High-frequency stimulation of nucleus accumbens changes in dopaminergic reward circuit

Deep brain stimulation (DBS) of the nucleus accumbens (NAc) is a potential remedial therapy for drug craving and relapse, but the mechanism is poorly understood. We investigated changes in neurotransmitter levels during high frequency stimulation (HFS) of the unilateral NAc on morphine-induced rats. Sixty adult Wistar rats were randomized into five groups: the control group (administration of saline), the morphine-only group (systematic administration of morphine without electrode implantation), the morphine-sham-stimulation group (systematic administration of morphine with electrode implantation but not given stimulation), the morphine-stimulation group (systematic administration of morphine with electrode implantation and stimulation) and the saline-stimulation group (administration of saline with electrode implantation and stimulation). The stimulation electrode was stereotaxically implanted into the core of unilateral NAc and microdialysis probes were unilaterally lowered into the ipsilateral ventral tegmental area (VTA), NAc, and ventral pallidum (VP). Samples from microdialysis probes in the ipsilateral VTA, NAc, and VP were analyzed for glutamate (Glu) and caminobutyric acid (GABA) by high-performance liquid chromatography (HPLC). The levels of Glu were increased in the ipsilateral NAc and VP of morphine-only group versus control group, whereas Glu levels were not significantly changed in the ipsilateral VTA. Furthermore, the levels of GABA decreased significantly in the ipsilateral NAc, VP, and VTA of morphineonly group when compared with control group. The profiles of increased Glu and reduced GABA in morphine-induced rats suggest that the presence of increased excitatory neurotransmission in these brain regions. The concentrations of the Glu significantly decreased while the levels of GABA increased in ipsilateral VTA, NAc, and VP in the morphine-stimulation group compared with the morphine-only group. No significant changes were seen in the morphine-sham stimulation group compared with the morphine-only group. These findings indicated that unilateral NAc stimulation inhibits the morphineinduced rats associated hyperactivation of excitatory neurotransmission in the mesocorticolimbic reward circuit

