2011.10.15.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 1 Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework** Consortium leader PETER PAZMANY CATHOLIC UNIVERSITY Consortium members SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund *** **Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben ***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg. PETER PAZMANY CATHOLIC UNIVERSITY SEMMELWEIS UNIVERSITY sote_logo.jpg dk_fejlec.gif INFOBLOKK 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 2 Peter Pazmany Catholic University Faculty of Information Technology BEVEZETÉS A FUNKCIONÁLIS NEUROBIOLÓGIÁBA INTRODUCTION TO FUNCTIONAL NEUROBIOLOGY www.itk.ppke.hu By Imre Kalló Contributed by: Tamás Freund, Zsolt Liposits, Zoltán Nusser, László Acsády, Szabolcs Káli, József Haller, Zsófia Maglóczky, Nórbert Hájos, Emilia Madarász, György Karmos, Miklós Palkovits, Anita Kamondi, Lóránd Erőss, Róbert Gábriel, Zoltán Kisvárdai, Zoltán Vidnyánszky Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 3 www.itk.ppke.hu Epilepsy and neurodegenerative disorders Imre Kalló & Zsófia MaglóczkyPázmány Péter Catholic University, Faculty of Information Technology I. Epilepsy as a disease. II. Functional morphological changes in the epileptic hippocampus. III. Experimental models of epilepsy. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 4 www.itk.ppke.hu Functional morphological alterations in epileptic diseases: cell death and reorganisation kischrist1750 EPILEPSY: It is a chronic functional disturbance characterized by spontaneously recurrent seizures and different etiology. FUNCTIONAL BACKGROUND: Large number of cells fire synchronously. EPIDEMIOLOGY: About 2% of the population is affected. MOST FREQUENT: Focal epilepsy with temporal lobe origin (TLE). Questions arise: What is the mechanism of the synchronous discharges? What is the structural basis of this functional disturbance? Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 5 www.itk.ppke.hu Classification of epileptic fits 1.Partial (focal, local) fits -simple partial seizures(no disturbance of consciousness) with motor, somatosensory, autonomic, psychic symptoms -complex partial fits (there is disturbance of consciousness) it may start with a simple partial onset, which is followed by the disturbance of consciousness with automatisms or it is dominated by the disturbance of consciousness from the beginning THEY CAN GENERALISE SECONDARILY 2. Generalized fits -absence (with disturbance of consciousness) (PM)it may be accompined by automatism, clonus, atonia, tonus, autonomiccomponents -tonic-clonicseizures, (GM)(onlytonus, onlyclonus, onlyatonia) -myoclonus (involuntary muscle contractions, localised or generalised, upper limb is more frequently affected) 3. Non-classified seizures -e.g.febrile seizure S T A T U S E P I L E P T I C U S Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 6 www.itk.ppke.hu Etiology of epileptic seizures -perinatal anomalies -brain injury (infarcts) -tumor, pressure injury of the brain, head trauma -unknown reason -neuronal infection -vascular malformation -developmental malformation of the nervous system (dysplasia, migrational disturbances, microgyria, heterotopia etc.) -genetic errors -intoxications (alcohol, medicines, drugs, herbicidesetc.) Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 7 www.itk.ppke.hu Cortical and temporal epilepsy is often accompanied by developmental malformations –dual pathology Malformation of Cortical Development (MCD) Types of MCD - proliferation-related (reduced, increased, time-shifted) - migration-related (e.g. heterotopia) - organization-related (polimicrogyria, microdysgenesis, schizencephalia) - others Focal appearance: Focal Cortical Dysplasia (FCD) Ectopic neurons, immature neurons, giant cells, abnormal layer formation In general, there are fewer inhibitory cells in MCD, consequently its epileptogenic state is hipothetised! Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 8 www.itk.ppke.hu Frontal focal dysplasia Focal.jpg Cells in the white matter Hypertophic neurons SMI 32 staining Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 9 www.itk.ppke.hu Epilepsy as a disease kiswentz1983 Epilepsy is frequently accompanied by other psychiatric diseases: depression, psychotic symptoms, personality changes, decay of cognitive capabilities, anxiety, increased rate of suicidesetc. