El proyecto BRAIN: mapeo de la conectividad
neuronal y su relevancia clínica
Rafael Yuste
Neurotechnology Center
Kavli Institute of Brain Science
Columbia University
Dense
Text
Text
COlumbia
Photo by D. Peterka
Neurotechnology Center at Columbia
Welcome'all'!!!!'
El desafío de entender la corteza cerebral
Doctrina'Neuronal'Anatómica'
Cajal'
!
Doctrina'Neuronal'Fisiológica'
Sherrington'
Doctrina'Neuronal'Psicológica'
Hubel'and'Wiesel'
Barlow'
Hay propiedades emergentes en los circuitos cerebrales?
Las propiedades emergentes son comunes
What are the emergent diseases of the brain?
Systems
Circuits
Cellular
Synapses
Molecules
Lorente'de'Nó'
Circuits in brain have recurrent
connections to sustain reverberating
activity
EEG: All parts of brain have ongoing intrinsic activity
Hans Berger
Neuronal ensembles: an emergent functional group of neurons
Individual'neurons'
Ensemble'formed'
Hebb
How to study emergent levels of brain circuits?
La actividad neuronal general cambios en la concentración de calcio
Yuste and Katz, 1991
Mapa de actividad de circuitos neuronales
Yuste and Katz, 1991
Two-photon microscopy to image neurons deep in brain circuits
1-photon
fluorescence
2-photon
fluorescence
Excited
Excited
uv hν
visible hν
Ground
Ground
Fluor. + 1 uv hν
visible hν
ir hν
Fluor. + 1 visible hν
Fluor. + 2 ir hν
Fluor. + 1 visible hν
Denk et al, 1990; Yuste and Denk, 1995'
Mapeo de la actividad neuronal en ratones vivos
Miller et al., 2014
Cor$cal(neurons(are(ac$vated(in(ensembles(
Spike''
probability' ∆F/F'
50µm'
Spontaneous(and(evoked(ac$vity(from(the(same(neurons(
50µm'
Spontaneous'acMvity'
Evoked'acMvity'
Calcium(movies(sped(up(by(3x(
Visual(s$muli(recruit(intrinsically(generated(cor$cal(ensembles(
Controlling cortical ensembles:
Does the circuit complete patterns?
Image ensemble
Stimulate to induce observed ensemble
Behaviour?
?
?
BRIEF COMMUNICATIONS
e
b
AAV-CaMKII-C1V1
(E162T)-p2A-EYFP
Two photon optogenetics in 3D
P0 P21–P24 P57–P117
C
1
1P
0 0.4 0.8 1.2 1.6 2.0
One-photon current (nA)
h
i
d
600
E122T = C1V1T
0
h
50 pA
50 ms
P photocurrent (norm)
1.0
0.8
0.6
0.4
0.2
R
H
2
hR
1V
1
T/
T
T
C
20
15
10
5
0
0
5
Inters
C
T/
T
T
1V
1
1V
1
C
1V
1
2P photocurrent (pA)
C
g
C
20 ms
(R2 = 0.8; Fig. 1i). Prolonged photostimulation produced APs at
frequencies exceeding those of four times rheobase (Fig. 1j).
