Anatomy in the resting state? Taking a look at receptor patterns

Transcription

1 Anatomy in the resting state? Taking a look at receptor patterns Karl Zilles Institute of Neuroscience and Medicine INM- Research Centre Jülich and University Hospital of Psychiatry, Psychotherapy and Psychosomatics, RWTH Universität Aachen

2 Regionally and functionally specific endowment of the brain with neurotransmitter receptors. How to visualize receptors at high spatial resolution throughout the entire brain Zilles, K., Schleicher, A., Palomero-Gallagher, N., Amunts, K.: Quantitative analysis of cyto- and receptorarchitecture of the human brain, pp In: Brain Mapping: The Methods, 2nd edition (A.W. Toga and J.C. Mazziotta, eds.). Academic Press (22)

3 Quantitative in vitro Receptor Autoradiography: Method () MRI autopsy in situ post mortem MRI 3D MR volume unfixed post mortem brain mounting on the cryomicrotome chuck dissection into slabs

4 Serial sections (2 µm) mounted onto glass slides

5 Preincubation: re-hydrate sections. Remove endogenous substances Main incubation: buffer solution with [ 3 H]-ligands which specifically bind to a given receptor type Washing step: terminate binding procedure and eliminate surplus [ 3 H]- ligand and buffer salts Exposition of labeled sections and scales of known radioactivity concentrations to tritiumsensitive film Digitization of the films, measurement of receptor densities in fmol/mg protein, and color coding Histological staining of alternating sections for cell bodies and myelinated fibers Examined receptor binding sites Neurotransmitter Receptor [ 3 H] Ligand Glutamate Kainate MK-8 GABA GABA A muscimol, GABA B CGP GABA A BZ Flumazenil Acetylcholine M pirenzepine M 2 M 3 oxotremorine-m, AF-DX DAMP nicotinic a 4 b 2 epibatidine Noradrenaline a prazosin a 2 UK 4,34 RX-822 Serotonin 5-HT A 8-OH-DPAT 5-HT 2 ketanserin Dopamine D SCH 2339

6 Grey values Image processing C S a K L L D [fmol/mg protein] 4 5-HT A Radioactivity concentration (cpm) 5 Linearised and color-coded autoradiograph grey values or colors encode receptor densities

7 Regionally and functionally specific endowment of the brain with neurotransmitter receptors 2. Regional and laminar distribution of single receptor types in the cerebral cortex

8 The amygdala must be further subdivided based on it s distinct and heterogenous receptor distribution l bm bl basomedial basolateral lateral Structural MRI at 4 Tesla: spatial resolution 35 mm isotrop GABA muscarinic receptor M A receptor 2 Receptor autoradiography

9 The termination fields of the trisynaptic pathway in the hippocampus show distinct regional and laminar expression patterns of different glutamatergic receptors Perforant path to CA and molecular layer of DG Mossy fibers from DG to CA3 Schaffer collaterals from CA3 to CA fmol/mg protein 82 CA sub CA2 633 CA3 464 mol 295 fmol/mg protein Glutamatergic terminals of the perforant path and the Schaffer collaterals fmol/mg protein CA mol Kainate Glutamatergic terminals of the perforant path and the Schaffer collaterals Glutamatergic terminals of the mossy fibers

10 Cholinergic muscarinic M 2 receptor and primary sensory areas: Matching receptor- and myeloarchitecture Receptor autoradiography Myelin staining fmol/mg protein V layers II-IVa layer IVc layer IVb Gennari stripe = layer IVb V V V V V

11 Cholinergic muscarinic M 2 receptor and primary sensory areas: Receptorarchitecture sc motor cortex primary somatosensory cortex area 3b motor cortex primary somatosensory cortex area 3b sc primary auditory cortex BA4 primary auditory cortex BA4 Human Brain 84 fmol/mg protein 42 Macaque Brain

