Charles H. Keith

Glutamate‐induced changes in the pattern of hippocampal dendrite outgrowth: A role for calcium‐dependent pathways and the microtubule cytoskeleton

Glutamate regulation of a variety of aspects of dendrite development may be involved in neuronal plasticity and neuropathology. In this study, we examine the calcium-dependent pathways and alterations in the microtubule (MT) cytoskeleton that may mediate glutamate-induced changes in the pattern of dendrite outgrowth. We used Fura-2 AM and inhibitors of the calcium-dependent proteins, calmodulin and calpain, to identify the role of specific calcium-dependent pathways in glutamate-regulated dendrite outgrowth. Additionally, we used a quantitative fluorescence technique to correlate changes in MT levels with glutamate-induced changes in dendrite outgrowth. We show that the intracellular calcium concentration ([Ca(2+)](i)) changes in a biphasic manner over a 12-h period in the presence of glutamate. A transient increase in [Ca(2+)](i) over the first hour of glutamate exposure correlated with a calmodulin-associated increase in the rate of dendrite outgrowth, whereas a sustained increase in [Ca(2+)](i) was correlated with calpain-associated dendrite retraction. Quantitative fluorescence measurements showed no net change in the level of MTs during calmodulin-associated increases in dendrite outgrowth, but showed a significant decline in the level of MTs during calpain-associated dendrite retraction. These findings provide insights into the intracellular mechanisms involved in activity-dependent regulation of dendrite morphology during development and after pathology.

Glutamate modulation of dendrite outgrowth: alterations in the distribution of dendritic microtubules.

Glutamate can both facilitate and inhibit dendrite outgrowth in vitro. The major effects of low levels of glutamate occur only on the dendrites (not the axon) of pyramidal neurons and may be important for modulating dendrite outgrowth during neuronal development in vivo. Cytoskeletal changes resulting from glutamate exposure must underlie these changes in dendrite outgrowth. In the present study, hippocampal neuron cultures were used to measure the outgrowth of both axons and immature dendrites in the presence or absence of 50 μM glutamate. Subsequently, neurons were extracted and fixed for immunofluorescent labeling of microtubules and rhodamine phalloidin labeling of microfilaments. Additionally, neurons were prepared for electron microscopy to examine dendritic microtubules at the ultrastructural level. Glutamate led to increased dendrite outgrowth in the short term (4 hr) and dendrite retraction in the long term (8 hr). After short‐term glutamate exposures, no obvious morphological changes occur in either the microtubules or microfilaments. However, longer glutamate exposure causes a decrease in the number of microtubules in the distal region of retracting dendrites, and causes an increase in microtubule number in the dendritic shaft of both retracting and growing dendrites. Thus, the microtubule cytoskeleton may be involved in producing the changes in dendrite outgrowth caused by glutamate exposure. J. Neurosci. Res. 52:599–611, 1998.

Estimating weak ratiometric signals in imaging data. I. Dual-channel data

Ratiometric fluorescent indicators are becoming increasingly prevalent in many areas of biology. They are used for making quantitative measurements of intracellular free calcium both in vitro and in vivo, as well as measuring membrane potentials, pH, and other important physiological variables of interest to researchers in many subfields. Often, functional changes in the fluorescent yield of ratiometric indicators are small, and the signal-to-noise ratio (SNR) is of order unity or less. In particular, variability in the denominator of the ratio can lead to very poor ratio estimates. We present a statistical optimization method for objectively detecting and estimating ratiometric signals in dual-wavelength measurements of fluorescent, ratiometric indicators that improves on standard methods. With the use of an appropriate statistical model for ratiometric signals and by taking the pixel-pixel covariance of an imaging dataset into account, we are able to extract user-independent spatiotemporal information that retains high resolution in both space and time.

Neurite Development in PC12 Cells on Flexible Micro-Textured Substrates under Cyclic Stretch

We investigated the combined effect of micro‐texture and mechanical strain on neuronal cell development such as neurite length and neurite density in a rat pheochromocytoma cell line (PC12 cells). Cells were seeded on flexible silicone substrates with micro‐texture or no texture (smooth) and cultured under static and dynamic conditions. In the static condition substrates were not stretched and in the dynamic conditions substrates were subjected to cyclic uniaxial stretching at three different strain levels of 4%, 8%, and 16% with each at three different strain rates at 0.1, 0.5, and 1.0 Hz. Results showed that of all cell cultures there was no significant difference in neurite development between cells on smooth and textured substrates, except in the static and 4% at 0.1 Hz conditions, where micro‐texture induced significantly longer neurites. With both types of substrates, a lower mechanical condition (4% at 1.0 Hz or 16% at 0.1 Hz) resulted in more and longer neurites and lower cell density, and a higher mechanical condition (16% at 1.0 Hz) resulted in fewer and shorter neurites and lower cell density as compared to the static condition. These findings suggest that the effect of the micro‐texture on neurite development is more prominent in low mechanical conditions than in high mechanical conditions and that the strain level and strain rate have an interrelated effect on neurite development: a higher strain level at a lower strain rate has a similar effect as a lower strain level at a higher strain rate in terms of promoting neurite development.

