Transcript Khodakhah Lab - Albert Einstein College of Medicine

Khodakhah Lab and the Cerebellum: A Love Story
AECOM, Kennedy 506. Beautiful view of parking lot.
Karina Alviña, Paola Calderón, Johanna Dizon, Kathryn Harper, Caroline Hoang, Joy Walter, Kamran Khodakhah
Introduction
Cerebellar dysfunction induces Dystonia
The cerebellum integrates sensory and cortical information in order to generate the
timing signals required for motor coordination. Purkinje cells (PCs), the sole output of the
cerebellar cortex, are the primary sites of integration of sensory and cortical information
within the cerebellum. Therefore, the algorithm with which PCs integrate the information
they receive is fundamental for cerebellar function. However, the input-output relationship
of PCs has not been determined.
Sensory and cortical information is relayed to PCs by over 150,000 excitatory
granule cell (GC) synaptic inputs. We set out to determine the relationship between the
strength of excitatory GC input and PC output. This was accomplished by electrically
stimulating GCs and recording the response of PCs with single-unit extracellular
recordings in acute cerebellar slices (see below). The granule cell stimulation strengths
ranged from a minimum intensity that evoked the smallest detectable increase in the
spontaneous firing rate, to 10 or 20 times this minimum intensity. Experiments were done
in the presence of blockers of inhibitory synaptic transmission (picrotoxin, a GABAA
antagonist, and CGP, a GABAB antagonist).
Dystonia is a neurological disorder characterized by an excessive cocontraction of agonist and antagonist muscles. It has been suggested that
malfunction of the basal ganglia is the unique origin of this disease. However,
symptoms from different types of dystonia have shown that dysfunction of the
cerebellum may contribute to this disease as well.
One type of dystonia that shows cerebellar symptoms is called Rapid-onset
dystonia-parkinsonism (RDP). Interestingly, this genetic disease manifests a
specific mutation that reduces the function of the Na/K ATPase pump α3 isoform
which is the exclusive isoform expressed in Purkinje cells.
It is thought that the information needed for cerebellar motor coordination is
encoded in the rate and pattern of spontaneous activity of Purkinje cells. A
reduction of the current generated by the electrogenic Na/K ATPase may affect the
regulation of the spontaneous activity of Purkinje cells and therefore the
generation of precise timing signals that allow motor coordination. We propose
that disruption of this activity may be the mechanism by which the cerebellum
induces dystonia.
Typical PC response to a single pulse electrical GC layer stimulation
stimulus
Firing Rate (spikes/s)
The cerebellum coordinates movement and maintains balance by generating
precise timing signals for the proper contraction of agonist and antagonist muscles.
Failure of the cerebellum to generate precise timing signals results in movement
disorders. Our lab is interested in determining how the cerebellum generates timing
signals and how dysfunction of these signals leads to motor impairments.
In order to generate precise timing signals, the cerebellum receives and
integrates information from cortical areas and all sensory modalities. Information
entering the cerebellum is processed primarily by the circuitry of the cerebellar
cortex. The sole output of the cerebellar cortex, Purkinje cells, relay processed
information to the deep cerebellar nuclei (DCN). After further processing, the DCN
then sends signals to various target areas.
Our lab focuses on examining the cerebellum from the single cell level up to
the behaving animal. More specifically, we are interested in examining the intrinsic
properties and information processing of Purkinje cells and DCN neurons under
normal and pathological conditions. Currently, our lab is interested in understanding:
• How alterations in calcium homeostasis modulate Purkinje cell activity.
• How Purkinje cells integrate and encode synaptic input.
• Information transfer between the cerebellar cortex and the DCN.
• The role/contribution of the cerebellum in dystonia.
• The mechanisms underlying cerebellar ataxia.
To address these issues, we utilize a variety of techniques that include
electrophysiology, photolysis, imaging, modeling, and various behavioral paradigms.
The granule cell input - Purkinje cell output function
90 µA
stimulatio
n
Anatomy of the Cerebellum
200 µV
50 ms
300
Maximum firing
rate (spikes/s)
Stimulus intensity (A)
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15
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0
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Ouabain 10nM
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20
0
Average PC Response vs.
