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Matrix metalloproteinase-9 (MMP-9) mediates cholinergic-induced plasticity of the excitatory input to hippocampal fast spiking interneurons

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Salamian A. , Beroun A. , Kaczmarek L.

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nr Suppl.1

EN

INTRODUCTION: MMP-9 is an extracellular protease involved in modification of synaptic functions. Cholinergic receptors activation in the hippocampus, that is mostly mediated by projections from the medial septum, plays an important role in the hippocampal synaptic plasticity. Additionally, activation of muscarinic cholinergic receptors can be involved in synaptic transmission between parvalbumin positive cells, known as fast spiking inhibitory interneurons, and pyramidal cells. AIM(S): The aim of this study was to evaluate the effect of MMP-9 on the cholinergic-induced synaptic plasticity. METHOD(S): To induce synaptic plasticity, carbachol, a cholinergic agonist, triggering rhythmic activity, which causes a lasting synaptic enhancement, was applied to cultured hippocampal organotypic slices. MMP-9 activity was either genetically or pharmacologically blocked. Using whole-cell patch-clamp technique, AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) were recorded. Enzymatic activity of MMP-9 was assessed by gelatin zymography. RESULTS: One hour of carbachol treatment, followed by an overnight carbachol‑free incubation, produced significant increase in frequency of mEPSCs. Treatment with either MMP-9 inhibitor I or genetic ablation of MMP-9 along with carbachol further enhanced frequency of mEPSCs. Recording mEPSCs from fast spiking GABAergic interneurons located at the stratum radiatum showed enhancement of mEPSCs frequency by carbachol. The increased excitatory inputs to those inhibitory interneurons were impaired while MMP-9 activity was inhibited. Evaluation of gelatinase activity in conditioned cultured medium indicated remarkable increase in the level of MMP-9 compared with control around 24 hours after carbachol treatment. CONCLUSIONS: MMP-9 proteolytic activity can have a marked impact on the cholinergic-induced synaptic plasticity and transmission between pyramidal neurons and inhibitory interneurons. FINANCIAL SUPPORT: ITN training network EU FP7 grant “EXTRABRAIN”.

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Synaptic plasticity in the central amygdala during addictive and natural learning

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Bijoch L. , Pekala M. , Kaczmarek L. , Beroun A.

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nr Suppl.1

EN

Drug addiction has been proposed as a form of Hebbi‑ an learning, as it creates changes in neural networks by strengthening or weakening some synapses. Similar chang‑ es occur during natural reward learning and it is believed that they are stored as encoded information-engrams. It has been suggested that drugs of abuse hijack these en‑ grams to create extremely durable forms of memories. Therefore, in this study, we aimed to test for similarities between initial exposure to an addictive substance and a natural form of learning. As a model of “addictive” learn‑ ing we chose intraperitoneal (IP) cocaine injections, while sucrose self-administration served to represent a natural form of learning. To distinguish different neuronal popu‑ lations, transgenic mice with labeled GABAergic neurons were used. All experiments were performed with contra‑ distinction between inhibitory and excitatory neurons. A series of electrophysiology experiments were performed on a specific brain pathway: the connection between pos‑ terior basolateral amygdala (pBLA) and the central medi‑ al amygdala (CeM). This pathway was recently shown to process positive memories. To ensure pathway specificity, viruses were injected into pBLA allowing for channeloro‑ dopsin2 expression in neurons. Synaptic changes were tested by whole-cell patch clamp electrophysiological re‑ cordings with the use of optogenetic stimulation. Results from electrophysiological recordings were confirmed by confocal microscopy. For this purpose brain slices were im‑ munolabeled with an antibody against c-Fos protein, which is a marker of neural plasticity. Our results indicated that indeed both cocaine IP injection and sugar administration changed the pBLA-to-CeM pathway in the same manner. In these structures we observed generation of silent synaps‑ es—immature synaptic contacts. Silent synapses contain mostly NMDA receptors(and not AMPA receptors) and may function as substrates for increased learning. Expression of c Fos protein also indicated that both sugar and cocaine contributed to structural changes in neurons in CeM. Addi‑ tionally, these changes were independent of cell-type (in‑ hibitory or excitatory). Thus, drug exposure affects a path‑ way that processes positive memories and engages similar neurons that natural learning does. Our results shed light on the debate surrounding of addiction as a form of simple, appetitive learning.

