Voditelj:
prof. dr. sc. Zdravko Petanjek, dr. med.
redoviti profesor anatomije i neuroznanosti
Suradnici
doc. dr. sc. Sanja Darmopil, dipl. ing. biologije
Ana Hladnik, dr. med.
Domagoj Džaja, dr. med.
Ivana Bičanić, dr. med.
Dora Mandić, dr. med.
Istraživački interesi:
U laboratoriju se istražuju organizacija i molekularna svojstva neurona i njihovih veza s naglaskom na neuralne sustave i područja posebno razvijena kroz evoluciju čovjeka: frontalni asocijativni korteks, te asocijativni projekcijski neuroni i kalretininski interneuroni. Navedena područja i neuroni zbog svoje brojnosti predstavljaju glavni biološki elementi u procesuiranju najkompleksnijih kognitivnih funkcija kod čovjeka te stoga imaju glavnu ulogu u etiopatogenezi važnih psihijatrijskih i neuroloških poremećaja.
Iako su osnovni principi organizacije neuralne mreže kore velikoga mozga zajednički svim sisavcima, neke vrste neurona, kao što su veliki kortiko-kortikalni piramidni neuroni s paralelnim projekcijama prema više kortikalnih područja, ali i izrazito bogatim intra-arealnim projekcijama, jasno su izražene tek kod majmuna i čovjeka. Drugim skupinama, kao što su kalretininski GABA-ergički interneuroni, broj se eksponencijalno povećava i vjerojatno se unutar ove skupine pojavljuju strukturno i funkcionalno nove populacije neurona.
Glavne teme istraživanja:
- Kvantitativna (utvrđivanje broja i distribucije) i kvalitativna (utvrđivanje morfoloških i molekularnih obilježja) analiza piramidnih neurona sloja III i GABA-ergičkih interneurona te regionalna komparativna analiza navedenih obilježja između multimodalnih, unimodalnih i primarnih kortikalnih područja. Također se provodi i komparativna analiza navedenih obilježja između čovjeka, majmuna i štakora.
- Usporedba parametara stanične organizacije normalnog mozga čovjeka i makaki majmuna s mozgovima osoba oboljelih od psihijatrijskih i neuroloških poremećaja (npr. tkivo dobiveno prilikom operacija pacijenata s teškim oblicima epilepsije), kao i s podacima iz eksperimentalnih modela (model epilepsije frontalnog režnja majmuna). Ovaj dio istraživanja provodi se u suradnji s laboratorijem dr. Monique Esclapez u Marseilleu (Brain Dynamic Institute Marseille (BDI: http://ins.medecine.univmed.fr/).
- Istraživanje genetički modificiranog (“humaniziranog”) FoxP2 miša u svrhu utvrđivanja kako promjene u strukturi gena (koje su bile selekcionirane tijekom evolucije čovjeka i smatraju se jednom on najvažnijih promjena u razvoju govora) utječu na organizaciju kortikalnih veza. Ovaj dio istraživanja provodi se u suradnji s laboratorijem prof. Svante Paabo u Leipzigu (Max Planck Institute for Evolutionary Anthropology Leipzig; MPI-EVA: http://www.eva.mpg.de/).
- Razvojne studije u majmuna i čovjeka usmjerene na istraživanja za primate specifičnih razvojnih događanja, kao što su obrazac rasta dendritičkog stabla asocijativnih piramidnih neurona, mjesto stvaranja kalretininskih neurona i specifični putovi migracije neurona.
METODOLOGIJA:
KVANTITATIVNA ANALIZA HISTOLOŠKIH PREPARATA; morfometrija i stereologija
U laboratoriju se koriste općeprihvaćene i dobro definirane kvantitativne metode analize obojenih histoloških preparata:
- stereologija (optički frakcionator)
- rekonstrukcija serijskih rezova i 3D mapiranje mozga
- rekonstrukcija neurona, anatomsko mapiranje
- morfometrija (rekonstrukcija i analiza grananja dendritičkog stabla i aksona te utvrđivanje broja i položaja dendritičkih trnova)
Kvantitativna analiza histoloških preparata provodi se uz korištenje Neurolucida i Stereoinvestigator programskih paketa (MicroBrightField, Williston, USA) na dva video-računarsko-mikroskopska sustava s 3D motoriziranim stolićem kontroliranih elektroničkim upravljačem. Stariji sustav sastoji se od Hitachi 3CCD video kamere u boji HV-C20M, Lucivid mikromonitora spojenog na Olympus BX50 mikroskop s 3D motoriziranim stolićem kontroliranim s elektroničkim upravljačem MAC 2000 (Ludl Electronic Products Ltd.). Noviji sistem (2010) sastoji se od MBF-DV-46 digitalne kamere koja se nalazi na Olympus BX61 mikroskopu motoriziranom za kretanje u dubinu, te motoriziranog stolića za pokretanje u x-y smjeru i koji je kontroliran MAC 5000 elektroničkim upravljačem (Ludl Electronic Products Ltd.).
HISTOLOŠKE TEHNIKE
- Golgi metode: Golgi-Cox, Rapid Golgi
- Histokemija: AchE, PAS-Alcian, NADPH
- Imunohistokemija za identifikaciju interneurona (GAD, kalretinin, somatostatin, parvalbumin, kalbindin), mapiramidnih neurona (SMI32, MAP2) te neurotransmitera i aksonskih završetaka (GABA, GAD; 65 i 67, VGAT, VGLUT1, VGLUT2). Imunohistokemijska bojenja provode se kao jednostruka bojenja korištenjem biotiniliranih protutijela i metode označavanja bazirane na DAB-u, te kao višestruka obilježavanja korištenjem fluorescentnih protutijela.
