of neurons. Specifically, the universality of ER-dependent calcium signalling ensures that its critical influence extends to regulating the physiology of astrocytes, an abundant form of glial cells in the hippocampus. Due to the presence of calcium release channels on ER membrane, astrocytes are calcium excitable, whereby they respond to neuronal activity by increase in their cytosolic calcium levels. Specifically, astrocytes respond to the release of neurotransmitters from neuronal presynaptic terminals through activation of metabotropic receptors expressed on their plasma membrane. Such activation results in the mobilization of cytosolic InsP3 and subsequent release of calcium through InsP3 on the astrocytes ER membrane. These ER-dependent [Ca2+]c elevations in astrocytes then result in the release of gliotransmitters from astrocytes, which bind to corresponding receptors located on neuronal plasma membrane resulting in voltage-deflections and/or activation of signaling pathways in the neuron. Although it is well established that gliotransmission constitutes an important communication channel between astrocytes and neurons, the impact of gliotransmission on neurons have largely been centered at the cell body of the neurons. Consequently, the impact of the activation of astrocytic InsP3R on neuronal dendrites, and the role of dendritic active conductances in regulating this impact have been lacking. This lacuna in mapping the spatial spread of gliotransmission in neurons is especially striking because most afferent synapses impinge on neuronal dendrites, and a significant proportion of information processing in neurons is performed in their dendritic arborization. Additionally, given that active dendritic conductances play a pivotal role in regulating the impact of fast synaptic neurotransmission on neurons, we hypothesized that such active-dendritic regulation should extend to the impact of slower extrasynaptic gliotransmission on neurons. The second part of the thesis is devoted to testing this hypothesis using dendritic and paired astrocyte-neuron electrophysiological recordings, where we also investigate the specific roles of active dendritic conductances in regulating the impact of gliotransmission initiated through activation of astrocytic InsP3Rs. In testing this hypothesis, in the second part of the thesis, we first demonstrate a significantly large increase in the amplitude of astrocytically originating spontaneous slow excitatory potentials (SEP) in distal dendrites compared to their perisomatic counterparts. Employing specific neuronal infusion of pharmacological agents, we show that blocking HCN channels increased the frequency, rise-time and width of dendritically-recorded spontaneous SEPs, whereas blockade of A-type potassium channels enhanced their amplitude. Next, through paired neuron-astrocytes recordings, we show that our conclusions on the differential roles of HCN and A-type potassium channels in modulating spontaneous SEPs also extended to SEPs induced through infusion of InsP3 in a nearby astrocyte. Additionally, employing subtype-specific receptor blockers during paired neuron-astrocyte recordings, we provide evidence that GluN2B-and GluN2D-containing NMDARs predominantly mediate perisomatic and dendritic SEPs, respectively. Finally, using morphologically realistic conductance-based computational models, we quantitatively demonstrate that dendritic conductances play an active role in mediating compartmentalization of the neuronal impact of gliotransmission. These results unveil an important role for active dendrites in regulating the impact of gliotransmission on neurons, and suggest astrocytes as a source of dendritic plateau potentials that have been implicated in localized plasticity and place cell formation. This thesis is organized into six chapters as follows: Chapter 1 lays the motivations for the questions addressed in the thesis apart from providing the highlights of the results presented here. Chapter 2 provides the background literature for the thesis, introducing facts and concepts that forms the foundation on which the rest of the chapters are built upon. In chapter 3, we present quantitative analyses of the physiological interactions between A-type potassium conductances and InsP3Rs in CA1 pyramidal neurons. In chapter 4, using electrophysiological recordings, we investigate the role of calcium released through InsP3Rs in induction of plasticity of intrinsic response dynamics, and demonstrate that this form of plasticity is consequent to changes in neuronal HCN channels. In chapter 5, we systematically map the spatial dynamics of the impact of gliotransmission on neurons across the somato-apical trunk, also unveiling the role of neuronal HCN and A-type potassium channels in compartmentalizing such impact. Finally, chapter 6 concludes the thesis highlighting its major contributions and discussing directions of future research.