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 10 www.itk.ppke.hu Treatment for epilepsy kishrondius1642 There is no causal therapy. Either patients get over the epilepsy„spontaneously” Or receive treatments, which aim to prevent seizures. -antiepileptic drug treatment -antiepileptic surgery Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 11 www.itk.ppke.hu Antiepileptic surgery kisboschxv It is recommended only, if the source of the epileptic seizure (the focus) is known. Most frequently the temporal lobe is targeted, and portions are removed such as the hippocampus, subiculum, entorhinal cortex or temporal cortex. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 12 www.itk.ppke.hu Epileptic surgery.png Antiepileptic surgery In case of focal epileptic seizures and drug therapy resistant epilepsy, the epileptic focus can be removed. Photo: István Ulbert Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 13 www.itk.ppke.hu Localization of the epileptic focus - Focus in the cerebral cortex: Usually it is associated to developmental abnormalities e.g. dysgenezis, dysplasia, migrational disturbances, abnormal gyrification, etc . - Focus in the temporal pole: Affected areas are the hippocampus, amygdala, subiculum, entorhinal, perirhinal, piriform corticies, temporal cortex, insula. The focus can be one of these regions, or even more of them. Sometimes the focus migrates from one place to the other. Dual pathology is also possible, e.g. when the focus is in the entorhinal cortex, the subiculum or the amygdala, dysplasia or minor abnormalities might be also present in cortical areas. -Tumor may also cause seizures -Febrile seizure, head trauma may also cause recurrent seizures Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 14 www.itk.ppke.hu Functional neuromorphological studies on the epileptic reorganisation of the hippocampus sampled from patients with temporal lobe epilepsy Most frequently affected brain region is the hippocampus, which is partially removed from the brain of drug therapy resistant patients. Molecular biological, cellular and/or neuronal network studies (licenced!) can be carried out on the tissue samples removed. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 15 www.itk.ppke.hu Levels of epileptic reorganisation • Intracellular changes (receptors, ion channels, gene transcription, second messenger systems, enzyme activity, cellular organelles etc.) • Cellular events (cell death, cell division, cell migration, morphological deformations, gliosis, quantitative and qualitative alterations in neurochemical markers) • Changes at neuronal network level (changes of intercellular connections, axonal decay/ sprouting) • Changes in the activity of cells/cell groups • Alterations in large pathways connecting brain regions (decay or sprouting in neuronal pathways) • Changes affecting the whole CNS (hormonal or metabolic alterations, synthesis/degradation of neurotransmitters, changes in the EEG pattern etc.) Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 16 www.itk.ppke.hu khh67nisslegybeszabott Structure of the human hippocampus (Nissl-staining) Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 17 www.itk.ppke.hu khkgolgi1 Golgi-staining, human dentate gyrus Camillo Golgi (1843-1926) 1873: discovery of staining 1906: Nobel prize, shared with Ramon y Cajal Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 18 www.itk.ppke.hu Golgi-staining, human gyrus dentatus, granule cells khkgolgigdgranule_cell Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 19 www.itk.ppke.hu The hippocampal trisynaptic loop hc entorhinal input Schaffer- collaterals Mossy fibres Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 20 www.itk.ppke.hu 3-step immunostaining applied in the studies ICC.png Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 21 www.itk.ppke.hu Principal cells (human control tissue) hkhippoc3 Perforantpathway Mossy fibres Schaffer collaterals csikohal Drawing was made by Lucia Wittner (PhD thesis, 2004) Granule cell Mossy cell CA3 pyramidal cell CA1, CA2 pyramidal cell Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 22 www.itk.ppke.hu Principal cells of the hippocampus AREA PRINCIPALCELL TRANSMITTER NEUROCHEMICAL MARKER Cornu Ammonis-CA1 Pyramidal cell glutamate Calbindin, GluR2/3-R, NeuN CA2 Pyramidal cell glutamate Calbindin, GluR2/3-R, NeuN CA3 Pyramidal cell glutamate GluR2/3-R, NeuN CA3c Pyramidal cell glutamate GluR2/3-R, NeuN Hilus Mossy cell glutamate CART peptide,GluR2/3-R CGRP,(calretinin in mouse, partially in monkey) Gyrus dentatus Granule cell glutamate, GABA (No GABA transporter) Calbindin, GluR2/3-R, Dynorphin,CART