Balancing a retroreflector to minimize rotation errors using a pendulum and quadrature interferometer.

A corner-cube retroreflector has the property that the optical path length for a reflected laser beam is insensitive to rotations about a mathematical point called its optical center (OC). This property is exploited in ballistic absolute gravity meters in which a proof mass containing a corner-cube retroreflector is dropped in a vacuum, and its position is accurately determined with a laser interferometer. In order to avoid vertical position errors when the proof mass rotates during free fall, it is important to collocate its center of mass (COM) with the OC of the retroreflector. This is commonly done using a mechanical scale-based balancing procedure, which has limited accuracy due to the difficulty in finding the exact position of the COM and the OC. This paper describes a novel way to achieve the collocation by incorporating the proof mass into a pendulum and using a quadrature interferometer to interrogate its apparent translation in its twist mode. The mismatch between the COM and OC generates a signal in a quiet part of the spectrum where no mechanical resonance exists. This allows us to tune the position of the COM relative to the OC to an accuracy of about 1 µm in all three axes. This provides a way to directly demonstrate that a rotation of the proof mass by several degrees causes an apparent translation in the direction of the laser beam of less than 1 nm. This technique allows an order of magnitude improvement over traditional methods of balancing.

On the intensity distribution function of blazed reflective diffraction gratings.

We derive from first principles the expression for the angular/wavelength distribution of the intensity diffracted by a blazed reflective grating, according to a scalar theory of diffraction. We considered the most common case of a groove profile with rectangular apex. Our derivation correctly identifies the geometric parameters of a blazed reflective grating that determine its diffraction efficiency, and fixes an incorrect but commonly adopted expression in the literature. We compare the predictions of this scalar theory with those resulting from a rigorous vector treatment of diffraction from one-dimensional blazed reflective gratings.

Computer analysis of organelle translocation in primary neuronal cultures and continuous cell lines.

Organelle translocation in a number of cell types in tissue culture as seen by high-resolution Zeiss-Nomarski differential interference contrast optics was filmed and analyzed by computer. Principal cell types studied included primary chick spinal cord, chick dorsal root ganglion, ratbrain, and various clones of continuous cell lines. Organelle translocations in all cell types studied exhibited frequent, large changes in velocity during any one translocation. The appearance of particles as seen with Nomarski optics was correlated with their fine structures in one dorsal root ganglion neurite by fixing the cell as it was being filmed and obtaining electron micrographs of the region filmed. This revealed the identity of several organelles as well as the presence of abundant neurotubules but no neurofilaments. Primary cell cultures exhibited more high-velocity organelle movements than continuous cell lines. The net progress of an organelle in a given direction was greater in primary neuronal cells than in fibroblasts or continuous cell lines. These findings are correlated with the literature on organelle translocation and axoplasmic transport.

Fate of tetanus toxin bound to the surface of primary neurons in culture: evidence for rapid internalization.

We examined the nature of the tetanus toxin receptor in primary cultures of mouse spinal cord by ligand blotting techniques. Membrane components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled tetanus toxin. The toxin bound only to material at or near the dye front, which was lost when the cells were delipidated before electrophoresis. Gangliosides purified from the lipid extract were separated by thin-layer chromatography and the chromatogram was overlaid with 125I-toxin. The toxin bound to gangliosides corresponding to GD1b and GT1b. Similar results were obtained with brain membranes; thus, gangliosides rather than glycoproteins appear to be the toxin receptors both in vivo and in neuronal cell cultures. To follow the fate of tetanus toxin bound to cultured neurons, we developed an assay to measure cell-surface and internalized toxin. Cells were incubated with tetanus toxin at 0 degree C, washed, and sequentially exposed to antitoxin and 125I-labeled protein A. Using this assay, we found that much of the toxin initially bound to cell surface disappeared rapidly when the temperature was raised to 37 degrees C but not when the cells were kept at 0 degree C. Some of the toxin was internalized and could only be detected by our treating the cells with Triton X-100 before adding anti-toxin. Experiments with 125I-tetanus toxin showed that a substantial amount of the toxin bound at 0 degree C dissociated into the medium upon warming of the cells. Using immunofluorescence, we confirmed that some of the bound toxin was internalized within 15 min and accumulated in discrete structures. These structures did not appear to be lysosomes, as the cell-associated toxin had a long half-life and 90% of the radioactivity released into the medium was precipitated by trichloroacetic acid. The rapid internalization of tetanus toxin into a subcellular compartment where it escapes degradation may be important for its mechanism of action.