1.0
We then used two-photon illumination
of C1V1 to stimu = 1,040 nm T
late single dendrites and spines. We selected cells exhibiting
C1V1
0.8
high EYFP expression and raster-scanned
individual dendritic
C1V1
T
processes, using the same patterns used to successfully generC1V1
T/T
0.6o 11 pA (mean o s.d.), range
ate photocurrents in somata (23
C1V1
7–49 pA, n = 21 dendrites and 8 neurons; Fig. 2a). Dendrites
0.4
located further from the soma yielded lower currents (R =C1V1
0.45;T
Supplementary Fig. 2). We also targeted spines and dendrites
C1V1T/T
i
P photocurrent (norm)
0
1
C1V1
T/
T
40
1V
1
C1V1T
80
20 ms
25
f
C
C1V1T/T
Latency
1s
Figure 1 | Two-photon activation of individual neurons with C1V1 T in mouse brain slices. (a) Experimental st
700somatosensory cortex of* a mouse. Several weeks later, brain slices wer
and EYFP genes were injected into the
*
(b) Two-photon fluorescence image of a living cortical brain slice expressing EYFP (940-nm excitation, 15 m
600
* images from b showing C1V1T-expressing cells in upper (c) an
scale bar, 100 Mm). (c,d) Higher-magnification
and 10 Mm (d)). (e) Distribution of 500
steady-state currents elicited by one-photon (1P) wide-field stimulation
from C1V1T-expressing cells (mercury arc lamp, band-pass 470–490 nm, 40×
20×/0.5-NA objective, 300 MW mm−2
400
0.8with
NA voltage clamp in a C1V1 T-expr
illustrates stimulated area. (f) Two-photon
(2P) photocurrents measured
light powers. The raster-scan pattern
(inset,
gray
lines)
across
a
neuronal
cell body had 32 lines, 2 ms per lin
20×
300
1–41 mW on sample, 20×/0.5-NA objective). (g) Two-photon photocurrents
0.5 NAinduced in C1V1 T-expressing neu
2004 ms per line; experimental parameters as in f). (h) Top, current-clam
(gray lines correspond to 0.5, 1, 2 and
neurons during two-photon illumination (stimulated at tick marks; experimental parameters as in f). Bottom
100
illumination (gray bar). (i) Quantification of AP latency changes from experiments as in h with different stim
resulting from either a current injection
0 at four times rheobase (top) or from an optical stimulation produce
body for the same time duration (bottom; other experimental parameters as in f).
T
120
C
© 2012 Nature America,
Inc. All rights reserved.
1V
e
off (ms)
d
C
C
1V
1
E122T/E162T = C1V1T/T
10 µm
10 mV
Jitter
Packer et al, 2012'
sus TPLSM
C1V1T-ts-EYFP
1,200
1V
1
E122
c
*
14
12
10
8
6
4
2
0
10 mV
E162
c
1,800
1P photocurrent (pA)
N
b
ChR1
VChR1
C
1V
1
a
100 pA
iking with
mutants
and ChR2HR
1,075 o
A (n = 18);
R2HR,
see
n-Whitney
C1V1T-ts. Purple
ocurrents.
Is were
values
60 o 1.9 ms
)).
NA
7), 20×/0.5,
1V1T:
/0.5,
1V1T/T:
/0.5,
enotes
C1V1T
ferent light
tra
n = 6).
mum
Number of cells
Infection Slicing
f
Latency change (%)
a
and monitored time-lock
(EPSCs) while raster scann
fluorescent cells to identif
(Supplementary Fig. 4).
photostimulating a neuron
patched cell (Fig. 2c). Inc
time-locked EPSCs from n
erate them, presumably by
spike (Supplementary Fig.
neurons, of which 8 were ide
Collaborator: Deisseroth(
0.2
Activacion sistematica de neuronas con luz de laser
Fino and Yuste, 2011
Packer and Yuste, 2011
Playing the piano in awake mice with optogenetics
(thin skull experiments C1V1+GCaMP6s)
OptogeneMc'sMmulaMon'
Evoked'calcium'
transients'
Luis'CarrilloNReid'
Single(cell(optogene$c(s$mula$on(
Thin'skull'experiments'C1V1+GCaMP6s'visual'sMmulaMon'
OpMcal'sMmulaMon'one'cell'
Evoked'calcium'transients'
Entraining an ensemble and triggering it by one neuron
Evoked'calcium'transients''
targeted'cell'
Overall'acMvity'
Experimental'protocol'
Neuronal'ensemble'acMvated'by'one'cell'
Luis'CarrilloNReid'
Breaking the neural code
THE BRAIN ACTIVITY MAP
Paul Alivisatos
Berkeley
!