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Abnormal AMPA receptor trafficking in a mouse model of fragile X syndrome

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Chojnacka M. , Beroun A. , Kuzniewska B. , Kaczmarek L. , Dziembowska M.

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nr Suppl.1

EN

INTRODUCTION: Fragile X syndrome (FXS) a common inherited form of mental retardation and autism is caused by lack of expression of fragile X mental retardation protein (FMRP). FMRP is RNA-binding protein that regulates local translation of many synaptic proteins, including AMPA-type glutamate receptors subunits. Accumulated evidence indicate that proper rates of exocytosis and endocytosis of glutamate receptors play a key role in synaptic plasticity. However, current state of knowledge of AMPA receptor trafficking in FXS models is incomplete. AIM(S): The aim of this study was to analyze AMPA receptor trafficking in a mouse model of fragile X syndrome. METHOD(S): We used synaptoneurosomes (SN) isolated from Fmr1 KO and wild-type (WT) mice and stimulated them in vitro with NMDA/glutamate. To determine levels of surface and intracellular GluR1, GluR2 and GluR3 we used crosslinking of SN with BS3 reagent followed by western blot analysis. To confirm our biochemical results we investigated the synaptic calcium-permeable AMPA receptors using whole-cell patch-clamp recordings. RESULTS: We found that SN stimulation produced an increase in the surface glutamate receptor subunits only in WT mice. We also found that surface GluR2 protein level was significantly higher in Fmr1 KO SN in basal conditions, when compared to WT. The electrophysiological experiments confirmed higher abundance of GluR2‑containing AMPA receptors in the hippocampus of Fmr1 KO mice. CONCLUSIONS: Our results indicate that Fmr1 KO mice exhibit abnormal AMPA receptor trafficking and it is demonstrated by elevated amount GluR2.

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C-Fos driven modulation of appetitive behavior in the central nucelus of amygdala

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Lebitko T. , Madej H. , Jaworski T. , Beroun A. , Kondrakiewicz K. , Knapska E. , Kaczmarek L.

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nr Suppl.1

EN

INTRODUCTION: The central nucleus of the amygdala (CeA) has primarily been studied as a structure involved in processing of aversive behaviors, whereas its role in appetitively-motivated learning is less understood. The published data show involvement of the basolateral amygdala (BL), which sends projections to the CeA, in encoding sensory‑specific features during appetitive learning. In contrast, the CeA was implicated in modulation of incentive motivation to pursue an associated external reward. Previously we reported that after appetitive, but not aversive learning, expression of c-Fos, a protein closely linked to synaptic plasticity, is significantly increased in the CeA. AIM(S): We aimed at testing the hypothesis that appetitive learning depends on c-Fos expressing neural circuits in the CeA. METHOD(S): We first compared c‑Fos expression pattern in the amygdala following place preference and place avoidance training and examined inputs from the BL on the activated CeA neurons. Then we used c-fos-driven targeting of channelrhodopsin and trained the animals in an operant conditioning task, in which they learned to associate auditory stimulus with food reinforcement. To further test the role of c-fos-expressing neurons in appetitive learning, we locally blocked behaviorally-induced c-fos expression using a shRNA. RESULTS: The c‑Fos expression in the CeA wassignificantly higher following place preference than place avoidance training, with over 90% of the c‑Fos positive cells receiving projections from the BL. Optogenetic stimulation of the neurons increased bar-pressing responses but only when the conditioned stimulus was present. Blocking c-fos expression resulted in impairment of appetitively but not aversively motivated discrimination learning and decreased motivation to seek reward. CONCLUSIONS: The results reveal that c-fos expression in the CeA neurons is necessary for appetitively but not aversively motivated learning, modulating of incentive motivation but not reward consumption. FINANCIAL SUPPORT: Tomasz Lebitko was supported by NCN grant Sonata UMO-2012/05/D/NZ3/02085 to Tomasz Jaworski.