Intracellular horseradish peroxidase injection for correlation of light and electron microscopic anatomy with synaptic physiology of cultured mouse spinal cord neurons

Neale, Elaine A.; Macdonald, Robert L.; Nelson, Phillip G.

doi:10.1016/0006-8993(78)90255-x

Cited by 72 publications

(20 citation statements)

References 28 publications

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“…Specific binding of biotin-MAP2 with MTs was revealed under electron microscopy (Fig. 1C) (19,28). We examined the native pattern of MAP2 distribution in this culture system.…”

Section: Resultsmentioning

confidence: 99%

Rapid turnover of microtubule-associated protein MAP2 in the axon revealed by microinjection of biotinylated MAP2 into cultured neurons.

Okabe

Hirokawa

1989

Proc. Natl. Acad. Sci. U.S.A.

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We studied the mechanism of compartmentation of microtubule-associated protein 2 (MAP2) in the dendrites and cell bodies by using microinjection of biotinlabeled MAP2 into mature spinal cord neurons in culture. MAP2 molecules microinjected into the nerve cell body were distributed not only throughout the cytoplasm of the cell body and dendrites, but also in the axon as far as a few millimeters from the cell body within 24 hr after injection. However, when injected cells were incubated for more than 3 days, the amount of biotin-labeled MAP2 in the axon decreased remarkably compared with that in the dendrites. This indicates that there is no sorting mechanism in the cell body for the transport of MAP2 selectively into the dendrites but that the turnover rate of MAP2 in the axons differs from that in the dendrites. To further characterize the mechanism of MAP2 compartmentation, we performed immunoelectron microscopy of injected cells and detergent extraction of microinjected cells prior to immunocytochemistry with anti-biotin. The results strongly suggest that a large part of axonal MAP2 is not associated with cytoskeleton and that this weak association of MAP2 favors selective loss of MAP2 from the axon.The formation of two distinct classes of neuronal processes, axons and dendrites, is a fundamental step in neuronal morphogenesis because the functional polarity of nerve cells is established through this event. The cellular mechanism that determines the distinctive shapes of dendrites and axons is not clear, but several lines of evidence indicate that the cytoskeleton is one ofthe important endogeneous factors that control the elaborate shape of neuronal processes (1-3). Furthermore, recent studies have shown that axons and dendrites are not identical in the composition of their cytoskeletal proteins (4-6). Since microtubule-associated protein 2 (MAP2), one of the major microtubule-associated proteins in nervous tissue, is preferentially localized in dendrites and cell bodies, it is possible that this protein may be an intrinsic factor that regulates the shape of neurites as dendrites.Although biochemical properties and immunocytochemical distribution of MAP2 have been thoroughly investigated (7)(8)(9)(10)(11)(12), the molecular mechanism of MAP2 compartmentation is not known. The technique developed for the microinjection of haptenized cytoskeletal proteins combined with hapten-mediated immunocytochemistry has enabled us to examine directly the pattern of incorporation and turnover of cytoskeletal proteins in living cells (13)(14)(15). Thus, to directly answer the problem of MAP2 compartmentation, we microinjected biotin-labeled MAP2 (biotin-MAP2) into mouse spinal cord neurons, which were fully differentiated for 2-4 weeks in culture, and examined the distribution of injected molecules by using anti-biotin antibody. Our results indicate that injected MAP2 molecules are moved into both the dendrites and axons but are not associated tightly with the axonal cytoskeleton and are eliminated rapidly from the a...

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“…Specific binding of biotin-MAP2 with MTs was revealed under electron microscopy (Fig. 1C) (19,28). We examined the native pattern of MAP2 distribution in this culture system.…”

Section: Resultsmentioning

confidence: 99%

Rapid turnover of microtubule-associated protein MAP2 in the axon revealed by microinjection of biotinylated MAP2 into cultured neurons.

Okabe

Hirokawa

1989

Proc. Natl. Acad. Sci. U.S.A.