- Identifikacija i morfološka analiza neurona obilježenih tehnikama praćenja aksonskih projekcija te neurona obilježenih tijekom elektrofiziološkog eksperimenta: analiza neurona obilježenih tehnikama aksonskog transporta (biotin-dekstran-amin; pšenična klica konjugirana s aglutininom u koloidnom zlatu), uključujući i metode retrogradnog trans-sinaptičkog prijenosa biljega (rabies virus) te analiza morfologije neurona koji su po završetku elektrofiziološkog eksperimenta injicirani biocitinom.
Head:
Zdravko Petanjek, MD, PhD
Professor of Gross Anatomy and Neuroscience
Members:
Sanja Darmopil, PhD, MSc, Assistant Professor of Neuroscience
Ana Hladnik, MD, PhD student
Domagoj Džaja, MD, PhD student
Ivana Bičanić, MD, PhD student
Dora Mandić, MD, PhD student
The goal of our laboratory is to study organization and molecular properties of microcircuitry with an emphasis on neuronal elements that became particularly expressed during evolution of the human neocortex. Therefore, we specifically focus on the human associative frontal cortex as well as on the associative layer III of projecting neurons and calretinin expressing GABA-interneurons. Due to their expansion and unique features they are considered to be a main biological substrate of the most complex cognitive functions and involved in pathophysiology of various psychiatric and neurological disorders.
Despite common principles in connectivity, some neuron types are specific for monkey and human cerebral cortex, such as large cortico-cortical projecting neurons with parallel projection to several cortical areas and extremely reach local intra-areal connections. Also, a certain class of GABA-interneurons, calretinin expressing, shows a supralinear increase in number suggesting an appearance of new neuron types inside this group. These neurons are the key elements of human microcircuitry which molecular specificity and neuronal interaction need to be determined in order to assess how human cortex processes information.
RESEARCH TOPICS:
- Quantitative (number and distribution) and qualitative (morphological and chemical properties) analysis of human specific neuron subclasses (layer IIIC pyramidal neurons, calretinin expressing GABA-interneurons) with comparative analysis of regional differences comparing multimodal, unimodal and primary cortical areas, as well as analysis of species differences (human, monkey and rat).
- Comparison of cellular organization parameters in the normal human and monkey brain with morphology and chemical properties of specific neuron subclasses in various psychiatric and neurological disorders (analysis of cortical resections obtained after surgery of patients with severe epilepsy), including experimental models (model of frontal lobe epilepsy in monkey). This part of the research is performed in collaboration with dr.Monique Esclapez at the Brain Dynamic Institute Marseille (BDI: http://ins.medecine.univmed.fr/).
- Examination of genetically modified mice model (“humanized” Foxp2 mice) in order to show how changes in the structure of a gene (that was positively selected during human evolution and related to the appearance of language) will affect the organization of cortical circuitry (this research is performed in collaboration with prof. Svante Paabo at the Max Planck Institute for Evolutionary Anthropology Leipzig; MPI-EVA: http://www.eva.mpg.de/).
- Developmental studies in monkey and human with an emphasis on studying primate specific developmental events, as well as a pattern of dendritic growth in associative pyramidal neurons, place of origin of calretinin neurons and specific neuronal migratory routes.
TOOLS:
QUANTITATIVE ANALYSIS OF HISTOLOGICAL SECTIONS; morphometry and stereology
In our laboratory we are using well established qualitative methods for analysis of stained brain tissue:
- stereology (optical fractionator method),
- serial section reconstruction with three-dimensional brain mapping,
- neuron tracing, anatomical mapping and
- morphometry (reconstruction and analysis of branching pattern of the axon and dendrites together with spine counting).
These analyses are performed using Neurolucida and Stereoinvestigator software (MicroBrightField, Williston, USA) on two computer based automatic measuring microscope-video system connected to three-dimensional motorized stages controllers. An older system consists of Hitachi 3CCD color video camera HV-C20M placed on the Olympus BX50 microscope and connected to the MAC 2000 stage controller, (Ludl Electronic Products Ltd). A newer system (2010) is equipped with MBF digital camera placed on the Olympus BX61 microscope and connected to the MAC 5000 stage controller (Ludl Electronic Products Ltd).
TISSUE STAINING TECHINQUES
- Golgi methods: Golgi-Cox, Rapid Golgi method
- Histochemistry: AchE, PAS-Alcian, NADPH etc.
- Immunohistochemistry for identification of local circuit neuron markers (GAD, calretinin, somatostatin, parvalbumin, calbindin), pyramidal neuron markers (SMI32, MAP2) and markers of neurotransmitters and neurotransmitter terminals (GABA, GAD 65 and 67, VGAT, VGLUT1, VGLUT2). Immunohistochemistry is performed as a single labeling; using mostly byotinilated antibodies proceeded by DAB protocol, and as multiple labeling using mostly fluorescent labels.
- Identification and morphological analysis of neurons label by tracing experiments and during electrophysiological recording; neurons labeled by axon transport methods (biotin dextran amine, wheat germ agglutinin-conjugated colloidal gold), including methods which allow retrograde transynaptic/transneuronal transfer (rabies virus), as well as neurons injected with biocytin at the end of the electrophysiological recording.