Ion-regulatory proreins in neuronal development and communication

Brain function is critically dependent on the ionic homeostasis in both the extra- and intracellular compartment. The regulation of brain extracellular ionic composition mainly relies on active transport at blood brain and at blood cerebrospinal fluid interfaces whereas intracellular ion regulation is based on plasmalemmal transporters of neurons and glia. In addition, the latter mechanisms can generate physiologically as well as pathophysiologically significant extracellular ion transients. In this work I have studied molecular mechanisms and development of ion regulation and how these factors alter neuronal excitability and affect synaptic and non-synaptic transmission with a particular emphasis on intracellular pH and chloride (Cl-) regulation. Why is the regulation of acid-base equivalents (H+ and HCO3-) and Cl- of such interest and importance? First of all, GABAA-receptors are permeable to both HCO3- and Cl-. In the adult mammalian central nervous system (CNS) fast postsynaptic inhibition relies on GABAA-receptor mediated transmission. Today, excitatory effects of GABAA-receptors, both in mature neurons and during the early development, have been recognized and the significance of the dual actions of GABA on neuronal communication has become an interesting field of research. The transmembrane gradients of Cl- and HCO3- determine the reversal potential of GABAA-receptor mediated postsynaptic potentials and hence, the function of pH and Cl- regulatory proteins have profound consequences on GABAergic signaling and neuronal excitability. Secondly, perturbations in pH can cause a variety of changes in cellular function, many of them resulting from the interaction of protons with ionizable side chains of proteins. pH-mediated alterations of protein conformation in e.g. ion channels, transporters, and enzymes can powerfully modulate neurotransmission. In the context of pH homeostasis, the enzyme carbonic anhydrase (CA) needs to be taken into account in parallel with ion transporters: for CO2/HCO3- buffering to act in a fast manner, CO2 (de)hydration must be catalyzed by this enzyme. The acid-base equivalents that serve as substrates in the CO2 dehydration-hydration reaction are also engaged in many carrier and channel mediated ion movements. In such processes, CA activity is in key position to modulate transmembrane solute fluxes and their consequences. The bicarbonate transporters (BTs; SLC4) and the electroneutral cation-chloride cotransporters (CCCs; SLC12) belong the to large gene family of solute carriers (SLCs). In my work I have studied the physiological roles of the K+-Cl- cotransporter KCC2 (Slc12a5) and the Na+-driven Cl--HCO3- exchanger NCBE (Slc4a10) and the roles of these two ion transporters in the modualtion of neuronal communication and excitability in the rodent hippocampus. I have also examined the cellular localization and molecular basis of intracellular CA that has been shown to be essential for the generation of prolonged GABAergic excitation in the mature hippocampus. The results in my Thesis provide direct evidence for the view that the postnatal up-regulation of KCC2 accounts for the developmental shift from depolarizing to hyperpolarizing postsynaptic EGABA-A responses in rat hippocampal pyramidal neurons. The results also indicate that after KCC2 expression the developmental onset of excitatory GABAergic transmission upon intense GABAA-receptor stimulation depend on the expression of intrapyramidal CA, identified as the CA isoform VII. Studies on mice with targeted Slc4a10 gene disruption revealed an important role for NCBE in neuronal pH regulation and in pH-dependent modulation of neuronal excitability. Furthermore, this ion transporter is involved in the basolateral Na+ and HCO3- uptake in choroid plexus epithelial cells, and is thus likely to contribute to cerebrospinal fluid production.Väitöskirjassani tarkastelen hermosolujen ionisäätelyyn osallistuvien proteiinien toiminnan merkitystä hermosolujen välisessä kommunikaatiossa. Tutkimuksellinen painopiste kohdistuu kloridi-, protoni- ja bikarbonaatti-ioneihin sekä niiden säätelyyn osallistuvien solukalvon ionikuljettajiin ja hiilidioksidi-bikarbonaatti tasapainon säätelyyn osallistuvaan soluliman karboanhydraasi-entsyymiin. Tutkimuksessa on tarkasteltu näiden ionisäätelyproteiinien ilmentymistä yksilönkehityksen aikana, niiden vaikutusta hermosolujen ja hermosoluverkkojen synaptiseen ja ei-synaptiseen kommunikaatioon sekä kyseisten signalointimekanismien biofysikaalisia mekanismeja. Gamma-aminovoihappo (GABA) on täysikasvuisen nisäkkään aivojen pääasiallinen hermoimpulsseja ehkäisevä välittäjäaine. Siihen pohjautuva hermosolujen välinen nopea viestintä perustuu ns. A-tyypin GABA kanavien läpi kulkeviin kloridi- ja bikarbonaatti-ionien kantamiin sähkövirtoihin. Näiden ionien solunsisäisiä pitoisuuksia säätelevät proteiinit muovaavat GABA-välitteistä hermosoluviestintää. Väitöskirjassani esitetyt tutkimustulokset osoittavat, että tietyn kaliumvaraisen kloridikuljettajan (KCC2:n) ilmentyminen kehittyvissä aivoissa on edellytys hermoimpulsseja ehkäisevien, GABAA-kanavien kautta solusta ulos suuntautuvien virtojen synnylle. Myöhemmässä kehitysvaiheessa GABAA-kanavien voimakas aktivoituminen voi, esimerkiksi patofysiologisissa tilanteissa, johtaa solunsisäisen ja -ulkoisen ionitasapainon tilapäiseen häiriintymiseen, jolloin GABAA-kanavien tuottamat vasteet paradoksaalisesti synnyttävät hermoimpulsseja. Tällainen GABAA-välitteinen hermoärsytys edellyttää bikarbonaatti-ionien nopeaa tuottoa solunsisäisen karboanhydraasi-entsyymin avulla, mikä johtaa hermosoluja ärsyttävään hetkelliseen soluvälitilan kalium-pitoisuuden kasvuun. Tutkimukseni perusteella voidaan todeta, että karboanhydraasi-entsyymin ilmentyminen rotan hippokampus-aivoalueen pyramidihermosoluissa lisääntyy vasta syntymänjälkeisen kehityksen aikana ja on ratkaiseva tekijä tämän aivoalueen hermoverkon ärtyvyyden kasvussa. Lisäksi tutkin geenin Slc4a10 koodaamaa, natriumista riippuvaista bikarbonaatti-kuljettajaa (NCBE). Tulosten mukaan NCBE:lla on tärkeä osuus hippokampuksen pyramidisolujen pH-, ja sitä kautta myös ärtyvyyden, säätelyssä: poistogeenisten NCBE-hiirten hippokampuksen pyramidisolujen kyky poistaa protoni-ylijäämää oli selvästi heikentynyt ja vastaavasti kynnys epileptisen kohtauksen laukeamiselle oli huomattavasti kohonnut. Tutkimustulokset viittaavat pH-säätelymekanismien tärkeään rooliin hermosolujen ärtyvyyden kontrolloinnissa. On myös erittäin todennäköistä, että poistogeenisissä NCBE-hiirissä havaittu aivokammioiden tilavuuden radikaali pienentyminen on seurausta aivo-selkäydinnesteen tuotannon heikentymisestä. Havainnot tukevat ajatusta, että NCBE kuljettajaproteiinista riippuvaisella natriumin ja bikarbonaatin yhteiskuljetuksella aivojen suonipunosten epiteelisoluissa olisi merkittävä osuus aivo-selkäydinnesteen tuotannossa