Normalized stimulus intensity
Raster plot of PC responses to
different GC stimulation intensities
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0
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200
Time (min)
Figure 1. The Na/K ATPase blocker, ouabain, disrupts the regular firing frequency
of Purkinje cells at very low concentrations. By using extracellular recordings in
cerebellar acute slices, a bath application of 10 nM ouabain increased the firing rate
up to causing cell-bursting.
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0
-100
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5x 10x 15x 20x
Normalized stimulus intensity
0
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Time (ms)
- each line represents an action potential
- stimulation occurs at time point 0
Peak Current and Total Charge vs. Stimulus Intensity
InsP3-mediated Ca2+ release in Purkinje cells
Cytosolic calcium is tightly regulated such that the concentration inside the cell is
thousands-folds lower compared to outside the cell. This enables small increases in
cytosolic calcium to act as a signaling mechanism within cells. Activation of inositol (1,4,5)trisphosphate (InsP3) receptors releases calcium from the endoplasmic reticulum, thereby
increasing cytosolic calcium levels. Calcium release from these receptors can signal for
diverse cellular processes such as gene transcription, fertilization, and transmitter release.
The absence of a functional InsP3 receptor in mice results in motor dis-coordination
(ataxia) which is associated with impairments in cerebellar Purkinje cells. Ideally, if calcium
release through InsP3 receptors is used as a signaling mechanism in Purkinje cells then
they should be suited to respond to the fast pace of neuronal activity. In non-neuronal
tissues InsP3 signaling is slow, requiring hundreds of seconds between release periods
due to calcium inactivation. Currently, the time course for recovery between release
periods for InsP3 receptors in intact Purkinje cells is not known.
Question: Has InsP3 signaling in Purkinje cells adapted to cope with the fast pace of
neuronal activity?
0.8
Figure 2. Dystonic postures after chronic perfusion of 100 µM ouabain into the
cerebellum.
100 nA
25 ms
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Q (-pC)
Peak EPSC (-nA)
Kandel, Schwartz and Jessell. Principles of Neural Science.
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Mechanisms underlying cerebellar ataxia
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Stim int (A)
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Stimulus intensity (A)
PCs linearly encode the strength of GC synaptic input in their maximum firing rate.
Mapping granule cell-to-Purkinje cell connectivity
We are characterizing the functional connectivity between GCs and PCs, thereby
addressing three controversial questions: 1) Does activity in this pathway propagate in a
dispersed or patchy manner? 2) Do the ascending axon and parallel fiber regions of the
GC provide differential input to PCs? and 3) How do molecular layer inhibitory
interneurons between GCs and PCs modulate PC output? In ongoing experiments, PC
electrical activity in response to stimulation in multiple underlying GC patches is
monitored via extracellular recording. Future experiments include paired intracellular
recording, and sodium and chloride imaging to measure and trace the currents involved.
Methods
rate of release, d[Ca2+]/dt
50 ms
B
1 M
Figure 1. Caged glutamate is released by a
1 ms-40 µm diameter UV pulse at 63 sites
on a 160 x 200 µm granule cell region
underlying the Purkinje cell being assessed.
UV
pulse
A. Schematic diagram illustrating the whole-cell patch clamp technique. Purkinje cells from cerebellar slices were voltageclamped at -60 mV. The Ca2+ indicator fura-6f and caged InsP3 were included in the pipette solution. B. Photoreleased
InsP3 evokes a transient increase in cytosolic Ca2+. The slope of the rising phase of the transient was measured to give the
change in Ca2+ over time, or rate of release. This rate is proportional to the number of open InsP3 channels and is used for
comparison within and between cells.
The goal of this project is to examine whether cerebellar mGluR1 receptors are required
for motor coordination, or for motor learning. Signaling via the mGluR1 receptors is
considered to be involved in motor learning. This is because of its role in long-term
depression, which is thought to be the learning mechanism of the cerebellum. Additionally,
mGluR1 knock-outs are unable to do complex motor tasks and have problems with their gait.
These complex motor tasks, such as the rotorod task, require motor coordination and
learning. To determine whether motor learning or coordination is affected by mGluR1s, we
train our mice to do these tasks while being chronically perfused with a mGluR1 blocker. If
mGluR1s are important for motor learning then a mouse receiving blockers will show a slower
learning curve on the task. On the other hand, if mGluR1s are really more important for motor
coordination, the mice receiving blockers should show a normal learning curve, but would be
unable to maintain as a high a performance on the task compared to a control mouse.
d[Ca2+]/dt = 1.4 μM/s
UV
UV
d[Ca2+]/dt = 81.6 μM/s
1 M
2.5 s
1 M
1s
Vcmd
UV
d[Ca2+]/dt = 166.6 μM/s
2.0
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Figure1. A: The mice receiving mGluR1 blockers are showing a motor learning impairment compared to control mice on the
rotorod task. B: The mice receiving mGluR1 blockers are showing a motor coordination impairment compared to control mice
on the rotorod task.