The biophysical properties and distribution of voltage gated ion channels shape the spatio-temporal pattern of synaptic inputs and determine the input-output properties of the neuron. Of the various voltage-gated ion channels, persistent Na⁺ current (INaP) is of interest because of its activation near rest, slow inactivation kinetics, and consequent effects on excitability. Overshadowed by transient Na⁺ current (INaT) of large amplitude and fast inactivation, various quantitative characterizations of INaP have yet to provide a clear understanding of their role in neuronal excitability. We addressed this question using quantitative electrophysiology to compare somatic INaP and INaT in 4–7 week old Sprague-Dawley rat hippocampal CA1 pyramidal neurons. INaP was evoked with 0.4 mV/ms ramp voltage commands and INaT with step commands in hippocampal neurons from in vitro brain slices utilizing nucleated patch-clamp recording. INaP was found to have a density of 1.4 ± 0.7 pA/pF in the soma. Compared to INaT, it has a much smaller amplitude (2.38% of INaT) and distinct voltage dependence of activation (16.7 mV lower half maximal activation voltage and 41.3% smaller slope factor than those of INaT). The quantitative measurement of INaT gave the activation time constant ([tau]m) of 22.2 ± 2.3 [mu]s at 40 mV. Hexanol, which has anesthetic effects, was shown to preferentially block INaP compared to INaT with a significant voltage threshold elevation (4.6 ± 0.7 mV) and delayed 1st spike latency (221 ± 54.6 ms) suggesting reduced neuronal excitability. The number of spikes evoked by either given step current injections or [alpha]-EPSP integration was also significantly decreased. The differential blocking of INaP by halothane, a popularly used volatile anesthetic, further supports the critical role of INaP in setting voltage threshold. Taken together, the presence of INaP in the soma demonstrates an intrinsic mechanism utilized by hippocampal CA1 pyramidal neurons to regulate axonal spike initiation through different biophysical properties of the Na⁺ channel. Furthermore, INaP becomes an interesting target of intrinsic plasticity because of its profound effect on the input-output function of the neuron.<br>text

In response to an action potential (AP), a transient rise in the intracellular calcium concentration ([Ca2+]i) causes transmitter release from nerve terminals. As the spatiotemporal dynamics of this calcium rise can affect the efficacy and plasticity of synaptic connections, it is essential to understand their determinants. To characterise factors that shape calcium transients in neocortical synaptic boutons, layer 5 pyramidal cells in the rat somatosensory cortex were filled through the patch pipette with a fluorescent calcium indicator for the measurement of [Ca2+]i. For accurate calculation of [Ca2+]i from the fluorescence intensity, the calcium binding affinities (Kd) of the indicators were measured in vitro, in solutions that were similar to the patch-clamp internal solution. These solutions were made with various concentrations of calcium chloride, but a constant concentration of a calcium buffer. The resultant free [Ca2+] was measured with a calcium-selective macroelectrode. It was found that the Kd values of the calcium indicators were considerably different from those previously published or provided by the manufacturers. Two main determinants of the intracellular calcium dynamics are the capacity of endogenous calcium buffers and the activity of calcium sequestration mechanisms. By measuring the peak amplitude of single AP-evoked calcium transients with different concentrations of OGB-1 or OGB-6F, a value of 7 was estimated for the calcium-binding ratio of endogenous buffers. Thus, in response to a single AP and in the absence of exogenous buffers, [Ca2+]i was raised by 5.3 microM, with a total change of approximately 50 microM. The rate constant of calcium sequestration (0.60 per s) was estimated from the slow decay time constant of the measured transients. The initial fast decay did not prolong when intracellular calcium uptake was inhibited, or speed up during repetitive stimulation. These findings suggest that calcium-induced calcium release (CICR), buffer saturation, and a non-linear calcium transporter were not the main cause of the bi-exponential decay. A 3D model of a bouton en passant showed that diffusion of calcium into the axon was likely the underlying mechanism. During high-frequency stimulation, CICR contributed to a supralinear summation of [Ca2+]i. Spontaneous increases in [Ca2+]i have been observed in several nerve terminals. They have been implicated in a number of cellular processes, including calcium homeostasis and spontaneous transmitter release. Here, the high-affinity calcium indicator OGB-1 was used to monitor small changes in [Ca2+]i. Spontaneous calcium transients (sCaTs) were observed at a frequency of around 0.2 per min. The increase in [Ca2+]i associated with each sCaT was 1.4–2.3 microM, in the absence of exogenous buffers. It was hypothesised that sCaTs arose from calcium release from presynaptic stores. In support of this, caffeine increased the average frequency of sCaTs by approximately 90%. The amplitude and kinetics of sCaTs identified in caffeine and in the control condition were not different from each other, suggesting that the majority of sCaTs might have been a result of calcium release through ryanodine receptors. The functional consequence(s) of sCaTs in neocortical synaptic boutons remains to be determined.