Investigation of the role of nicotinic acetylcholine receptors in modulating epileptiform activity

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that have been implicated in a variety of brain functions as well as pathological states. In the Mppocampus, nAChRs appear to modulate both excitatory and inhibitory circuits. The numerous subunits that make up nAChRs result in a great diversity of functional receptors, equipping them with different pharmacological and biophysical properties. It has recently been found that certain forms of epilepsy may arise from mutation in the genes responsible for encoding of nAChR subunits. Moreover, many reports have shown that high doses of nicotine induce seizures in animals, which are blocked by different nAChR antagonists. However, the mechanism underling the role of nAChRs in patterning epileptiform activity is poorly understood. This project aims to establish the role that nAChRs may play in experimental models of epilepsy and to assess whether pharmacological agents acting at these receptors might represent a novel avenue for developing future anticonvulsants. To assess the possible modulatory influence of nAChRs on epileptiform activity, a range of nAChR ligands were applied during experimentally induced epileptiform activity in hippocampal slices prepared from wistar rats (2-6 weeks). Extracellular recordings were obtained in the stratum pyramidale of the area CAS (n=280). Initial experiments investigated the effects of nAChR ligands on 4-aminopyridine (4AP)-induced epileptiform activity. The work presented in the rest of the thesis was focused to establish the mechanisms by which nAChRs mediate their pro-epileptogenic actions. This study demonstrates that nAChRs regulate epileptiform discharges generated by a number of different pharmacological manipulations. The cellular mechanisms generating each pattern of epileptiform activity are quite distinct involving complex interactions between synaptic and non-synaptic elements of different neuronal circuits. Since nAChRs produce a similar phenotype of modulation in each epileptiform model it is possible that nAChRs target a common cellular mechanism that is prevalent in each model and which mediates the increase in burst frequency in these models

Sensitivity to Adenosine of Hippocampal Pyramidal Neurones in Calcium-Free Medium

