Identified Cellular Correlates of Neocortical Ripple and High-Gamma Oscillations during Spindles of Natural Sleep.

Ultra-high-frequency network events in the hippocampus are instrumental in a dialogue with the neocortex during memory formation, but the existence of transient ∼200 Hz network events in the neocortex is not clear. Our recordings from neocortical layer II/III of freely behaving rats revealed field potential events at ripple and high-gamma frequencies repeatedly occurring at troughs of spindle oscillations during sleep. Juxtacellular recordings identified subpopulations of fast-spiking, parvalbumin-containing basket cells with epochs of firing at ripple (∼200 Hz) and high-gamma (∼120 Hz) frequencies detected during spindles and centered with millisecond precision at the trough of spindle waves in phase with field potential events but phase shifted relative to pyramidal cell firing. The results suggest that basket cell subpopulations are involved in spindle-nested, high-frequency network events that hypothetically provide repeatedly occurring neocortical temporal reference states potentially involved in mnemonic processes.

Human neuronal changes in brain edema and increased intracranial pressure.

Functional and molecular changes associated with pathophysiological conditions are relatively easily detected based on tissue samples collected from patients. Population specific cellular responses to disease might remain undiscovered in samples taken from organs formed by a multitude of cell types. This is particularly apparent in the human cerebral cortex composed of a yet undefined number of neuron types with a potentially different involvement in disease processes. We combined cellular electrophysiology, anatomy and single cell digital PCR in human neurons identified in situ for the first time to assess mRNA expression and corresponding functional changes in response to edema and increased intracranial pressure. In single pyramidal cells, mRNA copy numbers of AQP1, AQP3, HMOX1, KCNN4, SCN3B and SOD2 increased, while CACNA1B, CRH decreased in edema. In addition, single pyramidal cells increased the copy number of AQP1, HTR5A and KCNS1 mRNAs in response to increased intracranial pressure. In contrast to pyramidal cells, AQP1, HMOX1and KCNN4 remained unchanged in single cell digital PCR performed on fast spiking cells in edema. Corroborating single cell digital PCR results, pharmacological and immunohistochemical results also suggested the presence of KCNN4 encoding the α-subunit of KCa3.1 channels in edema on pyramidal cells, but not on interneurons. We measured the frequency of spontaneous EPSPs on pyramidal cells in both pathophysiological conditions and on fast spiking interneurons in edema and found a significant decrease in each case, which was accompanied by an increase in input resistances on both cell types and by a drop in dendritic spine density on pyramidal cells consistent with a loss of excitatory synapses. Our results identify anatomical and/or physiological changes in human pyramidal and fast spiking cells in edema and increased intracranial pressure revealing cell type specific quantitative changes in gene expression. Some of the edema/increased intracranial pressure modulated and single human pyramidal cell verified gene products identified here might be considered as novel pharmacological targets in cell type specific neuroprotection.

Digital PCR to determine the number of transcripts from single neurons after patch-clamp recording.

Whole-cell patch-clamp recording enables detection of electrophysiological signals from single neurons as well as harvesting of perisomatic RNA through the patch pipette for subsequent gene expression analysis. Amplification and profiling of RNA with traditional quantitative real-time PCR (qRT-PCR) do not provide exact quantitation due to experimental variation caused by the limited amount of nucleic acid in a single cell. Here we describe a protocol for quantifying mRNA or miRNA expression in individual neurons after patch-clamp recording using high-density nanocapillary digital PCR (dPCR). Expression of a known cell-type dependent marker gene (gabrd), as well as oxidative-stress related induction of hspb1 and hmox1 expression, was quantified in individual neurogliaform and pyramidal cells, respectively. The miRNA mir-132, which plays a role in neurodevelopment, was found to be equally expressed in three different types of neurons. The accuracy and sensitivity of this method were further validated using synthetic spike-in templates and by detecting genes with very low levels of expression.