Khodakhah Lab and the Cerebellum: A Love StoryAECOM, Kennedy 506. Beautiful view of parking lot.Karina Alviña, Paola Calderón, Johanna Dizon, Kathryn Harper, Caroline Hoang, Joy Walter, Kamran Khodakhah

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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?

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A. Schematic diagram illustrating the whole-cell patch clamp technique. Purkinje cells from cerebellar slices were voltage-clamped 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.

Recovery from Ca2+ inactivation

The rapid recovery from Ca2+ inactivation suggests that InsP3 receptors in intact Purkinje cells are suited for use during fast synaptic signaling.

The granule cell input - Purkinje cell output function

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 thespontaneous 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).

Typical PC response to a single pulse electrical GC layer stimulation

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Raster plot of PC responses to different GC stimulation intensities

Average PC Response vs. Normalized stimulus intensity

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PCs linearly encode the strength of GC synaptic input in their maximum firing rate.

Mapping granule cell-to-Purkinje cell connectivity

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.

Mechanisms underlying cerebellar ataxia

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).

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 small-conductance calcium-activated potassium channels.

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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

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.

Anatomy of the Cerebellum

IntroductionThe 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.

Kandel, Schwartz and Jessell. Principles of Neural Science.

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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.

Figure 2. Dystonic postures after chronic perfusion of 100 µM ouabain into the cerebellum.

Dystonia is a neurological disorder characterized by an excessive co-contraction 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.

Cerebellar dysfunction induces Dystonia

The Role of Cerebellar mGluR1 Receptors in Motor Function

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.

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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.

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.

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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.