Fragile X Syndrome is the most common form of heritable cognitive disability. It is caused by a genetic mutation that leads to a lack of protein from the FMR1 gene. This protein (FMRP) is used to regulate the translation of many other proteins, thereby leading to a wide range of effects. Because the origin of this disease is based on the lack of a single protein, an animal model with construct validity can be used to investigate the potential effects leading to the symptoms of the disease. Many studies have investigated the synaptic plasticity differences of CA1 pyramidal neurons between a mouse model of fragile X syndrome (KO) and a wild type mouse (WT). This study investigates the differences in firing properties of a CA1 pyramidal neuron between the KO and WT. Specifically, contributions of two ion channels are investigated: the Ca2+ and voltage activated potassium channel (BK) and the potassium channel (M) inhibited by the muscarinic acetylcholine receptor. This study finds some differences that warrant further investigation, including differences in spike timing, spike width and the initial rate of rise of an action potential. However, several areas of investigation yield subtle or confounding results, which may indicate that the CA1 pyramidal neurons affected by the lack of FMRP may make up more than one population.<br>text

Epilepsy is characterized by the hyperexcitability of individual neurons and hyper synchronization of groups of neurons (networks). The acquired changes that take place at molecular, cellular and network levels are important for the induction and maintenance of epileptic activity in the brain. Epileptic activity is known to alter the intrinsic properties and signaling of neurons. Understanding acquired changes that cause epilepsy may lead to innovative strategies to prevent or cure this neurological disorder. Advances in in vitro electrophysiological techniques together with experimental models of epilepsy are indispensible tools to understand molecular, cellular and network mechanisms that underlie epileptiform activity. The aim of the study was to investigate the epileptiform activity induced alterations in Ca2+ dynamics in apical dendrites of hippocampal subicular pyramidal neurons in slices and changes in network properties of cultured hippocampal neurons. We have also made attempts to develop an in vitro model of epilepsy using organotypic hippocampal slice cultures. In the first part of the present study, investigations on the basic properties of dendritic Ca2+ signaling in subicular pyramidal neurons during epileptiform activity are described. Subiculum, a part of the hippocampal formation is present, adjacent to the CA1 subfield. It acts as a transition zone between the hippocampus and entorhinal cortex. It receives inputs directly from the CA1 region, the entorhinal cortex, subcortical and other cortical areas. Several forms of evidences support the role of subiculum in temporal lobe epilepsy. Pronounced neuronal loss has been reported in various regions of the hippocampal formation (CA1 and CA3) leaving the subiculum generally intact in human epileptic tissue. It has been observed that epileptic activity is generated in subiculum in cases where the CA3 and CA1 regions are damaged or even absent. However, it is not clear how subicular neurons protect themselves from epileptic activity induced neuronal death. It is widely accepted that epileptiform activity induced neuronal damage is a result of an abnormally large influx of Ca2+ into neuronal compartments. In the present study, combined hippocampus / entorhinal cortical brain slices were exposed to zero Mg2+ + 4-amino pyridine artificial cerebrospinal fluid (ACSF) to generate spontaneous epileptiform discharges. Whole cell current-clamp recordings combined with Ca2+ imaging experiments (by incorporating Oregon green BAPTA-1 in the recording pipette) were performed on subicular pyramidal neurons to understand the changes in [Ca2+]i transients elicited in apical dendrites, in response to spontaneous epileptic discharges. To understand the changes occurring with respect to control, experiments were performed (in both control and in vitro epileptic conditions) where [Ca2+]i transients in dendrites were elicited by back propagating action potentials following somatic current injections. The results show clear distance-dependent changes in decay kinetics of [Ca2+]i transients (τdecay), without change in the amplitude of the [Ca2+]i transients, in distal parts (95–110 µm) compared to proximal segments (30–45 µm) of apical dendrites of subicular pyramidal neurons under in vitro epileptic condition, but not in control conditions. Pharmacological agents that block Ca2+ transporters viz. Na+/Ca2+ exchangers (Benzamil), plasma membrane Ca2+-ATPase pumps (Calmidazolium) and smooth endoplasmic reticulum Ca2+-ATPase pumps (Thapsigargin) were applied locally to the proximal and distal part of the apical dendrites in both experimental conditions to understand the molecular aspects of the Ca2+ extrusion mechanisms. The relative contribution of Na+/Ca2+ exchangers in Ca2+ extrusion was higher in the distal apical dendrite in in vitro epileptic condition. Using computer simulations with NEURON, biophysically realistic models were built to understand how faster decay of [Ca2+]i transients in the distal part of apical dendrite associated with [Ca2+]i extrusion mechanisms affect excitability of the neurons. With a linear increase in the density of Na+/Ca2+ exchangers along the apical dendrite, the decrease in τ decay values of [Ca2+]i transients in distal regions seen in experimental epileptic condition was reproduced in simulation. This linear increase in Na+/Ca2+ exchangers lowered the threshold for firing in response to consecutive synaptic inputs to the distal apical dendrite. Our results thus, show the existence of a novel neuroprotective mechanism in distal parts of the apical dendrite of subicular pyramidal neurons under in vitro epileptic condition with the Na+/Ca2+ exchangers being the major contributors to this mechanism. Although the enhanced contribution of Na+/Ca2+ exchangers helps the neuron in removing excess [Ca2+]i loads, it paradoxically makes the neuron hyperexcitable to synaptic inputs in the distal parts of the apical dendrites. Thus, the Na+/Ca2+ exchangers may actually protect subicular pyramidal neurons and at the same time contribute to the maintenance of epileptiform activity. In the second part of the study, neuronal network topologies and connectivity patterns were explored in control and glutamate injury induced epileptogenic hippocampal neuronal networks, cultured on planar multielectrode array (8×8) probes. Hyper synchronization of neuronal networks is the hallmark of epilepsy. To understand hyper synchronization and connectivity patterns of neuronal networks, electrical activity from multiple neurons were monitored simultaneously. The electrical activity recorded from a single electrode mainly consisted of randomly fired single spikes and bursts of spikes. Simultaneous measurement of electrical activity from all the 64 electrodes revealed network bursts. A network burst represents the period (lasting for 0.1–0.2 s) of synchronized activity in the network and, during this transient period, maximum numbers of neurons interact with each other. The network bursts were observed in both control and in vitro epileptic networks, but the frequency of network bursts was more in the latter, compared to former condition. Time stamps of individual spikes (from all 64 electrodes) during such time-aligned network burst were collected and stored in a matrix and used to construct the network topology. Connectivity maps were obtained by analyzing the spike trains using cross-covariance analysis and graph theory methods. Analysis of degree distribution, which is a measure of direct connections between electrodes in a neuronal network, showed exponential and Gaussian distributions in control and in vitro epileptic networks, respectively. Quantification of number of direct connections per electrode revealed that the in vitro epileptic networks showed much higher number of direct connections per electrode compared to control networks. Our results suggest that functional two-dimensional neuronal networks in vitro are not scale-free (not a power law degree distribution). After brief exposure to glutamate, normal hippocampal neuronal networks became hyperexcitable and fired a larger number of network bursts with altered network topology. Quantification of clustering coefficient and path length in these two types of networks revealed that the small-world network property was lost once the networks become epileptic and this was accompanied by a change from an exponential to a Gaussian network. In the last part of the study, we have explored if an excitotoxic glutamate injury (20 µM for 10 min) that produces spontaneous, recurrent, epileptiform discharges in cultured hippocampal neurons can induce epileptogenesis in hippocampal neurons of organotypic brain slice cultures. In vitro models of epilepsy are necessary to understand the mechanisms underlying seizures, the changes in brain structure and function that underlie epilepsy and are the best methods for developing new antiseizure and antiepileptogenic strategies. Glutamate receptor over-activation has been strongly associated with epileptogenesis. Recent studies have shown that brief exposure of dissociated hippocampal neurons in culture to glutamate (20 µM for 10 min) induces epileptogenesis in surviving neurons. Our aim was to extend the in vitro model of glutamate injury induced epilepsy to the slice preparations with intact brain circuits. Patch clamp technique in current-clamp mode was employed to monitor the expression of spontaneous epileptiform discharges from CA1 and CA3 neurons using several combinations of glutamate injury protocols. The results presented here represent preliminary efforts to standardize the glutamate injury protocol for inducing epileptogenesis in organotypic slice preparations. Our results indicate that glutamate injury protocols that induced epileptogenesis in dissociated hippocampal neurons in culture failed to turn CA1 and CA3 neurons of organotypic brain slice cultures epileptic. We also found that the CA1 and CA3 neurons of organotypic brain slice cultures are resilient to induction of epileptogenesis by glutamate injury protocols with 10 times higher concentrations of glutamate (200µM) than that used for neuronal cultures and long exposure periods (upto 30 min). These results clearly show that the factors involved in induction of epileptiform activity after glutamate injury in neuronal cultures and those involved in making the neurons in organotypic slices resilient to such insults are different, and understanding them could give vital clues about epileptogenesis and its control. The resilience of CA1 and CA3 neurons seen could be due to differences in homeostatic plasticity that operate in both these experimental systems. However, further studies are required to corroborate this hypothesis.

