Working memory represents the ability of the brain to hold externally or internally driven information for relatively short periods of time. Persistent neuronal activity is the elementary process underlying working memory but its cellular basis remains unknown. The most widely accepted hypothesis is that persistent activity is based on synaptic reverberations in recurrent circuits. The entorhinal cortex in the parahippocampal region is crucially involved in the acquisition, consolidation and retrieval of long-term memory traces for which working memory operations are essential. Here we show that individual neurons from layer V of the entorhinal cortex-which link the hippocampus to extensive cortical regions-respond to consecutive stimuli with graded changes in firing frequency that remain stable after each stimulus presentation. In addition, the sustained levels of firing frequency can be either increased or decreased in an input-specific manner. This firing behaviour displays robustness to distractors; it is linked to cholinergic muscarinic receptor activation, and relies on activity-dependent changes of a Ca2+-sensitive cationic current. Such an intrinsic neuronal ability to generate graded persistent activity constitutes an elementary mechanism for working memory.

SummaryIntracellular recording shows how differences in single cell subthreshold oscillation frequency could directly underlie the differences in spacing of grid cell firing locations shown previously in awake, behaving animals.Grid cells in layer II of entorhinal cortex fire to spatial locations in a repeating hexagonal grid with smaller spacing between grid fields for neurons in more dorsal anatomical locations. Data from in vitro whole-cell patch recordings show a corresponding difference in frequency of subthreshold membrane potential oscillations in entorhinal neurons at different positions along the dorsal to ventral axis, supporting a model of physiological mechanism for grid cell responses.The entorhinal cortex plays an important role in encoding of spatial information (1-3) and episodic memory (4). Many layer II neurons of rat entorhinal cortex are "grid cells," firing when the rat is in an array of spatial locations forming a hexagonal grid within the environment (5-7). The spacing of firing fields in the grid varies with anatomical position of the cell along the dorsal to ventral axis of entorhinal cortex, as measured by distance from the postrhinal border (5). Neurons closer to the dorsal border of entorhinal cortex have shorter distances between firing fields. Computational models explicitly predict that differences in grid field spacing should correspond to differences in intrinsic frequencies of neurons along the dorsal to ventral axis (3,8). This could provide systematic variation in the gain of a movement-speed signal for path integration (2,3,9).Subthreshold membrane potential oscillations in entorhinal cortical stellate cells (10) arise from a single-cell mechanism involving voltage-sensitive currents (11-13) and could contribute to network dynamics (14). We recorded subthreshold oscillations from 57 stellate cells in layer II of medial entorhinal cortex (Fig. S1) in slices from different anatomical positions along the dorsal to ventral axis, using whole-cell patch clamp techniques (15). The position of individual horizontal slices was measured relative to the dorsal surface of the brain (Fig. 1A).Stellate cells in dorsal entorhinal cortex show higher temporal frequencies of subthreshold membrane potential oscillations compared to lower frequencies in cells from more ventral entorhinal slices (Fig. 1B). Dorsal cells (n = 30) are defined as cells recorded in slices taken between 3.8 mm (the border with postrhinal cortex (16)) and 4.9 mm from the dorsal surface of the brain. Ventral cells (n = 27) are defined as cells recorded in slices between 4.9 and 7.1 mm from the dorsal surface. Fig. 1B shows the group means of the frequency of subthreshold oscillations recorded from these populations. Because frequency of subthreshold oscillations can depend upon the mean membrane potential voltage, we performed this analysis separately for data gathered at different approximate holding membrane potentials of −50 mV and −45 mV. The mean frequency in dorsal cells was significantly higher than the mean frequ...

Various subsets of brain neurons express a hyperpolarization-activated inward current ( I h) that has been shown to be instrumental in pacing oscillatory activity at both a single-cell and a network level. A characteristic feature of the stellate cells (SCs) of entorhinal cortex (EC) layer II, those neurons giving rise to the main component of the perforant path input to the hippocampal formation, is their ability to generate persistent, Na+-dependent rhythmic subthreshold membrane potential oscillations, which are thought to be instrumental in implementing theta rhythmicity in the entorhinal-hippocampal network. The SCs also display a robust time-dependent inward rectification in the hyperpolarizing direction that may contribute to the generation of these oscillations. We performed whole cell recordings of SCs in in vitro slices to investigate the specific biophysical and pharmacological properties of the current underlying this inward rectification and to clarify its potential role in the genesis of the subthreshold oscillations. In voltage-clamp conditions, hyperpolarizing voltage steps evoked a slow, noninactivating inward current, which also deactivated slowly on depolarization. This current was identified as I h because it was resistant to extracellular Ba2+, sensitive to Cs+, completely and selectively abolished by ZD7288, and carried by both Na+and K+ ions. I h in the SCs had an activation threshold and reversal potential at approximately −45 and −20 mV, respectively. Its half-activation voltage was −77 mV. Importantly, bath perfusion with ZD7288, but not Ba2+, gradually and completely abolished the subthreshold oscillations, thus directly implicating I h in their generation. Using experimentally derived biophysical parameters for I h and the low-threshold persistent Na+ current ( I NaP) present in the SCs, a simplified model of these neurons was constructed and their subthreshold electroresponsiveness simulated. This indicated that the interplay between I NaP and I h can sustain persistent subthreshold oscillations in SCs. I NaP and I h operate in a “push-pull” fashion where the delay in the activation/deactivation of I h gives rise to the oscillatory process.

