Jackie Schiller

Synaptic Integration in Tuft Dendrites of Layer 5 Pyramidal Neurons: A New Unifying Principle

Fine Dendrites Fire Differently The pyramidal neuron is the basic computational unit in the brain cortex. Its distal tuft dendrite is heavily innervated by horizontal fibers coursing through cortical layer-I providing long-range corticocortical and thalamocortical associational input. Larkum et al. (p. 756) investigated whether the apical tuft dendrites of layer-5 neocortical pyramidal neurons, like basal dendrites, generate n-methyl-d-aspartate (NMDA) spikes using two-photon–guided direct dendritic recording, glutamate uncaging, and modeling. NMDA spikes could be evoked in the distal tuft dendrites, while Ca2+ spikes could be triggered at the bifurcation points. Block of the hyperpolarization-activated current enhanced these NMDA spikes. Thus, the generation of NMDA spikes is a general principle of thin, basal, and tuft dendrites. Thin tuft and basal dendrites of pyramidal neurons use N-methyl-d-aspartate spikes to sum up synaptic inputs in semi-independent compartments. Tuft dendrites are the main target for feedback inputs innervating neocortical layer 5 pyramidal neurons, but their properties remain obscure. We report the existence of N-methyl-d-aspartate (NMDA) spikes in the fine distal tuft dendrites that otherwise did not support the initiation of calcium spikes. Both direct measurements and computer simulations showed that NMDA spikes are the dominant mechanism by which distal synaptic input leads to firing of the neuron and provide the substrate for complex parallel processing of top-down input arriving at the tuft. These data lead to a new unifying view of integration in pyramidal neurons in which all fine dendrites, basal and tuft, integrate inputs locally through the recruitment of NMDA receptor channels relative to the fixed apical calcium and axosomatic sodium integration points.

Properties of basal dendrites of layer 5 pyramidal neurons: a direct patch-clamp recording study.

Basal dendrites receive the majority of synapses that contact neocortical pyramidal neurons, yet our knowledge of synaptic processing in these dendrites has been hampered by their inaccessibility for electrical recordings. A new approach to patch-clamp recordings enabled us to characterize the integrative properties of these cells. Despite the short physical length of rat basal dendrites, synaptic inputs were electrotonically remote from the soma (>30-fold excitatory postsynaptic potential (EPSP) attenuation) and back-propagating action potentials were significantly attenuated. Unitary EPSPs were location dependent, reaching large amplitudes distally (>8 mV), yet their somatic contribution was relatively location independent. Basal dendrites support sodium and NMDA spikes, but not calcium spikes, for 75% of their length. This suggests that basal dendrites, despite their proximity to the site of action potential initiation, do not form a single basal-somatic region but rather should be considered as a separate integrative compartment favoring two integration modes: subthreshold, location-independent summation versus local amplification of incoming spatiotemporally clustered information.

NMDA receptors amplify calcium influx into dendritic spines during associativepre- and postsynaptic activation

Long-term potentiation (LTP) of synaptic strength can be induced by synchronous pre- and post-synaptic activation, and a rise in postsynaptic calcium is essential for induction of LTP. Calcium can enter through both voltage-dependent Ca2+ channels and NMDA-type glutamate receptors, but the relative contributions of these pathways is not known. We have examined this issue in layer V cortical pyramidal neurons, using focal flash photolysis of caged glutamate to mimic synaptic input and two-photon, laser-scanning microscopy to measure calcium levels in dendritic spines. Most of the calcium entry in response to glutamate alone was via voltage-dependent Ca2+ channels, and NMDA receptors accounted for less than 20% of total Ca2+ entry. When glutamate was paired with postsynaptic action potentials, however, the NMDA-receptor-dependent component was selectively amplified. The same is likely to occur during paired physiological pre- and postsynaptic activation, providing a mechanism for the input specificity and Hebbian behavior of LTP.

Calcium Handling in Human Embryonic Stem Cell-Derived Cardiomyocytes

The objective of the current study was to characterize calcium handling in developing human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs). To this end, real‐time polymerase chain reaction (PCR), immunocytochemistry, whole‐cell voltage‐clamp, and simultaneous patch‐clamp/laser scanning confocal calcium imaging and surface membrane labeling with di‐8‐aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC‐CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor‐2, and inositol‐1,4,5‐trisphosphate (IP3) receptors. Store calcium function was manifested as action‐potential‐induced calcium transients. Time‐to‐target plots showed that these action‐potential‐initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC‐CMs also exhibited local calcium events (“sparks”) that were localized to the surface membrane. The presence of caffeine‐sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early‐stage (with the initiation of beating), intermediate‐stage (10 days post‐beating [dpb]), and late‐stage (30–40 dpb) hESC‐CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3‐releasable calcium pool was also demonstrated in the hESC‐CMs in experiments using caged‐IP3 photolysis and antagonist application (2 μM 2‐Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early‐stage hESC‐CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC‐CMs both for developmental studies and for the development of future myocardial cell replacement strategies.

Plasticity Compartments in Basal Dendrites of Neocortical Pyramidal Neurons

Synaptic plasticity rules widely determine how cortical networks develop and store information. Using confocal imaging and dual site focal synaptic stimulation, we show that basal dendrites, which receive the majority of synapses innervating neocortical pyramidal neurons, contain two compartments with respect to plasticity rules. Synapses innervating the proximal basal tree are easily modified when paired with the global activity of the neuron. In contrast, synapses innervating the distal basal tree fail to change in response to global suprathreshold activity or local dendritic spikes. These synapses can undergo long-term potentiation under unusual conditions when local NMDA spikes, which evoke large calcium transients, are paired with a “gating molecule,” BDNF. Moreover, these synapses use a new temporal plasticity rule, which is an order of magnitude longer than spike timing dependent plasticity and prefers reversed presynaptic/postsynaptic activation order. The newly described plasticity compartmentalization of basal dendrites expands the networks plasticity rules and may support different learning and developmental functions.