Biochemical studies have shown that divalent cations modulate adenosine receptor binding. In this electrophysiological study the effect of calcium on the postsynaptic sensitivity to adenosine was investigated. Extracellular recordings were made in the CA1 pyramidal cell layer of rat hippocampal slices following orthodromic stimulation of Schaffer collateral fibres in the stratum radiatum or antidromic stimulation of the alveus. Field excitatory postsynaptic potentials (fEPSPs) were recorded in stratum radiatum. In antidromic stimulation experiments, CaCl2 was omitted (calcium-free medium) or reduced to 0.24 mM (low calcium medium) and in some experiments MgSO4 was increased to 2 mM or 4 mM. Adenosine and baclofen induced a concentration- dependent reduction in the amplitude of orthodromic potentials with maximum effects at 20 and 5 muM , respectively. In nominally Ca2+ free medium, multiple population spikes were obtained in response to antidromic stimulation. Adenosine had little effect on reducing the secondary spike amplitude. Kynurenic acid, an excitatory amino acid antagonist, at high concentration had no effect on secondary spikes in calcium-free or low calcium medium. 2-Chloroadenosine (1-500 muM) and R-PIA (50 muM) , which are not substrates for either the nucleoside transporters or adenosine deaminase were inactive in the absence of calcium. S-(2-Hydroxy-5 nitrobenzy1)-6-thioinosine , an adenosine uptake blocker, at a concentration of 100 M-M had no effect on secondary potential size and did not restore adenosine sensitivity in calcium free medium. Sensitivity to adenosine in calcium-free medium was restored by 240 muM calcium medium or by raising magnesium (0.8-2.8 mM) . Raising the divalent cations concentration increased the inhibitory effect of adenosine and desensitisation was seen. Thapsigargin, which discharges intracellular calcium stores, at 1 muM had no significant effect on the bursts and did not change the effect of 0.5 mM adenosine in calcium free medium. Unlike adenosine, baclofen concentration-dependently reduced the secondary spike size in calcium free medium and at maximum effect (0.5 mM) or above no sign of recovery was observed during maintained superfusion for up to 45 minutes. The activity of adenosine was restored in the presence of the stabilizer agents procaine or carbamazepine, known inhibitors of sodium channels. The GABAg agonist baclofen did not restore sensitivity to adenosine. The xanthines theophylline and eyelopentyltheophylline , the latter of which is selective for Aj purine receptors, depressed the excitability of hippocampal pyramidal neurones in calcium-free media. Chelating residual calcium with EGTA reduced excitability which was additive with the xanthine effect, while 100 muM calcium depressed the response to theophylline. The inhibition by xanthines was prevented by adenosine, which had no effect by itself, but was not reproduced or modified by adenosine deaminase. The xanthine effects were also prevented by baclofen and carbamazepine. Tolbutamide 1 mM blocked the inhibitory effect of adenosine on the size of orthodromic population spikes but had no effect on the inhibitory action of adenosine on field EPSPs. Tolbutamide did not change the inhibitory action of baclofen on the population spikes or antidromic secondary spikes induced in calcium-free media with high magnesium but dramatically blocked the effect of adenosine in the calcium-free media. Levcromakalim 100 muM potentiated the inhibitory effect of adenosine, but not baclofen, on orthodromic population spikes. The results of this study showed that the mechanisms of postsynaptic activity of adenosine and baclofen are different and at postsynaptic, but not presynaptic, sites adenosine may activate an ATP-(tolbutamide or levcromakalim) sensitive potassium channel. Loss of postsynaptic sensitivity to adenosine in calcium-free solution may result from increased sodium conductances. A common feature of adenosine, baclofen and carbamazepine which may account for their antagonism of the xanthines is the blockade of calcium fluxes. It is proposed that in the presence of low external concentrations of calcium xanthines can reduce excitability by promoting the mobilisation and trans-membrane movement of residual calcium in the medium or neuronal membranes

Modelling emergent rhythmic activity in the cerebal cortex

A la portada consta: IDIBAPS Institut d'Investigacions Biomèdiques August Pi i SunyerThe brain, a natural adaptive system, can generate a rich dynamic repertoire of spontaneous activity even in the absence of stimulation. The spatiotemporal pattern of this spontaneous activity is determined by the brain state, which can range from highly synchronized to desynchronized states. During slow wave sleep, for example, the cortex operates in synchrony, defined by low-frequency fluctuations, known as slow oscillations (<1Hz). Conversely, during wakefulness, the cortex is characterized mainly by desynchronized activity, where low-frequency fluctuations are suppressed. Thus, an inherent property of the cerebral cortex is to transit between different states characterized by distinct spatiotemporal complexity patterns, varying in a large spectrum between synchronized and desynchronized activity. All these complex emergent patterns are the product of the interaction between tens of billions of neurons endowed with diverse ionic channels with complex biophysical properties. Nevertheless, what are the mechanisms behind these transitions? In this thesis, we sought to understand the mechanisms and properties behind slow oscillations, their modulation and their transitions towards wakefulness by employing experimental data analysis and computational models. We reveal the relevance of specific ionic channels and synaptic properties to maintaining the cortical state and also get out of it, and its spatiotemporal dynamics. Using a mean-field model, we also propose bridging neuronal spiking dynamics to a population description.El cerebro, un sistema adaptativo natural, es capaz de generar un amplio repertorio dinámico de actividad espontánea, incluso en ausencia de estímulos. La patrón espacio-temporal de esta actividad espontánea viene determinada por el estado cerebral, el cual puede variar de estados altamente sincronizados hasta estados muy desincronizados. Cuando en el sueño se entra en la fase de ondas lentas, por ejemplo, la corteza opera en sincronía, cuya actividad es definida por fluctuaciones de baja frecuencia, conocidas como oscilaciones lentas (<1Hz). En cambio, durante la vigilia, el córtex se caracteriza principalmente por tener una actividad desincronizada, donde las fluctuaciones de baja frecuencia desaparecen. Por lo tanto, una propiedad inherente de la corteza cerebral es transitar entre diferentes estados caracterizados por distintos patrones de complejidad espacio-temporal, los cuales se sitúan dentro del amplio espectro marcado por la actividad sincronizada y la desincronizada. Estos patrones emergentes son el producto de la interacción entre decenas de miles de millones de neuronas dotadas de múltiples y distintos canales iónicos con complejas propiedades biofísicas. Sin embargo, ¿cuáles son los mecanismos que regulan estas transiciones? En esta tesis tratamos de entender los mecanismos, propiedades y sus transiciones hacia la vigilia, que están detrás de las oscilaciones lentas a través del uso y análisis de datos experimentales y modelos computacionales. En ella describimos la importancia de los canales iónicos específicos y sus propiedades sinápticas tanto para mantener el estado cortical como para salir de él, estudiando así su dinámica espacio-temporal. Además, mediante el uso de un modelo de campo medio, proponemos establecer un puente que pueda describir la dinámica de disparos neuronales con una descripción general de la población neuronal.Postprint (published version