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“…The number of connections a neuron makes is AP A , where A is the area of the axonal arborization, is the number of neurons per area, and P A is the probability that a neuron connects to another neuron within its axonal arborization. We measured axonal arborization from neurons visualized after intracellular injection with horseradish peroxidase (Neale et al 1978), and found it to be 2.0 Ϯ 0.86 mm 2 (mean Ϯ SD, n ϭ 12 neurons). The density of our cultures, , was 23.7 Ϯ 10.2 neurons/mm 2 (n ϭ 10 culture dishes).…”

Section: Choice Of Simulation Parametersmentioning

confidence: 99%

Intrinsic Dynamics in Neuronal Networks. I. Theory

Latham

Richmond

Nelson

et al. 2000

Journal of Neurophysiology

303

278

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in the mammalian nervous system remain active in the absence of stimuli. This activity falls into two main patterns: steady firing at low rates and rhythmic bursting. How are these firing patterns generated? Specifically, how do dynamic interactions between excitatory and inhibitory neurons produce these firing patterns, and how do networks switch from one firing pattern to the other? We investigated these questions theoretically by examining the intrinsic dynamics of large networks of neurons. Using both a semianalytic model based on mean firing rate dynamics and simulations with large neuronal networks, we found that the dynamics, and thus the firing patterns, are controlled largely by one parameter, the fraction of endogenously active cells. When no endogenously active cells are present, networks are either silent or fire at a high rate; as the number of endogenously active cells increases, there is a transition to bursting; and, with a further increase, there is a second transition to steady firing at a low rate. A secondary role is played by network connectivity, which determines whether activity occurs at a constant mean firing rate or oscillates around that mean. These conclusions require only conventional assumptions: excitatory input to a neuron increases its firing rate, inhibitory input decreases it, and neurons exhibit spike-frequency adaptation. These conclusions also lead to two experimentally testable predictions: 1) isolated networks that fire at low rates must contain endogenously active cells and 2) a reduction in the fraction of endogenously active cells in such networks must lead to bursting.

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“…The method provides possibilities for quantitative light and electron microscopical analyses of the differences between neurons which by electrophysiological techniques have been classified as functionally alike (Czarkowska et al, 1976a, b;Brown, 1976Brown, , 1977Brown et al, 1977;Rastad et al, 1977;Cullheim, 1978;Cullheim & Kellerth, 1978a, b,c;Lagerback et al, 1978;Neale et al, 1978;Rastad, 1978). The present ultrastructural investigation was performed on the axodendritic and axosomatic collateral terminals of two neurons functionally identified as spinocervical tract cells.…”

Section: Introductionmentioning

confidence: 98%

Quantitative analysis of axodendritic and axosomatic collateral terminals of two feline spinocervical tract cells

Rastad

1981

J Neurocytol

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The initial axon collaterals of two feline spinocervical tract cells have been ultrastructurally investigated after intracellular injection with horseradish peroxidase. A total of 200 axodendritic and 43 axosomatic terminals were analysed in serial section. The following variables were measured: the diameter and area of the bouton profiles, the areal densities of synaptic vesicles and mitochondria of the bouton profiles, the width and length of the synaptic clefts, the width of the postsynaptic densities, the width of the postsynaptic dendrites, and the width and length of axons between the boutons. Ten boutons were reconstructed for an investigation of the relation between measurements in two and three dimensions. All the measured variables showed wide ranges of variation. They were analysed statistically for significant differences between the cells and between the axodendritic and axosomatic boutons of each and both cells. Significant differences were mainly observed in the former comparison but they were also found between the axodendritic and axosomatic terminals of the individual cells.

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Intracellular horseradish peroxidase injection for correlation of light and electron microscopic anatomy with synaptic physiology of cultured mouse spinal cord neurons

Cited by 72 publications

References 28 publications

Rapid turnover of microtubule-associated protein MAP2 in the axon revealed by microinjection of biotinylated MAP2 into cultured neurons.

Rapid turnover of microtubule-associated protein MAP2 in the axon revealed by microinjection of biotinylated MAP2 into cultured neurons.

Intrinsic Dynamics in Neuronal Networks. I. Theory

Quantitative analysis of axodendritic and axosomatic collateral terminals of two feline spinocervical tract cells

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