Calcium is an important second messenger that participates in triggering and regulating numerous neuronal processes. Action potentials (APs or AP) initiate rapid changes of intracellular Ca2+-concentration ([Ca2+];) in both soma and dendrites of central neurons. We addressed two major questions about the origin and modulation of these changes in [Ca2 +]i. Firstly, how does a neurotransmitter, serotonin (5-HT), modulate the backpropagation of APs and associated changes in the [Ca2+]; in CA1 hippocampal pyramidal neurons? Secondly, do APs trigger Ca2+-induced Ca2+-release (CICR) from internal stores in these neurons? We used whole-cell somatic or dendritic patch-clamp recordings combined with high-speed imaging of [Ca2+]; and analyses of the responses to applications of pharmacological agents, studying the changes in electrical membrane properties and [Ca2+]i. The experiments were conducted in the CA1 pyramidal neurons of in vitro slices from the rat hippocampus (11 day- to 5 week-old). Changes in [Ca2+]; were measured in neurons filled with bisfura- 2, Calcium Green-1 or fura-2-AM. Bath applications of 5-HT increased membrane conductance and hyperpolarized both soma and apical dendrites. They also lowered peak potentials of antidromically-activated, backpropagating APs in the dendrites. In the soma, 5-HT applications increased the absolute AP-amplitude while slightly decreasing peak potentials. 5-HT reduced the amplitude of the AP-evoked changes in [Ca2+]i at all locations along the apical dendrites and soma. The application of 5-HT and antidromically evoked APs generated, through synergistic actions, increases in [Ca2+]j that propagated along dendrites (Ca2 + waves). Such waves originated in the proximal or middle apical dendrites and were not accompanied by a significant change in somatic membrane potential. A minimum of five APs was required to evoke the waves. According to these new observations and supporting literature, the waves are a likely consequence of Ca2+-induced Ca2+-release (CICR) from internal stores through (inositol-1,4,5- triphosphate) IP3-sensitive channels. In the absence of 5-HT, APs evoked CICR. Caffeine application increased the amplitude of AP-induced changes in [Ca2+]i. During simultaneous calcium imaging, the whole-cell recordings showed that caffeine application did not significantly change either the resting membrane potential or amplitude and shape of APs. The enhancement of AP-evoked Ca2+-transients due to caffeine application could not be attributed to protein phosphorylation or modulation of high-threshold Ca2+-channels. Applications of IBMX, a non-specific inhibitor of phosphodiesterases, forskolin, an activator of adenylyl cyclase, H-89, an inhibitor of PKA and PKG, or nifedipine, a blocker of high-threshold Ca2 + channels, did not mimic or prevent the caffeine effect. Pretreatment of neurons with thapsigargin or cyclopiazonic acid (CPA) -- substances that facilitate depletion of intracellular Ca2+-stores by blocking endoplasmic reticulum specific Ca2+-ATPases -- precluded this effect. Similar pretreatment with ryanodine, a blocker of 'ryanodine-sensitive' channels, also precluded the caffeine effect. Despite a presence or absence of caffeine, applications of thapsigargin, ryanodine or CPA reduced the AP-evoked changes in [Ca2+]i. From these new experimental observations, we can conclude that the CICR through ryanodine-sensitive channels contributes to the AP-induced changes of [Ca2+]; in hippocampal CA1 pyramidal neurons. [Scientific formulae used in this abstract could not be reproduced.]

