Costa M. Colbert

K+ channel regulation of signal propagation in dendrites of hippocampal pyramidal neurons

Pyramidal neurons receive tens of thousands of synaptic inputs on their dendrites. The dendrites dynamically alter the strengths of these synapses and coordinate them to produce an output in ways that are not well understood. Surprisingly, there turns out to be a very high density of transient A-type potassium ion channels in dendrites of hippocampal CA1 pyramidal neurons. These channels prevent initiation of an action potential in the dendrites, limit the back-propagation of action potentials into the dendrites, and reduce excitatory synaptic events. The channels act to prevent large, rapid dendritic depolarizations, thereby regulating orthograde and retrograde propagation of dendritic potentials.

Dendritic potassium channels in hippocampal pyramidal neurons

Potassium channels located in the dendrites of hippocampal CA1 pyramidal neurons control the shape and amplitude of back‐propagating action potentials, the amplitude of excitatory postsynaptic potentials and dendritic excitability. Non‐uniform gradients in the distribution of potassium channels in the dendrites make the dendritic electrical properties markedly different from those found in the soma. For example, the influence of a fast, calcium‐dependent potassium current on action potential repolarization is progressively reduced in the first 150 μm of the apical dendrites, so that action potentials recorded farther than 200 μm from the soma have no fast after‐hyperpolarization and are wider than those in the soma. The peak amplitude of back‐propagating action potentials is also progressively reduced in the dendrites because of the increasing density of a transient potassium channel with distance from the soma. The activation of this channel can be reduced by the activity of a number of protein kinases as well as by prior depolarization. The depolarization from excitatory postsynaptic potentials (EPSPs) can inactivate these A‐type K+ channels and thus lead to an increase in the amplitude of dendritic action potentials, provided the EPSP and the action potentials occur within the appropriate time window. This time window could be in the order of 15 ms and may play a role in long‐term potentiation induced by pairing EPSPs and back‐propagating action potentials.

Ion channel properties underlying axonal action potential initiation in pyramidal neurons

A high density of Na+ channels in the axon hillock, or initial segment, is believed to determine the threshold for action potential initiation in neurons. Here we report evidence for an alternative mechanism that lowers the threshold in the axon. We investigated properties and distributions of ion channels in outside-out patches from axons and somata of layer 5 pyramidal neurons in rat neocortical slices. Na+ channels in axonal patches (〈30 μm from the soma) were activated by 7 mV less depolarization than were somatic Na+ channels. A-type K+ channels, which were prominent in somatic and dendritic patches, were rarely seen in axonal patches. We incorporated these findings into numerical simulations which indicate that biophysical properties of axonal channels, rather than a high density of channels in the initial segment, are most likely to determine the lowest threshold for action potential initiation.

Regulation of back−propagating action potentials in hippocampal neurons

Protein kinase C has recently been shown to modulate the slow recovery from inactivation of Na+ channels in apical dendrites of hippocampal CA1 pyramidal neurons. Moreover, dendritic, A-type K+ channels have been found to be modulated by protein kinases A and C and by mitogen-activated protein kinase. The electrical signalling ability of these dendrites is thus highly regulated by a number of neurotransmitters and second-messenger systems.

Different Mechanisms Exist for the Plasticity of Glutamate Reuptake during Early Long-Term Potentiation (LTP) and Late LTP

Regulation of glutamate reuptake occurs along with several forms of synaptic plasticity. These associations led to the hypothesis that regulation of glutamate uptake is a general component of plasticity at glutamatergic synapses. We tested this hypothesis by determining whether glutamate uptake is regulated during both the early phases (E-LTP) and late phases (L-LTP) of long-term potentiation (LTP). We found that glutamate uptake was rapidly increased within minutes after induction of LTP and that the increase in glutamate uptake persisted for at least 3 h in CA1 of the hippocampus. NMDA receptor activation and Na+-dependent high-affinity glutamate transporters were responsible for the regulation of glutamate uptake during all phases of LTP. However, different mechanisms appear to be responsible for the increase in glutamate uptake during E-LTP and L-LTP. The increase in glutamate uptake observed during E-LTP did not require new protein synthesis, was mediated by PKC but not cAMP, and as previously shown was attributable to EAAC1 (excitatory amino acid carrier-1), a neuronal glutamate transporter. On the other hand, the increase in glutamate uptake during L-LTP required new protein synthesis and was mediated by the cAMP–PKA (protein kinase A) pathway, and it involved a different glutamate transporter, GLT1a (glutamate transporter subtype 1a). The switch in mechanisms regulating glutamate uptake between E-LTP and L-LTP paralleled the differences in the mechanisms responsible for the induction of E-LTP and L-LTP. Moreover, the differences in signaling pathways and transporters involved in regulating glutamate uptake during E-LTP and L-LTP indicate that different functions and/or sites may exist for the changes in glutamate uptake during E-LTP and L-LTP.