Studies of Adenosine and GABAA Receptor Functions in Rat Hippocampal Slices

Recent evidence has indicated that adenosine, in addition to potassium and calcium currents, may also affect chloride movement in hippocampal neurones. This project was undertaken to determine the possible role of adenosine on chloride channels and synaptic plasticity in comparison with a selective GABA[A] agonist, muscimol. Extracellular recordings were made from the CA1 pyramidal cell layer of hippocampal slices in response to stimulation of Schaffer collateral fibres in stratum radiatum (0.01 Hz). Adenosine and muscimol induced concentration dependent reductions in the amplitude of orthodromically induced population potentials. In order to eliminate effects of these agents on potassium channels, experiments were performed in the presence of barium, 1mM (in some experiments in the presence of 1 mM tolbutamide). This concentration increased potential size, and reduced the inhibitory effect of adenosine on population spike size, but not synaptic potential size. This profile is consistent with the blockade of potassium channels associated only with the postsynaptic effects of adenosine. However, muscimol responses were unaffected. Adenosine potentiated the ability of muscimol to inhibit evoked potentials in the absence or presence of barium. The potentiation was prevented by the Al selective antagonist 8-cyclopentyltheophylline. The effects of adenosine, as well as muscimol, were reduced by the chloride channel blocker DIDS, which also prevented the adenosine potentiation of muscimol. The results indicate the ability of adenosine to operate chloride channels in hippocampal neurones, and suggest a potentiative interaction between adenosine and muscimol which also involve chloride channels. The second part of this study was to examine neurosteroids which have been reported to be positive modulators of the GABAA receptors. Alphaxalone and 5alpha-pregnan-3alpha-o1-20-one potentiated the inhibitory effect of muscimol on the population spike size at low concentrations (0.5 and 1muM) that had no significant effect on the spike size by themselves. This profile is in agreement with other reports which have described the effect of these neurosteroids as barbiturate-like. Alphaxalone and 5alpha-pregnan-3alpha-o1-20-one also at low concentrations potentiated the inhibitory effect of adenosine alone and in the presence of barium 1 mM which blocked adenosine activated potassium channels. Alphaxalone failed to potentiate the inhibitory effect of adenosine in the presence of bicuculline 1muM. It is concluded that these neurosteroids enhanced the potentiative interaction between adenosine and muscimol in the presence of barium. The results indicate that adenosine's effects are normally enhanced by virtue of the potentiative interaction occurring with endogenous GABA. In addition to this, the results show the chloride channels activated by adenosine to be different from those operated by the GABA[A] receptor. The third part of this project was to investigate the role of GABA[A] and adenosine receptors on long-term depression (LTD) and synaptic plasticity. Unlike long-term potentiation, LTD in the central nervous system remains poorly understood. Muscimol induced a time and concentration-dependent LTD in the amplitude of orthodromic potentials. Increasing the stimulation frequency from 0.01 Hz to 1 Hz for 10 seconds reversed the LTD induced by muscimol. Although adenosine decreased the spike size in a concentration-dependent manner, it failed to induce LTD. Muscimol also induced LTD in the absence of electrical stimulation. Alphaxalone and 5alpha-pregnan-3alpha-o1-20-one at concentrations that did not have any effect themselves on the population spike (0.5 and 1 muM), potentiated the inhibitory effect of muscimol on the population spike size. These neurosteroids at high concentrations (5 and 10 muM) decreased the spike size by themselves. On the other hand, at the low concentrations both steroids were able to potentiate the ability of muscimol to induce LTD. Moreover, muscimol 1 muM which is not able to induce LTD, alphaxalone and 5alpha-pregnan-3alpha-o1-20-one 1 muM maintained the LTD induced by muscimol 10 muM. Bicuculline 5 muM reversed the LTD induced by muscimol 10 muM. To examine the possible role of glutamate receptors in LTD induced by muscimol a number of NMDA and metabotropic receptors agonists and antagonists were used. The NMDA receptor antagonist 2-AP5, the NMDA/metabotropic antagonist 2-AP3 and selective metabotropic antagonist L(+)-AP3 failed to modify the LTD. Quisqualic acid and (1S,3R)- aminocyclopentane dicarboxylic acid (ACPD), a selective agonist at metabotropic receptors, did not induce LTD or short-term depression, whereas kynurenic acid prevented the reversal of the LTD obtained at 1 Hz. It is concluded that LTD can be induced by the selective activation of GABA[A] receptors and the lack of involvement of glutamate receptors in the protocol which is presented in this study confirms the unique role of classical GABA[A] receptors in the effect of muscimol and may indicate a novel type of long-lasting depression. Furthermore, the failure of adenosine to induce LTD taken together with the earlier results, suggests that the adenosine activated chloride channel differs from the GABA[A] receptor chloride channel