SummarySynapses are found in vast numbers in the brain and contain complex proteomes. We developed genetic labeling and imaging methods to examine synaptic proteins in individual excitatory synapses across all regions of the mouse brain. Synapse catalogs were generated from the molecular and morphological features of a billion synapses. Each synapse subtype showed a unique anatomical distribution, and each brain region showed a distinct signature of synapse subtypes. Whole-brain synaptome cartography revealed spatial architecture from dendritic to global systems levels and previously unknown anatomical features. Synaptome mapping of circuits showed correspondence between synapse diversity and structural and functional connectomes. Behaviorally relevant patterns of neuronal activity trigger spatiotemporal postsynaptic responses sensitive to the structure of synaptome maps. Areas controlling higher cognitive function contain the greatest synapse diversity, and mutations causing cognitive disorders reorganized synaptome maps. Synaptome technology and resources have wide-ranging application in studies of the normal and diseased brain.

Working memory is an emergent property of neuronal networks, but its cellular basis remains elusive. Recent data show that principal neurons of the entorhinal cortex display persistent firing at graded firing rates that can be shifted up or down in response to brief excitatory or inhibitory stimuli. Here, we present a model of a potential mechanism for graded firing. Our multicompartmental model provides stable plateau firing generated by a nonspecific calcium-sensitive cationic (CAN) current. Sustained firing is insensitive to small variations in Ca2+ concentration in a neutral zone. However, both high and low Ca2+ levels alter firing rates. Specifically, increases in persistent firing rate are triggered only during high levels of calcium, while decreases in rate occur in the presence of low levels of calcium. The model is consistent with detailed experimental observations and provides a mechanism for maintenance of memory-related activity in individual neurons.

A multicenter study was set up to elucidate the environmental and medical risk factors contributing to age-related hearing impairment (ARHI). Nine subsamples, collected by nine audiological centers across Europe, added up to a total of 4,083 subjects between 53 and 67 years. Audiometric data (pure-tone average [PTA]) were collected and the participants filled out a questionnaire on environmental risk factors and medical history. People with a history of disease that could affect hearing were excluded. PTAs were adjusted for age and sex and tested for association with exposure to risk factors. Noise exposure was associated with a significant loss of hearing at high sound frequencies (>1 kHz). Smoking significantly increased high-frequency hearing loss, and the effect was dose-dependent. The effect of smoking remained significant when accounting for cardiovascular disease events. Taller people had better hearing on average with a more pronounced effect at low sound frequencies (<2 kHz). A high body mass index (BMI) correlated with hearing loss across the frequency range tested. Moderate alcohol consumption was inversely correlated with hearing loss. Significant associations were found in the high as well as in the low frequencies. The results suggest that a healthy lifestyle can protect against age-related hearing impairment.Electronic supplementary materialThe online version of this article (doi: 10.1007/s10162-008-0123-1) contains supplementary material, which is available to authorized users.

BackgroundCell viability and motility comprise ubiquitous mechanisms involved in a variety of (patho)biological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions.Methodology/Principal FindingsDynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB) assay in a series of four cell concentrations, yielding fair to good correlations (Spearman's Rho 0.688 to 0.964). Cytotoxic action by paclitaxel (0–100 nM) correlated well with SRB (Rho>0.95) with similar IC50 values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90) and optical density (OD) measurement of extracted dye (Rho>0.95). Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95). Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method.Conclusions/SignificanceThe xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on different platforms applying only adapted matrix surface densities. The increased sensitivity however implies standardized experimental conditions to minimize technical-induced variance.

Age-related hearing impairment (ARHI), or presbycusis, is the most prevalent sensory impairment in the elderly. ARHI is a complex disease caused by an interaction between environmental and genetic factors. Here we describe the results of the first whole genome association study for ARHI. The study was performed using 846 cases and 846 controls selected from 3434 individuals collected by eight centers in six European countries. DNA pools for cases and controls were allelotyped on the Affymetrix 500K GeneChip for each center separately. The 252 top-ranked single nucleotide polymorphisms (SNPs) identified in a non-Finnish European sample group (1332 samples) and the 177 top-ranked SNPs from a Finnish sample group (360 samples) were confirmed using individual genotyping. Subsequently, the 23 most interesting SNPs were individually genotyped in an independent European replication group (138 samples). This resulted in the identification of a highly significant and replicated SNP located in GRM7, the gene encoding metabotropic glutamate receptor type 7. Also in the Finnish sample group, two GRM7 SNPs were significant, albeit in a different region of the gene. As the Finnish are genetically distinct from the rest of the European population, this may be due to allelic heterogeneity. We performed histochemical studies in human and mouse and showed that mGluR7 is expressed in hair cells and in spiral ganglion cells of the inner ear. Together these data indicate that common alleles of GRM7 contribute to an individual's risk of developing ARHI, possibly through a mechanism of altered susceptibility to glutamate excitotoxicity.