12 Multiple receptors and anterior cingulate cortex p32 p32 Glutamate receptor Noradrenaline a 2 receptor pacc p32 Glutamate 32 24c d receptor pd24 cv/cd amcc * p24a/b 24c v 24ab pv24c s24a s24b s32 sacc 25 pmcc p24b 24dd 24dv p24ab 33 Palomero-Gallagher et al., unpublished p24a p24b pd24cd pd24cv 33 Acetylcholine Glutamate M 2 receptor receptor pd24cv pd24cd Dopamine D receptor p24a 33 Palomero-Gallagher, Mohlberg, Zilles, Vogt (28) J Comp Neurol 58: Palomero-Gallagher & Zilles (29) In: Cingulate Neurobiology & Disease. Oxford University Press, pp

13 Regionally specific endowment of the brain with neurotransmitter receptors 3. Regional and laminar distribution of multiple receptor types in the cerebral cortex - RECEPTOR FINGERPRINTS -

14 5-HT A, 5-HT 2 serotonin glutamate,, GABA B GABA dopamine D, D 2, D 4 a, a 2 noradrenaline glutamate,,,, glutamate GABA GABA A, GABA A /BZ nicotinic, M, M 2, M 3 acetylcholine noradrenaline a, a 2 GABA A, GABA A /BZ GABA acetylcholine nicotinic, M, M 2, M 3 D, D 2, D 4 dopamine GABA GABA B GABA B GABA glutamate,, 5-HT A, 5-HT 2 serotonin GABA GABA A, GABA A /BZ GABA A, GABA A /BZ GABA GABA GABA A, GABA A /BZ axon terminal receptor

15 WT KO On the way to understand specificity and mechanisms behind receptor fingerprints: Conditional 5-HT A receptor knockout 5-HT A receptor a receptor 64 fmol/mg protein fmol/mg protein 9

16 V2 V V2 mglur2/3 V2 low Human primary visual cortex * * * GABA A GABA B M nic a 4 b 2 V V * * * V2 high Zilles, Palomero Gallagher: Comparative analysis of receptor subtypes that identify primary cortical sensory areas. In (Kaas, Striedter, Krubitzer, Herculano-Houzel, Preuss, eds.) Evolution of the Nervous System. Elsevier, Oxford (27) V2 V2 a 5-HT A 5-HT 2 D * * * *

17 Fingerprints: functional specificity primary somatosensory primary motor primary auditory 3b 5-HT A 5-HT 2 D a GABA A GABA B 4 5-HT A 5-HT 2 D a GABA A GABA B 4 5-HT A 5-HT 2 D a GABA A GABA B a BZ a BZ a BZ N M 3 M 2 M N M 3 M 2 M N M 3 M 2 M primary (V), and higher (V2, V3) visual areas V 5-HT 2 D 3 2 V2 5-HT 2 D 3 2 V3 5-HT 2 D HT A GABA A 5-HT A GABA A 5-HT A GABA A a 2 GABA B a 2 GABA B a 2 GABA B a BZ a BZ a BZ N M N M N M M 3 M 2 M 3 M 2 M 3 M 2

18 Ventral visual areas in human extrastriate cortex (FG2, FG2) FG2 FG FG2 FG FG2 FG FG2 FG FG FG2 FG FG2 FG FG2 FG FG2 (pfus) Weiner, K., Golarai, G., Caspers, J., Mohlberg, H., Zilles, K., Amunts, K., Grill-Spector, K.: The mid-fusiform sulcus: A landmark identifying both cytoarchitectonic and functional divisions of the human fusiform gyrus. Neuroimage 84: (24) Gomez, J., Barnett, M.A., Natu, V.S., Mezer, A., Palomero-Gallagher, N., Weiner, K.S., Amunts, K., Zilles, K., Grill-Spector, K. (26). Growth of tissue in human cortex is coupled with the development of face processing. Science 355: 68-7 (27)

