Zayd M. Khaliq - US grants

DESCRIPTION (provided by applicant): As the sole output from the cerebellar cortex, Purkinje cells are responsible for processing information from diverse brain regions and delivering this information to the cells of the cerebellar nucleus. Although signal processing is known to occur in the dendrites and the soma, little is known about the processing that may also occur in the axons of Purkinje cells. Specifically, we are interested in the pattern of axonal action potentials that result from the massive depolarization observed in Purkinje cell somata and dendrites during stimulation of climbing fibers (i.e. complex spike). Moreover, high-frequency firing and extensive branching may reduce the reliability of transmission in axons. Using electrophysiological and imaging techniques, we propose to determine the transfer function of Purkinje cell axons in periods of high-frequency and burst firing. We also propose to test the reliability of action potential propagation throughout the highly ramifying collateral axons and terminal projections to the deep cerebellar nucleus.

Dopamine neurons are key players in the reward and motor systems. They fire tonically at low rates of 2-5 Hz and fire bursts of action potentials at 15-20 Hz during reward-relevant behaviors. Bursting is not an intrinsic property of dopamine neurons but depends critically on synaptic inputs. How excitatory and modulatory inputs facilitate burst firing in dopamine neurons is unknown. We are testing the hypothesis that burst firing depends on both fast synaptic transmission and slow, metabotropic glutamatergic transmission coming from the subthalamic nucleus as well cholinergic inputs from the brainstem pedunculopontine nucleus. Because the lab is newly established, we have only started to make progress on the proposed aims. We have made good progress in setting up and staffing the laboratory. There are currently four (4) members of the lab including the Principal Investigator (one postdoctoral fellow, a graduate fellow, and a Post-Baccalaureate IRTA). We have constructed three patch-clamp setups which we use daily for brain slice recordings. We also have a separate setup dedicated to two-photon laser scanning microscopy (2PLSM) which we use for simultaneous calcium-imaging of the dendrites and patch-clamp recordings from dopamine neurons. Our initial studies test the contribution of excitatory input from the subthalamic nucleus (STN) to synaptically evoked high-frequency firing in substantia nigra pars compacta (SNc) dopamine neurons. To start, we are examining short-term synaptic plasticity of the STN to SNc synapse. STN neurons fire repetitively at high tonic rates (20-30 Hz) but fire bursts of spikes up to 200 Hz for short periods of time (100-200 ms). We discovered that there is little short-term plasticity across a broad range of frequencies (2-100 Hz), with synapses depressing only slightly (max 35%) for short bursts of 5 stimuli. We have also shown that slightly more depression (about 40% on average) occurs during long trains of 60 stimuli at 20 Hz. Despite this initial depression, however, synaptic release is reasonably maintained at tonic rates, suggesting that tonic, sustained input from STN neurons can possibly have a significant impact on the excitability of SNc dopamine neurons. The postbacc fellow involved with this project presented a poster of this material at the Summer Intern Poster Day 2012 in Natcher Auditorium. The brainstem pendunculopontine nucleus (PPN) provides the sole source of cholinergic input to SNc and activation this nucleus stimulates burst firing in dopamine neurons. Likewise, lesioning the PPN limits bursting in dopamine neurons suggesting that cholinergic input is necessary for burst firing in SNc dopamine neurons. Therefore, it is possible that cholinergic input enhances the impact of excitatory input from other sources such as the STN, for example, through depolarization and relief of Mg2+ block of NMDA receptors. To test this hypothesis, we have obtained transgenic mice that express the light-activated molecule, channelrhodopsin, only in cholinergic neurons (ChAT-ChR2). We are current breeding the mice and these studies are just starting to get underway. Dopamine neurons are capable of releasing dopamine not only from terminals but also from dendrites into the neighboring substantia nigra pars reticulata (SNr), the main output nucleus of the basal ganglia. As such, dendrites of dopamine neurons are positioned to have a major impact on basal ganglia function. Somatodendritic release of dopamine occurs in a calcium-dependent manner and calcium influx is likely determined by the interaction of synaptic inputs and dendritic excitability. We are beginning to examine calcium signals in the dendrites dopamine neurons using two-photon imaging techniques. So far, we have probed calcium signals in proximal to distal dendrites during spontaneous action potential firing, which we record simultaneously from whole-cell somatic recordings. In our limited number of preliminary experiments, we have consistently found measurable calcium oscillations linked to somatic firing throughout the proximal as well distal dendrites at distances up to 500 um away from the soma. This suggests that there is reliable back-propagation of action potential even throughout the distal dendrites, thus maximizing the influence of somatodendritically released dopamine in the SNr. Since high-frequency burst firing in dopamine neurons is thought to enhance dopamine released from terminals, we have also begun to examine the activity-dependence of calcium signals in the dendrites of dopamine neurons during high-frequency firing.