Cellular mechanisms and function of hippocampal gamma oscillations

Gamma oscillations (30 - 120 Hz) are a common feature of active cortical networks and changes in these rhythms can act as a useful marker of circuit dysfunction in psychiatric disorders and neurological disease. While their function remains debated, there has been converging evidence regarding their mechanism of generation. These rhythms depend upon the spiking of inhibitory interneurons, which synchronise the firing of excitatory pyramidal cells via fast synaptic inhibition. Specifically, parvalbumin-expressing interneurons, which target the perisomatic domain of pyramidal neurons, are thought to play the key role in generating and maintaining gamma oscillations in the brain. However, it was recently demonstrated that somatostatin-expressing interneurons, which target the dendritic domains of pyramidal neurons, are responsible for the generation of lowfrequency gamma oscillations in the primary visual cortex of awake mice. It is not yet known if the involvement of somatostatin-expressing interneurons in gamma rhythmogenesis is specific to visually-induced oscillations in V1, or if it is general phenomenon that occurs across brain areas. Here, we took advantage of optogenetic techniques to test the involvement of parvalbumin- and somatostatinexpressing interneurons in a well studied model of carbachol-induced gamma oscillations in the hippocampal CA3 in vitro. For both classes of interneurons, we found that rhythmic photo-activation was sufficient to entrain ongoing gamma oscillations, and that the predominant effect of sustained photo-activation was to decrease the power and increase the frequency of gamma oscillations. Importantly, there was a functional distinction between the interneuronal classes. While photoinhibition of parvalbumin-expressing interneurons decreased the oscillation power, photo-inhibition of somatostatin-expressing interneurons both decreased the oscillation power and increased its frequency. These experiments suggest that the activity of both interneuron classes are important for carbachol-induced oscillations in hippocampal area CA3. They also indicate that somatostatin-expressing interneurons may have a more intimate role in the control of gamma oscillation frequency. Interestingly, sustained photo-activation of somatostatin-expressing interneurons was suficient to induce de novo high-frequency gamma oscillations. Our experiments reveal that the de novo gamma oscillations may have different properties than cchinduced oscillations, but may also depend on synaptic excitation. Our data suggest that several different interneuronal subtypes may be necessary to generate and/or maintain oscillations across the gamma range, but are also consistent with the idea that rhythmic perisomatic inhibition is a critical feature of low-frequency gamma oscillations in the hippocampus. In order to further understand the function of rhythmic perisomatic inhibition, we used dynamic clamp to inject inhibitory conductance trains into pyramidal neurons, and examined the effects of disturbing rhythmicity and/or synchrony on pyramidal cell spiking. Previous evidence using this approach has highlighted a role for gamma frequency synchronisation of excitatory inputs in multiplicative gain enhancement of principal cell output. Here, we show that rhythmic inhibition can also induce multiplicative increases in the gain of principal cell output. Moreover, we found that reductions in either rhythmicity or synchrony of the input trains impaired that multiplicative gain enhancement in principal cells. These experiments suggest that inputs at gamma frequency are ideally suited for multiplicatively enhancing gain, and highlight an important implication for gamma oscillations at the single cell level. i