1.0
0.5
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B
Control
mGluR1 blocker
A
0
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Time (s)
Vcmd
UV
The rapid recovery from Ca2+ inactivation suggests that InsP3 receptors in intact Purkinje
cells are suited for use during fast synaptic signaling.
Control
mGluR1 blocker
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70
Latency to Fall (sec)
Vcmd
d[Ca ]/dt (normalized)
~1 μM
2+
[Ca2+]i
d[Ca2+]/dt = 155.7 μM/s
1s
Figure 2. Plots of instantaneous firing rate vs. time after stimulus onset
corresponding to the 63 stimulation sites are overlayed on a coordinate map of
the PC and the surrounding layers. These data were obtained from the same
cell, in the absence (2a) or presence (2b) of picrotoxin, a GABAA receptor
blocker. Blue and red traces denote PC excitation and inhibition, respectively.
The Role of Cerebellar mGluR1 Receptors in Motor Function
Recovery from Ca2+ inactivation
1 M
2b
A
1s
Vcmd = - 60 mV
2a
Latency to Fall (sec)
B
free cytosolic Ca2+
A
1
Cerebellar ataxia is a disorder characterized by poorly coordinated movements
and impairment of balance and gait. Mutations in the P/Q-type calcium channel cause
ataxia in both humans and mice. These mutations result in decreased calcium
currents in Purkinje cells of ataxic animals. However, the mechanisms by which these
alterations produce ataxia are largely unknown.
Previous work in our lab has shown that P/Q-type calcium channels are required
to sustain the normal intrinsic activity of cerebellar Purkinje cells. For that reason, we
are evaluating the hypothesis that disruptions in Purkinje cells’ normal firing result in
the motor impairment observed in P/Q-type calcium channel ataxic mutants. We
monitored the spontaneous activity of mutant Purkinje cells using extracellular
recording in cerebellar slices (Figure 1). Compared to normal littermates, mutants
showed an increased variation between action potentials.
In ducky mutants, the observed reduction in the density of calcium current could
result in decreased activation of the calcium-activated potassium channels that set the
interspike interval. Therefore, we decided to test whether the activation of them could
recover the regular firing rate in mutant Purkinje cells (Figure 2). In addition, we
perfused chronically the cerebellum of ducky heterozygous and tested the motor
performance using two common paradigms to assess cerebellar-mediated motor
coordination, accelerating rotarod and balance beam (Figure 3).
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Sessions (days)
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A
Figure 2. Activation of calcium-activated potassium
channels increases the regularity of Purkinje cell
intrinsic activity. EBIO is a specific activator of smallconductance calcium-activated potassium channels.
C
B
Figure 3. In vivo perfusion of EBIO into the cerebellum of ducky mice increases motor performance. (a) An osmotic
pump was implanted in the midline cerebellum. (b) motor performance was tested daily on an accelerating rotarod.
After 8 days of training, the pump was implanted. © ducky mice receiving EBIO significantly increased their
performance on the rotarod. The bar shows the time during the pump was perfusing the drug (red circles) or the
vehicle (black circles). After the pump stops perfusion, the performance returned to the vehicle level. (*) p<0.0001
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The irregular firing could contribute to the ataxic phenotype by preventing the normal
processing of information within the cerebellar cortex. Recovering the normal
Purkinje cell activity could be a promising method of recovering the normal
phenotype in P/Q-type ataxic individuals. Activation of calcium-activated potassium
channels may be a potential therapeutic target for P/Q-type related ataxias.
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Figure 1. Purkinje cells of ducky mutants have a very erratic
spontaneous activity. (a) Activity of Purkinje cells of normal
littermates (+/+) and ducky mutants (du/du). Each line represents
an action potential. Red lines indicate an action potential that
was not within 2 standard deviations from the mean control
interspike interval. (b) Average of the coefficient of variation (left)
and predominant firing rate (right).
0
5
Sessions (days)
10