Revolutionary Insights into Neural Networks and Motor Control from Jaeger-Dieter Lab Research!

The Jaeger-Dieter Lab combines neurophysiology and computer simulations to investigate the function of neural networks within the basal ganglia and cerebellum, as well as their projections to the cortex via the motor thalamus. The ultimate goal is to understand and replicate the computational algorithms employed by these brain structures. The lab uses behaving mice to record and manipulate brain activity during motor tasks using optogenetics, shedding light on how inhibitory and excitatory signals are integrated and how dynamic brain networks are flexibly engaged during task performance. These insights are applied to the study of dynamic dysfunction of brain circuits in Parkinson's disease using Parkinsonian mouse models.

Investigating Cerebellar Pathways with Optogenetics

The cerebellum, traditionally viewed as processing GABAergic input from Purkinje cells (PCs) in the cerebellar cortex, also receives substantial glutamatergic inputs from mossy fiber and inferior olive (IO) axon collaterals. While the role of mossy fiber input has been integrated into functional models, the function of climbing fiber collaterals remains largely unknown.

To address this gap, the lab uses optogenetic techniques to selectively stimulate climbing fiber collateral input to cerebellar nuclei (CN) neurons. This approach allows for characterization of this cerebellar pathway in vitro and in vivo. Channelrhodopsin-2 (ChR2) expression in the IO is achieved using the Ptf1a gene as a Cre driver, crossing Ptf1a-Cre mice with a Rosa Ai27 ChR2-tdTomato reporter line. This method ensures ChR2 expression is restricted to the IO and cerebellar PCs.

The lab studies the effects of olivary input to CN neurons in vivo by direct optical stimulation of the IO in anesthetized mice. Electrophysiological recordings are then performed to measure the neural responses.

Methods

Animals: Two to six-month old male transgenic mice are anesthetized with isoflurane and injected with urethane.
Surgery: The skin over the cerebellum and brainstem is incised, and muscles are separated to expose the foramen magnum. The obex is used as a reference point.
Light Stimulation: A blue laser (473 nm) delivers single light pulses of 10 or 50 ms duration to the IO (0.5 mm lateral from the midline and 2.5 mm in depth).
Electrophysiology: Glass pipettes are used to record single unit and local field potential (LFP) responses in the cerebellar cortex and CN. A blue dye is injected at the recording site for later identification.
Brain Slice Preparation: Animals are perfused with a sodium-replaced ice-cold solution, and the cerebellum is removed and sectioned. Slices are superfused with artificial cerebrospinal fluid (ACSF) during recording.
Patch-Clamp Recording: Whole-cell patch-clamp recordings are performed on CN neurons using borosilicate glass electrodes. Light stimulation is achieved with full-field flashes from a mercury bulb using a FITC filter.
Histology: Brains are sectioned, and ChR2 expression is verified using confocal microscopy to identify tdTomato-labeled neuronal structures.
Data Analysis: Spike detection is performed using a threshold based on the standard deviation of the recording traces. Spike rates are calculated by convolving spikes with a Gaussian kernel. Responses to optogenetic stimuli are determined by comparing spike rates before and after stimulation.

Selective Optogenetic Stimulation and Expression Verification

The study carefully verified the selective expression of ChR2 in the IO and the absence of expression in mossy fibers, which would confound the results. Confocal microscopy confirmed that tdTomato fluorescence was localized to the IO, with fibers forming bundles between olivary neuron cell bodies. Brighter staining was observed in putative local collateral terminals, suggesting enrichment of ChR2 in these areas. In the cerebellum, strong expression was found near Purkinje cell somas, indicative of climbing fibers synapsing onto proximal dendrites.

In Vivo Findings: Local Field Potential Responses

Local field potential (LFP) recordings in vivo revealed that optical stimulation of the IO evoked responses that varied with depth. The amplitude of the LFP responses increased with depth, reaching a maximum around 1500 μm, and then decreased at the depth of the CN. The LFP responses typically contained multiple peaks. The depth of the CN was confirmed to be between 2000 and 2500 μm through blue dye injections at the recording site. The peaks of LFP responses in the CN occurred 20-30 ms after stimulation. LFP responses recorded in the cerebellar cortex contained earlier peaks, suggesting population climbing fiber responses to the optogenetic stimulation. The later event observed in the CN region may represent delayed Purkinje cell input.

In Vivo Findings: Single-Unit Responses

Single-unit recordings from 29 CN neurons revealed baseline firing rates ranging from 4 to 64 Hz. Only one neuron showed a statistically significant increase in spike rate after olivary stimulation, consisting of a single added spike. Nearly half of the recorded neurons showed no response to stimulation. The remaining neurons showed a decrease in spike rate of variable strength and duration.

Voltage-Sensitive Dye Imaging: Expanding Research Capabilities

The lab requests funding to establish a voltage-sensitive dye (VSD) imaging setup to enhance research capabilities. This setup will enable the study of cortical activity modulation resulting from basal ganglia and cerebellar feedback loops. VSD imaging of mouse cortical activity in vivo will be crucial for determining the temporal and spatial effects of basal ganglia and cerebellar activation on cortical activity.

This technology will also benefit other research areas, such as the study of cortical activity patterns following social ultrasound signaling in mice. VSD imaging would allow for a better understanding of spatial and temporal signal flow beyond primary auditory cortex.

The equipment will also be used to study invertebrate pattern generation circuits, such as the leech heartbeat circuit and crab/lobster stomatogastric ganglion. These ganglia consist of a spatial network with complex activity patterns that can be surveyed by voltage-sensitive dye imaging techniques.

Equipment and Resources

The setup will include a dual CMOS camera system mounted on an in vivo imaging setup and an Olympus BX50 microscope. Both setups will share manipulators and amplifiers for simultaneous electrophysiological recordings. The Department of Biology will contribute a substantial amount of basic equipment, including an Olympus BX50 microscope, vibration isolation table, and recording amplifiers. A dedicated room will also be provided. The core item requested is the dual camera CMOS system, along with the necessary optical equipment, software, and auxiliary hardware.

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