Fiberoptic system for recording dendritic calcium signals in layer 5 neocortical pyramidal cells in freely moving rats

Calcium influx into the dendritic tufts of layer 5 neocortical pyramidal neurons modifies a number of important cellular mechanisms. It can trigger local synaptic plasticity and switch the firing properties from regular to burst firing. Due to methodological limitations, our knowledge about Ca2+ spikes in the dendritic tuft stems mostly from in vitro experiments. However, it has been speculated that regenerative Ca2+ events in the distal dendrites correlate with distinct behavioral states. Therefore it would be most desirable to be able to record these Ca2+ events in vivo, preferably in the behaving animal. Here, we present a novel approach for recording Ca2+ signals in the dendrites of populations of layer 5 pyramidal neurons in vivo, which ensures that all recorded fluorescence changes are due to intracellular Ca2+ signals in the apical dendrites. The method has two main features: 1) bolus loading of layer 5 with a membrane-permeant Ca2+ dye resulting in specific loading of pyramidal cell dendrites in the upper layers and 2) a fiberoptic cable attached to a gradient index lens and a prism reflecting light horizontally at 90 degrees to the angle of the apical dendrites. We demonstrate that the in vivo signal-to-noise ratio recorded with this relatively inexpensive and easy-to-implement fiberoptic-based device is comparable to conventional camera-based imaging systems used in vitro. In addition, the device is flexible and lightweight and can be used for recording Ca2+ signals in the distal dendritic tuft of freely behaving animals.

Dendritic spikes in apical dendrites of neocortical layer 2/3 pyramidal neurons

Layer 2/3 (L2/3) pyramidal neurons are the most abundant cells of the neocortex. Despite their key position in the cortical microcircuit, synaptic integration in dendrites of L2/3 neurons is far less understood than in L5 pyramidal cell dendrites, mainly because of the difficulties in obtaining electrical recordings from thin dendrites. Here we directly measured passive and active properties of the apical dendrites of L2/3 neurons in rat brain slices using dual dendritic-somatic patch-clamp recordings and calcium imaging. Unlike L5 cells, L2/3 dendrites displayed little sag in response to long current pulses, which suggests a low density of I(h) in the dendrites and soma. This was also consistent with a slight increase in input resistance with distance from the soma. Brief current injections into the apical dendrite evoked relatively short (half-width 2-4 ms) dendritic spikes that were isolated from the soma for near-threshold currents at sites beyond the middle of the apical dendrite. Regenerative dendritic potentials and large concomitant calcium transients were also elicited by trains of somatic action potentials (APs) above a critical frequency (130 Hz), which was slightly higher than in L5 neurons. Initiation of dendritic spikes was facilitated by backpropagating somatic APs and could cause an additional AP at the soma. As in L5 neurons, we found that distal dendritic calcium transients are sensitive to a long-lasting block by GABAergic inhibition. We conclude that L2/3 pyramidal neurons can generate dendritic spikes, sharing with L5 pyramidal neurons fundamental properties of dendritic excitability and control by inhibition.

Comparison between confocal scanning laser tomography, scanning laser polarimetry and optical coherence tomography on the ability to detect localised retinal nerve fibre layer defects in glaucoma patients

BACKGROUND/AIM: To compare the ability of confocal scanning laser tomography (CSLT), scanning laser polarimetry (SLP) and optical coherence tomography (OCT) in recognising localised retinal nerve fibre layer (RNFL) defects. METHODS: 51 eyes from 43 patients with glaucoma were identified by two observers as having RNFL defects visible on optic disc photographs. 51 eyes of 32 normal subjects were used as controls. Three masked observers evaluated CSLT, SLP and OCT images to determine subjectively the presence of localised RNFL defects. RESULTS: Interobserver agreement was highest with OCT, followed by SLP and CSLT (mean kappa: 0.83, 0.69 and 0.64, respectively). RNFL defects were identified in 58.8% of CSLT, 66.7% of SLP and 54.9% of OCT (p = 0.02 between SLP and OCT) by at least two observers. In the controls, 94.1% of CSLT, 84.3% of SLP and 94.1% of OCT scans, respectively, were rated as normal (p = 0.02 between CSLT and SLP, and SLP and OCT). CONCLUSION: Approximately 20-40% of localised RNFL defects identified by colour optic disc photographs are not detected by CSLT, SPL or OCT. SLP showed a higher number of false-positive results than the other techniques, but also had a higher proportion of correctly identified RNFL defects in the glaucoma population.

Hop-to-Hop Reliability in IP-Based Wireless Sensor Networks - A Cross-Layer Approach

To interconnect a wireless sensor network (WSN) to the Internet, we propose to use TCP/IP as the standard protocol for all network entities. We present a cross layer designed communication architecture, which contains a MAC protocol, IP, a new protocol called Hop-to-Hop Reliability (H2HR) protocol, and the TCP Support for Sensor Nodes (TSS) protocol. The MAC protocol implements the MAC layer of beacon-less personal area networks (PANs) as defined in IEEE 802.15.4. H2HR implements hop-to-hop reliability mechanisms. Two acknowledgment mechanisms, explicit and implicit ACK are supported. TSS optimizes using TCP in WSNs by implementing local retransmission of TCP data packets, local TCP ACK regeneration, aggressive TCP ACK recovery, congestion and flow control algorithms. We show that H2HR increases the performance of UDP, TCP, and RMST in WSNs significantly. The throughput is increased and the packet loss ratio is decreased. As a result, WSNs can be operated and managed using TCP/IP.