Real-time imaging of hippocampal network dynamics reveals trisynaptic induction of CA1 LTP and "circuit-level" effects of chronic stress and antidepressants

Today’s pervasive presence of stress renders stress-related psychiatric disorders (SRPDs), a relevant global health problem. Memory impairment is a major symptom likely mediated by the hippocampus (HIP), a limbic brain region highly vulnerable to stress. Recent evidence suggests that information processing problems within specific neuronal networks might underlie SRPDs. However, the precise functional neurocircuitry that mediates hippocampal CA1 long-term potentiation (LTP), a putative correlate of mammalian learning and memory, remains unknown at present. Furthermore, valuable assays for studying stress and drug effects on polysynaptic activity flow through the classical input/output circuit of the HIP are missing. To engage a circuit-centered approach, voltage-sensitive dye imaging was applied in mouse brain slices. Single pulse entorhinal cortex (EC) to dentate gyrus (DG) input, evoked by perforant path stimulation, entailed strong neuronal activity in the DG, but no distinct neuronal activity in the CA3 and CA1 subfield of the HIP. In contrast, a thetafrequency (5 Hz) stimulus train induced waves of neuronal activity percolating through the entire hippocampal trisynaptic circuit (HTC-waves). Spatially restricted blocking of glutamate release at CA3 mossy fiber synapses caused a complete disappearance of HTC-waves, suggesting frequency facilitation at DG to CA3 synapses the pivotal gating mechanism. In turn, non-theta frequency stimulations (0.2/1/20 Hz) proved much less effective at generating HTC-waves. CA1 long-term potentiation (CA1 LTP) is the best understood form of synaptic plasticity in the brain, but predominantly at the monosynaptic level. Here, HTC-waves comprise high-frequency firing of CA3 pyramidal neurons (>100 Hz), inducing NMDA receptordependent CA1 LTP within a few seconds. Detailed examination revealed the existence of an induction threshold for LTP. Consequently, baseline recordings with a reduced number of HTC-waves were carried out to test the effects of memory enhancing drugs and HPA axis hormones on hippocampal network dynamics. Bath application of caffeine (5 mM), corticosterone (100 nM) and corticotropin-releasing hormone (5 & 50 nM) rapidly boosted HTC-waves. Cognitive processes taking place within the HIP are challenged by stress exposure, but whether and how chronic stress shapes "net" neuronal activity flow through the HIP remains elusive. The HTC-wave assay, refined for group comparisons, revealed that chronic stress markedly lowers the strength of evoked neuronal activity propagation through the hippocampal trisynaptic circuit. In contrast, antidepressants (ADs) of several classes, the mood stabilizer lithium, the anesthetic ketamine, and the neurotrophin brainderived neurotrophic factor amplified HTC-waves. An opposite effect was obtained with the antipsychotic haloperidol and the anxiolytic diazepam. The tested ADs exert this effect at low micromolar concentrations, but not at 100 nM, and nearly always, also not at 500 nM. Furthermore, the AD fluoxetine was found to facilitate LTP of HTC-waves. Finally, pharmacological blockade of the tyrosine-related kinase B receptor abolished fluoxetine effects on HTC-waves. These results highlight a circuit-centered approach suggesting evoked synchronous theta rhythmical firing of EC principal cells as a valuable tool to investigate several aspects of neuronal activity flow through the HIP. The physiological relevance is emphasized by the finding that the resulting HTC-waves, which likely occur during EC theta oscillations, evoke NMDA receptor-dependent CA1 LTP within a few seconds. Furthermore, HTC-waves allow to integrate molecular, cellular and structural adaptations in the HIP, pointing to a monoaminergic neurotransmission-independent, "circuit-level" mechanism of ADs, to balance the detrimental effects of chronic stress on HIP-dependent cognitive abilities

Activity-dependent regulation of GABA release at immature mossy fibers-CA3 synapses: role of the Prion protein

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