A few axonal proteins distinguish ventral spinal cord neurons from dorsal root ganglion neurons.

A series of proteins putatively involved in the generation of axonal diversity was identified. Neurons from ventral spinal cord and dorsal root ganglia were grown in a compartmented cell-culture system which offers separate access to cell somas and axons. The proteins synthesized in the neuronal cell somas and subsequently transported into the axons were selectively analyzed by 2-dimensional gel electrophoresis. The patterns of axonal proteins were substantially less complex than those derived from the proteins of neuronal cell bodies. The structural and functional similarity of axons from different neurons was reflected in a high degree of similarity of the gel pattern of the axonal proteins from sensory ganglia and spinal cord neurons. Each axonal type, however, had several proteins that were markedly less abundant or absent in the other. These neuron-population enriched proteins may be involved in the implementation of neuronal diversity. One of the proteins enriched in dorsal root ganglia axons had previously been found to be expressed with decreased abundance when dorsal root ganglia axons were co-cultured with ventral spinal cord cells under conditions in which synapse formation occurs (P. Sonderegger, M. C. Fishman, M. Bokoum, H. C. Bauer, and P.G. Nelson, 1983, Science [Wash. DC], 221:1294-1297). This protein may be a candidate for a role in growth cone functions, specific for neuronal subsets, such as pathfinding and selective axon fasciculation or the initiation of specific synapses. The methodology presented is thus capable of demonstrating patterns of protein synthesis that distinguish different neuronal subsets. The accessibility of these proteins for structural and functional studies may contribute to the elucidation of neuron-specific functions at the molecular level.

Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium.

Activity of the enzyme choline acetyltransferase (CAT), which mediates the synthesis of the neurotransmitter, acetylcholine, was increased up to 20- fold in spinal cord (SC) cells grown in culture with muscle cells for 2 wk. This increase was directly related to the duration of co-culture as well as to the cell density of both the SC and muscle involved and was not affected by the presence of the acetylcholine receptor blocking agent, alpha-bungarotoxin. Glutamic acid decarboxylase (GAD) activity was often markedly decreased in SC-muscle cultures while the activities of acetylcholinesterase and several other enzymes were little changed. Increased CAT activity was also observed when SC cultures were maintained in medium which had been conditioned by muscle cells or by undifferentiated cells from embryonic muscle. Muscle-conditioned medium (CM) did not affect the activities of SC cell GAD or acetylcholinesterase. Dilution or concentration of the CM directly affected its ability to increase SC CAT activity , as did the duration and timing of exposure of the SC cells to the CM. The medium could be conditioned by muscle cells in the presence or absence of serum, and remained effective after dialysis or heating to 58 degrees C. Membrane filtration data were consistent with the conclusion that the active material(s) in CM had a molecular weight in excess of 50,000 daltons. We conclude that large molecular weight material that is released by muscle cells is capable of producing a specific increase in CAT activity of SC cells.

Acetylcholine responses in L cells.

L cells, a family of continuous cell lines of mouse fibroblastic origin, generate a prolonged active membrane hyperpolarization (the hyperpolarizing activation response) when stimulated mechanically or electrically. lontophoretically applied acetylcholine elicits a similar response; atropine blocks the acetylcholine but not the electrically or mechanically elicited responses. The hyperpolarizing activation response can also be elicited by electrical, mechanical, or acetylcholine stimulation of cells adjacent to the recorded cell. Propagation of the response from one cell to another is not dependent on direct electrical coupling between cells and is not blocked by application of a bath containing atropine or curare. These results show that L cells are capable of generating an active electrical response. that they are sensitive to at least one neurotransmitter (acetylcholine), and that humorally mediated interaction (probably noncholinergic) between L cells occurs.

Synaptic activity in motoneurons during natural stimulation of muscle spindles.

Synaptic activity evoked in cat motoneurons during stretch stimulation of muscle spindles in the homonymous muscle has been studied by intracellular recording. A class of miniature excitatory postsynaptic potentials evoked by such physiologic stimulation results from activity in single group la spindle afferent fibers, and the criterion for identification is a predictable pattern of rhythmic occurrence of the synaptic potentials. Statistical examination of the amplitude distributions of this class of miniature synaptic potentials suggests that some group Ia fibers liberate a relatively large number of quantal excitatory postsynaptic potential units per fiber impulse.

