Spontaneous activity (SA) in the developing brain is responsible for neural network formation processes such as proliferation, migration, axon extension and pathfinding, and synapse formation and maintenance. In embryonic mouse hindbrain, SA role in forming networks tends to be the most critical at specific developmental stage E11.5, when the activity is most robust and synchronous. The SA is thought to initiate in a group of serotonergic raphe nuclei located in the midline former rhombomere 2 (r2) region of the hindbrain. Voltage-dependent calcium waves propagate rostrocaudally in cells along the midline and laterally into the surrounding tissue (Hunt et al. 2005). Over time, SA decreases in frequency, later disappearing by stage E15. This project aims to investigate the mechanisms for this disappearance. Our experiments are done using electrophysiology and calcium imaging. Electrophysiology has shown that SA disappearance correlates in time with an increase in conductance as well as a hyperpolarization of the membrane potential. Calcium imaging has shown that application of BaCl2, a potassium channel blocker, can induce activity at stage E15. Activity at this stage can also be induced by raising extracellular potassium concentration, which effectively depolarizes membrane potential. This evidence suggests the up regulation of potassium channels in the mechanism of SA disappearance.

The inferior olive is heavily involved in implicit timing, the unconscious encoding of timing of sensory or motor events. Performance of these tasks relies on synchronous output from ensembles of neurons in the inferior olive, which are electrotonically coupled to one another via gap junctions. Previous attempts to understand coupling properties in the inferior olive have relied on technically challenging dual whole-cell recordings. Here, I demonstrate an optogenetic approach which provides rapid evaluation of electrotonic coupling in the inferior olive using femtosecond pulsed infrared stimulation of olivary cells expressing the light-activated cation channel channelrhodopsin-2 (ChR2 H134R). Targeted expression of ChR2 in olivary neurons was achieved through the Ai32 mouse line engineered at the Allen Institute for Brain Science, which were crossed with mice expressing Cre-recombinase targeted to the pancreatic transcription factor 1a (Ptf1a) locus. Parasagittal brainstem slices were prepared from young (P20-P40) Ptf1a-Ai32 positive mice. Electrodes were guided under IR-DIC for whole-cell recording of a single olivary neuron. Neighboring cells were stimulated using 920 nm light (<1 mW/µm² average power), in which a single infrared beam was split into 64 beamlets and distributed evenly over the neighboring cell's cross sectional area (100-400 µm²). Coupling properties determined by optogenetic stimulation of neighboring cells replicated previous results produced by dual whole-cell recordings (CC=1-10%). Furthermore, optogenetic evaluation of coupling following pharmacological manipulation of olivary neurons were consistent with previous findings. This optogenetic approach will provide a rapid and efficient method for future exploration of coupling properties in the inferior olive, which may accelerate the study of their involvement in pathologies such alcoholism, autism and sudden infant death syndrome.

Spinocerebellar ataxia type 7 (SCA7) belongs to a family of neurodegenerative diseases characterized by degeneration of Purkinje cell neurons (PC) within the cerebellum, which leads to motor dysfunction. The disease results from a CAG tract expansion mutation of the ataxin-7 gene. This type of expansion is common within the SCA family along with other neurological disorders like Huntington’s disease. Recent research has provided evidence for a non-cell autonomous degeneration of PCs, which means that other cell types cause the PCs to degenerate. One population of cells that may be involved is the inferior olive (IO). The IO, which is known to degenerate in humans, is made up of neurons and is located within the brainstem. The axons of the IO, known as climbing fibers, extend to the cerebellum and synapse with PCs. These climbing fibers transport neurotrophic factors to the PCs. We hypothesize that loss of neurotrophic factors due to the degeneration of climbing fibers may contribute to the disease state of SCA7.
To support this belief, we seek to characterize the climbing fibers by immunohistochemistry. I have stained cerebellar sections of non-transgenic and SCA7 positive mice for VGLUT2, a marker of climbing fiber terminals. In order to understand whether a change in climbing fibers occurs before or after presentation of the disease I have also stained sections from multiple time points. To analyze the differences between the groups I have focused on measuring the amount of VGLUT2 positive puncta in regions proximal and distal to the Purkinje cell soma. Preliminary results indicate that a change in distribution of puncta occurs in mice in the late stages of disease.
 