19 Receptor fingerprint of FG Hierarchical Cluster Analysis ventral stream Receptor fingerprint of FG2 auditory somatosensory motor Caspers, J., Palomero-Gallagher, N., Caspers, S., Schleicher, A., Amunts, K., Zilles, K.: Receptor architecture of cytoarchitectonic visual areas FG and FG2 of the posterior fusiform gyrus. Brain Struct. Funct. 22: (25)

20 FG GABA B FG2 (pfus) GABA 5-HT A bz b.s. A

21 /BZ V 4 2 /BZ V2v 4 2 /BZ V3v 4 2 /BZ 3b 4 2 /BZ /BZ /BZ /BZ /BZ /BZ /BZ L 4 2 /BZ /BZ 4 2 /BZ /BZ supragranular granular infragranular /BZ PFm 4 2 Zilles K, & Palomero-Gallagher N: Transmitter receptor fingerprints in regions and layers of the human cerebral cortex. Frontiers Neuroanat (submitted)

22 /BZ V /BZ V2v /BZ V3v /BZ 3b /BZ /BZ /BZ /BZ /BZ PFm /BZ L /BZ /BZ /BZ /BZ /BZ /BZ supragranular granular infragranular Zilles K, & Palomero-Gallagher N: Transmitter receptor fingerprints in regions and layers of the human cerebral cortex. Frontiers Neuroanat (submitted)

23 Dimension Multi-receptor fingerprints systematically differ between cortical layers V2d 3b V2v V V2d V3A V3d V3d V3v 4 V4v V3A 4 V2v V4v V3v 5M 4 PGp b 23 5L PGa V3d V3A 3a 4 FG2 5L V4v FG 44 3a FG2 3 5M PGa 3b PGa PFm FG 2 PFt PFt 37M PFm B 3 5M 37B 37M 4 PGp 37L 38 5L 37L 22 PFm 6 47 PFt 6 38 FG 22 L L 3a 37B 9 M L M 2 37M PGp 9 M FG L Dimension Zilles K, & Palomero-Gallagher N: Transmitter receptor fingerprints in regions and layers of the human cerebral cortex. Frontiers Neuroanat (submitted) 32 V V3v V2v V2d V Strata supragranular (layers I-III) granular (layer IV) infragranular (layers V-VI)

24 Receptors and the Default Mode Network

25 L 3a,3b 2 PFt PFm PGa PGp L L V4v V3d L 5M V3A V3d V V2d V2v V3v V4v FG 37M 37B 36 Hi EC 38 s24 p24 32 M DMN areas defined by Buckner et al. (28) Ann NY Acad Sci 24: -38 Cytoarchitectonic areas defined by Brodmann (99) and JuBrain

26 2nd Principal Component Principal component analysis of receptor densities (averaged over all cortical layers) and default mode network DMN FG 9 8 FG p24 M EC 36 37L L PGp 37M 4 PFt PFm 3 37B 38 PGa 5L s24 3a 5M DMN 24 Broca V3A 3b V3v early visual primary sensory V2 V posterior cingulate cortex (BA23) precuneus (BA3) anterior cingulate cortex (p24, s24) ventral medial prefrontal cortex (m, BA32, BA) dorsal medial prefrontal cortex (medial part of BA9) inferior parietal lobule (PGa, PGp, PFm, PFt) lateral temporal cortex (BA2, BA2) anterior temporal pole (BA38) hippocampus, entorhinal (EC), and parahippocampal (BA36) cortex 6 45 V4v motor V3d -3 HIP st Principal Component

27 SCORE Discriminance analysis of the densities (averaged over all cortical layers) of all receptor types between default mode network DMN areas and Non-DMN areas DMN Networks Non-DMN p =.2 Contribution of each receptor type to the segregation between DMN and Non-DMN areas Receptor Standardized score M Kainate a HT M GABA B.695 D a 4 b HT A a GABA A /BZ GABA A.235 M -.22