Specific-opiate-induced depression of transmitter release from dorsal root ganglion cells in culture.

The opiate etorphine depresses monosynaptic excitatory postsynaptic potentials (EPSP's) elicited in spinal cord cells by activation of dorsal root ganglion cells in murine neuronal cell culture. The depression is reversed by naloxone. Statistical analysis of the synaptic responses reveals that the opiate reduces EPSP quantal content at this synapse without altering quantal size. Therefore, the opiate action is presynaptic and affects transmitter release rather than postsynaptic responsiveness.

Synapse formation between two clonal cell lines.

Clonal neuroblastoma x glioma hybrid cells frequently formed synapses with clonal mouse striated muscle cells. Clonal myotubes were similar to cultured mouse embryo myotubes with respect to acetylcholine sensitivity and other membrane properties examined. However, acetylcholine sensitivity measurements indicate that acetylcholine receptors of clonal myotubes are distributed more uniformly over the cell surface than the receptors of cultured mouse embryo myotubes.

Choline acetyltransferase activity is increased in combined cultures of spinal cord and muscle cells from mice.

The activity of choline acetyltransferase was more than tenfold greater in combined cultures of spinal cord and muscle cells than in cultures of spinal cord cells alone. This increase was associated with the formation of functional neuromuscular junctions in culture. Counts of silver-stained cells and determinations of other enzyme activities indicated that the increased choline acetyltransferase activity was not due to nonspecific neuronal survival but reflected greater activity in the surviving neurons. Hence, muscle had a marked, highly specific trophic effect on the cholinergic neurons that innervated it.

A coeruleo-spinal system in culture.

In combined cultures of dissociated spinal neurons and explants from the region of locus coeruleus, rich catecholamine-containing fiber projections from the explant to the surrounding regions of spinal neurons were demonstrated by fluorescence histochemistry. Electrical stimulation of the explant resulted in slow depolarizing responses in many of the spinal neurons. Cells exhibiting this type of response were also usually depolarized by local application of noradrenaline, whereas other, unresponsive neurons usually were not. The depolarizing responses to electrical stimulation and to noradrenaline were both increased by depolarizing current injection and decreased by hyperpolarizing current. These and other data suggest that the depolarizing responses of the spinal neurons to explant stimulation are mediated by noradrenaline released from axons of locus coeruleus neurons.

Axonal proteins of presynaptic neurons during synaptogenesis.

Changes occur in the synthesis and axonal transport of neuronal proteins in dorsal-root ganglia axons as a result of contact with cells from the spinal cord during synapse formation. Dorsal-root ganglia cells were cultured in a compartmental cel culture system that allows separate access to neuronal cell bodies and their axons. When cells from the ventral spinal cord were cultured with the dorsal-root ganglia axons, synapses were established within a few days. Metabolic labeling and two-dimensional electrophoresis revealed that four of more than 300 axonal proteins had changed in their expression by the time synapses were established. The highly selective nature of these changes suggests that the proteins involved may be important in the processes of axon growth and synapse formation and their regulation by the regional environment.

Synaptic connections in vitro: modulation of number and efficacy by electrical activity.

The functional architecture of synaptic circuits is determined to a crucial degree by the patterns of electrical activity that occur during development. Studies with an in vitro preparation of mammalian sensory neurons projecting to ventral spinal cord neurons slow that electrical activity induces competitive processes that regulate synaptic efficacy so as to favor activated pathways over inactive convergent pathways. At the same time, electrical activity initiates noncompetitive processes that increase the number of axonal connections between these sensory and spinal cord neurons.

Opposing actions of protein kinase A and C mediate Hebbian synaptic plasticity.

A compartmental nerve-muscle tissue culture system expresses Hebbian activity-dependent synapse modulation. Protein kinase C (PKC) mediates a heterosynaptic loss of efficacy, and we now show that protein kinase A (PKA) is involved in homosynaptic stabilization. Both work through postsynaptic changes in the acetylcholine receptor (AChR) as measured electrophysiologically and by imaging techniques.

Transmission on an active electrical response between fibroblasts (L cells) in cell culture.

