searching of membrane target for circadian clock
DESCRIPTION
AACIMP 2011 Summer School. Neuroscience Stream. Lecture by Nikolai Kononenko.TRANSCRIPT
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Searching of a membrane target for mammalian circadian clock responsible for
circadian modulation of firing rate
Nikolai I. Kononenko
Department of General Physiology of Nervous System, Bogomoletz Institute of Physiology,
Kiev, Ukraine
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1. The suprachiasmatic nucleus (SCN) of the hypothalamus is the primary biological clock regulating circadian
rhythms in mammals
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2. Individual SCN neurons express self-sustained circadian oscillations. As a result of internal coupling, the SCN generates a coherent output signal
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Circadian-clockcore
Messenger
Membrane target
0
6
Hz
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There are two common features for presented above hypotheses:
1. All experiments were done employing whole cell recordings
2. Spike-associated currents are key targets for circadian modulation of firing rate
Our approach was based on on-cell (cell-attached) recordings of electrical events in SCN neurons
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Multielectrode array dish
0
6
Hz
0 1 2 3 4 5(Days)
1 min
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Spike-associated currents are key targets for circadian modulation of firing rate
First of all, we asked whether spike-associated currents (i.e., currents
active only during action potentials) are indeed primary membrane target(s)
responsible for circadian modulation of firing rate (CMFR).
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Time (ms)
0 1 2 3 4 5
uV
-20
-10
0
10
222 APs averagedThreshold255 APs averaged
Time (hrs)
0 12 24
Firi
ng r
ate
(Hz)
0
2
4
6Circadian rhythmInterval of averagingInterval of averaging
BAAction potentials across the circadian cycle
Thus, result allowed preliminarily to suggest that spike-associated currents are not a key targets
for circadian modulation of firing rate.
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Nature, 2002, 16, 286-290
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Effect of nifedipine on circadian firing rhythms in SCN neurons
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Nature Neuroscience, 2005, 8, 650-656
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Effect of 4-aminopyridine on circadian rhythms of firing rate in SCN neurons
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A B
Time (hrs)
0 20 40 60 80 100
Firi
ng r
ate
(Hz)
0
2
4
6
8100 M Cd2+
Cd2+ does not suppress immediately spontaneous activity and its circadian modulation
(n=13 neurons)
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Spike-associated channels are not the principal determinants of circadian
modulation of firing rate
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Two acutely isolated SCN neurons on the bottom of a Petri dish 6 days after isolation
100 m
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On-cell recording of single channels
2 pA
100 ms
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Spontaneous firing of isolated SCN neurons and circadian modulation of their firing rate
C
D
30 s
B
A
E
Firing rate (Hz)
0 1 2
Nu
mb
er
of
ne
uro
ns
0
2
4
6
80.87 ± 0.12 Hz
Firing rate (Hz)
0 1 2
Num
ber
of n
euro
ns
0
6
12
18 0.24 ± 0.06 Hz
Firing rate (Hz)
0 1 2
Nu
mb
er
of
ne
uro
ns
0
2
4
6
80.68 ± 0.11 Hz
Noon Midnight Noon of the next day
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2 s (A)5 s (B)
7 pA20 mV
A B
0 pA
C
Vh = -90 mV
Vh = -65 mV
2 s
5 pA
Fluctuation of membrane potential produces spontaneous activity of SCN neuron
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The numerous single-channel inward currents we observed during on-cell recording of spontaneous electrical firing in isolated SCN neurons led us to study the properties of the corresponding channels in their relation to spontaneous electrical firing
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20 ms
2 pA
8 pA
20 mV
A1
A2
A3
Closed time (ms)0 5 10 15 20 25
Num
ber
of e
vent
s
0
200
400
600
800 5 mVf = 0.76 ms
s = 13.4 ms
25 mVf = 0.90 ms
s = 5.31 ms
B
CPatch membrane potential (mV)
-25 0 25 50
Cha
nnel
cur
rent
(pA
)
-4
-2
0
2
57.5 pSErev = 33 mV
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We have revealed a novel set of subthreshold, voltage-dependent cation
(SVC) channels that are active at resting potential and increase their
open probability with membrane depolarization
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A CB
20 pA0.016
2 s 10 s
10 pA0.032
-15 s 15 s0 -100 s 0 100 s
* *
2 pA
200 ms
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Correlation between Po of single-channel activity andspontaneous firing in isolated SCN neuron
1 s10 pA
2 pA0.06
200 ms
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Voltage dependence of persistent single channel
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We hypothesize that a sufficient number of SVC channel openings
would result in threshold depolarization of the SCN neuronal membrane and
spontaneous electrical firing
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Time (min)
0 5 10 15 20 25F
iring
rat
e (H
z)0
1
2
Time (min)
0 5 10 15 20 25
Firi
ng r
ate
(Hz)
0
1
2
A B
db-cGMP application evokes spontaneous electrical activity in acutely isolated SCN neurons
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gC=40 pS; EC=0 mV
o=0.2 ms; fc=1 ms
Single-channel openings (N=1)
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36 channels~0.017 Hz
44 channels~3.2 Hz
55 channels~10 HzB
A
40 mV
Number of channels
40 60 80 100
Fir
ing
ra
te (
Hz)
0
5
10
15
res
=4 ms5 6
7
8 9
Single-channel openings produce spontaneous firing in a model SCN neuron
1 s
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Minimal mammalian circadian clockwork model (circadian oscillator)
From Scheper et al., J Neurosci 1999
Period
Period
Period*
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Time (hrs)
0 5 10 15 20 25 30
Firi
ng r
ate
(Hz)
0
5
10
15
Num
ber
of c
hann
els
0
20
40
60
1
2 2
A B
Circadian modulation of the number of single channels produces modulation of the action potential firing rate
Circadian peaks of 3 intact SCN neurons in MED
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Conclusions
1. The present findings bring together several lines of study seeking a membrane target of the circadian clock, and bridge the gap between single-channel physiology and features of circadian rhythms of firing rate.
2. The SVC single channels described here are proposed to be a key membrane target mediating the effects of circadian clock protein concentration on electrical firing rate.
3. Concomitantly, these data delineate a novel pathway that links the core circadian clock with membrane events regulating spontaneous firing in SCN neurons.
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Acknowledgments:
The author thanks Dr. F.E. Dudek (University of Utah School of Medicine, Salt Lake City, USA) and Dr. S. Honma (Hokkaido University Graduate School of Medicine, Sapporo, Japan), in whose laboratories experimental data were obtained, and Dr. N.M. Berezetskaya (Institute of Physics, Kiev, Ukraine), who converted differential equations into computer programs.