Nonlinear dendritic processing determines angular tuning of barrel cortex neurons in vivo

Layer 4 neurons in primary sensory cortices receive direct sensory information from the external world. A general feature of these neurons is their selectivity to specific features of the sensory stimulation. Various theories try to explain the manner in which these neurons are driven by their incoming sensory information. In all of these theories neurons are regarded as simple elements summing small biased inputs to create tuned output through the axosomatic amplification mechanism. However, the possible role of active dendritic integration in further amplifying the sensory responses and sharpening the tuning curves of neurons is disregarded. Our findings show that dendrites of layer 4 spiny stellate neurons in the barrel cortex can generate local and global multi-branch N-methyl-d-aspartate (NMDA) spikes, which are the main regenerative events in these dendrites. In turn, these NMDA receptor (NMDAR) regenerative mechanisms can sum supralinearly the coactivated thalamocortical and corticocortical inputs. Using in vivo whole-cell recordings combined with an intracellular NMDAR blocker and membrane hyperpolarization, we show that dendritic NMDAR-dependent regenerative responses contribute substantially to the angular tuning of layer 4 neurons by preferentially amplifying the preferred angular directions over non-preferred angles. Taken together, these findings indicate that dendritic NMDAR regenerative amplification mechanisms contribute markedly to sensory responses and critically determine the tuning of cortical neurons.

Calcium handling in human induced pluripotent stem cell derived cardiomyocytes.

Background The ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca2+-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs). Methodology/Principal Findings RT-PCR and immunocytochemistry experiments identified the expression of key Ca2+-handling proteins. Detailed laser confocal Ca2+ imaging demonstrated spontaneous whole-cell [Ca2+]i transients. These transients required Ca2+ influx via L-type Ca2+ channels, as demonstrated by their elimination in the absence of extracellular Ca2+ or by administration of the L-type Ca2+ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca2+ store, contributing to [Ca2+]i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca2+) and ryanodine (decreasing [Ca2+]i). Similarly, the importance of Ca2+ reuptake into the SR via the SR Ca2+ ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca2+]i transients elimination. Finally, the presence of an IP3-releasable Ca2+ pool in hiPSC-CMs and its contribution to whole-cell [Ca2+]i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phosopholipase C inhibitor U73122. Conclusions/Significance Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca2+ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca2+]i transients in hiPSC-CMs on both sarcolemmal Ca2+ entry via L-type Ca2+ channels and intracellular store Ca2+ release.

NMDA receptor-mediated dendritic spikes and coincident signal amplification.

Dendrites of cortical neurons possess active conductances, which contribute to the nonlinear processing of synaptic information. Recently it has been shown that basal dendrites can generate highly localized spikes mediated by NMDA receptor channels. These spikes may serve as a powerful mechanism to detect and amplify synchronously activated spatially clustered excitatory synaptic inputs in individual dendritic segments, and may enable parallel processing in several integrative dendritic subunits.

Encoding and decoding bursts by NMDA spikes in basal dendrites of layer 5 pyramidal neurons.

Bursts of action potentials are important information-bearing signals in the brain, although the neuronal specializations underlying burst generation and detection are only partially understood. In apical dendrites of neocortical pyramidal neurons, calcium spikes are known to contribute to burst generation, but a comparable understanding of basal dendritic mechanisms is lacking. Here we show that NMDA spikes in basal dendrites mediate both detection and generation of bursts through a postsynaptic mechanism. High-frequency inputs to basal dendrites markedly facilitated NMDA spike initiation compared with low-frequency activation or single inputs. Unlike conventional temporal summation effects based on voltage, however, NMDA spike facilitation depended mainly on residual glutamate bound to NMDA receptors from previous activations. Once triggered by an input burst, we found that NMDA spikes in turn reliably trigger output bursts under in vivo-like stimulus conditions. Through their unique biophysical properties, NMDA spikes are thus ideally suited to promote the propagation of bursts through the cortical network.

Dynamics of Excitability over Extended Timescales in Cultured Cortical Neurons

Although neuronal excitability is well understood and accurately modeled over timescales of up to hundreds of milliseconds, it is currently unclear whether extrapolating from this limited duration to longer behaviorally relevant timescales is appropriate. Here we used an extracellular recording and stimulation paradigm that extends the duration of single-neuron electrophysiological experiments, exposing the dynamics of excitability in individual cultured cortical neurons over timescales hitherto inaccessible. We show that the long-term neuronal excitability dynamics is unstable and dominated by critical fluctuations, intermittency, scale-invariant rate statistics, and long memory. These intrinsic dynamics bound the firing rate over extended timescales, contrasting observed short-term neuronal response to stimulation onset. Furthermore, the activity of a neuron over extended timescales shows transitions between quasi-stable modes, each characterized by a typical response pattern. Like in the case of rate statistics, the short-term onset response pattern that often serves to functionally define a given neuron is not indicative of its long-term ongoing response. These observations question the validity of describing neuronal excitability based on temporally restricted electrophysiological data, calling for in-depth exploration of activity over wider temporal scales. Such extended experiments will probably entail a different kind of neuronal models, accounting for the unbounded range, from milliseconds up.