博士<br>長庚大學<br>臨床醫學研究所<br>93<br>Ischemic stroke is a serious neurological disease. It has been the second leading cause of death in Taiwan after 1983 and the leading killer for those aged over 65. The patients with stroke often have variable functional disability, resulting in many socioeconomic problems. Thus, how to minimize the injury induced by cerebral ischemia and promote functional recovery after stroke has been a great challenge for clinical practice. In the past years, the therapy for stroke is restricted to secondary prevention. Recent advances in searching neuroprotective agents provide a new target to ameliorate the sequelae caused by ischemic injury. Therefore, to find a more effective drug, it is important to investigate and understand the cellular and molecular mechanisms of cerebral ischemia. The cerebral ischemia caused by occluding the blood vessel results in neuronal death. Substantial studies indicated that excitotoxicity induced by glutamate is important for neuronal injury caused by cerebral ischemia. L-glutamate is the major excitatory neurotransmitter in the mammalian brain and plays essential roles in neural plasticity, neural development and neurodegeneration. Glutamate activates ionotropic glutamate receptors (iGluRs) to open cationic channels. While larger amount of glutamate released, postsynaptic metabotropic glutamate receptors (mGluRs) including mGluR1 and mGluR5, which are localized at the perisynaptic junction in brain, are activated and subsequently induce G-protein linked secondary messenger cascades. The released glutamate is reuptaked by astroglial glutamate transporters GLT-1 or GLAST. Overactivation of ionotropic and metabotropic glutamate receptors causes enhancement of cell excitability as well as substantial increase of intracellular Ca2+ and finally results in delayed neuronal death. Accordingly, excessive extracellular glutamate caused by dysfunction of astroglial glutamate transporters could deteriorate the ischemic neuronal injury. Thus, the aims of this project are to investigate functional change of glutamate transporters in astrocytes and of group I metabotropic glutamate receptor in CA1 pyramidal neurons after global ischemia. Astroglial glutamate transporters, GLT-1 and GLAST, play a crucial role in removing released glutamate from the extracellular space and are essential for maintaining a low concentration of extracellular glutamate in the brain. It was hypothesized that impaired function of glial glutamate transporters induced by cerebral ischemia may lead to an elevated level of extracellular glutamate and subsequent excitotoxic neuronal death. Since glutamate transporters mediate transport of glutamate accompanied by the cotransport of 3 Na+ and 1 H+, and the countertransport of 1 K+, the function of astroglial glutamate transporter can be investigated by recording the transporter current. In the present study we performed whole-cell patch-clamp recording of hippocampal CA1 astrocytes in control or postischemic slices, and measured glutamate transporter activity by recording glutamate-evoked transporter currents. Six to twenty-four hours after global ischemia, maximal amplitude of glutamate transporter currents recorded from postischemic CA1 astrocytes was significantly reduced. Western blotting analysis indicated that transient global ischemia decreased the protein level of GLT-1 in the hippocampal CA1 area without affecting GLAST protein level. Further TaqMan real-time quantitative RT-PCR assays showed that global ischemia resulted in a decrease in GLT-1 mRNA level of hippocampal CA1 region. Global ischemia-induced reduction in GLT-1 expression and glutamate transporter function of CA1 astrocytes precedes the initiation of delayed neuronal death in CA1 pyramidal layer of the rat hippocampus. The present study provides the evidence that transient global ischemia downregulates glutamate transporter function of hippocampal CA1 astrocytes by decreasing mRNA and protein levels of GLT-1, which could lead to delayed neuronal death. The activation of postsynaptic mGluR1 or mGluR5 increases the neuronal excitability, elevates intracellular Ca2+ concentration, and potentiates NMDA receptor-mediated response. It was hypothesized that overactivation of postsynaptic mGluRs results in excitotoxic neuronal death following the transient global ischemia. Within the hippocampus, electrophysiological and immunohistochemical studies showed that mGluR5 is the major postsynaptic mGluR expressed in CA1 pyramidal neurons. To better understand the role of mGluR5 in ischemia-induced neuronal death, we investigated the functional change of mGluR5 in CA1 pyramidal neurons of control and postischemic hippocampal slices using whole-cell patch-clamp recordings. Our results indicated that 6 to 24 hours after transient global ischemia, mGluR5-induced cationic currents and mGluR5-mediated enhancement of NMDA-evoked currents in CA1 pyramidal neurons were significantly reduced. Further TaqMan real-time quantitative RT-PCR assay showed that mGluR5 mRNA expression in hippocampal CA1 region or single CA1 pyramidal neurons was markedly downregulated following ischemic insults. Global ischemia-induced reduction in mGluR5 mRNA levels and function precedes the initiation of delayed neuronal death in CA1 pyramidal layer. The present study suggests that transient global ischemia downregulates mGluR5 function of CA1 pyramidal neurons by decreasing mGluR5 mRNA and that the resulting reduced mGluR5-mediated excitotoxicity could contribute to the survival of CA1 pyramidal neurons after ischemic insult. In conclusion, the present results demonstrate that global ischemia downregulates functions of GLT-1 glutamate transporter in CA1 astrocytes and of postsynaptic mGluR5 in CA1 pyramidal neurons. These findings indicate that astroglial GLT-1 glutamate transporter and mGluR5 in CA1 pyramidal neurons play important roles in delayed neuronal death following ischemic insults. Our results will provide a novel target for developing new neuroprotective agents for stroke therapy in the future.