28 Ratio between and GABA A receptor densities Receptor density (fmol/mg protein) EC s24 /GABA A Hippocampus M 8 L p24 2 PFt PFm 5M PGa 3 PGp y = -.844x R² =.52 p= Cerebral metabolic rate of oxygen consumption (mmol/g/min) ( ) and GABA A ( ) 5 Hip 38 M EC L s24 PFm PFt p24 5M PGa 3 PGp Cerebral metabolic rate of oxygen consumption (mmol/g/min) areas of the DMN non-dmn areas Metabolic data from: Hyder F, Herman P, Bailey CJ, Møller A, Globinsky R, Fulbright RK, Rothman DL, Gjedde A (26) Uniform distributions of glucose oxidation and oxygen extraction in gray matter of normal human brain: No evidence of regional differences of aerobic glycolysis. J Cerebral Blood Flow & Metabolism 36(5): 93 96

29 Thanks to: Institute of Neuroscience and Medicine (INM-), Research Center Jülich: Katrin Amunts Mareike Bacha-Trams Sebastian Bludau Julian Caspers Svenja Caspers Nicola Palomero-Gallagher Filip Scheperjans Axel Schleicher Stanford University: Kalanit Grill-Spector Kevin Weiner Institute of Developmental Genetics, German Research Center for Environmental Health München: Wolfgang Wurst Max Planck Institute Leipzig: Angela Friederici Brain Mapping Center, UCLA: John C. Mazziotta Arthur Toga

30 A receptor fingerprint is 5-HT 2 D HT A a 2 5 GABA A GABA B a BZ caudate nach M 3 M 2 M putamen area 4

31 Receptor Fingerprints of the primary visual cortex: layer specificity L. I 5-HT 2 D 3 2 Ll. II-III 5-HT 2 D 3 2 L. IV 5-HT 2 D HT A GABA A 5-HT A GABA A 5-HT A GABA A a 2 GABA B a 2 GABA B a 2 GABA B a BZ a BZ a BZ N M N M N M M 3 M 2 M 3 M 2 M 3 M 2 In/output Synapse L. V D 3 L. VI D 5-HT HT I II I II I II 5-HT A a 2 a N M GABA A BZ GABA B 5-HT A a 2 a N M GABA A BZ GABA B III IV V III III IV V M 3 M 2 M 3 M 2 VI VI

32 Dimension Multidimensional scaling analysis of receptor densities (averaged over all cortical layers) and default mode network DMN 24 EC 6 motor 32 p L PGp M 37M L 42 5L 5M 2 2 PGa 22 PFt 3 s24 37B PFm 3a 4 3b FG2 V3v FG V4v Broca V3d V3A 4 primary sensory V2 early visual V posterior cingulate cortex (BA23) precuneus (BA3) anterior cingulate cortex (p24, s24) ventral medial prefrontal cortex (m, BA32, BA) dorsal medial prefrontal cortex (medial part of BA9) inferior parietal lobule (PGa, PGp, PFm, PFt) lateral temporal cortex (BA2, BA2) anterior temporal pole (BA38) hippocampus, entorhinal (EC), and parahippocampal (BA36) cortex -4 HIP Dimension

33 Receptor density (fmol/mg protein) Receptor density (fmol/mg protein) Receptor density (fmol/mg protein) Receptor density (fmol/mg protein) 5 y = -57.8x R² =.3239 p= Cerebral metabolic rate of oxygen consumption (mmol/g/min) y = 255.6x R² =.656 p= Cerebral metabolic rate of oxygen consumption (mmol/g/min) 3 2 y = -.2x GABA A 5-HT A R² =.4852 y = 22.6x R² =.422 p=. y = 278.8x R² =.432 p= Cerebral metabolic rate of oxygen consumption (mmol/g/min) 5 5 p= Cerebral metabolic rate of oxygen consumption (mmol/g/min) Metabolic data from: Hyder F, Herman P, Bailey CJ, Møller A, Globinsky R, Fulbright RK, Rothman DL, Gjedde A (26) Uniform distributions of glucose oxidation and oxygen extraction in gray matter of normal human brain: No evidence of regional differences of aerobic glycolysis. J Cerebral Blood Flow & Metabolism 36(5): 93 96