An active electrical response, the hyperpolarizing activation or H.A. response, is characteristic of L cells (a continuous line of fibroblasts) and is transmitted in a decremental manner between contiguous cells. Direct electrical coupling between pairs of L cells occurs occasionally, but transmission of the active electrical response is not dependent on such electrical connections. Some L cells are sensitive to acetylcholine but the transmitted response is not dependent on a cholinergic mechanism. 5-Iodosalicylate blocks the active electrical response. The response can be elicited readily by mechanical stimuli, and thus can serve both as a mechanical and chemical receptor mechanism and as a means of communication between cells.

Evidence for an intracellular calcium store releasable by surface stimuli ifibroblasts (L cells).

A spontaneously occurring or electrically elicited hyperpolarizing activation (HA) in L cells was previously shown to be due to a specific increase in the membrane K+ permeability (Nelson et at. 1972. J. Gen. Physiol. 60:58--71). Intracellular injection of Ca++ elicits an identical hyperpolarizing response which suggests that the increased K+ permeability associated with the HA is mediated by an increase in cytoplasmic Ca++. In zero-Ca, EGTA-containing saline the proportion of cells in which HA's can be evoked decreases, but the amplitude of those HA's that are produced is comparable to that of HA's in normal Ca saline. Co++ does block the HA but only after a period of 2 h or longer; D-600 does not affect the HA. The observations, with others, suggest that the primary source of the Ca mediating the HA response is intracellular. In L cells the endoplasmic reticulum forms morphologically specialized appositions with the surface membrane which resemble structures at the triads of muscle that are thought to mediate coupling between surface membrane electrical activity and contraction via Ca release from the sarcoplasmic reticulum. The similar structures in L cells may mediate coupling between surface membrane electrical, mechanical, or chemical stimuli and the HA response via release of Ca from the endoplasmic reticulum. Surface-coupled release of Ca from intracellular stores might also regulate a number of other intracellular functions in nonmuscle cells.

An active electrical response in fibroblasts.

L cells have a resting potential of about -16 mv (internal negative) at 37 degrees C in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and a potassium concentration of 5.4 mM. Membrane resistivity is about 20,000 ohmcm(2) when the surface filopodia described by others are taken into account. Mechanical and electrical stimuli can evoke an active response from mouse L cells, cells of the 3T3 line, and normal fibroblasts which we have termed hyperpolarizing activation or the H.A. response. This consists of a prolonged (3-5 sec) increase in the membrane permeability by a factor of 2-10 with a parallel increase in membrane potential to about -50 mv. The reversal potential for the H.A. response is -80 mv. The resting cells are depolarized to about -12 mv when the external medium contains 27 mM potassium, and the potential reached at the peak of the H.A. response is about -30 mv. The reversal potential for the H.A. response is about -40 mv in 27 mM external potassium. This effect of potassium ions on the reversal potential of the H.A. response leads us to conclude that the response represents an increase in membrane permeability, predominantly to potassium, by at least a factor of five. This increase must be greater than 20-fold if previous measurements of the ratio of potassium permeability to chloride permeability in L cells are valid for the preparation used in the present study.

Adult-onset acid maltase deficiency. Electrophysiological properties of aneurally cultured muscle.

Electrophysiological studies were performed on aneurally cultured muscle cells from one patient with adult-onset acid maltase deficiency (AAMD) and from controls. The cells from the patient with AAMD had a higher mean resting membrane potential, a lower input resistance, and a higher incidence of action potentials at resting membrane potential than the control cells. Therefore, sarcolemma maturation was not adversely affected. The AAMD cells had membrane thresholds and action potential amplitudes similar to those of the control cells, and rarely produced repetitive action potentials. Therefore, the membrane instability noted in adult muscle fibers from the patient with AAMD was not present in cultured cells. This study does not support the suggestion that the biochemical and morphological abnormalities present in muscle fibers of patients with AAMD are sufficient to cause the electrical abnormalities.

Electrophysiologic properties of aneurally cultured muscle from patients with myotonic muscular atrophy.

Electrophysiologic studies were performed on aneurally cultured human muscle cells from seven patients with myotonic muscular atrophy and seven controls. There was no significant difference in resting membrane potential. When the cells were hyperpolarized to -80 mV, there was no significant difference in effective membrane resistance, effective membrane capacitance, normalized membrane conductance, membrane threshold, action potential amplitude, or maximum rate of rise of the action potential. Repetitive discharges were elicited by anodal-break excitation in a few cells from each group. We found no evidence that cultured myotonic atrophy muscle cells are electrically different from control cells.