Over time astrocytes have been thought to function in an auxiliary manner, providing neurons with metabolic and structural support. However, recent research suggests they may play a fundamental role in the generation and propagation of focal epileptic seizures by causing synchronized electrical bursts in neurons. It would be helpful to have a simple mathematical model that represents this dynamic and incorporates these updated experimental results. We have created a two-compartment model of a typical neuron found in the hippocampal CA1 region, an area often thought to be the origin of these seizures. The focus is on properly modeling the astrocytic input to examine the pathological excitation of these neurons and subsequent transmission of the signals. In particular, we consider the intracellular astrocytic calcium fluctuations which are associated with slow inward currents in neighbouring neurons. Using our model, a variety of experimental results are reproduced, and comments are made about the potential differences between graded and “all-or-none” astrocytes.

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia<br>Os mecanismos neurobiológicos subjacentes ao desenvolvimento cerebral dependem da constante harmonia entre os fatores endógenos e exógenos que os coordenam. Em circunstâncias em que o ambiente fetal se encontre alterado, como no caso da exposição a glucocorticoides, o desenvolvimento neurológico poder-se-á desviar do seu curso normal, culminando na formação de cérebros anómalos e disfuncionais.De facto, está extensivamente descrito que a exposição a elevadas concentrações de glucocorticoides (devido a tratamentos farmacológicos ou stress) pode ter efeitos deletérios no cérebro. Recentemente, demonstrámos que ratos expostos a dexametasona (glucocorticoide sintético) durante o período pré-natal, apresentam comportamento tipo ansioso. Por sua vez, estas modificações comportamentais encontram-se positivamente correlacionadas com alterações estruturais nas células da microglia do córtex pré-frontal. Curiosamente, a remodelação citoarquitectural das células da microglia apresentou efeitos dependentes do sexo, uma vez que a dexametasona promoveu modificações morfológicas diferentes entre machos e fêmeas. De modo a superar os défices comportamentais observados, o bloqueio farmacológico crónico dos recetores de adenosina A2A, importantes reguladores fisiológicos da microglia, provou ser eficaz,apenas em machos. A melhoria a nível comportamental também se refletiu numa melhoria a nível estrutural nas células da microglia, comprometendo ainda mais a morfologia destas células em fêmeas.Relativamente ao hipocampo, uma região cerebral intimamente ligada ao comportamento, observámos que a dexametasona no período pré-natal induz alterações estruturais a longo prazo na microglia de fêmeas. Estas modificações foram acompanhadas por défices de conectividade entre o córtex pré-frontal e o hipocampo, sugerindo possíveis alterações na integridade estrutural do hipocampo.No presente trabalho, de forma a explorar a possível especificidade de género na região do hipocampo e a extensão das alterações induzidas pela exposição a dexametasona, analisou-se a estrutura celular na formação hipocampal (CA1 e giro denteado) em ratos machos adultos submetidos a dexametasona in utero. Modelos tridimensionais de neurónios piramidais da região CA1 reconstruídos manualmente apresentaram um aumento nas ramificações dendríticas basais e apicais, com um ligeiro aumento no número de ramificações. Esta restruturação também foi notável a nível sinático. Em culturas organotípicas de hipocampo, um breve estímulo com dexametasona mostrou tendência a promover a maturação de espículas dendríticas na mesma população neuronal.Quanto à morfologia da microglia, a dexametasona promoveu um ligeiro aumento no comprimento e número de processos no giro denteado. Após o bloqueio crónico dos recetores adenosinérgicos A2A na idade adulta, animais expostos a dexametasona durante o período gestacional apresentaram células da microglia hipertróficas, com um aumento acentuado no comprimento e número de processos. Estes resultados contrastaram com os observados em ratos fêmeas, nos quais o bloqueio crónico dos recetores A2A induziu uma recuperação parcial na morfologia da microglia no hipocampo. A implicação dos recetores adenosinérgicos A2A como moduladores morfológicos da microglia foi igualmente validada em murganhos knockout para este recetor, onde