peptide, NeuN Blue:RAT. RED: HUMAN. BLACK: BOTH Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 23 www.itk.ppke.hu Common neurochemical features paticbtelj Calbindin-immunostaining cbsclerko2 human rat Granule cells, CA1 and CA2 pyramidal cells are CB-immunoreactive Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 24 www.itk.ppke.hu Schaffer collaterals CA3 CA1 pyramidal cell axons towards subiculum Only CA3 mossy terminals Septal+comissural fibers Schaffer-collaterals Perforant pathway (ecx) Septal + comissural fibers Str. pyramidale Str. lucidum Str. radiatum CA1 CA3 Sulcus Layer-specific input of principal cells –pyramidal cells Str. lacunosum- moleculare Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 25 www.itk.ppke.hu Layer-specific input of principal cells –granulecells Comissural fibers SUM input Mossy fibers GD HILUS Str. moleculare Str. garnulosum Sulcus Perforant pathway (ecx) Local interneurons Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 26 www.itk.ppke.hu Granule cells khkgolgigd kapkogdh rat Mossy terminals 15-20 terminals 30-50 active zones there are no recurrents Basal dendrites in the hilus 20% human Acsády et al. , 1998, J. Neurosci Seress & Ribak, 1992, Brain Res. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 27 www.itk.ppke.hu Mossy cells hkmoharajz developing adult Complex spines (mossy fibers terminate on it) SeressL.Azemberihippocampus születésutánifejlődése. LegeArtisMedicine, 2000. 10 (6):489, 5. ábra Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 28 www.itk.ppke.hu Functional types of inhibitory cells according to their targets hippoc_in 1 1 2 2 2 2 1. Perisomatic inhibitory cells: terminate on principal cells bodies, proximal dendrites and axon initial segments; regulate the output activity(basket and chandelier or axo-axonic cells) 2. Dendritic inhibitory cells:terminate on distal dendrites of principal cells; regulate the input activity 3. Interneuron selective cells: regulate the activity of interneurons Freund and Buzsaki, Hippocampus, 1996, 6,347-470. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 29 www.itk.ppke.hu Role of dendritic and perisomatic inhibition HippSema.png DENDRITIC TREE: Input plasticity CELL BODY: Generation of output signal AXON: Signal transmission STIMULI OF THE EXTERNAL WORLD EFFECTS OF OUR INTERNAL WORLD Dendritic inhibition Perisomatic inhibition Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 30 www.itk.ppke.hu Function of neurochemically different inhibitory cells in the human hippocampus Parvalbumin-containing interneurons basket and axo-axoniccells, perisomatic inhibition(+ any species examined) Calbindin-containing interneurons dendriticinhibition, + axo-axonic cell, perisomaticinhibition (rat: only dendritic) Calretinin-containing interneurons Dendriticandinterneuron specific inhibition (rat: different) Cholecystokinin-containing interneurons Perisomaticanddendriticinhibition (+rat) Somatostatin-containing interneurons Dendritic inhibition (+rat) NeuropeptidY-containing interneurons Dendritic inhibition (+rat) Substance P receptor expressinginterneurons Dendritic inhibition (rat: different) Red: calcium binding proteins; Blue: neuropeptides; Orange: receptor Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 31 www.itk.ppke.hu Pathological types of TLE patients regarding theprincipal cell loss TLE types1.jpg hk10hcteljkicsi Control TLE types2.jpg 40hccbteljeskicsi 1: “mild” group, similar tothe control Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 32 www.itk.ppke.hu Pathological types of TLE patients regarding theprincipal cell loss TLE types3.jpg 47mca110xglurkicsi 2: “patchy” type patchy pyramidalcell loss TLE types4.jpg 75mca1cbkicsi 3: “sclerotic” type Profound CA1 pyramidal cell loss TLE types5.jpg 7Omcbgd4Okicsi 4. “gliotic” type loss of all cell types including the resistant cells (granule cells, CB-immunostained interneurons) Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 33 www.itk.ppke.hu Hippocampal sclerosis so:stratum oriens;sp: stratumpyramidale; sr: stratumradiatum;sl-m: stratumlacunosum-moleculare; DG: dentate gyrus episcler 75mcbhcteljeskicsi Control CA1 EpilepticCA1 Calbindin-immunostaining G.D. s.l-m. s.r. s.p. G.D. s.l-m. s.o.,p.,r. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 34 www.itk.ppke.hu Gliosis gliokicsi2 Control Epileptic, sclerotic Maglóczky Zs: A hippocampálisneuronhálózatokátalakulásakrónikustemporálislebenyepilepsziában. In: HalászP (ed.)Hippocampus, mint neuropszichiátriaibetegségekközösnevezője. Budapest: Melinda Kiadó, 2005. pp. 61-101. Theamountofglialfibersincreasessignificantlyinthehippocampusofepilepticpatients.GliosisisverycharacteristicforthescleroticCA1region,andalsopresentinthedentategyrus.GFAPimmunostaining. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 35 www.itk.ppke.hu Gliosis TLE_EM.jpg TLE_EM2.jpg Magloczky et al. Neuroscience 2000, Wittner et al. Neuroscience, 2002 Epileptic Dentate Gyrus Epileptic CA1 region Theamountofglialfibersincreasessignificantlyinthehippocampusofepilepticpatients.LargeamountofglialfibresisverycharacteristicforthescleroticCA1region,andalsopresentinthedentategyrus.Thereisalsoincreasedamountofglial fibersvisibleinthehippocampusofnon-scleroticpatients. Calbindin immunostaining Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 36 www.itk.ppke.hu Mossy fiber sprouting –enhanced internal excitatory pathway cbemmf The number of granule cell axons terminals increases in the str. moleculare of dentate gyrus and CA3 region. These fibers terminate primarily on principal cells. Ann.Neurol., 26321-330, Epilepsia, 36543-558 J Neurosci, 10267-82., J Neurosci, 131511-22. Neuroscience, 42351-364. sprepiem Neuroscience, 1997.76377-85. Large fraction of the fibers terminatesalso on dendrites of local interneurons. If the inhibitory cells receive excess stimulation, many of those will dye. A subset of these neurons, however will survive and transmit a more effective inhibition. Maglóczky, Neuroscience 2000. 96: 7-25 Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 37 www.itk.ppke.hu Sprouting of excitatory input pathways Thesupramammillarypathway (SUM) innervates the granule cells of the DG with excitatory terminals, which are arranged in a thin layer in controls (arrows). In contrast, in epileptic patients this layer occupies the whole stratum moleculare, where the axons terminate mainly on granule cells. The SUM contains calretinin. The number of local calretinin-containinginhibitory cells is reduced. crfmdg cremgdsm Control Epileptic Maglóczky et al Neuroscience 2000. 96: 7-25 SUM fibers form asymmetric synapses (C,E), whereas the axon terminals of the localinterneuronsestablish symmetricsynapses (D,F). Calretinin immunostaining. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 38 www.itk.ppke.hu Abnormal localization of interneurons EpilepSPr1.jpg EpilepSPr2.jpg Maglóczky könyvfejezet Gabro kiadó. Alterations of input characteristics of the interneurons. Receptor mis-match in the controls.Abnormallocalization of interneurons (migration; arrows). Substance P receptor-immunoreactiveinhibitory cells can be detected in thestratummoleculare(sm)of the epileptichippocampus,in turn, such neurons are localised mainly in the hilus (H) of the control hippocampi.The number of cells are reduced in the hippocampus. Control Epileptic Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 39 www.itk.ppke.hu Dendritic morphology of Substance P receptor-expressing neurons sprsejtekfoto sprsejtekrajz Number of ramifications/cell (meanstdev) Number of cells studied Stratum oriens Stratumpyramidaleandradiatum Control (n=33) 4.25 1.04 10.52 3.28 Mild(n=30) 6.63 2.88 11.59 4.62 Patchy, non sclerotic(n=28) 7.25 1.91 21.15 4.68 Sclerotic (n=18) Stratumoriens, pyramidaleandradiatum= 3.22 1.26 Tóth K. et al. 2007 Neuroscience Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 40 www.itk.ppke.hu Axonal sprouting of local interneurons diaf1monPVfmem Number of patients (Number of AIS) Total length of the studied AISs Synaptic coverage (µm synapse/100 µm AIS) Control n=10 (n=95) 335.4 0.55 Control n=5 (n=88) 249.3 0.49 Patient n=21 (n=43) 222.7 3.05 Patient n=9 (n=47) 269.8 2.64 Patient n=22 (n=58) 355.33 1.08 Non-sclerotic patient n=15; (n=74) 1.22 Mean of control 0.52 Mean of epileptic patients 1.77 -Changes in the neurochemical markers (e.g. parvalbumin (PV) disappears from the inhibitory cells, number of immunoreactive (IR) cell bodies decreases, but the IR terminals remain visible) -Axonal sprouting of interneurons. PV-containing axo-axonic cells establish more synapses on the AIS of granule cells of epileptic patients than in controls. Parvalbumin immunostaining. Wittner et al. Neuroscience, 2001 Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 41 www.itk.ppke.hu Reduction of the number of local interneurons and their axonal sprouting somhkdgfm somsarjdgepi sm sm sg sg Control Epileptic - The number of somatostatin-immunoreactive (SOM-IR)cells is reduced, and the SOM-IR axons show sprouting in the dentate