Automatic centerline extraction of irregular tubular structures using probability volumes from multiphoton imaging

In this paper, we present a general framework for extracting 3D centerlines from volumetric datasets. Unlike the majority of previous approaches, we do not require a prior segmentation of the volume nor we do assume any particular tubular shape. Centerline extraction is performed using a morphology-guided level set model. Our approach consists of: i) learning the structural patterns of a tubular-like object, and ii) estimating the centerline of a tubular object as the path with minimal cost with respect to outward flux in gray level images. Such shortest path is found by solving the Eikonal equation. We compare the performance of our method with existing approaches in synthetic, CT, and multiphoton 3D images, obtaining substantial improvements, especially in the case of irregular tubular objects.

Relationship between increase in astrocytic GLT-1 glutamate transport and late-LTP

Na⁺-dependent high-affinity glutamate transporters have important roles in the maintenance of basal levels of glutamate and clearance of glutamate during synaptic transmission. Interestingly, several studies have shown that basal glutamate transport displays plasticity. Glutamate uptake increases in hippocampal slices during early long-term potentiation (E-LTP) and late long-term potentiation (L-LTP). Four issues were addressed in this research: Which glutamate transporter is responsible for the increase in glutamate uptake during L-LTP? In what cell type in the hippocampus does the increase in glutamate uptake occur? Does a single type of cell contain all the mechanisms to respond to an induction stimulus with a change in glutamate uptake? What role does the increase in glutamate uptake play during L-LTP? We have confirmed that GLT-1 is responsible for the increase in glutamate uptake during L-LTP. Also, we found that astrocytes were responsible for much, if not all, of the increase in glutamate uptake in hippocampal slices during L-LTP. Additionally, we found that cultured astrocytes alone were able to respond to an induction stimulus with an increase in glutamate uptake. Inhibition of basal glutamate uptake did not affect the induction of L-LTP, but inhibition of the increase in glutamate uptake did inhibit both the expression of L-LTP and induction of additional LTP. It seems likely that heightened glutamate transport plays an ongoing role in the ability of hippocampal circuitry to code and store information.

Live Neuron Morphology Automatically Reconstructed From Multiphoton and Confocal Imaging Data

We have developed a fully automated procedure for extracting dendritic morphology from multiple three-dimensional image stacks produced by laser scanning microscopy. By eliminating human intervention, we ensure that the results are objective, quickly generated, and accurate. The software suite accounts for typical experimental conditions by reducing background noise, removing pipette artifacts, and aligning multiple overlapping image stacks. The output morphology is appropriate for simulation in compartmental simulation environments. In this report, we validate the utility of this procedure by comparing its performance on live neurons and test specimens with other fully and semiautomated reconstruction tools.

Towards automatic reconstruction of dendrite morphology from live neurons

The recent advent of optical imaging methods to capture both structural and functional data from living neurons holds the promise of improving our understanding of neuronal function. An important technical advance would be to use computer simulations to determine the optimal locations for high-speed functional imaging and electrical recording on an individual neuron. However, such simulations require a precise reconstruction of the dendritic morphology. Thus, the currently available time-consuming, semi-manual methods for morphological reconstruction prevent integration of cell-specific simulations into the experiments. Our work focuses on implementing a fast, robust method for extracting dendrite morphology from confocal or two-photon images to enable concurrent simulation and functional imaging of individual neurons. We discuss the current implementation of the morphological reconstruction, our approach to validation, and our initial results.

Preparation of Cortical Brain Slices for Electrophysiological Recording

Acute brain slices allow electrophysiological and imaging techniques to be applied in vitro to the study of neuronal ion channels, synaptic plasticity, and whole-cell function in juvenile and adult tissue. Ion channel recordings from small dendritic branches and axons in brain slices have demonstrated considerable functional differences in ion channel function within subregions of single cells. These findings have greatly increased our understanding of neuronal computation. This chapter presents methods for obtaining high-quality brain slices developed to aid visualization of small neuronal structures using differential interference contrast microscopy.