machos adultos exibiram microglia com ligeiras alterações estruturais.Este estudo detalhou a remodelação estrutural a nível celular no cérebro em desenvolvimento exposto a concentrações elevadas de glucocorticoides, enfatizando o sexo e as regiões cerebrais enquanto moduladores diferenciais destes efeitos. Demonstrámos queé essencial discriminar as repostas de cada sexo não apenas perante distúrbios mas também perante tratamentos farmacológicos aquando da avaliação de alterações estruturais e das suas implicações no funcionamento cerebral e no comportamento. Esta consciencialização é imperativa para o desenvolvimento de novas ferramentas farmacológicas, principalmente no tratamento de patologias com diferentes suscetibilidades entre sexos, como os distúrbios psiquiátricos.<br>The neurobiological mechanisms underlying brain development rely on the constant harmony between the endogenous and exogenous factors that coordinate them. In case of compromised fetal environment, the neurodevelopmental programming can deviate from its normal course, leading to dysfunctional brains with altered functionality, as is the case of glucocorticoid exposure.Indeed, it is widely reported that the exposure to high levels of glucocorticoids during development (due to pharmacological treatment or stress) can have deleterious effects in the brain. Recently, we demonstrated that rats prenatally exposed to dexamethasone, a synthetic glucocorticoid, present anxious like behaviour which positively correlates with morphological alterations in prefrontal cortex microglial cells. Interestingly, the cytoarchitectural remodelling had a strong gender-biased effect, since dexamethasone elicited different structural alterations according to sex. To overcome the behavioural deficits, a pharmacological chronic blockade of adenosine A2A receptors, important modulators of microglia morphology, proved to be efficient, but only in males. The improvement in behaviour was correlated with an amelioration regarding microglia structure, while further compromising microglia in females.Regarding the hippocampus, which has a central role in behaviour, we observed that antenatal dexamethasone also induces long-term structural alterations in microglia in females. These alterations were accompanied by connectivity deficits between the prefrontal cortex and the hippocampus, further suggesting that the structural integrity of the hippocampal region is compromised.In this work, to explore gender specificity regarding the hippocampus and the extent of its compromise upon dexamethasone exposure, we assessed the cellular structure in the hippocampal formation (CA1 and dentate gyrus) in adult male rats exposed to dexamethasone in utero. Manual reconstructed pyramidal neurons from the CA1 presented heightened dendritic length in both basal and apical arborization with a mild increase in dendritic ramification, showing an overall structural hypertrophy. This structure remodelling was also noticeable at the synaptic level. In organotypical hippocampal slices, acute dexamethasone stimulus showed minor tendencies in promoting spine maturation in the same neuronal population. Concerning microglia morphology, prenatal dexamethasone promoted a slight increase in the length and number of processes in the dentate gyrus. Upon a chronic blockade of adenosine A2A receptors in adulthood, dexamethasone exposed animals revealed a marked structural hypertrophy, with increased length and number of processes. These results contrasted with females, since adenosine A2A receptor blockade induced a partial recovery in microglia morphology in the hippocampus. The implication of the adenosine A2A receptors was furthered validate in knockout mice for this receptor, where adult male microglia exhibited some minor structural alterations.This study further portrays the cellular structural remodelling in the developing brain exposed to elevated glucocorticoid levels, clearly emphasizing the importance of both sex and brain region in the modulation of these effects. Thus, when accounting structural alterations and their impact in brain function and behaviour, it is essential to have in mind the differential responses of each gender not only towards the insult but also to the pharmacological treatments. Finally, this awareness is imperative in the development of new pharmacological treatments, particularly regarding disorders with gender-specific susceptibilities such as psychiatric disorders.<br>FCT