gyrus. Somatostatin immunosatining. De Lanerolle et al., Brain Res. 495: 387-95 Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 42 www.itk.ppke.hu CA1: The morphology of inhibitory cells undergoes changes, the principal cells show functional alterations cbfmca1 cbfmca1p Control Epileptic Control Non-sclerotic Sclerotic -Changes of neurochemical markers (calbindindisappears from the pyramidal cells in the non sclerotic CA1 region) -Deformation of interneurons (arrows, dendritic growth, spine formation, hypertrophy) Wittner et al. Neuroscience, 2002 Calbindin immunostaining Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 43 www.itk.ppke.hu DG: The morphology of inhibitory cells undergoes changes, the principal cells show functional alterations hkepicbca1x10kicsi -Dispersion of granule cell layer (sg: stratum granulosum) -Changes of neurochemicalmarkers(calbindindisappearsfrom the granule cells -Deformation of interneurons(arrows; dendritic growth, spine formation, hypertrophy) Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 44 www.itk.ppke.hu Axonal sprouting of local interneurons cbemepica1t Table1.png The majority of calbindin-containing inhibitory cells are preserved in the epileptic hippocampus. Contrasting the controls however, they do not project onto principal cells; they rather establish connections with each other resulting in disinhibition. Wittner et al. Neuroscience, 115. 961-978 Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 45 www.itk.ppke.hu Loss of interneurons –calretinin-containing cells, human TLE crsejtszamtipus sejteskdegisejtekdendrit2-sotetebb kcrdendrnagyangolflatten-color felekdegisejtekdendrit2-sotetebb K. Tóth et. al. Brain 2010 Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 46 www.itk.ppke.hu kdendritekCRmon3 CONTROL EPILEPTIC Toth K. et al. Brain 2010 Calretinin-immunostained dendrites Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 47 www.itk.ppke.hu CR IS CR IS CR IS CR IS CR IS CR IS CR IS CR IS CR IS CR IS Control Epileptic Interneuron-specific inhibitory cell Synchronized dendritic inhibitory cells Pyramidal cells, no plasticity in dendrites Degenerating interneuron-specific inhibitory cell Asynchronous dendritic inhibitory cells Pyramidal dendrites with associative plasticity Toth K. Et al. 2010 Brain Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 48 www.itk.ppke.hu DG: The morphology of inhibitory cells undergoes changes, the principal cells show functional alterations dgcbepi2 CONTROL EPILEPTIC -Dispersion of granule cell layer (sg: stratum granulosum) -Changes of neurochemicalmarkers(calbindin partially disappearsfrom the granule cells -Deformation of interneurons(arrows; dendritic growth, spine formation, hypertrophy) sg sg Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 49 www.itk.ppke.hu Functional morphological changes at neuronal network level –the degree of it is in correlation with the loss of principal cells 1. Cell death:Pyramidal cells of CA1 and CA3c regions, „sensitive” inhibitory cells (calretinin-, somatostatin-, neuropeptid Y-containing cells supplemented with parvalbumin-and Substance P receptor-containing cells of the CA1 region) and reduction of the number of mossy cells. 2. Migration of cells: Dispersion of granule cells, Substance P receptor-expressing inhibitory cells 3. Deformation of cellular morphology: Extra dendrites, formation of dendritic-and somatic spines, hypertrophy of cell body (calbindin-and Substance P receptor-containing inhibitory cells) 4. Neurochemical changes: Reduction of calbindin-level in the granule cells, and its increase in the interneurons, reduction of parvalbumin-level in the perisomatic inhibitory cells 5.Axonal sprouting –changes of external and internal neuronal connections -local principal cells: sprouting of mossy fibers and the axons of CA1 pyramidal cells -external input pathways: axons within the supramammillary pathway (and the subicular input) -local inhibitory interneurons a) enhancement of the perisomatic inhibition in the dentate gyrus b) axonal sprouting of calbindin-containing interneurons in the CA1 region, change in the target cells -dendritic inhibition of CA1 pyramidal cells is replaced with the inhibition of interneurons 6. Glial fibers -increased deposition Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 50 www.itk.ppke.hu Changes of neurochemical marker-content • Calbindin: may disappear from principal cells, but not from interneurons • Parvalbumin: may disappear from cells, dendrites, sometimes from