34 Receptor density (fmol/mg protein) Receptor density (fmol/mg protein) Receptor density (fmol/mg protein) y = x y = x M 2 R² =.5852 a R² =.92 p=.9 p= Cerebral metabolic rate of oxygen consumption (mmol/g/min) Cerebral metabolic rate of oxygen consumption (mmol/g/min) HT 2 y = x R² =.323 p= Cerebral metabolic rate of oxygen consumption (mmol/g/min) DMN: cerebral metabolic rate of oxygen consumption decreases with increasing, 5-HT A and a receptor densities, but increases with increasing GABA A receptor densities Non-DMN: cerebral metabolic rate of oxygen consumption increases with increasing, GABA A, M 2 and 5-HT 2 receptor densities Metabolic data from: Hyder F, Herman P, Bailey CJ, Møller A, Globinsky R, Fulbright RK, Rothman DL, Gjedde A (26) Uniform distributions of glucose oxidation and oxygen extraction in gray matter of normal human brain: No evidence of regional differences of aerobic glycolysis. J Cerebral Blood Flow & Metabolism 36(5): 93 96

35 Ratio between and GABA A Receptor density (fmol/mg protein) Receptor density (fmol/mg protein) receptor densities receptor densities /GABA A y = -.962x R² =.299 p= Cerebral metabolic rate of glucose consumption (mmol/g/min) ( ) and GABA A ( ) Cerebral metabolic rate of glucose consumption (mmol/g/min) Ratio between and GABA A /GABA A y = x R² =.538 p= Cerebral metabolic rate of oxygen consumption (mmol/g/min) ( ) and GABA A ( ) Cerebral metabolic rate of oxygen consumption (mmol/g/min) Metabolic data from: Hyder F, Herman P, Bailey CJ, Møller A, Globinsky R, Fulbright RK, Rothman DL, Gjedde A (26) Uniform distributions of glucose oxidation and oxygen extraction in gray matter of normal human brain: No evidence of regional differences of aerobic glycolysis. J Cerebral Blood Flow & Metabolism 36(5): 93 96

36 Receptor density (fmol/mg protein) Receptor density (fmol/mg protein) Receptor density (fmol/mg protein) y = x R² =.2679 p=.63 M y = 242.8x R² =.387 p= Cerebral metabolic rate of glucose consumption (mmol/g/min) 5-HT A y = -46.8x R² =.3729 p= Cerebral metabolic rate of glucose consumption Cerebral metabolic rate of glucose consumption DMN: cerebral metabolic rate of glucose consumption decreases with increasing and 5-HT A receptor densities Non-DMN: cerebral metabolic rate of glucose consumption increases with increasing and M receptor densities Metabolic data from: Hyder F, Herman P, Bailey CJ, Møller A, Globinsky R, Fulbright RK, Rothman DL, Gjedde A (26) Uniform distributions of glucose oxidation and oxygen extraction in gray matter of normal human brain: No evidence of regional differences of aerobic glycolysis. J Cerebral Blood Flow & Metabolism 36(5): 93 96

37 Summary High densities of receptors significantly correlate, or show a non-significant trend, with low cerebral metabolic rates of glucose or oxygen consumption in DMN areas. A notable exception is the positive correlation of the densities of inhibitory GABA A receptors with the cerebral metabolic rate of oxygen consumption. High densities of receptors significantly correlate, or show a non-significant trend, with high cerebral metabolic rates of glucose or oxygen consumption in non-dmn areas (mainly primary and higher sensory areas, motor areas).