Miyoung Chun !
Kavli Foundation !
!
George Church !
Har vard!
Ralph Greenspan
UCSD/Kavli!
Michael Roukes
Caltech
!
R a f a e l Yu s t e
Columbia/Kavli
WHY?
! Scientific goals
! Explore the emergent properties of brain circuits
! Decipher neural code
! Solve connectivity diagrams: Reverse engineer neural circuits
! Medical goals
! Develop novel assays for brain diseases
! Emergent hypotheses for pathophysiology of brain disease
! Novel therapeutics: Optomedicine, Brain-Computer Interfaces!
! Development of powerful new technology
! Economic benefits: Batelle report
! Training of a new generation of interdisciplinary scientists
! Historical Precedents
! Statistical mechanics, Magnetism, Dynamical Systems, Non-equilibrium thermodynamics
! Proven success of “big science” in molecular biology: The Human Genome Project
29
BRAIN ACTIVITY MAP (BRAIN
INITIATIVE)
Goal 1:
Goal 2:
Goal 3:
worm!
Interdisciplinary technology development
Measure every action potential for every neuron in brain circuits
Manipulate the activity of every neuron in these circuits
Computationally analyze/model these circuits
fly!
fish!
mouse!
30
Alivisatos et al, 2012, 2013
GOALS (DETAILS)!
Goal 1:
Measure every action potential for every neuron in complete
neural circuits
!! Optical approach: Image action potentials via calcium and voltage indicator!
! Electrophysiological approach: 10k channels and upward!
! Next-gen photonics-based silicon probes with nanoparticle indicators !
! Futuristic synthetic biology approaches for reconstructing action potentials!
16 Dec 2011
Alivisatos, Church, Greenspan, Roukes, Yuste, Chun - (c) 2011
31
EFFECT OF BAM ON HUMAN HEALTH
Donoghue, pers. comm.
Mapeo de actividad neuronal en pacientes?
Extroducer (Lundberg et al. 2010)
GOALS (DETAILS)!
Goal 1:
!Measure every action potential for every neuron in complete brain
“circuits”!
Goal 2:
Modulate the activity of every neuron in complete neural circuits
!
! Optogenetics & caged compounds!
! Nanoparticles coupled to nanoprobes!
! Local chemical modulation through probe-based microfluidics!
! Genetic strategies!
16 Dec 2011
Alivisatos, Church, Greenspan, Roukes, Yuste, Chun - (c) 2011
34
Quimica del Ruthenio: antenas de luz
(con Roberto Etchenique, U. Buenos Aires)
RuBiGlutamate: glutamato enjaulado
10 µm
Fino et al, 2009
Ruthenium uncaging of GABA
Parando la epilepsia en ratas opticamente con GABA enjaulado
Control 4-AP, no Mg2+
5 s blue light
0.5 mV
RuBi-GABA (10µM) 4-AP, no Mg2+
0.5 mV
Con Steve Rothman, U. Minnesota
Optical treatment of mental and neurological diseases
A DRAFT ROADMAP
5 years: !
50,000 neurons !
10 years:!
1 million neurons
15 years: !
Entire brains
behaving
16 Dec 2011
Alivisatos, Church, Greenspan, Roukes, Yuste, Chun - (c) 2011
39
COLLABORATION AMONG
FUNDING AGENCIES
! Coordinate funding support among various resources ranging from
Federal Funding Agencies, Private Foundations, and Industry
! Ethical and Legal supervision
Initiation
of the
Brain
Activity
Map!
DARPA!
NIH!
Project!
Completion!
NSF!
Others!
Private Foundations!
Industry!
!
•Optical methods for recording neuronal activity!
!
•Optical methods for controlling neuronal activity!
!
•Are ensembles an emergent level of cortical function?!
!
•Importance of methods development (neuron doctrine vs.
neural networks)!
NEI
MURI ARO
DARPA
0