terminals • Calretinin: seems to be stably present • SP: may APPEAR in principal cells • NPY: stably present in interneurons, mRNA may appear in granule cells. NPY appears in mossy fibres. • CCK: seems to be stably present Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 51 www.itk.ppke.hu Fate of inhibitory neurons in the epileptic hippocampus Interneuron types Black: looser Red: winner Green: looser&winner Non-sclerotic CA1 Sclerotic CA1 Non-sclerotic DG ScleroticDG Parvalbumin/ perisomatic Survive Vulnerable Survive/ sprouting Survive/ PV disappear, sprouting Calbindin/ dendritic (CCK) Survive/ sprouting Subsetof them survive/dendritic growth, spine formation, sprouting Survive Survive/ growth SPR/ dendritic Survive/ dendritic growing Vulnerable Survive Survive/ migration, dendritic growth Calretinin/ dendritic + interneuron specific Survive/dendritic degeneration Vulnerable Survive/ dendritic degeneration Subset ofthem vulnerable Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 52 www.itk.ppke.hu Epilepsy and the inhibitory neuronal network EPILEPTICREORGANISATION = Cell death+ sprouting: changes of the cellular connections and excitability Loss of interneuron specific inhibitory cells results in a reduction of the effectiveness of dendritic inhibition. Reduction of the dendritic inhibition and the sprouting of excitatory pathways result in an abnormal potentiation of the excitatory input. The increasedperisomatic inhibition may increase the probability of synchronised cellular activity. Theneuronal network becomes destabilised and consequently seizures develop more easily. Cellular death is induced by the increase of calcium levels deriving from the extracellular space, threshold phenomena. Loss of interneurons in the CA1 may depend on the survival of target cells. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 53 www.itk.ppke.hu Epileptic reorganisation Cell death + alterations in the neuronal connectivity and excitability 1. Reduction of dendriticinhibition. 2. Reduction of interneuron-specific inhibition 3. Increase of excitatory input onto dendrites 4. Increase of perisomaticinhibition Theneuronal network becomes destabilised, synchronisation is increased within. Seizures develop more easily. 1. Loss of inhibitory neurons develops in all types of epilepsy, independent of the sclerosis. 2. Axon sprouting (excitatory and inhibitory) develops in all types of epilepsy, independent of the sclerosis. 3. Loss of principal cells is likely to depend on the extent of excitation, threshold phenomena. EPILEPSY = SCLEROSIS and DUAL PATHOLOGY -other regions are also reorganised! Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 54 www.itk.ppke.hu Function of hippocampus (+ limbic system) • memory (transition of short-term and long-term) • learning • spatial orientation • emotional background of events, behavioral regulation explicit (declarative) -hippocampus dependent epizodic, semantic, visual Types of memory: implicit (procedural) –hippocampus independent Szirmai: Neurológia Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 55 www.itk.ppke.hu Participation of the left and right hippocampus in memory processing DOMINANT (left, by right-handed people) speach recognition word recognition memorising words echoing words object of tales SUBDOMINANT(right) vizual capabilities face recognition spatial rotation of images details of tales Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 56 www.itk.ppke.hu Experimental epilepsy models (according to the triggering methods) -Genetic modifications -Kindling(repeated small electric or chemical stimulation, till the level of spontaneously recurrent seizures) - Seizure induced by electricstimulation - Application of excitatory amino acid analogues - Alteration of the levels of inhibitory-excitatory amino acids -Alteration of the operation of ion channels - Alteration of the kationicconcentrations etc. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 57 www.itk.ppke.hu Experimental epilepsy models (according to the phenotype) 1. Acute seizure model(slice, cell culture) Tissue is sampled from control animal, and the seizure is triggered with a chemical agent. Epileptogenesis is studed, i.e. behavior of single cells in response to a seizure. There is no network effect and reorganisation. This is the model of synchronous activity. 2. Chronic epilepsy model It is studied in animals producing spontaneous seizures (such animals are produced by application of pilocarpine, kindling, or kainic acid). There is reorganization. The effect of long-term rearrangement and the network changes can be studied in this model. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 58 www.itk.ppke.hu Epilepsy models EVOKEDGENETIC CULTURED GENETICALLY MODIFIED IN VIVOIN VITRO TISSUE CULTURE, SLICE (control, chronic epilepsy) CHRONICACUTE (In animals producing(Seizure are induced by the manipulation acutely spontaneous seizures) there is no recurrent seizure) -kindling -4-amino piridin - pilocarpin -febrile seizure-model - kainic acid Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 59 www.itk.ppke.hu Experimental models of Temporal Lobe Epilepsy Kindling model -It provides a model only for seizure, there is no/a few/cell death -There is sprouting of mossy fiers, level of calbindin is reduced -It is a partial seizure model Pilocarpine (non-specific muscarin-receptor agonist) model -Administration of 300-380 mg/kg pilocarpine i.p. (+scopolamine to reduce peripheral cholinergic effects) - Acuteeffect is status epilepticus (24 h) then alatent period (days-week) -Chronically recurrent seizures -Cell death characteristic forTLEis in the hippocampus Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 60 www.itk.ppke.hu Experimental models of Temporal Lobe Epilepsy Kainic acid (glutamateanalogue,effects throughkainatereceptors)model - Highest density of receptorof the drug onthe pyramidal cells of CA3 region and the mossy fibers - direct effect trough its specific receptors - it spares theaxons, indirect effects through axonal pathways -can be administered: intraperitonially, subcutaneously,intracerebroventricularly,intracerebrally -resultant cell death varies, seizures are always similar to the one characterises theTLE, status epilepticusappeares if it applied in large dose. It is suitable also for chemical kindling. -ipsilateralkainateinjection in thehippocampus/entorhinalcortexresults in cell death in the contralateral hippocampus, the appearance of which is very similar to the one observed in the hippocampus ofTLE patients. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 61 www.itk.ppke.hu Kainatemodel, ipsilateralkainateinjection into the CA3 region kainatemodell kiskain38 kkontrarem20bead Magloczky and Freund, Neuroscience 1993. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 62 www.itk.ppke.hu Cell death in the contralateral hippocampus afteripsilateral kainate injection: Loss ofCA3 and CA1 pyramidal cells shows similar histology to the one observed in human hippocampal sclerosis. kiskain38 Gallyas silver impregnation. Magloczky et al. Neuroscience 1993. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 63 www.itk.ppke.hu Calbindin-immunoreactive cells in two models of epilepsy kkontrarem19cbca1 Kainate model (rat) Kpilo8_6cb Kpilo8_6cbdg PILO model (mouse) CA1 Gyrus dentatus Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 64 www.itk.ppke.hu Two types of cell loss in pilocarpine-induced epilepsy (weak-strong SE) kispiloepiweakstrongtabla Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 65 www.itk.ppke.hu Two types of cell loss in pilocarpine-induced epilepsy (weak-strong SE) kispiloeger_GluR23_20x_2 kispiloeger_W-2-6_ GluR23_ca3_40x kispiloeger_S8-4_ GluR23_ca3_40x CA1 CA3 Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 66 www.itk.ppke.hu AP model, calbindin-containingcells in the CA1region kapircbca1 Control 3-AP-treated Slezia et al., Neurobiol. Des. 2003. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 67 www.itk.ppke.hu Kainatemodel, calretinin-containing cells, CA1, rat kkainatecr Control Epileptic Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 68 www.itk.ppke.hu 4-AP model, calretinin-containing cellsin the ratCA1region kapirca1cr Control 4-AP-treated Slezia et al., Neurobiol. Des. 2003. Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 69 www.itk.ppke.hu Classification of TLE patients according to the extent of cell death(n=50 ) TLE types1.jpg Control TLE types2.jpg 1. non-sclerotic TLE types3.jpg TLE types4.jpg TLE types5.jpg crka01a kakontra kazsugor 2. non-sclerotic 3. sclerotic 4. gliotic 1:Kindling,low dose kainate 4-APinjection 3:Unilateral, medium dose kainate, Contralateral hc., pilocarpine 4: Icv, intrahippocampal or systemic injection of largedosekainate Introduction to functional neurobiology: Pathological Wiring 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 70 www.itk.ppke.hu Drawing of Escher may also demonstrate the relationship between the epileptic reorganization and the epileptic seizures escher_kez