Preferential initiation of PC12 neurites in directions of changing substrate adhesivity

When PC12 cells are grown on substrates showing a gradient of nonspecific adhesion, they preferentially initiate neurites in directions of changing adhesivity, whether that change is in the direction of increasing or decreasing adhesivity. This preference for changing adhesivity is ablated both by C. difficile toxin A, which inhibits all Rho‐family GTPases, and by C. botulinum C3 exoenzyme, which specifically inhibits Rho.

New statistical methods enhance imaging of cameleon fluorescence resonance energy transfer in cultured zebrafish spinal neurons

Cameleons are genetically encoded fluorescence resonance energy transfer (FRET)-based Ca(2+) indicators. Attempts to use cameleons to detect neural activity in vertebrate systems have been largely frustrated by the small FRET signal, in contradistinction to the higher signals seen in Drosophila and Caenorhabditis elegans. We have developed a statistical optimization method capable of detecting small ratiometric signals in noisy imaging data, called statistical optimization for the analysis of ratiometric signals. Using this method, we can detect and estimate anticorrelated ratiometric signals with subcellular resolution in cultured, dissociated zebrafish spinal neurons expressing cameleon or loaded with fluo-4 and fura-red. This method may make it possible to use yellow cameleons for measuring neural activity at high resolution in transgenic animals.

Adrenergic receptor-mediated increase of intracellular Ca2+ concentration in isolated bovine corneal epithelial cells

1. We determined if Ca2+ is a second messenger for adrenergic receptor-effector coupling in bovine corneal epithelial cells. 2. Methoxamine (10(-5) M) selectively increased intracellular Ca2+ concentration (Cai) by 65%. This increase was only partially suppressed through the removal of extracellular Ca2+ or pretreatment with 10(-6) M verapamil. 3. The beta-adrenergic-mediated increases in Cai were entirely dependent on extracellular Ca2+. These increases were directly elicited through stimulation of adenylate cyclase because 10(-6) M isoproterenol and the active analogues of forskolin (10(-5) M) all elevated Cai. 4. Therefore, increases in Cai serve a second messenger function for alpha-1 and beta-adrenergic receptor-effector coupling.

Quantitative fluorescence techniques for the determination of local microtubule polymerization equilibria in cultured neurons

The local control of intracellular microtubule polymerization equilibria has been hypothesized to be an important factor in the determination of neurite extension and other examples of cellular asymmetry. Provided that the quantum yield of the fluorophore remains constant, the combination of fluorescent analogue cytochemistry, differential extraction protocols, and quantitative video microscopy makes it possible to measure local fractions of cytoskeletal protein in polymer, even when it is impossible to resolve individual fibrils of the polymer. We have developed appropriate quantitative video microscopic techniques for measuring the fluorescence of a fluorescent analogue-injected neurite before and after extraction under microtubule-stabilizing conditions. We have used these methods to demonstrate that tetramethylrhodamine-n-hydroxysuccinimide tubulin is an appropriate fluorescent analogue, allowing us to measure fractions of tubulin in polymer locally within PC12 neurites. As would be expected, the fraction of tubulin fluorescent analogue in polymer approaches 1.0 in neurites exposed to the microtubule-stabilizing drug taxol and is close to 0 in neurites injected and extracted in the cold, or extracted under microtubule-destabilizing conditions. We have, therefore, developed a tool that allows us to measure microtubule polymerization equilibria out the neurites of cells in culture, which will allow us to test hypotheses that factors which affect neurite outgrowth do so by means of effects on microtubule polymerization equilibria.

Factors controlling axonal and dendritic arbors.

The sculpting and maintenance of axonal and dendritic arbors is largely under the control of molecules external to the cell. These factors include both substratum-associated and soluble factors that can enhance or inhibit the outgrowth of axons and dendrites. A large number of factors that modulate axonal outgrowth have been identified, and the first stages of the intracellular signaling pathways by which they modify process outgrowth have been characterized. Relatively fewer factors and pathways that affect dendritic outgrowth have been described. The factors that affect axonal arbors form an incompletely overlapping set with those that affect dendritic arbors, allowing selective control of the development and maintenance of these critical aspects of neuronal morphology.

Intracellular pH regulation by a Na+/H+ exchanger in cultured bovine trabecular cells

Abstract Intracellular pH (pHi) of cultured bovine trabecular cells was measured using video‐imaging techniques with a pH‐sensitive intracellular fluorescent dye, BCECF. In bicarbonate‐rich Ringer at pH 7.4, pHi was 7.29 ± 0.03 (± SEM, n = 12 monolayers, 120 cells sampled). Exposure to 20 mM NH4Cl immediately alkalinized pHi: replacement with a Na+‐rich solution acidified pHi before recovery to resting levels. When NH4Cl was replaced by a low Na+ solution, acidification was sustained but pHi recovery occurred after Na+‐rich solution. A pHi of 7.11 ± 0.02 (n = 2 monolayers, 20 cells) occurred in pH 6.8 and pHi was 7.72 ±0.03 (n = 2 monolayers, 20 cells) in pH 8.0. Amiloride (1 mM) acidified pHi but DIDS (1 mM) treatment, HCO3−‐free condition, 1 mM ouabain, 50 mM K+, and 2 mM BaCl2 failed to change pHi. Hydrogen peroxide (1 mM) acidified pHi but no change occurred with 50 μM. Trabecular cells possess an Na+/H+ exchanger similar to that in other cell types.