2011.11.25.. 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.
Neurobiológia alapjai - Módszerek
BASICS OF NEUROBIOLOGY - Methods
www.itk.ppke.hu
By Imre Kalló
2011.11.25. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 3
METHODS IN NEUROBIOLOGY IV.
Techniques to map neuronal connections
Imre Kalló
Pázmány Péter Catholic University, Faculty of Information Technology
I. Histology techniques: light microscopic studies II. Applications using fluorescent dyes
III. Histology techniques: electron microscopic studies IV. Techniques to map neuronal connections
V. Molecular biological techniques VI. Living experimental models VII. Electrophysiological approaches VIII. Behavioral studies
IX. Dissection, virtual dissection, imaging techniques
TECHNIQUES TO MAP NEURONAL CONNECTIONS
1. Correlated LM and EM processing
Transsection of neuronal pathways - neurodegeneration Multiple labelling techniques
2. Using neuronal tracers – Single cell filling and reconstruction
- Tracing neuronal pathways (anterograde, retrograde) 3. Application of transneuronal tracers
- Gap junctions - Lucifer Yellow
- Chemical synapses - neurotropic viruses – e.g. Bartha virus and its modifications
4. Latest approaches
- High-Resolution Molecular Imaging Tools (Brainbow, Rainbow transsynaptic viral tool)
- Automated Electron Microscopy (SSTEM, SSET, SBFSEM, FIBSEM)
TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 5 2011.11.25.
CORRELATED LM AND EM PROCESSING:
Transsection of neuronal pathways - neurodegeneration
Palkovits M, Léránth C, Jew JY, Williams TH.
Simultaneous characterization of pre- and
postsynaptic neuron contact sites in brain. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2705-8.
CORRELATED LM AND EM PROCESSING
a b
c d
PvN
V
At light microscopic level
At the electron microscopic level
Oxytocin cell (OT)
Oxytocin cell (OT)
Adrenergic axons (PNMT)
OT OT
PNMT PNMT
Adrenergic cells in the brainstem
(PNMT-IR) Oxytocin cells
in the PVH (OT-IR)
TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 7 2011.11.25.
USING NEURONAL TRACERS – Single cell filling and reconstruction
Neurobiotin-filled neuron in the prefrontal cortex,
40x
3D reconstruction of neurobiotin- filled neuron in the prefrontal
cortex
Neurolucida
Studying the afferent and efferent
connections of a cell group of the brain by using anterogradely and/or
retrogradely transported tracers
.USING NEURONAL TRACERS:
Tracing neuronal pathways (anterograde, retrograde)
TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 9 2011.11.25.
Chemical synapses used by neurotropic viruses
APPLICATION OF TRANSNEURONAL TRACERS:
Chemical synapses used by neurotropic viruses
Exp: Confocal laser microscopic images of transneuronal labeling of the parasympathetic preganglionic neurons of the salivatory (parotid and lacrimal) glands of the rat. Fluorescent micrographs of dual-viral-tracing. Green
fluorescent protein is expressed in the neurons of the inferior salivatory nucleus (ISN) while red fluorescent protein is expressed in the neurons of superior salivatory nucleus (SSN).
No staining.
By courtesy of Ida Tóth and Ida Gerendai
2nd Department of Anatomy, Semmelweis University, Budapest, Hungary
TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 11 2011.11.25.
Gap junctions used by Lucifer Yellow
Exp: The immortalised GnRH syntethising (GT1-7) cells establish often gap-junctions with each other, through which the tracer Lucipher Yellow injected in one cell will fill up also the coupled one (b and bb).
Wetsel WC, Valença MM, Merchenthaler I, Liposits Z, López FJ, Weiner RI, Mellon PL, Negro-Vilar A. Intrinsic pulsatile secretory activity of immortalized luteinizing hormone-releasing hormone-secreting neurons. Proc Natl Acad Sci U S A. 1992 May 1;89(9):4149-53.
High-Resolution Molecular Imaging Tools: Brainbow transgenes
Livet J, Weissman TA, Kang H, Draft RW, Lu J, Bennis RA, Sanes JR, Lichtman JW.
Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature. 2007 Nov 1;450(7166):56-62.
„The Cre/lox recombination was used to create a stochastic choice of expression between three or more fluorescent proteins (XFPs).
Integration of tandem Brainbow copies in transgenic mice yielded combinatorial XFP expression, and thus many colours, thereby providing a way to distinguish adjacent neurons and visualize other cellular interactions.”
TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 13 2011.11.25.
Multiple, colored PRVs used the demonstrate spatial organization of parallel circuits.
Different region are labelled transsynaptically with different colored Rainbow viruses.
Boldogkoi Z et al. Genetically timed, activity- sensor and rainbow transsynaptic viral tools.
Nat Methods. 2009 Feb;6(2):127-30.
Automated Electron Microscopy (SSTEM, SSET, SBFSEM, FIBSEM)
Serial Section Transmission Electron Microscopy (SSTEM)
Backscattered electron scanning electron microscopy (BsSEM) Serial Section Electron Tomography (SSET)
Serial Block Face Scanning Electron Microscopy (SBFSEM)
Focused Ion Beam Scanning Electron Microscope (FIBSEM).
(Exposure of block face either by diamond knife cutting or focused ion beam)