Parkinson’s disease is a motor disorder characterized by tremors, rigidity, and inability to initiate movement. One therapy for non-drug responsive cases is high amplitude, high frequency stimulation to the basal ganglia’s subthalamic nucleus (STN), via surgically implanted electrodes. While the mechanisms of how this therapy restores proper motor function remains under debate, one hypothesis is that the currents disrupt rhythmic, synchronous firing patterns present in basal ganglia structures of people with Parkinson’s disease. Our goal is to explore the effects of low amplitude, low frequency stimulation to the basal ganglia nucleus globus pallidus externus (Gpe), specifically whether this type of stimulation can disrupt pathological firing synchrony in GPe and STN. We use a conductance-based model of individual neurons in multicellular networks with varying patterns of short, depolarizing currents to mimic deep brain stimulation. Through this work we hope to discover novel patterns of deep-brain stimulation with the potential to eliminate pathological firing patterns of cells in the basal ganglia.

Alzheimer's disease (AD) is the leading cause of dementia, and its precise cause is still unknown. Mutations in the two Presenilin (PS) genes, Presenilin 1 and Presenilin 2 (PS2), cause AD. Understanding how AD mutations impact PS function may help us understand AD pathogenesis. One function of PS involves calcium (Ca2+) transfer between the endoplasmic reticulum (ER) and mitochondria. Presenilin 2 (PS2) and mutant PS2 (mtPS2) in particular have been shown to increase this transfer, possibly causing mitochondrial Ca2+ overload. This could lead to metabolic dysfunction, which has been suggested as a key pathogenic mechanism in AD. Establishing a link between PS mutations and mitochondrial dysfunction would support this hypothesis. One symptom of mitochondrial dysfunction is changes in mitochondrial fragmentation and localization. "Fragmented," rather than interconnected, "branchlike" mitochondrial networks are associated with increased ER-mitochondria Ca2+-transfer and apoptosis. Furthermore, while healthy mitochondria move towards energy-demanding neuronal processes, mitochondria with reduced ATP production stay near the nucleus. To determine the effects of PS2 and mtPS2 on mitochondrial fragmentation and localization, wild-type (wt) and mtPS2 (N141I) transgenic mouse cortical neurons were infected with a lentiviral vector expressing mitochondrial targeted red fluorescent protein (DsRed). Fixed cultures were imaged using confocal microscopy and analyzed using Huygens software. Currently, we have plated, infected, and analyzed sample preparations for mitochondrial length, sphericity, and other characteristics associated with fragmentation, as well as mitochondrial volume as a function of distance from the soma. Using this paradigm, we will also challenge cultured wt and mtPS2 neurons with camptothecin and staurosporine, inducers of mitochondrial fission and fusion, respectively. If mtPS2 induces mitochondrial dysfunction, mtPS2-expressing neurons should exhibit more mitochondrial fragmentation and mitochondria localized nearer to the soma. This would provide evidence for mitochondrial dysfunction in AD, supporting further research into mitochondrial changes in AD and a better understanding of AD’s etiology.

The effects of amphetamine on learning and memory are important areas of research due to the widespread use of amphetamine among people, ranging from prescription to recreational. It has been shown in animals that repeated treatment of amphetamine will result in sensitization to the drug. Sensitization is a process through which treated subjects develop an elevated response to a drug. Long term potentiation (LTP) is a physical change strengthening the connection between synapses that is thought to underlie learning and memory. Enhancement of LTP in the striatum has been shown to occur as a result of amphetamine use. The striatum is a brain region important in mediating a large number of behaviors including motor control and learning. For this reason it is possible that sensitization to amphetamine may affect learning in mice. To investigate this possibility, wild-type mice sensitized to amphetamine were tested in behavioral paradigms designed to measure learning ability. It was found that they do not show enhanced performance in motor learning, appetitive learning, or spatial learning, but they may show an improvement over controls when they are required to alter their strategy to escape from a water-maze. In order to explore the neurological pathway through which sensitization to amphetamine may affect learning, we also tested knockout mice that lack N-methyl-D–aspartate-type glutamate receptors (NMDARs) in medium spiny neurons, the primary neuron type in the striatum. In many brain regions, NMDARs are important for various forms of learning and memory, and it has been shown that these knockout mice display impaired motor and appetitive learning and yet are still able to sensitize to amphetamine. Using the same learning paradigms as with our wild-type mice, we found that knockout mice did not show any enhancement in motor or appetitive learning as a result of amphetamine sensitization.