Little is known about the physiological principles that govern large-scale neuronal interactions in the mammalian brain. Here, we describe an electrophysiological paradigm capable of simultaneously recording the extracellular activity of large populations of single neurons, distributed across multiple cortical and subcortical structures in behaving and anesthetized animals. Up to 100 neurons were simultaneously recorded after 48 microwires were implanted in the brain stem, thalamus, and somatosensory cortex of rats. Overall, 86% of the implanted microwires yielded single neurons, and an average of 2.3 neurons were discriminated per microwire. Our population recordings remained stable for weeks, demonstrating that this method can be employed to investigate the dynamic and distributed neuronal ensemble interactions that underlie processes such as sensory perception, motor control, and sensorimotor learning in freely behaving animals.
Key Words: Cav1.2 channels Ⅲ PKC Ⅲ calmodulin Ⅲ arrhythmias V oltage-gated L-Type (Cav1.2) Ca 2ϩ channels are expressed in the surface membrane of neurons and muscle cells, where they regulate multiple processes including excitability, contraction, gene expression, and memory storage. [1][2][3][4] Recent studies have revealed an unexpected feature of Cav1.2 channels: their activity (ie, open probability, P o ) varies within the cell membrane. [5][6][7] Low activity Cav1.2 channels open randomly and infrequently at rest, producing brief elevations in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) called "Ca 2ϩ sparklets." 8 In contrast, small clusters of Cav1.2 channels can function in a high open probability mode that generates localized zones of relatively high Ca 2ϩ influx ("persistent Ca 2ϩ sparklets"). Although targeting of protein kinase (PK)C␣ to the surface membrane by the kinase anchoring protein AKAP150 (the rodent ortholog of human AKAP79) increases the probability of persistent Cav1.2 channel activity, 9 the mechanisms by which small clusters of these channels open concertedly are unknown. Here, we used fluorescence resonance energy transfer (FRET) approaches in combination with optical and electrophysiological recordings of Ca 2ϩ influx via Cav1.2 channels to investigate this important issue. Our data indicate that activators of PKC␣, calmodulin (CaM) antagonists, or a specific Cav1.2 channel mutation that causes arrhythmias and autism in humans with the Timothy syndrome (TS) move the ubiquitous Ca 2ϩ -binding protein CaM from the IQ domain in the C-terminal tail of the channel. This induces functional interactions between nearby Cav1.2 channel via their C termini that could lead to coupled gating of these channels. MethodsAn expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Isolation of Arterial and Adult and Neonatal Cardiac MyocytesMice and rats were euthanized as approved by the University of Washington Institutional Animal Care and Use Committee. Adult arterial myocytes and neonatal cardiac myocytes were prepared as described previously. 5,10 Cav1.2 and Calmodulin Constructs and Inducible PKC␣ Translocation System and Their Expression in tsA-201 CellsWe transfected tsA-201 cells using JetPEI (Polyplus Transfection) with plasmids encoding calcium channel pore-forming and accessory subunits as well as CaM. We generated the rabbit homolog of the human TS Cav1.2 (G436R, rabbit; G406R human Coupled Markov Chain ModelWe implemented a coupled Markov chain model in Matlab to determine the coupling coefficient () among Cav1.2 channels underlying membrane currents and Ca 2ϩ sparklet sites. The model was originally developed by Chung and Kennedy 14 to analyze individual records of partially coupled GABA-activated chloride channels. Electrophysiology, Confocal, and Total Internal Reflection Fluorescence MicroscopyVoltage-clamp experiments were performed using standard patchclamp techniques. Total internal reflection fluorescence (TIRF) images were...
The mammalian olfactory system is able to discriminate among tens of thousands of odorant molecules. In mice, each odorant is sensed by a small subset of the approximately 1,000 odorant receptor (OR) types, with one OR gene expressed by each olfactory sensory neuron (OSN). However, the sum of the large repertoire of OR/OSN types and difficulties with heterologous expression have made it almost impossible to analyze odorant responsiveness across all OR/OSN types. We have developed a microfluidic approach that allowed us to screen over 20,000 single cells at once in microwells. By using calcium imaging, we were able to detect and analyze odorant responses of about 2,900 OSNs simultaneously. Importantly, this technique allows for both the detection of rare responding OSNs as well as the identification of OSN populations broadly responsive to odorants of unrelated structures. This technique is generally applicable for screening large numbers of single cells and should help to characterize rare cell behaviors in fields such as toxicology, pharmacology, and cancer research.
Gain adaptation of eye and head movement components of simian gaze shifts. J. Neurophysiol. 78: 2817-2821, 1997. To investigate the site of gaze adaptation in primates, we reduced the gain of large head-restrained gaze shifts made to 50 degrees target steps by jumping the target 40% backwards during a targeting saccade and then tested gain transfer to the eye- and head-movement components of head-unrestrained gaze shifts. After several hundred backstep trials, saccadic gain decreased by at least 10% in 8 of 13 experiments, which were then selected for further study. The minimum saccadic gain decrease in these eight experiments was 12.8% (mean = 18.4%). Head-unrestrained gaze shifts to ordinary 50 degrees target steps experienced a gain reduction of at least 9.3% (mean = 14.9%), a mean gain transfer of 81%. Both the eye and head components of the gaze shift also decreased. However, average head movement gain decreased much more (22.1%) than eye movement gain (9.2%). Also, peak head velocity generally decreased significantly (20%), but peak eye velocity either increased or remained constant (average increase of 5.6%). However, the adapted peak eye and head velocities were appropriate for the adapted, smaller gaze amplitudes. Similar dissociations in eye and head metrics occurred when head-unrestrained gaze shifts were adapted directly (n = 2). These results indicated that head-restrained saccadic gain adaptation did not produce adaptation of eye movement alone. Nor did it produce a proportional gain change in both eye and head movement. Rather, normal eye and head amplitude and velocity relations for a given gaze amplitude were preserved. Such a result could be explained most easily if head-restrained adaptation were realized before the eye and head commands had been individualized. Therefore, gaze adaptation is most likely to occur upstream of the creation of separate eye and head movement commands.
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