Transcript Khodakhah Lab - Albert Einstein College of Medicine

Khodakhah Lab and the Cerebellum: A Love Story
AECOM, Kennedy 506. Beautiful view of parking lot. http://www.aecom.yu.edu/kamlab
Karina Alviña, Paola Calderón, Johanna Dizon, Sung-Min Park, Esra Tara, Joy Walter, and 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
stimulation
Anatomy of the cerebellum
200 µV
50 ms
Average PC Response vs.
Normalized stimulus intensity
20
- each line
represents an
action potential
- stimulation
occurs at time
point 0
15
10
5
0
-100
300
Maximum firing
rate (spikes/s)
Stimulus intensity (A)
Raster plot of PC responses to
different GC stimulation intensities
200
140
Ouabain 10nM
120
100
80
60
40
20
0
0
50
100
150
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.
100
0
0
5x 10x 15x 20x
Normalized stimulus intensity
0
100
Time (ms)
Kandel, Schwartz and Jessell. Principles of Neural Science.
Examining inhibition’s role in Purkinje cell
firing rate modulation
0.8
100 nA
25 ms
0.6
Figure 2. Dystonic postures after chronic perfusion of 100 µM ouabain into the
cerebellum.
12
0.4
Q (-pC)
Peak EPSC (-nA)
Peak Current and Total Charge vs. Stimulus Intensity
0.2
0.0
0
9
6
3
0
0
Stim int (A)
20
40
60
80
Stimulus intensity (A)
The cerebellum coordinates the body through its principal neuron, the Purkinje cell.
PCs linearly encode the strength of GC synaptic input in their maximum firing rate.
The Purkinje cell integrates over 150,000 cortical and sensory inputs to generate the
signals for coordinated movement. Those signals are in the form of a rate code that
modulates from the intrinsic rate of ~50 Hz to span from ~1 to ~250 Hz. Firing rates under
Mapping granule cell-to-Purkinje cell connectivity
~50 Hz require some form of net inhibition from inhibitory interneurons: basket cells and
stellate cells. However, their strengths relative to each other and the role of a single
We are characterizing the functional connectivity between GCs and PCs, thereby
interneuron in Purkinje cell firing rate modulation remain unclear. We will clarify these
addressing three controversial questions: 1) Does activity in this pathway propagate in a
issues using a spike train stimulus protocol in basket and stellate cells while recording from dispersed or patchy manner? 2) Do the ascending axon and parallel fiber regions of the
a connected Purkinje cell.
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
Question: Can a single interneuron sustain a GABA conductance to shunt a Purkinje electrical activity in response to stimulation in multiple underlying GC patches is
cell’s intrinsic inward currents?
monitored via extracellular recording.
A
B
1
2a
Mechanisms underlying cerebellar ataxia
20 40 60 80
2b
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).
A
start of 100 Hz train stimulus
B
200 pA
5 ms
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.
A. Recording configuration: Purkinje cells are voltage-clamped at -60 mV with a high Cl internal solution. Interneurons are
stimulated either extracellularly or in a loose-patch configuration. B. PC IPSCs elicited from an interneuron (basket cell)
stimulated at 100 Hz for 1 second (only first four IPSCs shown). Note that the IPSCs can be evoked before the prior IPSC
fully decays—thereby maintaining a continuous GABAA shunt (baseline shown as dotted line).
Peaks and Valleys of the GABA shunt at different stimulation
frequencies
10 Hz
50 Hz
0.8
67 Hz
0.7
Filled data points represent the peaks of
each IPSC—that is the maximum amount
of GABA-mediated current. Open data
points represent the trough between two
IPSCs—that is the minimum amount of GABAmediated current. Data is an average of 10
trials. Error bars are equal to SEM. Dashed
line at y = 0 indicates baseline.
Stimulation frequencies were at 10, 50, and 67
Hz for 1 second. However data only to 250 ms
is shown.
0.6
IPSC Peak Amplitude (nA)
as from initial baseline
0.5
0.4
0.3
0.2
0.1
0.0
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24
Time (s)
A single basket cell may maintain a continuous GABA shunt over baseline for nearly 250
ms.
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.
Mechanisms underlying episodic neurological disorders
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).
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.
Episodic neurological disorders such as episodic ataxia and paroxysmal dyskinesia are
characterized by transient expression of symptoms superimposed on a normal baseline.
C
The episodic nature of these disorders is due to a transient dysfunction of ion channels.
While the symptoms of this family of disorders are different, they share common triggers
such as psychological stress, caffeine and alcohol.
A
B
We propose to investigate the mechanism by which these different triggers lead to the
expression of identical symptoms. The ataxic mouse tottering inherits a mutation in P/Q type
calcium channels, which results in a reduction in the P/Q channel current density in cerebellar
Purkinje cells and causes an ataxic baseline punctuated by episodes of severe dyskinesia.
We hypothesize that chemical, physical and psychological st
dyskinesia via a common physiological pathway, by altering the activity of Purkinje cells.
To address this question, we use the tottering mouse as a model of episodic neurological
disorders and record single unit Purkinje cell activity in vivo in awake behaving tottering mice
Figure 3. In vivo perfusion of EBIO into the cerebellum of ducky mice increases motor performance. (a) An osmotic
before, during and following an attack triggered by caffeine, ethanol or stress.
pump was implanted in the midline cerebellum. (b) motor performance was tested daily on an accelerating rotarod.
Figure 2. Purkinje cell
activity in awake
behaving mice
Figure 1. In vivo recording of
single unit activity in awake behaving mice
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
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.